CN110128665A - The Amphipathilic block polymer near infrared fluorescent probe of azo-based reductase response and application - Google Patents

The Amphipathilic block polymer near infrared fluorescent probe of azo-based reductase response and application Download PDF

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CN110128665A
CN110128665A CN201910395261.0A CN201910395261A CN110128665A CN 110128665 A CN110128665 A CN 110128665A CN 201910395261 A CN201910395261 A CN 201910395261A CN 110128665 A CN110128665 A CN 110128665A
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alab6azo
azo
block polymer
solution
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CN110128665B (en
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周年琛
郁佳玮
王雨晴
张正彪
张伟
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Suzhou University
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    • 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
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0082—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion micelle, e.g. phospholipidic micelle and polymeric micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Abstract

It include synthesizing the small molecule initiator containing azobenzene and near infrared fluorescent probe the invention discloses the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response and application, preparation method；Then reaction preparation amphiphilic polymer, i.e. the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response.Micellar solution generates fluorescence and as the time gradually increases after azo reductase is added, and the drug of package is also gradually released.By the way that with fluorescence spectrum test, the variation of fluorescence intensity can monitor drug release process under azo reductase effect.Azo reductase is primarily present in the colon of human body, and therefore, this Amphipathilic block polymer is a kind of potential colon specific drug controlled release carrier.

Description

Azo-based reductase response Amphipathilic block polymer near infrared fluorescent probe and Using
Technical field
The present invention relates to the medicine controlled releasing polymerizations that a kind of polymer biomaterial more particularly to azo-based reductase respond Object carrier, and the present invention is a kind of to be not only able to achieve drug release under azo reductase effect but also while to be able to achieve near-infrared glimmering The Amphipathilic block polymer of light probe effect.
Background technique
In order to make material have more general dynamics properties, in the past few decades, intelligent response profile material is answered It transports and gives birth to and ever-increasing trend is presented.Response type material is a kind of under the small stimulus effect of external environment, itself It is certain can physically or chemically occur corresponding reversible or irreversible change material (referring to Lu, C. and Urban, M. W. Stimuli-responsive polymer nano-science: Shape anisotropy, responsiveness, applications. Progress in Polymer Science2018,78:24-46.). Common stimulus has light, temperature, pH, ionic strength, redox, enzyme, electric and magnetic fields etc., the sound based on these multiplicity Answering property and the intellectual material designed has been widely used in every field, especially some biological responding materials are by widely Applied to drug controlled release, image-forming contrast medium, the fields such as carrier of gene and bioactive molecule.
It is well known that there are many microorganism and bacterium in human colon, wherein it is most of be anaerobic bacteria (people's in-vivo content: Every gram of alimentary canal content), these floras can secret out of a large amount of enzyme solution, such as beta -glycosidase, β-glucose Aldehydic acid enzyme, nitroreductase, nitrate reductase and azo reductase.Enzyme plays an important role in life entity, typically Enzyme for drug conveying response has protease, phosphatidase, oxidoreducing enzyme etc..In sick cell and tissue, many enzymes Content is than high more under normal circumstances and have very high activity.According to this feature, the enzyme of specific site can use Respond the transmission and controlled release to realize drug.In recent years, the drug delivery and releasing research of related enzyme response and control are drawn The concern of vast researcher has been played (referring to Hu, Q., Katti, P. S. and Gu, Z. Enzyme- responsive nanomaterials for controlled drug delivery. Nanoscale 2014, 6 (21): 12273-12286.).
Photobiology detector of the fluorescence probe as a kind of efficient and sensible, in bio-imaging and bio-sensing application field Show critical role.And the appearance of the fluorescence probe based on enzyme response is identification, cell classification, the tracking metabolism of enzyme Course and cell regulation/breeding, drug transmission provide the strong research means of one kind and have and widely answer with bio-imaging Use prospect.
The Amphipathilic block polymer of azo reductase response is reported disclosed in the prior art as fluorescence probe, Since the limitation of its structure causes micella that can not thoroughly be dissociated by azo reductase, drug release rate is lower, and it is absorbed and hair Ejected wave grows the application which greatly limits it in organism positioned at visible light region, and many organisms and its group are woven in visible It itself can emit fluorescence, the fluorescence detection of severe jamming biological sample and radiography under the excitation of light, so that visible region fluorescence point The sensitivity of analysis and accuracy receive very big influence.Therefore it is glimmering to solve existing polymer to need to research and develop more polymer Light probe cannot function as the problem of near infrared fluorescent probe, while the polymer researched and developed also is needed with azo reductase responsiveness Energy.
Summary of the invention
In summary situation, the invention discloses a kind of Amphipathilic block polymer near-infrared of azo reductase response is glimmering Light probe, the polymer can be used as pharmaceutical carrier, at the same there is fluorescent probe function, it can be achieved that monitoring drug in vivo Release process.
Maximum of the amphiphilic polymer of azo-based reductase response disclosed by the invention as near infrared fluorescent probe Absorbing wavelength and launch wavelength are 600 ~ 900 nm, can avoid background interference, and can penetrate into organization internal and obtain higher Sensitivity for analysis, have apparent superiority in biological sample analysis；It solves in the prior art and there was only near-infrared fluorescent dye Expect the medicine controlled releasing polymer near infrared fluorescent probe responded as near infrared fluorescent probe without azo-based benzene reductase The problem of；And the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response of the invention is glimmering using near-infrared Light group is connected with azobenzene, and as the bridge of connection hydrophobe segment, the block polymer is by being self-assembly of ball Shape micella, azo bond, which is reduced, under the action of azo reductase and coenzyme interrupts, and micella dissociation, drug discharges therewith, companion With gradually increasing for near-infrared fluorescent；Greatly expand its application in organism.
The present invention adopts the following technical scheme:
A kind of Amphipathilic block polymer near infrared fluorescent probe (PEG-alAB6Azo-PLA) of azo reductase response, it is described The general structure of the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response is as follows:
In formula, 50≤m≤200,40≤n≤400.
