WO2018205286A1 - 含荧光发色团的缀合物、嵌段共聚物及其制备方法和应用 - Google Patents

含荧光发色团的缀合物、嵌段共聚物及其制备方法和应用 Download PDF

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WO2018205286A1
WO2018205286A1 PCT/CN2017/084503 CN2017084503W WO2018205286A1 WO 2018205286 A1 WO2018205286 A1 WO 2018205286A1 CN 2017084503 W CN2017084503 W CN 2017084503W WO 2018205286 A1 WO2018205286 A1 WO 2018205286A1
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conjugate
antibody
drug
polymer
formula
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刘世勇
刘固寰
姜琰琰
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University of Science and Technology of China USTC
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University of Science and Technology of China USTC
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Priority claimed from CN201710317186.7A external-priority patent/CN106967167B/zh
Priority claimed from CN201710317704.5A external-priority patent/CN107056738B/zh
Priority claimed from CN201710316952.8A external-priority patent/CN107189775B/zh
Application filed by University of Science and Technology of China USTC filed Critical University of Science and Technology of China USTC
Priority to US16/611,191 priority Critical patent/US11129909B2/en
Publication of WO2018205286A1 publication Critical patent/WO2018205286A1/zh
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
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    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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Definitions

  • the invention relates to the technical field of organic bridging molecules, in particular to a conjugate containing a fluorescent chromophore, a block copolymer and a preparation method and application thereof.
  • ADCs antibody drug conjugates
  • Protein-polymer conjugates are prepared by grafting, graft to and macropolymer copolymer through.
  • the preparation of antibody-drug conjugates and protein-polymers relies on the selection of suitable highly efficient coupling reactions and linking motifs.
  • selection is best without affecting protein/drug activity and antibody function.
  • Modification of specific sites of the native protein by protein/antibody tissue engineering eg, reduction of disulfide bonds, modification of carbon ends, or oxidation of polysaccharides
  • direct reaction with specific amino acids can be achieved by direct reaction with specific amino acids.
  • Synthetic functional polymers/drugs covalently attached to proteins/antibodies primarily utilizing orthogonal click reactions such as the Staudinger reaction, copper-catalyzed azide-alkyne cycloaddition (CuAAC), and tension-promoted azide-rings Cycloaddition of alkynes (SPAAC), DA addition, Michael addition, and formation of ruthenium/iridium from aldehydes and ketones.
  • orthogonal click reactions such as the Staudinger reaction, copper-catalyzed azide-alkyne cycloaddition (CuAAC), and tension-promoted azide-rings Cycloaddition of alkynes (SPAAC), DA addition, Michael addition, and formation of ruthenium/iridium from aldehydes and ketones.
  • CuAAC copper-catalyzed azide-alkyne cycloaddition
  • SPAAC tension-promoted azide-rings Cycloaddition of alkynes
  • Targeted antibodies labeled with fluorescent probes can be imaged in real time with high resolution in vivo, and can be used for initial detection and monitoring of residual cancer tissue during surgery.
  • probes that have been fluorescent have inherent defects such as background interference and low signal to noise ratio.
  • Antibody-drug conjugates are capable of reducing systemic toxicity and enhancing the therapeutic efficiency of the coupled drug.
  • the design of the linkage in an antibody-drug conjugate is critical because it requires not only sufficient stability to provide adequate circulation to the masking drug, but also rapid and efficient release of the drug within the tumor cell.
  • Triggerable cleavable linkages are often used to link drugs and antibodies.
  • the current quantification of coupling efficiency and post-trigger release is largely dependent on ex situ techniques such as high performance liquid chromatography, gel electrophoresis, volume exclusion chromatography, and mass spectrometry. Real-time monitoring of the release process of antibody-drug conjugates at in vitro and cellular levels has not yet been achieved.
  • the technical problem to be solved by the present invention is to provide a conjugation containing a fluorescent chromophore.
  • the block, the copolymer and the preparation method and application thereof can monitor the conjugation efficiency of the conjugate in situ by infrared fluorescence emission intensity, and can be applied to target-mediated drug delivery, or transmission of polypeptide and anticancer drug.
  • the present invention provides a fluorochrome-containing conjugate having any of the following structures:
  • R 1 and R 2 are groups capable of quenching fluorescent chromophore fluorescence and capable of performing a "click" reaction.
  • the present invention provides a block copolymer having fluorescent emission properties, characterized by having the structure represented by Formula I-1:
  • n is 10 to 150.
  • the invention provides a preparation method of the above block copolymer having fluorescence emission properties, comprising the following steps:
  • the polymer B containing a thiol end group and the polymer C containing an azide group are subjected to a Michael reaction and a click reaction with the chemically conjugated molecule D to obtain a block copolymer represented by the formula I-1;
  • n is 10 to 150.
  • the conjugation efficiency of the block copolymer is monitored in situ by fluorescence emission intensity.
  • the present invention provides a targeted drug having fluorescent emission properties, having the structure shown in Formula II-1:
  • Target is a cyclic RGD polypeptide, folic acid or sulfonamide
  • Drug is doxorubicin, camptothecin or paclitaxel.
  • the invention provides a preparation method of the above targeted drug with fluorescent emission properties, comprising the following steps:
  • Targeting molecule F, drug molecule G and chemically conjugated molecule H are subjected to Michael reaction and reverse D-A addition reaction to obtain a targeted drug represented by formula II-1;
  • Target is a cyclic RGD polypeptide, folic acid or sulfonamide
  • Drug is doxorubicin, camptothecin or paclitaxel.
  • the conjugation efficiency of the drug molecule and the targeting molecule is monitored in situ by infrared fluorescence emission intensity.
  • the present invention provides the use of the above targeted drugs for targeted mediation of drug delivery.
  • the above fluorescent chromophore-containing conjugate provided by the present invention comprises a fluorescent chromophore and two highly reactive groups R 1 and R 2 attached to the fluorescent chromophore by a covalent bond.
  • the fluorescent chromophore in the complex initially has no or only weak fluorescence emission capability, and the fluorescent chromophore has strong fluorescence emission only when the two highly reactive groups are reacted together with the corresponding molecule, so Infrared Fluorescence Emission Intensity Monitors the conjugation efficiency of drug molecules and targeting molecules in situ and applies them to targeted mediation of drug delivery for real-time monitoring of polymer/drug conjugate release in vitro and at cellular levels .
  • the present invention provides an antibody-drug/probe conjugate having fluorescence emission properties having the structure of Formula I-2:
  • Ab is an antibody
  • DP is a fluorescent probe or a drug molecule.
  • the present invention provides an antibody-probe conjugate having fluorescence emission properties having the structure of Formula II-2:
  • Ab is a carcinoembryonic monoclonal antibody or Herceptin.
  • the invention provides a preparation method of the above antibody-probe conjugate, comprising the following steps:
  • the monoclonal antibody, the azide-containing fluorescent probe A and the bifunctional fluorescent molecule D are subjected to a Michael reaction and a click reaction to obtain an antibody-probe conjugate represented by the formula II-2;
  • the monoclonal antibody is a thiol-containing carcinoembryosin monoclonal antibody or Herceptin.
  • the conjugation efficiency of the antibody-probe conjugate is monitored in situ by fluorescence emission intensity.
  • the present invention provides the use of the above antibody-probe conjugate or the antibody-probe conjugate prepared by the above preparation method as an antigen and a quinone oxidoreductase reaction indicator.
  • the present invention provides an antibody-drug conjugate having fluorescence emission properties having the structure shown in Formula III:
  • DOX is doxorubicin
  • Ab is carcinoembryonic monoclonal antibody or Herceptin.
  • the invention provides a preparation method of the above antibody-drug conjugate, comprising the following steps:
  • the monoclonal antibody, the azide-containing prodrug molecule E and the bifunctional fluorescent molecule D undergo a Michael reaction and a click reaction to obtain an antibody-drug conjugate of the formula III;
  • DOX is doxorubicin
  • the monoclonal antibody is a thiol-containing carcinoembryonic monoclonal antibody or Herceptin.
  • the conjugation efficiency of the antibody-drug conjugate is monitored in situ by fluorescence emission intensity.
  • the present invention provides the use of the above antibody-drug conjugate or the antibody-drug conjugate prepared by the above preparation method as a fluorescent indicator for real-time monitoring of drug release.
  • the present invention provides the use of the above antibody-drug conjugate or the antibody-drug conjugate prepared by the above preparation method as a targeted drug release carrier.
  • the antibody-drug/probe conjugates provided by the present invention have self-reported conjugation efficiency characteristics, which are The fluorescent molecule bridges the antibody with the probe/drug molecule, and the difunctional fluorescent molecule itself has no or only weak fluorescence emission capability, and the fluorescent molecule is strong only when the antibody-probe/drug is conjugated Fluorescence emission, so the conjugation process can be monitored in situ by fluorescence monitoring and applied to the delivery of therapeutic peptides and anticancer drugs.
  • the present invention provides a protein/polypeptide-polymer conjugate having fluorescent emission properties having the structure of Formula I-3:
  • the POI is a protein or a polypeptide; and the polymer is a polymer.
  • the present invention provides a protein-polymer conjugate having fluorescent emission properties having the structure of Formula I-a:
  • the POI is bovine serum albumin or salmon calcitonin
  • the polymer is polyethylene glycol
  • the invention provides a preparation method of the above protein-polymer conjugate, comprising the following steps:
  • the thiol-containing bovine serum albumin, the azide-terminated polyethylene glycol, and the compound D are subjected to a Michael reaction and a click reaction to prepare a protein-polymer conjugate represented by the formula I-a;
  • the POI is bovine serum albumin, and the m is 23 to 445;
  • the thiol-containing salmon calcitonin, the azide-terminated polyethylene glycol, and the compound D are subjected to a Michael reaction and a click reaction to prepare a protein-polymer conjugate represented by the formula I-a;
  • POI salmon calcitonin
  • m 23 to 445.
  • the present invention provides a polypeptide-polymer conjugate having fluorescent emission properties having the structure of Formula I-b:
  • the POI is a matrix metalloproteinase to cleave the polypeptide
  • the Polymer is a polytrimethylene carbonate.
  • the invention provides a preparation method of the above polypeptide-polymer conjugate, comprising the following steps:
  • the matrix metalloprotease represented by Formula J can cleave the polypeptide, the azide-terminated polytrimethylene carbonate represented by Formula K, and Compound D for Michael reaction and click reaction to prepare a polypeptide represented by Formula I-b. Conjugate
  • the conjugation efficiency of the conjugate is monitored in situ by the fluorescence emission intensity.
  • the present invention provides a polymer vesicle composed of the above polypeptide-polymer conjugate or the polypeptide-polymer conjugate prepared by the above preparation method.
  • the polymer vesicle has a particle size of 60 to 150 nm.
  • the above polymer vesicles have matrix metalloenzyme response characteristics.
  • the present invention provides the use of the above polymer vesicles as a pharmaceutical carrier, or as a fluorescent indicator for real-time monitoring of drug release applications.
  • the protein/polypeptide-polymer conjugates provided by the present invention bridge proteins/polypeptides and polymers from bifunctional fluorescent molecules, which do not have or have only weak fluorescence emission capability, only when protein /Plaso-polymer conjugated fluorescent molecules have strong fluorescence emission, so the conjugation process of the protein/polypeptide-polymer conjugate can be monitored in situ by fluorescence changes, and the protein/polypeptide-polymer The conjugate is useful for the delivery of therapeutic polypeptides and anti-cancer drugs.
  • Figure 1 is a nuclear magnetic resonance spectrum of C1 prepared by the present invention
  • Figure 3 is an electrospray mass spectrum of C1 prepared according to the present invention.
  • Figure 5 is a graph showing the relationship between fluorescence change and conjugation efficiency in the preparation of C1 of the present invention.
  • Figure 6 is a gel permeation chromatogram of the block polymer prepared in Example 5 of the present invention.
  • Figure 7 is a graph showing the change in fluorescence during the reaction of preparing a block polymer according to Example 5 of the present invention.
  • Figure 8 is a graph showing the relationship between fluorescence change and conjugation efficiency during the conjugation of a block polymer according to Example 5 of the present invention.
  • Figure 10 is an electrospray mass spectrum of a prodrug molecule prepared in Example 6;
  • Figure 11 is a nuclear magnetic resonance spectrum of the fluorescent probe A prepared in Example 7.
  • Figure 12 is an electrospray mass spectrum of the fluorescent probe A prepared in Example 7.
  • Figure 13 is a graph showing the progress of fluorescence change during the reaction of the antibody-probe conjugate prepared in Example 8.