The preparation method of the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response of the present invention includes closing At the small molecule initiator containing azobenzene and near infrared fluorescent probe；Then reaction preparation amphiphilic polymer, i.e. azo restore The Amphipathilic block polymer near infrared fluorescent probe of enzyme response.Specifically includes the following steps:
(1) compound A is reacted to obtain compound B with al6Azo compound；
(2) compound B is reacted with propine compound, obtains alAB6Azo compound；
(3) cause lactide ring-opening polymerization using alAB6Azo compound, obtain alAB6Azo-PLA；
(4) by alAB6Azo-PLA and mPEG-N3Reaction obtains the Amphipathilic block polymer near-infrared of azo reductase response Fluorescence probe is denoted as PEG-alAB6Azo-PLA.
The invention discloses polymer micelle, the preparation method of the polymer micelle, comprising the following steps:
(1) compound A is reacted to obtain compound B with al6Azo compound；
(2) compound B is reacted with propine compound, obtains alAB6Azo compound；
(3) cause lactide ring-opening polymerization using alAB6Azo compound, obtain alAB6Azo-PLA；
(4) by alAB6Azo-PLA and mPEG-N3Reaction obtains the Amphipathilic block polymer of azo reductase response；
(5) under stirring, buffer is added dropwise in the Amphipathilic block polymer solution of azo reductase response, is mixed Liquid；Continue to stir after being added dropwise to complete, finally dialysis obtains polymer micelle.
The invention discloses drug-carrying polymer micelle, the preparation method of the drug-carrying polymer micelle, comprising the following steps:
(1) compound A is reacted to obtain compound B with al6Azo compound；
(2) compound B is reacted with propine compound, obtains alAB6Azo compound；
(3) cause lactide ring-opening polymerization using alAB6Azo compound, obtain alAB6Azo-PLA；
(4) by alAB6Azo-PLA and mPEG-N3Reaction obtains the Amphipathilic block polymer of azo reductase response；
(5) stir under, by buffer be added dropwise to containing azo reductase response Amphipathilic block polymer and drug it is molten In liquid, mixed liquor is obtained；Continue to stir after being added dropwise to complete, finally dialysis obtains drug-carrying polymer micelle.
In the present invention, the propine compound is propargyl bromide；Buffer is PBS buffer solution；Drug is adriamycin；The idol In the amphipathic block near-infrared polymer solution of nitrogen reductase response, solvent DMF；It is described containing azo reductase response In the solution of Amphipathilic block polymer and drug, solvent DMF；In the mixed liquor, water content 80wt%.Preferably, In the Amphipathilic block polymer solution of the azo reductase response, the Amphipathilic block polymer of azo reductase response In, the molecular weight ratio of hydrophilic segment and hydrophobic segment is 4.5~5.5: 6；It is described to contain the amphipathic embedding of azo reductase response In the solution of section polymer and drug, in the Amphipathilic block polymer of azo reductase response, hydrophilic segment and hydrophobic chain The molecular weight ratio of section is 4.5~5.5: 6.In the Amphipathilic block polymer of azo reductase response of the invention, hydrophilic segment For PEG chain segment, hydrophobic segment is PLA segment.
Method of the invention prepares the small molecule initiator (alAB6Azo) containing azobenzene Yu compound A first, uses it Ring-opening polymerisation LA, obtains hydrophobic segment, then by CuAAC click-reaction, by the alAB6Azo group in molecule be connected to PEG and Among PLA, the Amphipathilic block polymer PEG of azobenzene Yu near infrared fluorescent probe bridging is obtained398-alAB6Azo-PLA144。 Polymer and simulation anticancer drug are self-assembly of to the nanoparticle of packaging medicine by solution.Human body environment is simulated, in idol Under nitrogen reductase (DT-diaphorase, Human) effect, the azo bond in the junction PEG and PLA is disconnected, and causes to assemble Body dissociation, polymer are separated into PEG chain segment and PLA segment, and PLA forms aggregation, and compound A is then connected in PEG and is dissolved in solution It is dispersed and realizes fluorescence and discharge.Therefore, micellar solution generates fluorescence and as the time gradually increases after azo reductase is added By force, the drug of package is also gradually released.Pass through the variation with fluorescence spectrum test fluorescence intensity under azo reductase effect Drug release process can be monitored.Azo reductase is primarily present in the colon of human body, and therefore, this Amphipathilic block polymer is A kind of potential colon specific drug controlled release carrier.
The invention also discloses the Amphipathilic block polymer near infrared fluorescent probes of above-mentioned azo reductase response to make Application in standby near infrared fluorescent probe and/or anticancer drug；Or the amphipathic block polymerization of above-mentioned azo reductase response Application of the object near infrared fluorescent probe in the pharmaceutical carrier for preparing azo reductase response.
In the present invention, in step (1), the molar ratio of compound A and al6Azo compound is 1: 1；The reaction is in carbon It is carried out in the presence of sour potassium, DMF；The temperature of the reaction is 60 DEG C, and the time is 24 hours；Preferably, the dosage and change of potassium carbonate Close object A equivalent；
In step (2), the molar ratio of compound B and propine compound is 1: 4；It is described reaction in the presence of potassium carbonate, DMF into Row；The temperature of the reaction is 50 DEG C, and the time is 4 hours；Preferably, the dosage of potassium carbonate and compound B equivalent；
In step (3), ring-opening polymerization carries out in nitrogen, in the presence of DMAP；AlAB6Azo compound, DMAP, lactide Molar ratio be 0.05:0.20:2.5~10, preferably 0.05: 0.2: 10；The temperature of ring-opening polymerization is 40oC, time is 24~48 hours；
In step (4), reacts in the presence of toluene, CuBr and PMDETA, carried out in nitrogen；The temperature of reaction is 65 DEG C, the time It is 24 hours；alAB6Azo-PLA,mPEG-N3, CuBr, PMDETA molar ratio be 1.0~1.2:1.0:7.5:15.
In the present invention, in step (1), after reaction, reaction solution is by ethyl acetate extraction, saturated aqueous ammonium chloride It washes, then that organic layer is dry with anhydrous sodium sulfate, column Chromatographic purification obtains compound B, and eluant, eluent is dichloromethane when column chromatographs Alkane and ethyl acetate；
In step (2), after reaction, reaction solution is washed by ethyl acetate extraction, saturated aqueous ammonium chloride, then will be organic Layer is dry with anhydrous sodium sulfate, and column Chromatographic purification obtains alAB6Azo compound；Eluant, eluent is methylene chloride and second when column chromatographs Acetoacetic ester；
In step (3), after reaction, reaction solution is added dropwise in ether after being diluted with methylene chloride and precipitates, and obtains grass green Color fluffy solid；Grass green fluffy solid is dried under vacuum to constant weight, obtains alAB6Azo-PLA.