  • Figure 14 is a graph showing the progress of fluorescence change during the reaction of the antibody-drug conjugate prepared in Example 8.
  • Figure 15 is a graph showing the fluorescence change process of the enzyme-detecting process of the antibody-probe conjugate prepared in Example 8.
  • Figure 16 is a diagram showing the detection of intracellular carcinoembryonic antigen and quinone oxidoreductase of the antibody-probe conjugate prepared in Example 8;
  • 17 is a nitro-reductase of the antibody-drug conjugate prepared in Example 8 triggering a fluorescence change process during drug release;
  • Figure 19 is a graph showing the fluorescence change process of the conjugation process of the bovine serum albumin-polyethylene glycol conjugate prepared in Example 10;
  • Figure 20 is a graph showing the relationship between the fluorescence change and the conjugation efficiency of the bovine serum albumin-polyethylene glycol conjugate prepared in Example 10;
  • 21 is a gel electrophoresis pattern of sodium dodecyl sulfate-polyacrylamide prepared by preparing bovine serum albumin-polyethylene glycol conjugate of Example 10;
  • Figure 22 is a graph showing the fluorescence change process of the conjugation process of the salmon calcitonin-polyethylene glycol conjugate prepared in Example 11;
  • Figure 23 is a volume exclusion chromatogram of the salmon calcitonin-polyethylene glycol conjugate prepared in Example 11;
  • Figure 24 is a gel electrophoresis pattern of sodium dodecyl sulfate-polyacrylamide prepared by preparing the salmon calcitonin-polyethylene glycol conjugate of Example 11;
  • Figure 25 is a transmission electron micrograph of the vesicles prepared in Example 14 in water
  • Figure 26 is a graph showing the controlled release profile of the drug of Example 15.
  • the present invention provides a fluorochrome-containing conjugate having any of the following structures:
  • R 1 and R 2 are groups capable of quenching fluorescent chromophore fluorescence and capable of performing a "click" reaction.
  • the above fluorescent chromophore-containing conjugate provided by the present invention comprises a fluorescent chromophore and two highly reactive groups R 1 and R 2 attached to the fluorescent chromophore by a covalent bond.
  • the fluorescent chromophore in the complex initially has no or only weak fluorescence emission capability, and the fluorescent chromophore has strong fluorescence emission only when the two highly reactive groups are reacted together with the corresponding molecule.
  • the compound is represented by the above formulas C1 to C3, wherein the fluorescent chromophore in C1 is a coumarin element, the fluorescent chromophore in C2 is a naphthalene diimide element, and the fluorescent chromophore in C3 is Wujiachuan primitive.
  • a curved line indicates a connection key.
  • the molecular species reacted with the above conjugate differs depending on R 1 and R 2 , R1-1 and R2-3 correspond to a molecule containing a thiol group, and R1-2 corresponds to a trans-cycloxin-containing group.
  • the present invention employs a polymer, a targeting molecule or a drug molecule containing the above specific reactive group to react with the above conjugate to obtain a block polymer or a targeted drug having fluorescent emission properties.
  • the present invention provides a block copolymer having fluorescent emission properties, which is prepared by using the above conjugate as a bridging molecule, and has the structure represented by Formula I-1:
  • n is preferably 10 to 150.
  • the invention also provides a preparation method of the above block copolymer having fluorescent emission properties, comprising the following steps:
  • the polymer B containing a thiol end group and the polymer C containing an azide group are subjected to a Michael reaction and a click reaction with the chemically conjugated molecule D to obtain a block copolymer represented by the formula I-1;
  • n is 10 to 150.
  • the conjugation efficiency of the block copolymer is monitored in situ by the fluorescence emission intensity.
  • the invention also provides a targeted drug having fluorescent emission properties, which is prepared by using the above conjugate as a bridging molecule, and has the structure represented by the formula II-1:
  • Target is a targeting group, including: a cyclic RGD polypeptide, folic acid or a sulfonamide;
  • Drug is a drug group, including: doxorubicin, camptothecin or paclitaxel.
  • the invention also provides a preparation method of the above targeted drug with fluorescent emission properties, comprising the following steps:
  • Targeting molecule F, drug molecule G and chemically conjugated molecule H are subjected to Michael reaction and reverse D-A addition reaction to obtain a targeted drug represented by formula II-1;
  • Target is a targeting group, including: a cyclic RGD polypeptide, folic acid or a sulfonamide;
  • Drug is a drug group, including: doxorubicin, camptothecin or paclitaxel.
  • the conjugation efficiency of the drug molecule and the targeting molecule is monitored in situ by infrared fluorescence emission intensity.
  • the invention also provides the use of the above targeted drugs for targeted mediation of drug delivery.
  • the present invention provides an antibody-drug/probe conjugate having fluorescence emission properties having the structure of Formula I-2:
  • Ab is an antibody
  • DP is a fluorescent probe or a drug molecule.
  • the antibody-drug/probe conjugates provided by the present invention have a self-reported conjugation efficiency characteristic that bridges an antibody and a probe/drug molecule from a bifunctional fluorescent molecule that does not have or only Has a weak fluorescence emission capability, and only when the antibody-probe/drug is conjugated, the fluorescent molecule has strong fluorescence emission, so the conjugation process can be monitored in situ by fluorescence monitoring and applied to therapeutic peptides and anticancer drugs. Transmission.
  • the present invention provides an antibody-probe conjugate having fluorescence emission properties, having the structure shown in Formula II-2:
  • Ab is a carcinoembryonic monoclonal antibody or Herceptin.
  • the invention also provides a preparation method of the above antibody-probe conjugate, comprising the following steps:
  • the monoclonal antibody, the azide-containing fluorescent probe A, and the bifunctional fluorescent molecule D undergo a Michael reaction and a click reaction to obtain an antibody-probe conjugate represented by the formula II-2.
  • the monoclonal antibody is a thiol-containing carcinoembryonic monoclonal antibody or Herceptin.
  • the azide-containing fluorescent probe A is represented by the following formula A:
  • the difunctional fluorescent molecule D is represented by the following formula D:
  • the raw material ratio and reaction conditions of the above reaction are not particularly limited, and may be a ratio and a condition of a Michael reaction and a click reaction which are conventional in the art.
  • the conjugation efficiency of the antibody-probe conjugate is monitored in situ by fluorescence emission intensity.
  • the above antibody-probe conjugate provided by the present invention has a fluorescence change under the action of a quinone oxidoreductase, and thus can be used as an antigen and a quinone oxidoreductase reaction indicator.
  • the present invention provides an antibody-drug conjugate having fluorescence emission properties having the structure of Formula III:
  • DOX is doxorubicin
  • Ab is carcinoembryonic monoclonal antibody or Herceptin.
  • the invention also provides a preparation method of the above antibody-drug conjugate, comprising the following steps:
  • the monoclonal antibody, the azide-containing prodrug molecule E and the bifunctional fluorescent molecule D undergo a Michael reaction and a click reaction to obtain an antibody-drug conjugate of the formula III;
  • the monoclonal antibody is a thiol-containing carcinoembryonic monoclonal antibody or Herceptin.
  • the azide-containing prodrug molecule E is represented by the following formula E:
  • DOX is doxorubicin.
  • the amino group in formula E is an amino group on the doxorubicin tetrahydropyran ring.
  • the difunctional fluorescent molecule D is represented by the following formula D:
  • the raw material ratio and reaction conditions of the above reaction are not particularly limited, and may be a ratio and a condition of a Michael reaction and a click reaction which are conventional in the art.
  • the conjugation efficiency of the antibody-drug conjugate is monitored in situ by fluorescence emission intensity.
  • the above antibody-drug conjugate provided by the invention has the release of the original drug doxorubicin under the action of nitroreductase, and thus can be used as a fluorescent indicator to monitor drug release in real time or as a targeted drug release carrier.
  • the present invention provides a protein/polypeptide-polymer conjugate having fluorescence emission properties, Structure shown in Formula I-3:
  • the POI is a protein or a polypeptide; and the polymer is a polymer.
  • the protein/polypeptide-polymer conjugate bridges a protein/polypeptide with a polymer from a bifunctional fluorescent molecule that does not have or has only weak fluorescence emission capability, only when the protein/polypeptide -
  • the fluorescent molecule has a strong fluorescent emission after conjugation of the polymer, so the conjugation process of the protein/polypeptide-polymer conjugate can be monitored in situ by fluorescence changes and the protein/polypeptide-polymer conjugation
  • the substance can be applied to the delivery of therapeutic polypeptides and anticancer drugs.
  • the POI is bovine serum albumin
  • the Polymer is polyethylene glycol, the structure of which is as shown in Formula I-a:
  • the above protein-polymer conjugates are preferably prepared as follows:
  • the thiol-containing bovine serum albumin, the azide-terminated polyethylene glycol, and the compound D are subjected to a Michael reaction and a click reaction to prepare a protein-polymer conjugate represented by the formula I-a;
  • the poly(ethylene glycol) structure of the azide end group is as follows:
  • the raw material ratio and reaction conditions of the above reaction are not particularly limited, and may be a ratio and a condition of a Michael reaction and a click reaction which are conventional in the art.
  • the conjugation efficiency of the above protein-polymer conjugates is monitored in situ by fluorescence emission intensity.
  • the POI is salmon calcitonin
  • the polymer is polyethylene glycol
  • the structure is as shown in Formula I-a:
  • the above protein-polymer conjugates are preferably prepared as follows:
  • the thiol-containing salmon calcitonin, the azide-terminated polyethylene glycol, and the compound D are subjected to a Michael reaction and a click reaction to prepare a protein-polymer conjugate represented by the formula I-a.
  • the azido end group of polyethylene glycol and compound D are the same as above, and will not be described herein.
  • the raw material ratio and reaction conditions of the above reaction are not particularly limited, and may be a ratio and a condition of a Michael reaction and a click reaction which are conventional in the art.
  • the conjugation efficiency of the above protein-polymer conjugates is monitored in situ by fluorescence emission intensity.
  • the POI is a matrix metalloproteinase cleavable polypeptide, and the sequence thereof is ⁇ APVGLIG ⁇ AC-SH, wherein SH is a thiol group (the thiol group is located at a carbon terminal cysteine residue), and the above polypeptide is purchased.
  • the Polymer is polytrimethylene carbonate. Its structure is shown in formula I-b:
  • the preparation method is preferably:
  • the matrix metalloprotease represented by Formula J can cleave the polypeptide, the azide-terminated polytrimethylene carbonate represented by Formula K, and Compound D for Michael reaction and click reaction to prepare a polypeptide represented by Formula I-b. Conjugate
  • the raw material ratio and reaction conditions of the above reaction are not particularly limited, and may be a ratio and a condition of a Michael reaction and a click reaction which are conventional in the art.
  • the conjugation efficiency of the above protein-polymer conjugates is monitored in situ by fluorescence emission intensity.
  • the invention also discloses a vesicle composed of the above polypeptide-polymer conjugate or the polypeptide-polymer conjugate prepared by the above preparation method.
  • the preparation method of the vesicles of the present invention is not particularly limited, and may be a preparation method well known to those skilled in the art.
  • the above polypeptide-polymer conjugate is dissolved in DMSO, deionized water is added, and then dialyzed against deionized water.
  • the above polymer vesicles provided by the invention have uniform dispersion and uniform particle size distribution, and the particle size distribution thereof is 60-150 nm.
  • the above polymer vesicles have matrix metalloenzyme response characteristics and thus can be used as a drug carrier or as a fluorescent indicator to monitor drug release in real time.
  • the invention is provided below as a Bruton tyramine in combination with the examples.
  • fluorescent chromophore-containing conjugate, block copolymer, antibody-drug/probe conjugate, protein/polypeptide-polymer conjugate, target provided by the present invention are combined with the following examples.
  • the drug and its preparation method and application are described in detail.
  • the synthetic route is as follows:
  • dimethyl acrylamide (DMA) (2.0 g, 20.2 mmol)
  • 4-cyano-4-(propylthiothiocarbamoyl)pentanoic acid was added to a sealed tube with a stir bar (223 mg, 0.8 mmol), 2,2'-diazoisobutyronitrile (AIBN) (16 mg, 0.98 mmol) and dioxane (4 mL).
  • AIBN 2,2'-diazoisobutyronitrile
  • the sealing tube was degassed by freezing-pumping-thawing operation three times, then sealed under vacuum, and reacted at 70 ° C for 6 h in a constant temperature oil bath, the reaction was immediately quenched with liquid nitrogen, deblocked, and diluted with THF. The precipitation in excess diethyl ether was repeated three times and the product was dried in vacuo to give a yellow powder PDMA (1.63 g, 86% yield).
  • the actual degree of polymerization of the PDMA block was 38 by 1 H NMR, so the product was abbreviated as PDMA 38 .
  • PDMA 38 (500 mg, 0.22 mmol) was dissolved in dry CH 2 Cl 2 and NH 2 NH 2 ⁇ H 2 O (55 mg, 1.1 mmol) was added dropwise to the reaction tube for 2 h, then concentrated and concentrated with diethyl ether. Three times, drying in a vacuum oven gave white powder HS-PDMA 38 (420 mg, yield 93.8%).
  • HS-PDMA 38 35 mg, 17.5 ⁇ mol
  • PEG 45- N 3 350 mg, 175 ⁇ mol
  • PEG 227 external standard, 300 mg
  • C1 5.2 mg, 17.5) ⁇ mol, dissolved in 5 mL of DMSO
  • CuSO 4 /vitamin C 1/5 molar ratio
  • the reaction system was stirred at 25 ° C for different times. After reaching a predetermined time, about 1.0 mL of the reaction solution was sampled and diluted into 9.0 mL of THF. Copper ions were quickly added to the copper ion adsorption resin (American Marine Chemical Company, 200 mg) and then shaken for 5 min. The supernatant was filtered through a 0.22 ⁇ m sterile syringe filter prior to further fluorescence and GPC testing.
  • Fig. 6 is a gel permeation chromatogram
  • Fig. 7 is a fluorescence change curve during the reaction
  • Fig. 8 is a fluorescence during the reaction. The relationship between change and conjugation efficiency.
  • the present invention is capable of monitoring the conjugation efficiency of the above polymer in situ by infrared fluorescence emission intensity.
  • Example 6 C1 mediates the synthesis of targeted drugs
  • the synthetic route is as follows:
  • the experimental results show that the fluorescence enhancement of the fluorescent chromophore is linearly related to the conjugation efficiency of the targeted drug. Therefore, the conjugation efficiency of the targeted drug can be determined by monitoring the fluorescence change in situ.
  • the synthetic route is as follows:
  • Example 8 Anti-CEA antibody mediated by bifunctional fluorescent molecule C1 and fluorescent conjugate of DOX-pNB-N 3 or QNAM-N 3
  • Dithiothreitol (DTT, 154 mg, 100 ⁇ mol) was dissolved in phosphate buffered saline (PBS) (10 mL, pH 8.0, 50 mM), and then 10 ⁇ L of the above solution (containing 0.1 ⁇ mol DTT) was added to the antibody containing anti-CEA ( ACEA, 1 mg) in PBS (1 mL, pH 8.0, 50 mM), stirred at 37 ° C for 30 min, the product was purified by ultrafiltration (centrifugation -0.5, micropore, molecular weight cutoff 10 kDa), which was redissolved in PBS before use.
  • PBS phosphate buffered saline
  • the thiol content was determined using 5,5'-dimercapto(2-nitrobenzoic acid) (DNTB) as a probe, and the number of thiol groups per antibody molecule was about 4.
  • DNTB 5,5'-dimercapto(2-nitrobenzoic acid)
  • FIG. 13 is a process of fluorescence change during the reaction of preparing the antibody-probe conjugate.
  • FIG. 14 is a graph showing the fluorescence change process during the reaction of preparing the antibody-drug conjugate.
  • Figure 15 is a graph showing the fluorescence change process of the antibody-probe conjugate prepared in Example 8 during the enzyme detection process; QNAM-C1-ACEA antibody-probe conjugate was cultured in an aqueous system with NADPH/NQO1, and NAM emission intensity was observed. A significant increase ( ⁇ 530 nm) is accompanied by a large drop in the emission intensity of C1 at ⁇ 435 nm. A change of about 20 times the FRET ratio (two emission peak intensity ratios) was observed over a period of about 1 hour (see inset). The above results indicate the occurrence of an efficient FRET process between the C1 coumarin linkage and the enzymatically produced NAM residue. A mutation ( ⁇ 20-fold) in the FRET ratio after the action of the QNAM-C1-ACEA conjugate with NQO1 indicates that the antibody-probe conjugate can be used to detect the NQO1 enzyme concentration.
  • Figure 16 is a graph showing the detection of intracellular carcinoembryonic antigen and quinone oxidoreductase of the antibody-probe conjugate prepared in Example 8.
  • Live HepG2 cells (lacking CEA, lacking NQO1), LS180 cells (CEA normal, lacking NQO1) and HT29 cells (CEA normal, NQO1 normal) were co-cultured with QNAM-C1-ACEA antibody-probe conjugate.
  • CEA normal and NOQ1-deficient LS180 cells with confocal laser scanning microscopy (CLSM) images showed intermittent blue light-emitting points in C1 coumarin and very few NAM green emission signals in the cells.
  • CLSM confocal laser scanning microscopy
  • the NQO1 enzyme is mainly located in the cytoplasm of certain types of cancer cells, the above results indicate that the QNAM-C1-ACEA conjugate-probe can only display strong green channel emission in an "AND" logic gate type (ie, CEA and NQO1 exists at the same time).
  • Figure 17 is a diagram showing the process of nitro-reductase of the antibody-drug conjugate prepared in Example 8 triggering the fluorescence change process of the drug release process; when the nitroreductase/reduced coenzyme II is co-cultured with DOX-C1-ACEA, the coupled A The mycin drug is continuously released linearly over time.
  • the release process of doxorubicin was accompanied by a significant increase in the emission of the C1 linkage at 435 nm and a significant decrease in the emission of doxorubicin at 590 nm, which clearly indicates fragmentation of the drug-antibody conjugate and disruption of the FRET process.
  • the HT29or HepG2 cells ( ⁇ 10 5 ) were spread in a 35 mm glass bottom culture dish overnight, then DOX-C1-ACEA was co-cultured with the cells at 37 ° C for 24 h, and the cells were washed with PBS (3 ⁇ 1 mL) and DMEM medium.
  • Cellular fluorescence imaging needs to be performed under a Leica SP5 confocal microscope. The C1 group contained in the sample was excited at 405 nm, the acridine orange was excited at 488 nm, and the DOX excitation was at 543 nm.
  • Figure 18 is a diagram showing the intracellular nitroreductase-containing drug release process of carcinoembryonic antigen containing the antibody-drug conjugate prepared in Example 9.
  • DOX-C1-ACEA conjugate was incubated with CEA-containing HT29 cells at normal oxygen levels, a strong C1 green emission in the cytoplasm and a red dot emission of doxorubicin were observed by confocal microscopy.
  • the blue/green emissions can be co-located with each other.
  • the blue fluorescence intensity of the C1 coumarin linkage for HepG2 cells lacking CEA was only 22% of that in HT29.
  • the drug-antibody conjugate and the probe-antibody conjugate can be constructed by linking the molecule C1 and monitoring the conjugation efficiency in situ by fluorescence.
  • Bovine serum albumin BSA (498 mg, 7.5 ⁇ mol) was dissolved in phosphate buffered saline (PBS) (70 mL, pH 6.5, 0.1 M, containing 1 mM EDTA) while tris(2-chloroethyl)phosphate hydrochloride (TCEP) • HCl) (21.5 mg, 75 ⁇ mol) was dissolved in PBS (2.5 mL) and then added dropwise to the above BSA solution. After 4 h, the solution was dialyzed against deionized water for 24 h (2.0 kDa molecular weight cut off) and then lyophilized to obtain BSA red .
  • PBS phosphate buffered saline
  • TCEP tris(2-chloroethyl)phosphate hydrochloride
  • BSA or BSA red (4 mg, 0.06 ⁇ mol) was dissolved in PBS (0.9 mL, pH 7.0, 50 mM), C1 (0.6 ⁇ mol, dissolved in 0.1 mL DMSO), PEG 227- N 3 (6 mg, 0.6 ⁇ mol) and CuSO 4 /Na-ascorbate (1/5 molar ratio) was added to the solution, stirred at 25 ° C for different times, and the conjugate progress was detected by in situ observation of a change in fluorescence emission intensity at about 420 nm.
  • the gel electrophoresis pattern is shown in Fig. 21.
  • TCEP ⁇ HCl (14.2 mg, 50 ⁇ mol) was dissolved in PBS (2 mL, pH 7.0, 50 mM), and 40 ⁇ L of the above solution (containing 1 ⁇ mol of TCEP ⁇ HCl) was added to the sCT containing sputum calcitonin (sCT, 1.7 mg, 0.5 ⁇ mol PBS (9 mL).
  • sCT sputum calcitonin
  • the molecular weight of the prepared protein-polymer conjugate was determined by volume exclusion chromatography, and the volume exclusion chromatogram is shown in FIG.
  • the gel electrophoresis pattern is shown in Fig. 24.
  • TMC trimethylene carbonate
  • PTMC 14- N 3 (7.5 mg, 5.0 ⁇ mol) and C1 (1.5 mg, 5.1 ⁇ mol) were dissolved in DMSO (1.8 mL), PVGLIG (8.1 mg, 10.1 ⁇ mol) and CuSO 4 /ascorbic acid (1/5 molar ratio) After being dissolved in 0.2 mL of deionized water, the reaction system was stirred at 25 ° C, and the conjugation process was detected by fluorescence in situ. After co-culture for ⁇ 3h, copper ion adsorption resin (100mg) was quickly added to remove copper ions. After shaking for 5 minutes, the supernatant was filtered through a 0.22 ⁇ m sterile syringe filter. The filtrate was filtered with excess cold acetonitrile. The precipitate was collected, centrifuged, and the precipitate was dried in a vacuum oven overnight.
  • PVGLIG-C1-PTMC 14 (2mg) was dissolved in 1mL DMSO, added to a 15mL vial containing a magnetic stir bar, stirred at room temperature for 3h, and added 9mL deionized water ( ⁇ 500rpm) within ⁇ 20s under stirring conditions. Stirring was continued for 5 h and then dialyzed against deionized water (molecular weight cut off 3.5 kDa) for 24 h.
  • Example 15 PVGLIG-C1-PTMC 14 polymer vesicle-embedded doxorubicin hydrochloride
  • PVGLIG-C1-PTMC 14 (2mg) was dissolved in 1mL DMSO and added to a 15mL vial containing a magnetic stir bar. Add DOX ⁇ HCl-containing deionized water (5g/L, 2mL) with stirring. 7 mL of deionized water was added at the same speed, stirring was continued for 5 hours, and then dialyzed against deionized water (molecular weight cut off of 3.5 kDa) for 24 hours. The loading content of DOX is -8.0% by weight.
  • the drug controlled release application curve is shown in Figure 26.
  • the above protein/polypeptide-polymer conjugate prepared by the present invention can monitor the conjugation efficiency in situ by the fluorescence emission intensity, and is applied to the delivery of therapeutic polypeptides and anticancer drugs.