In the present invention, the chemical structural formula of compound A is as follows:
The chemical structural formula of compound B is as follows:
The chemical structural formula of al6Azo compound is as follows:
The chemical structural formula of alAB6Azo compound is as follows:
The chemical structural formula of alAB6Azo-PLA is as follows:
mPEG-N3Chemical structural formula it is as follows:
In the present invention, coupling reaction is carried out in ice water with p-aminophenyl methanol diazonium salt and phenol and obtains alAzo compound；Tool Body is that the mixed solution containing concentrated hydrochloric acid, p-aminophenyl methanol, ice water is added dropwise in sodium nitrite in aqueous solution；Then in stirring Lower aqueous solution of the dropwise addition containing phenol, potassium carbonate, continues to stir；Then adjusting pH is 4~5, is precipitated, then by taking out Filter, cleaning, drying are alAzo compound；Preferably, p-aminophenyl methanol, concentrated hydrochloric acid, sodium nitrite, phenol, potassium carbonate Molar ratio is 16.2: 40.8: 17: 17: 22.7.The chemical structural formula of alAzo compound is as follows:
In the present invention, reacted to obtain al6Azo compound with 6- bromohexane with alAzo compound；Specially by alAzo compound, 6- bromohexane, potassium carbonate are dissolved in acetone；It is stirred 16 hours in 65 DEG C；It then cools to room temperature, reaction solution passes through ethyl acetate Extraction, washing, obtained organic layer is dry with anhydrous sodium sulfate, and petrol ether/ethyl acetate is recycled to cross column purification, last vacuum It is dried to obtain orange/yellow solid, is al6Azo compound.
Above-mentioned reaction is schematically as follows:
In the present invention, with poly glycol monomethyl ether (mPEG) by its end group modification be azido group, obtain mPEG-N3；Specifically, Under the conditions of ice-water bath, the dichloromethane solution containing poly glycol monomethyl ether, triethylamine is added dropwise to containing to tosylate chloride Dichloromethane solution in；Then it is reacted 24 hours in 55 DEG C, obtains the PEG-OTs of paratoluensulfonyl chloride sealing end；Then will PEG-OTs、NaN3It is added in DMF, is then reacted 24 hours in 85 DEG C, obtain mPEG-N3.It reacts schematically as follows:
The present invention carries out coupling reaction with p-aminophenyl methanol diazonium salt and phenol in ice water and obtains azobenzene alAzo(yield: 92%) it reacts, then with 6- bromohexane to obtain al6Azo(yield 77%).Compound A successively occurs with al6Azo and 3- propargyl bromide anti- It answers, obtains end one end band alkynyl, small molecule compound B(two step gross production rate 49% of the one end with benzylalcohol).It is to draw with compound B Agent is sent out, under DMAP catalysis, causes lactide (LA) ring-opening polymerisation by solvent of methylene chloride, obtains alAB6Azo-PLA.Choosing With poly glycol monomethyl ether (mPEG) by its end group modification be azido group, obtain mPEG-N3.By alAB6Azo-PLA with mPEG-N3CuAAC is carried out under CuBr catalyst action to react to obtain Amphipathilic block polymer PEG-alAB6Azo-PLA.
The conjunction of the amphipathic block near-infrared polymer (PEG-alAB6Azo-PLA) of azo reductase response of the invention It is as follows at route:
As preferred example, the present invention selects the block polymer PEG that hydrophobe segment ratio (mPEG/PCL) is about 5:6398- alAB6Azo-PLA144Solution self assembly is studied, process is as follows: polymer is dissolved in anhydrous DMF, and it is 8 mg/mL that concentration, which is made, Solution, and using the preceding filter membrane dedusting for passing through 0.22 μm.Then, PBS solution (pH 7.4) is slowly dripped with syringe pump Be added in above-mentioned solution until water quality content be 80%, continually and steadily stirred during dropwise addition, 6 h be stirred for after being added dropwise, i.e., Obtain stable micellar structure.It is dialysed for 24 hours, organic solvent is removed, originally in PBS solution with dialysis (MWCO 3500) Every 4 h replaces a dialyzate, 6 dialyzates is at least replaced, so that micella be made to be stably dispersed in PBS buffer solution.Finally The micellar solution that constant volume is 1 mg/mL at concentration.
Above-mentioned micellar solution and azo reductase (DT-diaphorase, Human) and coenzyme (NADPH) are mixed, 24 h are cultivated under 37 DEG C of constant-temperatureanaerobic anaerobic environment.Under azo reductase effect, azo bond is interrupted, and polymer is separated into PEG sections (being dissolved in solution) and PLA sections, micella is caused to dissociate, fluorescence is activated.This process can have by the way that sepectrophotofluorometer is convenient The variation at fluorescent characteristics peak is detected to prove in effect ground.Simultaneously using transmission electron microscope (TEM) observation assembly before reduction The variation of pattern afterwards, dynamic light scattering (DLS) test assembly particle size and distribution, and ultraviolet-visible spectrum monitors azobenzene Absorption peak strength variation further prove azo bond fracture.The results show that blank micella solution and azo reductase and auxiliary Enzyme coexists 24 h of culture and can substantially completely restore the azobenzene in polymer, and micella cracks completely, and fluorescence obtains greatly Enhancing.Show that Inventive polymers are the near-infrared fluorescent polymer probes of azo reductase response.
Further by above-mentioned Amphipathilic block polymer and aids drug adriamycin co-dissolve in DMF, it is made into certain The polymer medicament carrying micelle PBS solution of 1 mg/mL is prepared by above-mentioned identical self-assembling method in the solution of concentration.