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Abstract

本发明提供了一种含荧光发色团的缀合物，具有C1～C3任一结构。本发明提供的上述含荧光发色团的缀合物，包含一个荧光发色团和通过共价键连接在荧光发色团上的两个高反应活性基团R1与R2，所述缀合物中的荧光发色团初始不具有或者仅具有弱的荧光发射能力，仅当两个高反应活性基团共同与相应分子反应后，荧光发色团才具有强的荧光发射，因此可以通过红外荧光发射强度原位监控药物分子与靶向分子缀合效率，并应用于靶向介导的药物传输。

Description

含荧光发色团的缀合物、嵌段共聚物及其制备方法和应用
本申请要求于2017年05月08日提交中国专利局、申请号为201710317704.5、发明名称为“含荧光发色团的缀合物、嵌段共聚物、靶向药物及其制备方法和应用”，和2017年05月08日提交中国专利局、申请号为201710316952.8、发明名称为“具有荧光发射性质的抗体-药物/探针缀合物及其制备方法和应用”，和2017年05月08日提交中国专利局、申请号为201710317186.7、发明名称为“具有荧光发射性质的蛋白质/多肽-聚合物缀合物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及有机桥接分子技术领域，尤其涉及一种含荧光发色团的缀合物、嵌段共聚物及其制备方法和应用。
背景技术
多肽、蛋白质及抗体与合成聚合物、药物和成像探针的共价功能化形成了重要的可应用于临床治疗的生物偶联物，与蛋白质-聚合物偶联物，抗体-药物偶联物一起，成为典型的例子。蛋白质-聚合物偶联物可追溯到1970年，Davis，Abuchowski和合作者报道了聚乙二醇(PEG)与牛血清白蛋白的偶联。这项技术现在被称为PEGylation且延伸到了许多聚合物类型上，如，响应性聚合物和两性离子聚合物。合成的聚合物接到蛋白质上如PEGylation能带来许多优点，包括增强蛋白质的溶解性和稳定性，减少免疫原性，增加血液循环半衰期，目前至少PEGylated蛋白质已被美国食品与药物管理局(FDA)认证。另一方面，抗体药物偶联物(ADCs)与能特异性靶向病变部位的单克隆抗体的结合能杀死癌细胞。两种FDA认证的ADCs，brentuximabvedotin(商品名，Adcetris)和曲妥单抗(商品名，Kadcyla)，是目前可商购的，目前临床上使用的ADCs约40种。
蛋白质-聚合物偶联物通过长出支链(graftfrom)、嫁接支链(graft to)和大单体共聚接枝(graft through)的方法来制备。抗体-药物偶联物和蛋白质-聚合物的制备都依赖于选择合适的高效的偶联反应和连接基元。对于蛋白质/抗体偶联物来说，选择不影响蛋白质/药物活性和抗体功能是最好的。典型的， 可以通过蛋白质/抗体组织工程天然蛋白质特异性位点的改性(如，二硫键的还原，碳末端的改性，或多糖的氧化)，和直接利用特定氨基酸的特异性反应来实现。合成的功能性聚合物/药物共价接到蛋白质/抗体上主要利用了正交的click反应，如Staudinger反应，铜催化的叠氮-炔环加成(CuAAC)，张力促进的叠氮-环炔烃的环加成(SPAAC)，D-A加成，迈克尔加成，和由醛和酮形成肟/腙。新的设计原则的引入，如，模块化设计，温和的合成方式，光学示踪和多功能集成的能力，进一步促进了该领域的发展。
值得注意的是，即使是最优设计的蛋白质-聚合物偶联物，蛋白质功能和活性的明显减弱是不可避免的。一种解决方法是制备可断裂的生物偶联物，在体内随着时间的增加释放出天然的蛋白质。在细胞内化的过程中，ADCs应能有效释放出其活性的药物负载，以展现出细胞毒性。然而，监测发现蛋白质-聚合物偶联物和抗体-药物偶联物的偶联和随后释放蛋白质/药物的程度主要依赖于传统的非原位技术，如，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)，质谱(MS)，高效液相色谱(HPLC)，和体积排除色谱(SEC)。这阻止了对聚合物/药物偶联物在体外和细胞水平上的释放过程的实时监测。
另外，抗体定向的光学分子的成像在医学诊断和治疗响应评价中展现出巨大的潜力；也可直接与手术或内窥镜过程结合。用荧光探针标记的靶向抗体可以在体内实时高分辨成像，可用于初期的检测和手术中残留癌症组织的监测。然而，一直有荧光的探针也有本质的缺陷，如背景干扰和低的信噪比。
抗体-药物缀合物能够降低体系毒性和增强偶联药物的治疗效率。在抗体-药物缀合物中连接键的设计至关重要，因为它不仅要求能够为遮蔽药物在体循环提供充足的稳定性，而且在肿瘤细胞内应能快速有效地释放药物。触发性可断裂连接键常被用来连接药物和抗体。然而，目前偶联效率和触发后释放程度的定量大多依赖于非原位技术，如高效液相色谱、凝胶电泳、体积排除色谱以及质谱。目前尚不能实现对抗体-药物缀合物在体外和细胞水平上的释放过程的实时监测。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种含荧光发色团的缀合 物、嵌段共聚物及其制备方法和应用，能够通过红外荧光发射强度原位监控缀合物的缀合效率，并应用于靶向介导的药物传输，或多肽与抗癌药物的传输。
本发明提供了一种含荧光发色团的缀合物，具有以下任一结构：
Figure PCTCN2017084503-appb-000001
其中，R1和R2为能够淬灭荧光发色团荧光并且能够进行“点击”反应的基团。
优选的，
R1为
Figure PCTCN2017084503-appb-000002
R2为
Figure PCTCN2017084503-appb-000003
本发明提供了一种具有荧光发射性质的嵌段共聚物，其特征在于，具有式Ⅰ-1所示结构：
Figure PCTCN2017084503-appb-000004
其中，
x为
Figure PCTCN2017084503-appb-000005
m为23～445，n为10～150。
本发明提供了上述具有荧光发射性质的嵌段共聚物的制备方法，包括以下步骤：
含有巯基端基的聚合物B和含有叠氮基的聚合物C与化学缀合分子D进行Michael反应与点击反应，得到式Ⅰ-1所示嵌段共聚物；
Figure PCTCN2017084503-appb-000006
其中，
x为
Figure PCTCN2017084503-appb-000007
m为23～445，n为10～150。
优选的，通过荧光发射强度原位监控嵌段共聚物的缀合效率。
本发明提供了一种具有荧光发射性质的靶向药物，具有式Ⅱ-1所示结构：
Figure PCTCN2017084503-appb-000008
其中，
Target为环状RGD多肽、叶酸或磺酰胺；
Drug为阿霉素、喜树碱或紫杉醇。
本发明提供了上述具有荧光发射性质的靶向药物的制备方法，包括以下步骤：
靶向分子F、药物分子G和化学缀合分子H进行Michael反应和反向D-A加成反应，得到式Ⅱ-1所示靶向药物；
Figure PCTCN2017084503-appb-000009
Figure PCTCN2017084503-appb-000010
其中，
Target为环状RGD多肽、叶酸或磺酰胺；
Drug为阿霉素、喜树碱或紫杉醇。
优选的，通过红外荧光发射强度原位监控药物分子和靶向分子的缀合效率。
本发明提供了上述靶向药物在靶向介导药物传输中的应用。
本发明提供的上述含荧光发色团的缀合物，包含一个荧光发色团和通过共价键连接在荧光发色团上的两个高反应活性基团R1与R2，所述缀合物中的荧光发色团初始不具有或者仅具有弱的荧光发射能力，仅当两个高反应活性基团共同与相应分子反应后，荧光发色团才具有强的荧光发射，因此可以通过红外荧光发射强度原位监控药物分子与靶向分子缀合效率，并应用于靶向介导的药物传输，用于对聚合物/药物偶联物在体外和细胞水平上的释放过程的实时监测。
本发明提供了一种具有荧光发射性质的抗体-药物/探针缀合物，具有式Ⅰ-2所示结构：
Figure PCTCN2017084503-appb-000011
其中，Ab为抗体，DP为荧光探针或药物分子。
本发明提供了一种具有荧光发射性质的抗体-探针缀合物，具有式Ⅱ-2所示结构：
Figure PCTCN2017084503-appb-000012
其中，Ab为癌胚抗原单抗或赫赛丁。
本发明提供了上述抗体-探针缀合物的制备方法，包括以下步骤：
单克隆抗体、含叠氮基元的荧光探针A与双官能荧光分子D进行Michael反应和点击反应，得到式Ⅱ-2所示抗体-探针缀合物；
Figure PCTCN2017084503-appb-000013
其中，所述单克隆抗体为含巯基的癌胚抗原单抗或赫赛丁。
优选的，通过荧光发射强度原位监控抗体-探针缀合物的缀合效率。
本发明提供了上述抗体-探针缀合物或上述制备方法制备的抗体-探针缀合物作为抗原与醌氧化还原酶反应指示剂的应用。
本发明提供了一种具有荧光发射性质的抗体-药物缀合物，具有式Ⅲ所示结构：
Figure PCTCN2017084503-appb-000014
其中，DOX为阿霉素，Ab为癌胚抗原单抗或赫赛丁。
本发明提供了上述抗体-药物缀合物的制备方法，包括以下步骤：
单克隆抗体、含叠氮基元的前药分子E与双官能荧光分子D进行Michael反应和点击反应，得到式Ⅲ所示抗体-药物缀合物；
Figure PCTCN2017084503-appb-000015
其中，DOX为阿霉素，单克隆抗体为含巯基的癌胚抗原单抗或赫赛丁。
优选的，通过荧光发射强度原位监控抗体-药物缀合物的缀合效率。
本发明提供了上述抗体-药物缀合物或上述制备方法制备的抗体-药物缀合物作为荧光指示剂实时监控药物释放的应用。
本发明提供了上述抗体-药物缀合物或上述制备方法制备的抗体-药物缀合物作为靶向药物释放载体的应用。
本发明提供的抗体-药物/探针缀合物具有自报告缀合效率特征，其由双官 能度荧光分子桥接抗体与探针/药物分子，所述的双官能度荧光分子本身不具有或者仅具有弱的荧光发射能力，仅当抗体-探针/药物缀合后荧光分子才具有强的荧光发射，因此能够通过荧光监测的方法原位监控缀合过程，并应用于治疗多肽与抗癌药物的传输。
本发明提供了一种具有荧光发射性质的蛋白质/多肽-聚合物缀合物，具有式Ⅰ-3所示结构：
Figure PCTCN2017084503-appb-000016
其中，POI为蛋白质或多肽；Polymer为聚合物。
本发明提供了一种具有荧光发射性质的蛋白质-聚合物缀合物，具有式Ⅰ-a所示结构：
Figure PCTCN2017084503-appb-000017
其中，所述POI为牛血清蛋白或鲑鱼降钙素，所述Polymer为聚乙二醇。
本发明提供了上述蛋白质-聚合物缀合物的制备方法，包括以下步骤：
含巯基的牛血清蛋白、叠氮端基的聚乙二醇和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-a所示的蛋白质-聚合物缀合物；
Figure PCTCN2017084503-appb-000018
Figure PCTCN2017084503-appb-000019
其中，POI为牛血清蛋白，m为23～445；
或者包括以下步骤：
含巯基的鲑鱼降钙素、叠氮端基的聚乙二醇和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-a所示的蛋白质-聚合物缀合物；
其中，POI为鲑鱼降钙素；m为23～445。
本发明提供了一种具有荧光发射性质的多肽-聚合物缀合物，具有式Ⅰ-b所示结构：
Figure PCTCN2017084503-appb-000020
其中，所述POI为基质金属蛋白酶可切断多肽，所述Polymer为聚三亚甲基碳酸酯。
本发明提供了上述多肽-聚合物缀合物的制备方法，包括以下步骤：
式J所示的基质金属蛋白酶可切断多肽、式K所示的叠氮端基的聚三亚甲基碳酸酯和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-b所示的多肽-聚合物缀合物；
βAPVGLIGβAC-SH  式J；
Figure PCTCN2017084503-appb-000021
其中，y为10～55。
优选的，上述制备方法中，通过荧光发射强度原位监控缀合物的缀合效率。
本发明提供了上述多肽-聚合物缀合物或上述制备方法制备的多肽-聚合物缀合物组成的聚合物囊泡。
优选的，所述聚合物囊泡粒径为60～150nm。