The holding of above-mentioned polypeptide drug-loaded micelle solution is protected from light and azo reductase (DT-diaphorase, Human) and coenzyme (NADPH) it mixes, is cultivated under 37 DEG C of constant-temperatureanaerobic anaerobic environment.Azo bond in micella nucleocapsid interface is gradually interrupted, micella It gradually dissociates, polymer is gradually disengaged into PEG sections (being dissolved in solution) and PLA sections, and fluorescence is gradually activated.Pass through fluorescence spectrophotometer light The variation that degree meter conveniently and effectively detects fluorescent characteristics peak and aids drug DOX fluorescent characteristics peak effectively monitors drug release feelings Condition.Simultaneously using the variation of transmission electron microscope (TEM) observation assembly pattern in reduction process, dynamic light scattering (DLS) assembly particle size and distribution are tested, drug is released in the absorption peak strength variation of ultraviolet-visible spectrum monitoring azobenzene Journey is let off to make further to prove.
Currently, the near-infrared fluorescent polymer probe and medicament slow release monitoring system of azo-based reductase yet there are no report Road, present invention design have synthesized a kind of polymer near infrared fluorescent probe of azo reductase response, are amphiphilic block Object is used as bridge by the near infrared fluorescent probe (650 nm of excitation wavelength) of azobenzene connection, connects hydrophilic segment polyethylene glycol (PEG) and hydrophobic segment polylactide (PLA).This amphiphilic polymer (pH=7.4) in PBS buffer solution solution can be assembled into Nanoparticle and packaging medicine.Compound A leads to fluorescent quenching due to being wrapped in micella core at this time, when in micellar solution After middle addition azo reductase, azo bond (- N=N-) is reduced disconnection, and assembly dissociates, and polymer is dissociated into PEG chain With PLA chain, compound A is connected to soluble in water in PEG chain segment after azo bond disconnects, and fluorescence restores.Therefore, which has The sensitivity and highly selective of azo reductase detection.Meanwhile drug being wrapped in micella by assembling, preparation carries medicine glue Beam.Since the dissociation drug of micella gradually discharges, and fluorescence constantly enhances in drug release process, therefore can be by glimmering The variation monitoring drug release process of luminous intensity.Therefore, which there is near infrared detection and medicament slow release to monitor dual function Energy.Azo reductase is primarily present in the colon of human body, and therefore, this polymer can be used for colon specific drug controlled release carrier, Medically there is actual application prospect.
The near-infrared fluorescent polymer probe of azo-based reductase of the invention has the advantage that (1) dyestuff has High fluorescence quantum yield, or even fluorescence quantum yield in water can achieve 1.0；(2) polarity of the fluorescence signal to solvent It is insensitive with pH；(3) there is relatively good photo and thermal stability；(4) fluorescence spectrum half-peak breadth is relatively narrow, has when as fluorescence labelling Good sensitivity；(5) high molar extinction coefficient, typically larger than 80,000 Lmol-1·cm-1, extinction efficiency is relatively high； (6) structure is easy to modify, and launch wavelength is adjustable to fade near infrared region.
The present invention has at least the following advantages:
Present invention firstly discloses the Amphipathilic block polymer near infrared fluorescent probes of azo-based reductase response.It is amphipathic Polymer can be self-assembly of micella, and the micella of amphiphilic polymer containing azobenzene can solve under the action of azo reductase From leading to drug release, while it being able to achieve fluorescence fluorescence response from scratch again.
Up to the present, the near infrared fluorescent probe of the enzyme responsive polymer of azo-based benzene has not been reported.The present invention The near infrared fluorescent probe for being connected to azobenzene is introduced into the amphiphilic polymer of structure exquisiteness using the highly effective reaction of chemistry Centre efficiently prepares a kind of azo-based reductase stimuli responsive type near-infrared polymer fluorescent probe.
Based on above-mentioned obtained polymer, further to the following research of the application performance of polymer expansion: by solution from group Fill the stable polymer micelle of preparation structure；Using polymer micelle can azo reductase effect under be destroyed decomposition and it is glimmering Polymer is further carried out drug package as the carrier of packaging medicine and released by the characteristics of light is activated and gradually increases It puts, drug release process is tracked by change in fluorescence.
The present invention is directed to by the studies above, design and construct novel and near-infrared polymer efficiently based on enzyme response Fluorescence probe detection architecture and drug delivery system expand azobenzene polymer in bio-sensing, load medicine and cell marking etc. Application in field provides necessary theoretical foundation for colon specific drug controlled release carrier and medical diagnosis on disease, fills up research neck The blank in domain.
Detailed description of the invention
Fig. 1 be the alAzo in the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure；
Fig. 2 be the al6Azo in the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure；
Fig. 3 be the alAB6Azo in the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure；
Fig. 4 be the alAB6Azo-PLA in the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure；
Fig. 5 be PEG-alAB6Azo-PLA in the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure；
Fig. 6 is PEG-N in the present invention3, gel chromatography (GPC) the outflow song of alAB6Azo-PLA and PEG-alAB6Azo-PLA Line；
Fig. 7 is PEG-N in the present invention3With infrared spectroscopy (FT-TR) figure of PEG-alAB6Azo-PLA；
Fig. 8 is critical micelle concentration (CMC value) of the PEG-alAB6Azo-PLA assembly in PBS solution in the present invention；
Fig. 9 be PEG-alAB6Azo-PLA in the present invention blank micella PBS solution (concentration be 0.3 mg mL-1) azo also Ultraviolet/visible absorption spectra the figure in protoenzyme reduction front and back；
Figure 10 be PEG-alAB6Azo-PLA in the present invention blank micella PBS solution (concentration be 0.3 mg mL-1) in azo Reductase reduction front and back fluorescent emission map figure of fluorescence intensity changes spectrum (excitation wavelength is 650 nm)；
Figure 11 be PEG-alAB6Azo-PLA in the present invention blank micella PBS solution (concentration be 0.3 mg mL-1) in azo Reductase reduction front and back transmission electron microscope (TEM) image；
Figure 12 be PEG-alAB6Azo-PLA in the present invention blank micella PBS solution (concentration be 0.3 mg mL-1) in azo Reductase reduction front and back dynamic light scattering (DLS) change of size figure；
Figure 13 be contained in the present invention DOX PEG-alAB6Azo-PLA assembly PBS solution (concentration be 0.3 mg mL-1) Ultraviolet in azo reductase reduction process/visible absorption spectra figure；
Figure 14 be contained in the present invention DOX PEG-alAB6Azo-PLA assembly PBS solution (concentration be 0.3 mg mL-1) Fluorescent emission map figure of fluorescence intensity changes spectrum in azo reductase reduction process (excitation wavelength is 650 nm)；
Figure 15 be contained in the present invention DOX PEG-alAB6Azo-PLA assembly (concentration be 0.3 mg mL-1) in azo Drug release result in the presence of reductase；
Figure 16 be contained in the present invention DOX PEG-alAB6Azo-PLA assembly (concentration be 0.3 mg mL-1) in azo Transmission electron microscope (TEM) image in reductase reduction process；
Figure 17 be contained in the present invention DOX PEG-alAB6Azo-PLA assembly (concentration be 0.3 mg mL-1) in azo Reductase reduction front and back dynamic light scattering (DLS) change of size figure；
Figure 18 is the thin of the PEG-alAB6Azo-PLA blank micella PBS solution in the present invention before and after 4 h of azo reductase reductase 12 Cellular toxicity test result.