上述聚合物囊泡具有基质金属酶响应特性。
本发明提供了上述聚合物囊泡作为药物载体的应用，或作为荧光指示剂实时监控药物释放的应用。
本发明提供的蛋白质/多肽-聚合物缀合物由双官能度荧光分子桥接蛋白质/多肽与聚合物，所述的双官能度荧光分子本身不具有或者仅具有弱的荧光发射能力，仅当蛋白质/多肽-聚合物缀合后荧光分子才具有强的荧光发射，因此所述蛋白质/多肽-聚合物缀合物的缀合过程可以通过荧光变化原位监控，并且所述蛋白质/多肽-聚合物缀合物可应用于治疗多肽与抗癌药物的传输。
附图说明
图1为本发明制备的C1的核磁氢谱图；
图2为本发明制备的C1的核磁碳谱图；
图3为本发明制备的C1的电喷雾质谱图；
图4为本发明制备C1过程中荧光变化与高效液相色谱曲线；
图5为本发明制备C1过程中荧光变化与缀合效率的关系曲线；
图6为本发明实施例5制备的嵌段聚合物的凝胶渗透色谱图；
图7为本发明实施例5制备嵌段聚合物反应过程中的荧光变化；
图8为本发明实施例5制备嵌段聚合物缀合过程中荧光变化与缀合效率的关系；
图9为实施例6制备的前药分子的核磁共振氢谱图；
图10为实施例6制备的前药分子的电喷雾质谱图；
图11为实施例7制备的荧光探针A的核磁共振氢谱图；
图12为实施例7制备的荧光探针A的电喷雾质谱图；
图13为实施例8制备抗体-探针缀合物反应过程中的荧光变化过程曲线图；
图14为实施例8制备抗体-药物缀合物反应过程中的荧光变化过程曲线图；
图15为实施例8制备的抗体-探针缀合物的酶检测过程荧光变化过程；
图16为实施例8制备的抗体-探针缀合物的细胞内癌胚抗原与醌氧化还原酶检测；
图17为实施例8制备的抗体-药物缀合物的硝基还原酶触发药物释放过程荧光变化过程；
图18为实施例9制备的抗体-药物缀合物的含癌胚抗原的细胞内硝基还原酶触发药物释放过程；
图19为实施例10制备牛血清蛋白-聚乙二醇缀合物的缀合过程荧光变化过程曲线图；
图20为实施例10制备牛血清蛋白-聚乙二醇缀合物的荧光变化与缀合效率之间关系图；
图21为实施例10制备牛血清蛋白-聚乙二醇缀合物的十二磺酸钠-聚丙烯酰胺凝胶电泳图；
图22为实施例11制备鲑鱼降钙素-聚乙二醇缀合物的缀合过程荧光变化过程曲线图；
图23为实施例11制备鲑鱼降钙素-聚乙二醇缀合物的体积排除色谱图；
图24为实施例11制备鲑鱼降钙素-聚乙二醇缀合物的十二磺酸钠-聚丙烯酰胺凝胶电泳图；
图25为实施例14制备的囊泡在水中的透射电镜图；
图26为实施例15的药物控释曲线图。
具体实施方式
本发明提供了一种含荧光发色团的缀合物，具有以下任一结构：
Figure PCTCN2017084503-appb-000022
其中，R1和R2为能够淬灭荧光发色团荧光并且能够进行“点击”反应的基团。
本发明提供的上述含荧光发色团的缀合物，包含一个荧光发色团和通过共价键连接在荧光发色团上的两个高反应活性基团R1与R2，所述缀合物中的荧光发色团初始不具有或者仅具有弱的荧光发射能力，仅当两个高反应活性基团共同与相应分子反应后，荧光发色团才具有强的荧光发射，所述缀合物如上式C1～C3所示，其中，C1中的荧光发色团为香豆素基元，C2中的荧光发色团为萘二酰亚胺基元，C3中的荧光发色团为五甲川基元。
优选的，
R1为
Figure PCTCN2017084503-appb-000023
R2为
Figure PCTCN2017084503-appb-000024
本发明中，弯线表示连接键。
本发明中，与上述缀合物反应的分子种类根据R1和R2的不同而有所区别， R1-1与R2-3对应于含有巯基的分子，R1-2对应于含反式环辛烯基的分子，R1-3对应于含炔基的分子，R1-4与R2-1、R2-2对应含叠氮基的分子，R2-4对应含丁二烯基的分子。
本发明采用含有上述特定反应基团的聚合物、靶向分子或药物分子与上述缀合物进行反应，得到具有荧光发射性质的嵌段聚合物或靶向药物。
具体的，本发明提供了一种具有荧光发射性质的嵌段共聚物，采用上述缀合物作为桥连分子制备而成，具有式Ⅰ-1所示结构：
Figure PCTCN2017084503-appb-000025
其中，
x为
Figure PCTCN2017084503-appb-000026
m优选为23～445，n优选为10～150。
本发明还提供了上述具有荧光发射性质的嵌段共聚物的制备方法，包括以下步骤：
含有巯基端基的聚合物B和含有叠氮基的聚合物C与化学缀合分子D进行Michael反应与点击反应，得到式Ⅰ-1所示嵌段共聚物；
Figure PCTCN2017084503-appb-000027
Figure PCTCN2017084503-appb-000028
其中，
x为
Figure PCTCN2017084503-appb-000029
m为23～445，n为10～150。
本发明优选的，上述反应过程中，通过荧光发射强度原位监控嵌段共聚物的缀合效率。
本发明还提供了一种具有荧光发射性质的靶向药物，采用上述缀合物作为桥连分子制备而成，具有式Ⅱ-1所示结构：
Figure PCTCN2017084503-appb-000030
其中，
Target为靶向基团，包括：环状RGD多肽、叶酸或磺酰胺；
Drug为药物基团，包括：阿霉素、喜树碱或紫杉醇。
本发明还提供了上述具有荧光发射性质的靶向药物的制备方法，包括以下步骤：
靶向分子F、药物分子G和化学缀合分子H进行Michael反应和反向D-A加成反应，得到式Ⅱ-1所示靶向药物；
Figure PCTCN2017084503-appb-000031
Figure PCTCN2017084503-appb-000032
其中，
Target为靶向基团，包括：环状RGD多肽、叶酸或磺酰胺；
Drug为药物基团，包括：阿霉素、喜树碱或紫杉醇。
本发明优选的，上述反应过程中，通过红外荧光发射强度原位监控药物分子和靶向分子的缀合效率。
本发明还提供了上述靶向药物在靶向介导药物传输中的应用。
本发明提供了一种具有荧光发射性质的抗体-药物/探针缀合物，具有式Ⅰ-2所示结构：
Figure PCTCN2017084503-appb-000033
其中，Ab为抗体，DP为荧光探针或药物分子。
本发明提供的抗体-药物/探针缀合物具有自报告缀合效率特征，其由双官能度荧光分子桥接抗体与探针/药物分子，所述的双官能度荧光分子本身不具有或者仅具有弱的荧光发射能力，仅当抗体-探针/药物缀合后荧光分子才具有强的荧光发射，因此能够通过荧光监测的方法原位监控缀合过程，并应用于治疗多肽与抗癌药物的传输。
具体的，当DP为荧光探针时，本发明提供了一种具有荧光发射性质的抗体-探针缀合物，具有式Ⅱ-2所示结构：
Figure PCTCN2017084503-appb-000034
其中，Ab为癌胚抗原单抗或赫赛丁。
本发明还提供了上述抗体-探针缀合物的制备方法，包括以下步骤：
单克隆抗体、含叠氮基元的荧光探针A与双官能荧光分子D进行Michael反应和点击反应，得到式Ⅱ-2所示抗体-探针缀合物。
所述单克隆抗体为含巯基的癌胚抗原单抗或赫赛丁。
所述含叠氮基元的荧光探针A如下式A所示：
Figure PCTCN2017084503-appb-000035
所述双官能荧光分子D如下式D所示：
Figure PCTCN2017084503-appb-000036
本发明对上述反应的原料配比、反应条件并无特殊限定，可以为本领域常规的Michael反应和点击反应的配比和条件。
本发明优选的，通过荧光发射强度原位监控抗体-探针缀合物的缀合效率。
本发明提供的上述抗体-探针缀合物具有醌氧化还原酶作用下的荧光变化，因此可以作为抗原与醌氧化还原酶反应指示剂应用。
当DP为药物分子时，本发明提供了一种具有荧光发射性质的抗体-药物缀合物，具有式Ⅲ所示结构：
Figure PCTCN2017084503-appb-000037
其中，DOX为阿霉素，Ab为癌胚抗原单抗或赫赛丁。
本发明还提供了上述抗体-药物缀合物的制备方法，包括以下步骤：
单克隆抗体、含叠氮基元的前药分子E与双官能荧光分子D进行Michael反应和点击反应，得到式Ⅲ所示抗体-药物缀合物；
所述单克隆抗体为含巯基的癌胚抗原单抗或赫赛丁。
所述含叠氮基元的前药分子E如下式E所示：
Figure PCTCN2017084503-appb-000038
式E中，DOX为阿霉素。
式E中的氨基为阿霉素四氢吡喃环上的氨基。
所述双官能荧光分子D如下式D所示：
Figure PCTCN2017084503-appb-000039
本发明对上述反应的原料配比、反应条件并无特殊限定，可以为本领域常规的Michael反应和点击反应的配比和条件。
本发明优选的，通过荧光发射强度原位监控抗体-药物缀合物的缀合效率。
本发明提供的上述抗体-药物缀合物具有硝基还原酶作用下的原药阿霉素释放，因此可以作为荧光指示剂实时监控药物释放，或者作为靶向药物释放载体。
本发明提供了一种具有荧光发射性质的蛋白质/多肽-聚合物缀合物，具有 式Ⅰ-3所示结构：
Figure PCTCN2017084503-appb-000040
其中，POI为蛋白质或多肽；Polymer为聚合物。
所述蛋白质/多肽-聚合物缀合物由双官能度荧光分子桥接蛋白质/多肽与聚合物，所述的双官能度荧光分子本身不具有或者仅具有弱的荧光发射能力，仅当蛋白质/多肽-聚合物缀合后荧光分子才具有强的荧光发射，因此所述蛋白质/多肽-聚合物缀合物的缀合过程可以通过荧光变化原位监控，并且所述蛋白质/多肽-聚合物缀合物可应用于治疗多肽与抗癌药物的传输。
在本发明的某些具体实施例中，所述POI为牛血清蛋白，所述Polymer为聚乙二醇，其结构如式Ⅰ-a所示：
Figure PCTCN2017084503-appb-000041
其中，m为23～445。
上述蛋白质-聚合物缀合物优选按照以下方法制备：
含巯基的牛血清蛋白、叠氮端基的聚乙二醇和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-a所示的蛋白质-聚合物缀合物；
Figure PCTCN2017084503-appb-000042
所述叠氮端基的聚乙二醇结构如下：
Figure PCTCN2017084503-appb-000043
本发明对上述反应的原料配比、反应条件并无特殊限定，可以为本领域常规的Michael反应和点击反应的配比和条件。
本发明优选的，通过荧光发射强度原位监控上述蛋白质-聚合物缀合物的缀合效率。
在本发明的另外一些具体实施例中，所述POI为鲑鱼降钙素，所述Polymer为聚乙二醇，其结构如式Ⅰ-a所示：
Figure PCTCN2017084503-appb-000044
其中，m为23～445。
上述蛋白质-聚合物缀合物优选按照以下方法制备：
含巯基的鲑鱼降钙素、叠氮端基的聚乙二醇和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-a所示的蛋白质-聚合物缀合物。
所述叠氮端基的聚乙二醇和化合物D同上，在此不再赘述。
本发明对上述反应的原料配比、反应条件并无特殊限定，可以为本领域常规的Michael反应和点击反应的配比和条件。
本发明优选的，通过荧光发射强度原位监控上述蛋白质-聚合物缀合物的缀合效率。
在本发明的另外一些具体实施例中，所述POI为基质金属蛋白酶可切断多肽，其序列为βAPVGLIGβAC-SH，其中，SH为巯基(巯基位于碳端半胱氨酸残基)，上述多肽购自上海强耀生物科技公司，所述Polymer为聚三亚甲基碳酸酯。其结构如式Ⅰ-b所示：
Figure PCTCN2017084503-appb-000045
其中，y为10～55。
其制备方法优选为：
式J所示的基质金属蛋白酶可切断多肽、式K所示的叠氮端基的聚三亚甲基碳酸酯和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-b所示的多肽-聚合物缀合物；
βAPVGLIGβAC-SH  式J；
Figure PCTCN2017084503-appb-000046
本发明对上述反应的原料配比、反应条件并无特殊限定，可以为本领域常规的Michael反应和点击反应的配比和条件。
本发明优选的，通过荧光发射强度原位监控上述蛋白质-聚合物缀合物的缀合效率。
本发明还公开了一种囊泡，由上述多肽-聚合物缀合物或上述制备方法制备的多肽-聚合物缀合物组成。
本发明对所述囊泡的制备方法并无特殊限定，可以为本领域技术人员熟知的制备方法。本发明优选的，将上述多肽-聚合物缀合物溶解于DMSO中，加去离子水，然后用去离子水透析即可。