Specific embodiment
With reference to the accompanying drawings and examples, specific implementation method of the invention is described in further detail.
Small molecule structure synthesized by the present invention is characterized by nuclear-magnetism；Synthesized polymer architecture passes through nuclear-magnetism, gel Chromatography, infrared spectrum characterization；The morphology and size of the polymer micelle of prepared polymer micelle and packaging medicine and also Variation passes through transmission electron microscope (TEM) and dynamic light scattering (DLS) detailed characterizations during former；Polymer micelle in reduction process Change in fluorescence passes through fluorescence spectrum tracking and testing；The structure change of polymer is characterized by ultraviolet-visible spectrum in reduction process.
Performance test methods are as follows in specific embodiment:
1. nucleus magnetic hydrogen spectrum (1H NMR) it is by 300 MHz nuclear magnetic resonance spectrometer of Bruker, tetramethylsilane (TMS) is internal standard, will Test sample is with CDCl3(or deuterated DMSO) is to be tested after solvent dissolves；
2. polymer number-average molecular weight (M n), weight average molecular weight (M w) and molecular weight distributing index (M w/M n) use is equipped with and shows It is measured on poor refraction detector and UV detector TOSOH HLC-8320 gel permeation chrommatograph (GPC), using two TSKgel The series connection of Super Mutipore HZ-N (3 μm of beads size) pillar, molecular weight ranges are 500 to 190,000 g/ Mol selects chromatographically pure DMF as mobile phase, and flow velocity is 0.35 mL/min, is tested at 40 DEG C, (narrow with polystyrene Distribution) it is that standard specimen is corrected polymer molecular weight；
3. the infrared conversion spectrum of Fourier (FT-TR) test is tested with 27 FT-IR of Bruker TENSOR-, KBr pressure The test of piece method；
4. transmission electron microscope (TEM) uses HITACHI HT7700 TEM, work acceleration voltage is 120 KV.
5. ultraviolet-visible absorption spectroscopy UV-2600 ultraviolet-visual spectrometer (Shimadzu, (Nakagyo-ku, Kyoto, Japan)) it is measured at 25 DEG C；
6. fluorescence emission spectrum is tested to obtain using Hitachi F-4600 type fluophotometer.
Embodiment one
The preparation method of the Amphipathilic block polymer near infrared fluorescent probe of azo-based reductase response, including following step It is rapid:
1. the synthesis of alAzo:
2.0 g (16.2 mmol, 1 eq) p-aminophenyl methanol is dissolved in 30 mL ice water, and the concentrated hydrochloric acid that 3.4 mL 37% are added stirs It mixes.It weighs 1.17 g (17.0 mmol, 1.05 eq) sodium nitrite and is dissolved in 8 mL water, be cooled to 0oIt is added dropwise after C to amino Benzyl alcohol solution stirs 1 h.Weigh 1.6 g (17.0 mmol, 1 eq) phenol and 3.14 g (22.7 mmol, 1.4 eq) Potassium carbonate is dissolved in 25 mL water, is cooled to 0oC is added dropwise in above-mentioned solution, and process keeps 0-5oC.Stirring 2 hours, use is dilute The acetic acid aqueous solution released adjusts pH to 4-5, until generating salmon precipitation.It filters, a small amount of methanol rinses, and filter cake drying obtains Orange red solid, 3.4 g, yield 92%；Attached drawing 1 be alAzo of the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure.
2. the synthesis of al6Azo:
912.4 mg (4 mmol, 1 eq) alAzo, 1.94 g (8 mmol, 2 eq) 6- bromohexane, 552 mg (4 Mmol, 1 eq) potassium carbonate, it is dissolved in 8 dry mL acetone.65oC is stirred 16 hours, is condensed back.It is cooled to room temperature, second Acetoacetic ester extraction, three times, organic layer is dry with anhydrous sodium sulfate, petroleum ether for washing, and ethyl acetate crosses column purification, is dried in vacuo To orange/yellow solid, 1.2g, yield 77%；Attached drawing 2 be al6Azo of the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure.
3. the synthesis of alAB6Azo:
The synthesis of intermediate alB6Azo: 392.4 mg (1 mmol, 1 eq) al6Azo, 529.17 mg (1 mmol, 1 eq) Existing compound A, 138 mg (1 mmol, 1 eq) potassium carbonate is dissolved in the dry DMF of 8.5 mL, and 60oC is stirred 24 hours.Acetic acid Ethyl ester extraction, saturated aqueous ammonium chloride wash away DMF, and organic layer is dry with anhydrous sodium sulfate, and column Chromatographic purification removes small polarity Impurity, eluant, eluent are methylene chloride and ethyl acetate, and product alB6Azo is put into together with remaining compound A to react in next step.Change The chemical structural formula for closing object A is as follows:
The synthesis of alAB6Azo: 150mg (0.18mmol, 1eq) alB6Azo, 84mg (0.72mmol, 4eq) propargyl bromide, 24.6mg (0.18mmol, 1eq) potassium carbonate is dissolved in the dry DMF of 10mL, and 50oC is stirred 5 hours.Ethyl acetate extraction, saturation Ammonium chloride solution washes away DMF, and organic layer is dry with anhydrous magnesium sulfate.Column chromatography, eluant, eluent are that methylene chloride and ethyl acetate obtain To dark green solid, 70mg, two step gross production rates: 49 %；Attached drawing 3 be alAB6Azo of the present invention nuclear magnetic resonance spectroscopy (1H NMR) figure.
4. the synthesis of alAB6Azo-PLA:
AlAZ6Azo-PLA is prepared with the method for ring-opening polymerisation.Using alAB6Azo as initiator, DMAP is catalyst, lactide (LA) ring-opening polymerisation is carried out for monomer.5mL ampoule bottle is used while vacuumizing heat gun heated baking body 20 minutes, it is cold But it is passed through nitrogen afterwards.LA(1.44g, 10mmol are weighed in glove box), DMAP(24.4mg, 0.2mmol), alAB6Azo (43.87mg, 0.05mmol) is placed in dry ampoule bottle, and the dissolution of 1mL methylene chloride is added, takes out tube sealing after glove box.IKA It is stirred 48 hours for 40 DEG C in blender.Crude product is diluted with q. s. methylene chloride after reaction, is added dropwise to excess Ether in precipitate, obtain grass green fluffy solid.Place the product in 40oIt dries in C vacuum drying oven to constant weight, obtains alAB6Azo-PLA；836 mg, yield: 58 %,M N, SEC=28300 g/mol,M w/M n=1.05.Attached drawing 4 is the present invention AlAB6Azo-PLA nuclear magnetic resonance spectroscopy (1H NMR) figure.
5. the synthesis of PEG-alAB6Azo-PLA:
The synthesis of PEG-OTs: 2.5 g (0.125 mmol) poly glycol monomethyl ether 20000 is dissolved in the dry dichloromethane of 10 mL In alkane, 1.01 g (10 mmol, 80 eq) triethylamine is added, ice bath is cooled to 0oC.By 0.19 g (1 mmol, 8 eq) Paratoluensulfonyl chloride is dissolved in the dry methylene chloride of 5mL, and PEG solution is added dropwise, and process keeps 0oC.It is warming up to 55oC, magnetic force Stirring 24 hours.It is washed three times with saturated sodium bicarbonate aqueous solution, methylene chloride extraction.Organic layer is dry with anhydrous sodium sulfate, takes out It is concentrated after filter, instills the sedimentation of ice ether.It filters, 40oC vacuum drying oven dries to constant weight.Obtain the PEG- of paratoluensulfonyl chloride sealing end OTs, 2.4 g, yield: 95 %.
PEG-N3Synthesis: weigh 1.015 g (0.05 mmol, 1 eq) PEG-OTs, ox horn spoon takes 260 mg (4 mmol, 80 eq) NaN3, it is dissolved in the dry DMF of 15 mL, 85oC stirs 24 h.It filters and removes excess NaN3, dichloro Methane extraction, saturated aqueous ammonium chloride wash away DMF and remaining NaN3, organic layer is dry with anhydrous sodium sulfate, dense after suction filtration Contracting instills the sedimentation of ice ether.It filters, 40 DEG C of vacuum drying ovens dry to constant weight.Product is white crystalline solid, 936 mg, yield: 93 %。
The synthesis of PEG-alAB6Azo-PLA: PEG-N is weighed3With alAB6Azo-PLA in 10mL polymerization bottle, add 6mL without Water-toluene dissolution, and CuBr and PMDETA is added, freezing is evacuated deoxygenation in triplicate, tube sealing.65 DEG C of stirrings, react 24 hours. A small amount of methylene chloride dilution is added after the reaction was completed, and appropriate resin of copper of inhaling is added and is stirred overnight at room temperature.It filters to remove and inhales copper tree Ether is added dropwise after concentrated by rotary evaporation in rouge.The PEG-alAB6Azo- of molecular weight required for obtaining is purified by preparation scale SEC PLA, yield: 53 %,M n,SEC= 37600 g/mol, M w/M n=1.09；Attached drawing 5 is PEG-alAB6Azo-PLA of the present invention Nuclear magnetic resonance spectroscopy (1H NMR) figure, wherein the repetitive unit number (m, n) of hydrophobe segment can pass through PEG- The nucleus magnetic hydrogen spectrum figure of alAB6Azo-PLA is obtained using following calculation formula:
m = (I 5.06-5.35/2)/(I 7.38-7.51/ 8) formula 1
n = (I 3.47-3.85/4)/(I 7.38-7.51/ 8) formula 2
I 5.06-5.35: the proton peak of the corresponding polylactic acid repetitive unit-CH- segment of 5.06-5.35 ppm in spectrogram；
I 3.47-3.85: repetitive unit-OCH on corresponding poly glycol monomethyl ether at 3.47-3.85 ppm in spectrogram2CH2O- piece The proton peak of section;
I 7.38-7.51: corresponding PEG at 7.38-7.51 ppm in spectrogramn-alAB6Azo-PLAmProton on middle compound A Peak (c, d, e, h).
By GPC elution curve (Fig. 6) as can be seen that polymers obtained PEG-alAB6Azo-PLA molecular weight compared with AlAB6Azo-PLA and PEG-N3 significant change.Find out simultaneously from attached drawing 7, in PEG-N3Infrared figure in it can be observed that 2098 cm-1There is nitrine characteristic peak, the stretching vibration corresponding to azido group N ≡ N.And it is infrared in PEG-alAB6Azo-PLA In spectrogram, it can directly observe the disappearance at this feature peak, illustrate PEG-N3Fully reacting.Pass through nuclear magnetic resonance spectroscopy Analyzing obtained PEG-alAB6Azo-PLA molecular weight is 39100 g/mol, wherein the polymer segment ratio of hydrophobe according to Above-mentioned calculation method is calculated as 5/6, m=144, and n=398 meet the molecular weight ratio of close and distant aqueous polymer used.It is possible thereby to Conclude that PEG398-alAB6Azo-PLA144Success is made.
Fig. 6 is PEG-N in the present invention3, exclusion chromatography (SEC) stream of alAB6Azo-PLA and PEG-alAB6Azo-PLA Curve out；Fig. 7 is PEG-N in the present invention3With infrared spectroscopy (FT-TR) figure of PEG-alAB6Azo-PLA.