本发明提供的上述聚合物囊泡分散均匀，且粒径分布均匀，其粒径分布为60～150nm。
上述聚合物囊泡具有基质金属酶响应特性，因此可以作为药物载体应用，或作为荧光指示剂实时监控药物释放。
为了进一步说明本发明，下面结合实施例对本发明提供的用作布鲁顿酪氨
为了进一步说明本发明，下面结合实施例对本发明提供的含荧光发色团的缀合物、嵌段共聚物、抗体-药物/探针缀合物，蛋白质/多肽-聚合物缀合物，靶向药物及其制备方法和应用进行详细描述。
双封的香豆素(C1-C3)的制备
合成路线如下：
Figure PCTCN2017084503-appb-000047
实施例1 C1的合成
4-溴水杨醛(1；3.11g,15.4mmol),Pd(PPh3)2Cl2(0.22g,0.31mmol),PPh3(0.061g,0.23mmol),无水四氢呋喃(50mL)加入到含有磁力搅拌子的反应烧瓶中，用干燥的氮气鼓起泡对反应体系脱气30min，然后新蒸的干燥Et3N(3.05g,30.0mmol)和三甲基乙炔基硅(1.67g,17.0mmol)在氮气氛围下加入，溶液变成橙色。搅拌20min后将助催化剂CuI(0.088g,0.46mmol)在氮气氛围下加入到反应体系，溶液变成暗棕色。室温下搅拌过夜，然后移除溶剂得到暗棕色固体，将其溶解到正戊烷中过滤得到黄色溶液。旋除所有溶剂，最后，正己烷中两次重结晶得到黄色晶体2(2.96g,收率:88.2％,>95％HPLC纯度)。
1H NMR(CDCl3,δ,ppm,TMS):11.0(s,1H,苯-OH),9.87(s,1H,-CHO),7.48(d,J＝8.4Hz,1H,芳香氢),7.07(m,2H,芳香氢),0.26(s,9H,-Si(CH 3)3)。
将2(2.85g,13.1mmol)溶于干燥的THF(40mL)中，然后加入20mL含有KOH(0.74g,13.2mmol)的MeOH溶液，反应体系在室温下搅拌过夜，然后旋发移除所有溶剂，残存物重新分散在水中，加入1.0mL乙酸并用3x 200mL氯仿萃取。合并有机相并用无水硫酸镁干燥，过滤后移除所有溶剂得到棕色固体，正己烷中重结晶两次得到黄色固体3(1.02g,收率:53.2％,>95％HPLC纯度)。
1H NMR(CDCl3,δ,ppm,TMS):11.0(s,1H,苯-OH),9.89(s,1H,-CHO),7.52(d,J＝8.4Hz,1H,芳香氢),7.12(m,2H,芳香氢),3.29(s,1H,-C≡CH)。
将苯磺酰氯(2.51g,14.3mmol),Et3N(2.15g,21.3mmol)和N-乙酰甘氨酸(0.88g,7.5mmol)溶于THF并在室温下搅拌过夜。移除不溶性盐后，化合物3(1.02g,7.0mmol)添加到反应体系中并在80℃搅拌10h，然后冷却到0℃，得到沉淀4(1.09g,收率:68.5％,>95％纯度)。
1H NMR(CDCl3,δ,ppm,TMS):9.80(s,1H,-CO-NH-),8.57(s,1H,芳香氢),7.67(d,J＝8.4Hz,1H,芳香氢),7.35(m,2H,芳香氢),4.40(s,1H,-C≡CH),2.14(s,3H,-COCH 3)。
化合物4(1.09g,4.8mmol),DMAP(0.11g,0.96mmol),(Boc)2O(2.11g,9.8mmol)溶解在40mL THF中，反应体系在70℃搅拌4h，然后冷却至室温，添加MeOH(10mL)和NH2NH2·H2O(0.96g,19.2mmol)，反应体系再搅拌4h。加入CH2Cl2，然后反应混合物用1M HCl溶液水洗，收集有机相并用无水MgSO4干燥。过滤除掉不溶性的MgSO4，所有溶剂都用旋转蒸发移除，残存物溶解到CH2Cl2(40mL)和TFA(10mL)的混合溶剂中，搅拌2h后用NaHCO3溶液水洗，有机相用MgSO4干燥，滤除MgSO4后，旋转蒸发除掉溶剂得到目标产物5(0.76g)，立刻用于合成C1，具体如下：
化合物5(0.76g,4.1mmol)和马来酸酐(2.01g,20.5mmol)溶解在100mL丙酮中，回流过夜，冷却到0℃得到黄色固体沉淀。上述黄色固体沉淀(1.03g)和一水合对甲苯磺酸(154mg)溶解在30mL甲醇中回流过夜，冷却到0℃得到粗产物黄色沉淀，用EtOAc/DCM(v/v＝1/2)作为洗脱剂通过柱层析进一步 纯化，得到黄色固体C1(480mg,收率:39.4％,>95％HPLC纯度)。
1H NMR(d6-DMSO,δ,ppm,TMS):10.28(s,1H,-CONH-),8.65(s,1H,芳香氢),7.73(d,J＝8.1Hz,1H,芳香氢),7.49(s,1H,芳香氢),7.38(s,1H,芳香氢),6.74(d,J＝9.3Hz,1H,-NHCOCH＝CH-),6.51(d,J＝8.7Hz,1H,-CH＝CH-COO-),4.43(s,1H,-C≡CH),3.66(s,3H,-COO-CH 3)，其核磁氢图谱见图1。
13C NMR(CDCl3,δ,ppm TMS):167.5,164.2,157.6,149.9,131.9,129.7,128.8,128.6,125.3,124.0,123.1,120.7,119.2,83.8,83.0,52.1，其核磁碳谱图见图2。
RP-HPLC分析:4.4min(流动相:MeOH/H2O v/v 4/1)。
ESI-MS:m/z calc.for C16H12NO5:298.06[M+H]+；found:298.0705，其电喷雾质谱图见图3。
在反应过程中，通过荧光发射强度监控嵌段共聚物的缀合效率，结果见图4和图5，其中，图4为上述反应过程中，荧光变化与高效液相色谱曲线；图5为荧光变化与缀合效率的关系曲线。
实施例2 C2的合成
2,4-二羟基苯甲醛(6,10.0g,72.4mmol)，溴丙炔(8.33g,70.0mmol)，K2CO3(19.35g,140.0mmol)加入到100mL丙酮中，回流过夜后恢复至室温，过滤并移除所有溶剂，残余物溶于CH2Cl2并用水洗，收集有机相并用无水MgSO4干燥，然后过滤旋发，粗产物利用EtOAc/PE(v/v＝1:2)作为洗脱剂通过柱层析进一步纯化，得到白色粉末7(3.2g,收率:25.9％,>95％HPLC纯度)。
1H NMR(CDCl3,δ,ppm,TMS):11.45(s,1H,苯-OH),9.75(s,1H,-CHO),7.46(d,J＝8.1Hz,1H,芳香氢),6.61(d,J＝8.1Hz,1H,芳香氢),6.53(s,1H,芳香氢),4.75(s,2H,-OCH 2C≡CH),2.58(s,1H,-OCH2C≡CH)。
N-乙酰甘氨酸(2.23g,19.1mmol)，无水乙酸钠(4.48g,54.6mmol)，化合物7(3.21g,18.2mmol)加入到100mL干的乙酸酐中，反应体系在150℃搅拌12h，冷却到室温后将反应混合物倒入到冰水里面(300mL)，得到黄色沉淀，过滤出黄色固体，在浓盐酸和乙醇的混合溶液中(v:v＝2:1)回流两个小时，反应体系然后倒入到冰水里面(100mL)，将pH值用NaOH溶液调到5～6，体系 浓缩到约30mL，得到粗产物沉淀。最后，从乙醇中两次重结晶得到产物8(3.49g,纯度:89.2％,>95％HPLC纯度)。
1H NMR(d6-DMSO,δ,ppm,TMS):10.22(s,1H,-CONH-),8.67(s,1H,芳香氢),7.69(d,J＝8.4Hz,1H,芳香氢),7.03(m,2H,芳香氢),6.63(d,J＝9.3Hz,1H,-COCH＝CH-),6.42(d,J＝9.0Hz,1H,-CH＝CHCOOH),4.92(s,1H,-OCH 2C≡CH),2.50(m,1H,-OCH2C≡CH)。
化合物8(2.15g,10.0mmol)和马来酸酐(2.01g,20.5mmol)溶解在CHCl3(50mL)中，反应回流6h后冷却至0℃，得到黄色固体(2.76g)，与一水合对甲苯磺酸(190mg)溶解在200mL甲醇中回流过夜，冷却至0℃得到粗产物沉淀，利用EtOAc/DCM(v/v＝1/3)作为洗脱剂通过柱层析进一步纯化，得到黄色固体C2(1.81g,纯度:55.4％,>95％HPLC纯度)。
1H NMR(CDCl3,δ,ppm,TMS):9.98(s,1H,-CONH-),8.75(s,1H,芳香氢),7.41(d,J＝8.4Hz,1H,芳香氢),6.93(m,2H,芳香氢),6.41(d,J＝9.3Hz,1H,-COCH＝CH-),6.25(d,J＝9.0Hz,1H,-CH＝CHCO-),4.74(d,2H,-OCH 2C≡CH),3.85(s,3H,-COOCH 3),2.55(m,1H,-OCH2C≡CH)。
13C NMR(d6-DMSO,δ,ppm TMS):167.7,163.8,159.3,157.9,151.8,131.7,129.8,129.6,126.0,122.2,113.8,113.6,102.1,79.4,79.0,56.5,52.2。
ESI-MS:m/z calc.for C17H14NO6:328.07[M+H]+，C17H13NO6Na:350.08[M+Na]+；理论值分别为:328.081，350.0916。
实施例3 C3的合成
乙酰乙酸乙酯(3.19g,24.5mmol)，化合物7(3.52g,20.0mmol)，哌啶催化剂(0.1g)和乙酸(0.1mL)添加到15mL无水乙醇中，反应混合物回流12h，冷却至室温得到亮黄色沉淀为粗产物，通过在无水乙醇中重结晶得到亮黄色晶体9(3.12g,收率:64.5％,>95％HPLC纯度)。
1H NMR(CDCl3,δ,ppm,TMS):8.49(s,1H,芳香氢),7.57(d,J＝8.1Hz,1H,芳香氢),6.97(m,2H,芳香氢),4.80(s,2H,-OCH 2C≡CH),2.71(s,3H,-COCH 3),2.61(t,1H,-OCH2C≡CH)。
化合物9(2.25g,9.3mmol)和2-吡啶甲醛(2.21g,20.6mmol)溶解在EtOH/CH3CN(80mL,1:1v/v)中，0.4g哌啶作为催化剂加入，在氮气氛围下 回流24后，旋发除掉溶剂，粗产物在无水乙醇中多次重结晶得到黄色固体C3(1.25g,收率:40.6％,>95％HPLC纯度)。
1H NMR(CDCl3,δ,ppm,TMS):8.68(d,J＝7.8Hz,1H,芳香氢),8.56(s,1H,芳香氢),8.30(d,J＝9.0Hz,1H,-COCH＝CH-),7.80(d,J＝9.3Hz,1H,-COCH＝CH-),7.70(m,1H,芳香氢),7.59(m,2H,芳香氢),7.28(m,1H,芳香氢),6.97(m,2H,芳香氢),4.80(s,2H,-OCH 2C≡CH),2.61(t,1H,-OCH2C≡CH)。
13C NMR(CDCl3,δ,ppm TMS):186.8,162.9,159.3,157.5,153.5,150.3,148.3,143.1,136.7,131.4,127.8,124.6,124.3,121.9,114.3,112.8,101.7,56.4。
ESI-MS:m/z calc.for C20H14NO4:332.09[M+H]+；found:332.0911。
实施例4 HS-PDMA38的合成
Figure PCTCN2017084503-appb-000048
在一个带有搅拌子的封管中加入带电荷的二甲基丙烯酰胺(DMA)(2.0g,20.2mmol)，4-氰基-4-(丙硫基硫代甲酰硫基)戊酸(223mg,0.8mmol)，2,2'-二偶氮异丁腈(AIBN)(16mg,0.98mmol)和二氧六环(4mL)。封管通过冻结-泵抽-解冻操作三次进行脱气，然后在处于真空下密封，在恒温油浴锅中70℃反应6h后，反应用液氮立即淬灭，解封，用THF稀释后在过量的乙醚中沉淀重复三次，将产物放到真空干燥箱中干燥得到黄色粉末PDMA(1.63g,86％收率)。
1H NMR(CDCl3,δ,ppm,TMS:2.8～3.2(N(CH 3)2),1.1～2.8(主链),1.0(-S-CH 2-)。
PDMA嵌段的实际聚合度通过1HNMR计算是38，因此将产物缩写为PDMA38。
PDMA38(500mg,0.22mmol)溶解在干的CH2Cl2中，将NH2NH2·H2O(55mg,1.1mmol)逐滴加入到反应管中2h后，浓缩溶液并用过量的乙醚沉淀三次，真空干燥箱干燥得到白色粉末HS-PDMA38(420mg,收率93.8％)。
实施例5通过C1介导的HS-PDMA38和PEG45-N3的荧光共轭物
HS-PDMA38(35mg,17.5μmol)，PEG45-N3(350mg,175μmol)， PEG227(external standard,300mg)溶解在去离子水中(45mL,pH 7.0)，然后加入C1(5.2mg,17.5μmol,溶解于5mL DMSO)和CuSO4/维生素C(1/5摩尔比)。反应体系在25℃搅拌不同的时间，达到既定时间后约1.0mL反应溶液被抽样取出，稀释到9.0mL THF中。快速加入铜离子吸附树脂(美国海洋化学公司,200mg)移除铜离子，然后震荡5min，在进一步荧光和GPC测试之前，上清液通过0.22μm无菌针筒过滤器滤出。
通过荧光发射强度监控嵌段共聚物缀合效率，结果见图6～图8，其中，图6为凝胶渗透色谱图，图7为反应过程中的荧光变化曲线，图8为反应过程中荧光变化与缀合效率的关系曲线。
由图6～图8可知荧光发色团荧光的增强与嵌段共聚物的缀合效率线性相关，因此，通过原位实时监控荧光的变化测定嵌段聚合物的缀合效率。
由上述实施例可知，本发明能够通过红外荧光发射强度原位监控上述聚合物的缀合效率。
实施例6 C1介导靶向药物的合成
合成路线如下：
Figure PCTCN2017084503-appb-000049
化合物10(4.16g,20.0mmol)和NEt3(3.22g,31.9mmol)加入到100mL干的DCM中，冷却至0℃，4-硝基氯甲酸苯酚酯(6.12g,30.4mmol,20mL DCM溶液)逐滴加入，在0℃反应12h。过滤后溶液用水洗，有机相用无水MgSO4干燥，滤除MgSO4后旋发得到粗产物，利用EtOAc/PE(v/v＝1:8)作为洗脱剂通过柱层析进一步纯化，得到白色粉末11(3.82g,收率:51.2％,纯度>95％，HPLC纯度)。
1H NMR(CDCl3,δ,ppm,TMS):8.29(t,4H,芳香氢),7.64(d,2H,芳香氢),7.38(d,2H,芳香氢),5.74(m,1H,>CH-CH2N3),3.5～3.8(m,2H,>CH-CH 2N3)。
化合物11(186mg,0.5mmol)，DOX-HCl(58mg,0.1mmol)和NEt3(120mg,1.18mmol)溶解在干的DMF(10mL)中，室温下搅拌24h，抽滤除去盐后，旋发掉溶剂得到粗产物，利用CHCl3/MeOH(v/v＝100:0到90:10)作为洗脱剂通过柱层析进一步纯化，得到红色粉末DOX-pNB-N3(43mg,收率:55.3％,纯度>95％)。
1H NMR(DMSO-d6,δ,ppm):7.6～8.2(broad,5H,芳烃氢),7.42(d,2H,芳烃氢),5.95(m,1H,>CH-CH2N3),5.49(d,2H,>CH-OH&C-OH),5.31(s,1H,-CH2OH),4.92(m,2H,>CH-O-CH<),4.62(d,2H,-CH 2OH),4.15(m,1H,>CH-OH),3.95(s,3H,-OCH 3),3.5～3.9(m,3H,>CH-CH2N3&>CH-NHCO-),2.8～3.1(m,1H,>CHCH3),1.6～2.2(m,4H,-CH 2-),1.12(d,3H,>CHCH 3)。其核磁共振氢谱图如图9所示。
RP-HPLC分析:3.7min(淋洗液:MeOH/H2O v/v 1/3)。
ESI-MS:m/z calc.for C36H36N5O15:778.21[M+H]+；found:778.2109。其电喷雾质谱图如图10所示。
200μLC1的DMSO溶液(含0.2μmolC1)，DOX-pNB-N3(10.0mg,1.0μmol)和CuSO4/Na-抗坏血酸盐(1/5摩尔比)添加HS-cRGD(0.1μmol,溶解于1.8mL PBS缓冲溶液)中，在25℃搅拌不同的时间，通过原位观测在～420nm处荧光发射强度的改变检测轭合进程，4h后，快速添加铜离子吸附树脂(美国海洋化学公司,100mg)移除铜离子，震荡5min后，上清液通过0.22μm无菌针筒过滤器滤出。
实验结果表明，荧光发色团荧光的增强与靶向药物的缀合效率线性相关，因此，可以通过原位实时监控荧光的变化测定靶向药物的缀合效率。
实施例7 荧光探针A(QNAM-N3)的合成
合成路线如下：
Figure PCTCN2017084503-appb-000050
Q3PA(0.51g,2.0mmol)和三乙胺(0.61g,6.0mmol)溶解在60mL的二氯甲烷中，将混合物降温到0℃，然后缓慢滴加氯甲酸异丁酯(0.31g,2.2mmol)，反应1小时后，加入NAM-N3(0.49g,1.75mmol)。反应体系在0℃下继续反应5小时。反应结束后，通过抽滤除去所有的无机盐，旋蒸除去所有的溶剂后得到粗产物。纯产物通过柱色谱分离(使用乙酸乙酯/正己烷(v/v＝1/5)为流动相)得到。
1H NMR(CDCl3,δ,ppm):8.37(d,1H,芳烃氢),8.22(d,1H,芳烃氢),7.92(d,1H,芳烃氢),7.24(t,1H,芳烃氢),7.04(broad,1H,-CONH-),6.29(d,1H,芳烃氢),3.81(t,2H,-CH 2-CH2N3),3.47(t,2H,-CH 2N3),2.82(m,9H,醌-(CH 3)3),1.95(s,2H,-CH 2-CONH-),1.38(s,6H,>C(CH 3)2)，其核磁共振氢谱图见图11。
RP-HPLC分析:10.7min(流动相:MeOH/H2O v/v 7/3)。
ESI-MS:m/z calc.for C28H28N5O5:514.20[M+H]+；found:514.22，其电喷雾质谱图见图12。
实施例8 通过双官能荧光分子C1介导的Anti-CEA抗体和DOX-pNB-N3或QNAM-N3的荧光缀合物
二硫苏糖醇(DTT,154mg,100μmol)溶解在磷酸缓冲液(PBS)中(10mL,pH 8.0,50mM)，然后取10μL上述溶液(含0.1μmol DTT)添加到含有anti-CEA抗体的(ACEA,1mg)PBS(1mL,pH 8.0,50mM)溶液中，在37℃搅拌30min，产物通过超滤纯化(离心-0.5,微孔,截留分子量10kDa)，使用之前要将其重新溶解到PBS中(2.0mg/mL,pH 7.0,50mM)，利用5,5’-二巯基(2-硝基苯甲酸)(DNTB)作为探针确定巯基含量，每个抗体分子中巯基数约为4。
10μL双官能荧光分子C1的DMSO溶液(0.5μmolC1)，10μL DOX-pNB-N3(0.5μmol)或QNAM-N3溶液(0.5μmol)和CuSO4/Na-抗坏血酸盐(1/5摩尔比)添加到ACEA溶液中(0.2mg,in 180μL PBS,pH 7.0)，温度保持在25℃，共轭合过程利用荧光原位监测，共培养6h后，共轭物利用超滤进一步纯化(Amicon Ultra-0.5,Millipore,截留分子量10kDa)，得到抗体-探针缀合物或抗体-药物缀合物。
在反应过程中，通过荧光发射强度监控嵌段共聚物的缀合效率，结果见图13和图14，其中，图13为制备抗体-探针缀合物反应过程中的荧光变化过程 曲线图，图14为制备抗体-药物缀合物反应过程中的荧光变化过程曲线图。
图15为实施例8制备的抗体-探针缀合物的酶检测过程荧光变化过程；QNAM-C1-ACEA抗体-探针缀合物用NADPH/NQO1在水体系中培养，观察到NAM发射强度(～530nm)显著的增加，并伴随着C1在～435nm发射强度的大幅下降。在约1小时的培养时间内，观察到约20倍FRET比率(两发射峰强度比值)的变化(见插图)。上述结果表明，C1香豆素连接键和酶促产生的NAM残基之间的有效的FRET过程的发生。在QNAM-C1-ACEA偶联体与NQO1作用后FRET比率的突变(～20倍)，表明抗体-探针缀合物可用于检测NQO1酶浓度。
图16为实施例8制备的抗体-探针缀合物的细胞内癌胚抗原与醌氧化还原酶检测。将活的HepG2细胞(缺少CEA，缺少NQO1)、LS180细胞(CEA正常，缺少NQO1)和HT29细胞(CEA正常，NQO1正常)与QNAM-C1-ACEA抗体-探针缀合物共培养。共培养后，CEA正常和NOQ1缺乏的LS180细胞共聚焦激光扫描显微镜(CLSM)图像显示在细胞内存在C1香豆素的间断蓝色发光点和极少的NAM绿色发射信号。这些结果表明QNAM-C1-ACEA能显著的被细胞摄取和其具有胞内稳定性，这是由于胞质中缺乏NQO1。对于存在CEA和NQO1的结肠癌细胞(HT29)，绿色通道NAM发射明显增强，几乎能与C1香豆素连接键的蓝色通道发射共定位。后者的强度由于FRET过程发生被显著减小。然而，用QNAM-C1-ACEA处理缺乏CEA和NQO1的HepG2细胞显示了非常低的蓝色和绿色发射。由于NQO1酶主要位于特定类型癌细胞的胞质中，以上结果表明，QNAM-C1-ACEA偶联体-探针只能以“AND”逻辑门型方式显示强烈的绿色通道发射(即需要CEA和NQO1同时存在)。
图17为实施例8制备的抗体-药物缀合物的硝基还原酶触发药物释放过程荧光变化过程；当用硝基还原酶/还原型辅酶Ⅱ共培养DOX-C1-ACEA，偶联的阿霉素药物随着时间线性持续释放。阿霉素的释放过程伴随着C1连接键在435nm处发射的显著增强和阿霉素在590nm处发射的明显降低，这清楚地表明药物-抗体偶联体的断裂和FRET过程破坏。
实施例9 胞内细胞成像
HT29or HepG2细胞(～105)培养液铺展在35mm玻璃底培养皿中培养过 夜，然后DOX-C1-ACEA与细胞在37℃共培养24h，细胞用PBS(3×1mL)和DMEM培养液冲洗，细胞荧光成像需要在Leica SP5共聚焦显微镜下进行。样品中含有的C1基团激发在405nm，吖啶橙激发在488nm，DOX激发在543nm。
图18为实施例9制备的抗体-药物缀合物的含癌胚抗原的细胞内硝基还原酶触发药物释放过程。当用DOX-C1-ACEA偶联体与含有CEA的HT29细胞在正常氧含量下培养时，共聚焦显微镜观察到在细胞质内有间断的强C1绿色发射，和阿霉素的红色圆点发射，而且蓝色/绿色发射能彼此共定位。与此相对的是，对于缺乏CEA的HepG2细胞C1香豆素连接键的蓝色荧光强度仅仅是HT29中的22％。若用DOX-C1-ACEA在缺氧条件下与HT29共培养4小时，明显的阿霉素红色发射能与吖啶橙染色的细胞核很好的共定位。除此之外，非连续的C1-ACEA残基的蓝色发射只共定位在细胞质内。以上结果表示某些癌细胞(如HT29)表面的CEA抗原有助于DOX-C1-ACEA偶联体的细胞摄取，并且在缺氧环境中胞内很容易酶触发阿霉素的释放，这能联系上固体瘤组织的微环境。
由上述实施例可知，药物-抗体缀合物与探针-抗体缀合物可以通过连接分子C1构建，并通过荧光的方法原位监控缀合效率。
实施例10 双官能荧光分子c1介导的BSA和PEG227-N3的荧光缀合物
牛血清蛋白BSA(498mg,7.5μmol)溶解在磷酸缓冲液(PBS)(70mL,pH6.5,0.1M,含1mM EDTA)中，同时将三(2-氯乙基)磷酸酯盐酸化物(TCEP·HCl)(21.5mg,75μmol)溶解在PBS(2.5mL)中，然后逐滴加入到上述BSA溶液中，4h后，溶液用去离子水透析24h(2.0kDa截留分子量)，然后冻干以获得BSAred。
BSA或BSAred(4mg,0.06μmol)溶解在PBS(0.9mL,pH 7.0,50mM)中，C1(0.6μmol，溶解于0.1mL DMSO)，PEG227-N3(6mg,0.6μmol)和CuSO4/Na-抗坏血酸盐(1/5摩尔比)加入到溶液中，25℃搅拌不同的时间，通过原位观测在约420nm处荧光发射强度的改变检测轭合进程。达到既定时间后，80μL的样品溶液被取出，然后用2mL PBS稀释，快速添加铜离子吸附树脂(美国海洋化学公司,100mg)，移除铜离子，震荡5min后，在进一步SDS-PAGE测试 之前，上清液通过0.22μm无菌针筒过滤器滤出。
反应过程中，其荧光变化图如图19所示，其荧光变化与缀合效率之间的关系如图20所示。
凝胶电泳实验：SDS-PAGE实验在凝胶电泳仪(Bio-Rad)上操作，含有BSA-PEG缀合物的溶液(80μL)与20μL SDS-PAGE加样缓冲液混合，根据标准方案使用15.0wt％聚丙烯酰胺凝胶，凝胶电泳带在紫外光(365nm)或者UVP EC3成像系统的白光辐照(染色用考马斯亮蓝)可以直接观察到。