Following test is done to the performance of polymer obtained above:
1. self assembly and characterization of the polymer in DMF/PBS solution
By polymer P EG398-alAB6Azo-PLA144It is dissolved in anhydrous DMF, the solution that concentration is 8 mg/mL is made, and The solution is passed through to 0.22 μm of filter membrane dedusting.Then, PBS solution (pH 7.4) is added drop-wise in above-mentioned solution with syringe pump Until water content is 80%, continually and steadily stirred during dropwise addition, 6 h are stirred for after being added dropwise, be added dropwise and kept during stirring 25oC to get arrive stable micellar structure.Then it is dialysed one day in PBS solution with bag filter (MWCO 3500), it will be organic molten Agent all removes, and has just started every 4 h and has changed a dialyzate, 6 dialyzates is at least replaced, so that micella be made to be stably dispersed in PBS In buffer solution.Finally it is configured to the assembling liquid solution that concentration is 1 mg/mL.
Amphiphilic polymer critical micelle concentration (CMC) measurement.Hydrophobic fluorescent dyes Nile red (Nile Red) is typical Intramolecular reverse electric charge transfer (TICT) probe, it is extremely sensitive to environmental change, and its maximum emission wavelength and polymer The maximum emission wavelength of near infrared fluorescent probe is non-overlapping, therefore the fluorescence that can be used as the CMC of test amphiphilic polymer is visited Needle.Test process is as follows: the Nile red solution (methylene chloride is solvent) of 30 μ L being taken to be added separately in 8 volumetric flasks.To solvent After volatilization is clean, it is separately added into concentration 1.0 × 10−3~2.0 × 10−1 2 mL of polymersome PBS solution of mg/mL, Stirrer is added, 25o(ultimate density of Nile red is 5 × 10 to progress fluorometric investigation in each sample after stirring 16 h under C−6M).For the fluorescence spectrum of sample using 550 nm as excitation wavelength, scanning range is 580-700 nm.Using the fluorescence at 630 nm The mapping of the concentration of intensity and micellar solution.Concentration is polymer critical micelle concentration (Fig. 8) at the inflection point of low concentration region in figure.
2. the characterization and fluorometric investigation of enzymatic hydrolysis front and back blank micella
Firstly, blank micella (not carrying medicine) PBS solution freezing-pumping-applying argon gas is repeated 4 times to remove oxygen, in 2 mL After the deoxygenation of 0.6 mL is added in reaction tube blank micella solution and 0.4 mg azo reductase (DT-diaphorase, Human) and 1 mg coenzyme (NADPH), PBS(PH=7.4 after logical argon gas deoxygenation is added) buffer is diluted to 2 mL, and it is closed anti- Ying Guan.Then 37o24 h are vibrated in C constant temperature oscillator, are tested under the excitation wavelength of 650 nm by fluorescence spectrum glimmering Light variation；With ultraviolet specrophotometer UV-Vis, azobenzene structure variation is characterized；With transmission electron microscope TEM and dynamic light scattering DLS characterizes the variation of assembly pattern and size.
Solution drop is dyed one minute on copper mesh using Salkowski's solution, with transmission electron microscope (TEM) image table Levy the morphology and size of assembly.As shown in Figure 11 (a), the Amphipathilic block polymer is in PBS buffer solution (pH 7.4) Can obtain stable nano spherical particle, dynamic light scattering DLS(Figure 12) show that the assembly diameter is about 35 nm, PDI is 0.130.It can be found that the azobenzene UV absorption after digesting 24 h in system is substantially complete in conjunction with ultraviolet spectra and fluorescence spectrum It totally disappeared mistake (Fig. 9), system fluorescence is substantially enhanced (Figure 10), in conjunction with transmission electron microscope (Figure 11 b) and dynamic light scattering (Figure 12) It can be found that great variation has occurred in micella pattern and partial size, thus judge that azo reductase can successfully restore the assembly In azo benzo successfully dissociate micella, discharge fluorescence.
Fig. 9 is PEG in the present invention398-alAB6Azo-PLA144The azo before and after azo reductase is added in blank micella solution Benzene ultraviolet monitoring map, it can be found that in 350 cm-1With 460 cm-1The azobenzene absorption peak of left and right disappears substantially, it was demonstrated that even Nitrogen key is interrupted.
Figure 10 is PEG398-alAB6Azo-PLA144It is glimmering under 650 nm excitation wavelengths before and after blank micella solution reduction Flash ranging test result.It can be seen from the figure that fluorescence only faint before azo reductase is added；And azo reductase cultivation is added After 24 h, fluorescence intensity substantially enhances.After N=N key in reduction process as connection hydrophobe segment bridge is interrupted, polymerization Object is separated into hydrophilic PEG chain segment and hydrophobic PCL segment, and fluorescence probe is connected to be scattered in solution in hydrophilic PEG chain segment and lead Cause fluorescence release.Figure 11 is PEG in the present invention398-alAB6Azo-PLA144Blank micella solution is before being added azo reductase (a) TEM image of (b), Figure 12 are that azo is being added in PEG398-alAB6Azo-PLA144 blank micella solution in the present invention afterwards DLS characterization before and after reductase；It can be seen that in conjunction with Figure 11 and Figure 12, assembly is become after reduction by the spherical nanoparticle of rule For irregular aggregation, partial size and dispersion degree also increased (Dh =227 nm, PDI=0.288).
3. assembly contains adriamycin (DOX)
In order to contain hydrophobic drug adriamycin (DOX), by doxorubicin hydrochloride (DOXHCl) (5.0 mg) and triethylamine (2.4 μ L) is added in the dimethyl sulfoxide of 1.0 mL, is protected from light and is stirred overnight, and excess of triethylamine revolving is removed to get arriving The DMSO solution of DOX.Then, by the DMF solution of the DOX solution of 60 μ L and polymer, (1mL, 8 mg/mL pass through before use 0.22 μm of filter membrane dedusting) it is uniformly mixed, it is slowly added to the PBS buffer solution (PH 7.4) of 5 mL with syringe pump, is held during dropwise addition It is continuous to stablize stirring, 6 h are stirred for after being added dropwise.The dialysis in PBS buffer solution (PH 7.4) using bag filter (MWCO 3500) For 24 hours, the DOX of organic solvent and unentrapped is removed, just starts every 4h and changes a dialyzate, at least replace 6 dialyzates, it is whole It is carried out in the dark.Final constant volume assembles liquid solution at the load medicine that concentration is 1 mg/mL.