其凝胶电泳图如图21所示。
实施例11 双官能荧光分子c1介导的鲑降钙素(sCT)和PEG227-N3的荧光缀合物
TCEP·HCl(14.2mg,50μmol)溶解在PBS(2mL,pH 7.0,50mM)中，取40μL上述溶液(含1μmolTCEP·HCl)添加到含有鲑降钙素(sCT,1.7mg,0.5μmol PBS(9mL,pH 7.0,0.05M))的小瓶中，在室温下搅拌，RP-HPLC分析显示Cys1-Cys7二硫键在～30min内定量还原。
200μLC1的DMSO溶液(含0.2μmolC1)，PEG227-N3(10.0mg,1.0μmol)和CuSO4/Na-抗坏血酸盐(1/5摩尔比)添加到还原的sCT(0.1μmol,溶解于1.8mL PBS缓冲溶液)中，在25℃搅拌不同的时间，通过原位观测在～420nm处荧光发射强度的改变检测轭合进程，4h后，快速添加铜离子吸附树脂(美国海洋化学公司,100mg)移除铜离子，震荡5min后，上清液通过0.22μm无菌针筒过滤器滤出。80μL溶液用于SDS-PAGE实验，其余溶液在做RP-HPLC实验之前用去离子水透析24h。
反应过程中，其荧光变化图如图22所示。
采用体积排除色谱法测定制备的蛋白质-聚合物缀合物分子量，其体积排除色谱图如图23所示。
凝胶电泳实验：SDS-PAGE实验在凝胶电泳仪(Bio-Rad)上操作，含有sCT-PEG缀合物的溶液(80μL)与20μL SDS-PAGE加样缓冲液混合，根据标准方案使用了15.0wt％聚丙烯酰胺凝胶，凝胶电泳带在紫外光(365nm)或者UVP EC3成像系统的白光辐照(染色用CoomassieBrilliant Blue)可以直接观察到。
其凝胶电泳图如图24所示。
实施例12 合成叠氮功能化的PTMC(PTMC-N3)
重结晶的三亚甲基碳酸酯(TMC)单体(500mg,4.9mmol)溶解在干的CH2Cl2中(1mL)，然后逐滴滴加3-叠氮基-1-丙醇(24mg,0.24mmol)和1,3-二环己基脲(DCU,8mg,0.05mmol)在干的CH2Cl2中(1mL)，反应混合物在室温氮气氛围下搅拌12h，用乙酸淬灭反应，四氢呋喃稀释，然后用过量的冷甲醇沉淀三次，放进真空干燥箱干燥得到白色粉末PTMC-N3(160mg,收率30.5％)，PTMC的实际聚合度由1H NMR计算得到，为14，因此缩写为PTMC14-N3。
实施例13 双官能荧光分子C1介导的PVGLIG多肽和PTMC14-N3的荧光缀合物
PTMC14-N3(7.5mg,5.0μmol)和C1(1.5mg,5.1μmol)溶解在DMSO中(1.8mL)，PVGLIG(8.1mg,10.1μmol)和CuSO4/抗坏血酸(1/5摩尔比)溶于0.2mL去离子水后加入，反应体系在25℃搅拌，缀合过程通过荧光原位检测。在共培养～3h后，快速添加铜离子吸附树脂(美国海洋化学公司,100mg)移除铜离子，震荡5min后，上清液通过0.22μm无菌针筒过滤器滤出，滤液用过量冷乙腈沉淀，离心收集沉淀后真空干燥箱干燥过夜。
实施例14 制备PVGLIG-C1-PTMC14聚合物囊泡
PVGLIG-C1-PTMC14(2mg)溶于1mL DMSO中，加入到一个含有磁力搅拌棒的15mL的小瓶中，室温下搅拌3h，搅拌条件下在～20s内加入9mL去离子水(～500rpm)，继续搅拌5h后用去离子水透析(截留分子量3.5kDa)24h。
制备的囊泡在水中的透射电镜图如图25所示。
实施例15 PVGLIG-C1-PTMC14聚合物囊泡包埋阿霉素盐酸盐
PVGLIG-C1-PTMC14(2mg)溶于1mL DMSO中，加入到一个含有磁力搅拌棒的15mL的小瓶中，搅拌条件下先加入含DOX·HCl的去离子水(5g/L,2mL)，剩余7mL去离子水同样速度加入，继续搅拌5h后用去离子水透析(截留分子量为3.5kDa)24h。DOX的负载含量～8.0wt％。
其药物控释应用曲线图如图26所示。
由上述实施例可知，本发明制备的上述蛋白质/多肽-聚合物缀合物可以通过荧光发射强度原位监控缀合效率，并且应用于治疗多肽与抗癌药物的传输。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (29)

  1. 一种含荧光发色团的缀合物，其特征在于，具有以下任一结构：
    Figure PCTCN2017084503-appb-100001
    其中，R1和R2为能够淬灭荧光发色团荧光并且能够进行“点击”反应的基团。
  2. 根据权利要求1所述的缀合物，其特征在于，
    R1为
    Figure PCTCN2017084503-appb-100002
    R2为
    Figure PCTCN2017084503-appb-100003
  3. 一种具有荧光发射性质的嵌段共聚物，其特征在于，具有式Ⅰ-1所示结构：
    Figure PCTCN2017084503-appb-100004
    其中，
    x为
    Figure PCTCN2017084503-appb-100005
    m为23～445，n为10～150。
  4. 权利要求3所述的具有荧光发射性质的嵌段共聚物的制备方法，其特征在于，包括以下步骤：
    含有巯基端基的聚合物B和含有叠氮基的聚合物C与化学缀合分子D进行Michael反应与点击反应，得到式Ⅰ-1所示嵌段共聚物；
    Figure PCTCN2017084503-appb-100006
    其中，
    x为
    Figure PCTCN2017084503-appb-100007
    m为23～445，n为10～150。
  5. 根据权利要求4所述的制备方法，其特征在于，通过荧光发射强度原位监控嵌段共聚物的缀合效率。
  6. 一种具有荧光发射性质的靶向药物，其特征在于，具有式Ⅱ-1所示结构：
    Figure PCTCN2017084503-appb-100008
    其中，
    Target为环状RGD多肽、叶酸或磺酰胺；
    Drug为阿霉素、喜树碱或紫杉醇。
  7. 权利要求6所述的具有荧光发射性质的靶向药物的制备方法，其特征在于，包括以下步骤：
    靶向分子F、药物分子G和化学缀合分子H进行Michael反应和反向D-A加成反应，得到式Ⅱ-1所示靶向药物；
    Figure PCTCN2017084503-appb-100009
    Figure PCTCN2017084503-appb-100010
    其中，
    Target为环状RGD多肽、叶酸或磺酰胺；
    Drug为阿霉素、喜树碱或紫杉醇。
  8. 根据权利要求7所述的制备方法，其特征在于，通过红外荧光发射强度原位监控药物分子和靶向分子的缀合效率。
  9. 权利要求6所述的靶向药物在靶向介导药物传输中的应用。
  10. 一种具有荧光发射性质的抗体-药物/探针缀合物，其特征在于，具有式Ⅰ-2所示结构：
    Figure PCTCN2017084503-appb-100011
    其中，Ab为抗体，DP为荧光探针或药物分子。
  11. 一种具有荧光发射性质的抗体-探针缀合物，其特征在于，具有式Ⅱ-2所示结构：
    Figure PCTCN2017084503-appb-100012
    其中，Ab为癌胚抗原单抗或赫赛丁。
  12. 权利要求11所述的抗体-探针缀合物的制备方法，其特征在于，包括以下步骤：
    单克隆抗体、含叠氮基元的荧光探针A与双官能荧光分子D进行Michael反应和点击反应，得到式Ⅱ-2所示抗体-探针缀合物；
    Figure PCTCN2017084503-appb-100013
    其中，所述单克隆抗体为含巯基的癌胚抗原单抗或赫赛丁。
  13. 根据权利要求12所述的制备方法，其特征在于，通过荧光发射强度原位监控抗体-探针缀合物的缀合效率。
  14. 权利要求11所述的抗体-探针缀合物或权利要求12～13任一项所述的制备方法制备的抗体-探针缀合物作为抗原与醌氧化还原酶反应指示剂的应用。
  15. 一种具有荧光发射性质的抗体-药物缀合物，其特征在于，具有式Ⅲ所示结构：
    Figure PCTCN2017084503-appb-100014
    其中，DOX为阿霉素，Ab为癌胚抗原单抗或赫赛丁。
  16. 权利要求15所述的抗体-药物缀合物的制备方法，其特征在于，包括以下步骤：
    单克隆抗体、含叠氮基元的前药分子E与双官能荧光分子D进行Michael反应和点击反应，得到式Ⅲ所示抗体-药物缀合物；
    Figure PCTCN2017084503-appb-100015
    其中，DOX为阿霉素，单克隆抗体为含巯基的癌胚抗原单抗或赫赛丁。
  17. 根据权利要求16所述的制备方法，其特征在于，通过荧光发射强度原位监控抗体-药物缀合物的缀合效率。
  18. 权利要求15所述抗体-药物缀合物或权利要求16～17任一项所述的制备方法制备的抗体-药物缀合物作为荧光指示剂实时监控药物释放的应用。
  19. 权利要求15所述抗体-药物缀合物或权利要求16～17任一项所述的制备方法制备的抗体-药物缀合物作为靶向药物释放载体的应用。
  20. 一种具有荧光发射性质的蛋白质/多肽-聚合物缀合物，其特征在于，具有式Ⅰ-3所示结构：
    Figure PCTCN2017084503-appb-100016
    其中，POI为蛋白质或多肽；Polymer为聚合物。
  21. 一种具有荧光发射性质的蛋白质-聚合物缀合物，其特征在于，具有式Ⅰ-a所示结构：
    Figure PCTCN2017084503-appb-100017
    其中，所述POI为牛血清蛋白或鲑鱼降钙素，所述Polymer为聚乙二醇。
  22. 一种权利要求21所述的蛋白质-聚合物缀合物的制备方法，其特征在于，包括以下步骤：
    含巯基的牛血清蛋白、叠氮端基的聚乙二醇和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-a所示的蛋白质-聚合物缀合物；
    Figure PCTCN2017084503-appb-100018
    Figure PCTCN2017084503-appb-100019
    其中，POI为牛血清蛋白，m为23～445；
    或者包括以下步骤：
    含巯基的鲑鱼降钙素、叠氮端基的聚乙二醇和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-a所示的蛋白质-聚合物缀合物；
    其中，POI为鲑鱼降钙素；m为23～445。
  23. 一种具有荧光发射性质的多肽-聚合物缀合物，其特征在于，具有式Ⅰ-b所示结构：
    Figure PCTCN2017084503-appb-100020
    其中，所述POI为基质金属蛋白酶可切断多肽，所述Polymer为聚三亚甲基碳酸酯。
  24. 权利要求23所述的多肽-聚合物缀合物的制备方法，其特征在于，包括以下步骤：
    式J所示的基质金属蛋白酶可切断多肽、式K所示的叠氮端基的聚三亚甲基碳酸酯和化合物D进行Michael反应和点击反应，制备得到式Ⅰ-b所示的多肽-聚合物缀合物；
    βAPVGLIGβAC-SH 式J；
    Figure PCTCN2017084503-appb-100021
    其中，y为10～55。
  25. 根据权利要求22或24所述的制备方法，其特征在于，通过荧光发射强度原位监控缀合物的缀合效率。
  26. 权利要求23所述的多肽-聚合物缀合物或权利要求24所述的制备方法制备的多肽-聚合物缀合物组成的聚合物囊泡。
  27. 根据权利要求26所述的聚合物囊泡，其特征在于，所述聚合物囊泡粒径为60～150nm。
  28. 根据权利要求26所述的聚合物囊泡，其特征在于，所述聚合物囊泡具有基质金属酶响应特性。
  29. 权利要求26～28任一项所述的聚合物囊泡作为药物载体的应用，或作为荧光指示剂实时监控药物释放的应用。
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