In order to measure the content of DOX, 3 mL are added after freeze-drying in the 100 μ L micellar solution for containing DOX DMSO dissolution destroys micella and discharges DOX.The fluorescence intensity at 590 nm of various concentration DOX/DMSO solution is measured to obtain Standard curve (excitation wavelength is 480 nm).According to containing fluorescence intensity of the DMSO solution of DOX micella at 590 nm from mark Its drugloading rate and encapsulation rate are determined on directrix curve.
Drugloading rate (DLC) and encapsulation rate (DLE) can be calculated according to formula below
Drugloading rate (w.t %)=(load weight/polymer of drug and load the total weight of drug) × 100%
Encapsulation rate (w.t %)=(the total input amount of weight/drug for loading drug) × 100%
Amphiphilic polymer PEG398-alAB6Azo-PLA144Drugloading rate to DOX is 0.69 w.t %, encapsulation rate 18.37%.
4. containing the drug release and its fluorescence detection of adriamycin (DOX) assembly
Firstly, packet medicine micellar solution freezing-pumping-applying argon gas is repeated 4 times to remove oxygen, in several 2 mL reaction tubes In be separately added into the packet medicine micellar solution after the deoxygenation of 0.6 mL and 0.4 mg azo reductase (DT-diaphorase, Human) and 1 mg coenzyme (NADPH), PBS(PH=7.4 after logical argon gas deoxygenation is added) buffer is diluted to 2 mL, and it is closed anti- Ying Guan.Then 37oOscillating reactions in C constant temperature oscillator, periodically takes pipe, by fluorescence spectrum respectively in 480 nm and 650 Change in fluorescence is tested under the excitation wavelength of nm to monitor the fluorescence and drug release process of its reduction response.
Solution drop is dyed one minute on copper mesh using Salkowski's solution, with transmission electron microscope (TEM) image (figure 16 a) and dynamic light scattering DLS(Figure 17) characterization assembly morphology and size.The Amphipathilic block polymer is buffered in PBS Stable nano spherical particle can be obtained in solution (pH 7.4), diameter is about 36 nm, PDI 0.142, with blank micella phase Than no notable difference.
Figure 13 is PEG in the present invention398-alAB6Azo-PLA144Packet medicine assembly is after being added azo reductase to azo The ultraviolet monitoring map of benzene, it can be found that in 350 cm-1With 460 cm-1The azobenzene absorption peak of left and right is with the reaction time Extension gradually weakens, it was demonstrated that N=N key is gradually interrupted.
Figure 14 is PEG398-alAB6Azo-PLA144During packet medicine assembly solution reduction under 650 nm excitation wavelengths Fluorometric investigation as a result, it can be seen from the figure that only faint fluorescence before azo reductase is added；And azo reductase is added Afterwards, fluorescence intensity is gradually increased with the variation of time.In reduction process gradually as N=N key of connection hydrophobe segment bridge After disconnection, polymer is separated into hydrophilic PEG chain segment and hydrophobic PCL segment, and fluorescence probe is connected in hydrophilic PEG chain segment gradually Being scattered in solution causes fluorescence to discharge, and gradually increases as the recovery time increases fluorescence, the fluorescence when 4 h of reductase 12 is small Intensity reaches maximum.It can be seen that from Figure 16 and 17, assembly is gradually become from the spherical nanoparticle of rule in reduction process Irregular aggregation, partial size and dispersion degree also have larger change (D after reductionh =279 nm, PDI=0.318).
Figure 15 is PEG398-alAB6Azo-PLA144During packet medicine assembly solution reduction under 480 nm excitation wavelengths Drug release situation.It can be seen from the figure that drug gradually discharges after azo reductase is added, 12 h are basically reached most later Big burst size (about 54%).It has done blank assay simultaneously to compare, has investigated packet medicine assembly in no azo reductase 37oC's Drug release situation, the results showed that only few drug release (< 6%) illustrates the packet medicine assembly when having no stimulation With preferable stability.Compare simultaneously it can be found that the azobenzene steric hindrance of Inventive polymers near infrared fluorescent probe is smaller, Azo reductase reducing degree is high, causes micella to dissociate more thorough, medicine-releasing performance is better than prior art tetraphenyl ethylene The polymer visible light wave range fluorescence probe of connection.
Figure 16 is PEG in the present invention398-alAB6Azo-PLA144Packet medicine assembly is after being added azo reductase, micella shape The TEM that looks change over time characterizes (a) 0 h(b) 3 h(c) 6 h(d) 9 h(e) 12 h(f) 24 h.
Figure 17 is PEG in the present invention398-alAB6Azo-PLA144Packet medicine assembly is dynamic before and after azo reductase is added State light scatters (DLS) characterization.
5, cytotoxicity test
For test polymer PEG398-alAB6Azo-PLA144Whether micella and enzymatic hydrolysis post-consumer polymer segment are toxic, use The PEG of 24 h is digested in the presence of the polymer P BS blank micella solution and enzyme of various concentration398-alAB6Azo-PLA144PBS glue Beam solution (50,100,150,200 μ g/mL) and two kinds of cells: it is small that culture 72 coexists in normal cell L929 and cancer cell Hela When, cytotoxicity test is carried out using MTT colorimetric method.Figure 18 is the cytotoxicity test results in the present invention, as shown in figure 18, Micellar solution after 24 h of polymer micelle or enzymatic hydrolysis of various concentration all shows lower cytotoxicity in two kinds of cells, Cell survival rate is all 90% or more.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Relative to small molecule, the big molecular dimension of polymer can effectively reduce the absorption of probe molecule, and polymer Load capacity with higher.Therefore, exploitation is had broad application prospects based on the macromolecular fluorescence probe that enzyme responds.To current Until, the medicine controlled releasing polymer near infrared fluorescent probe of azo-based benzene reductase response is rarely reported.What the present invention obtained Azo-based reductase response amphiphilic polymer, by solution self assembly can the stable polymer micelle of preparation structure go forward side by side One step is used as the carrier of packaging medicine；The polymersome of packaging medicine is destroyed point under azo reductase effect Solution, thus freeing that drug；Simultaneously as drug release fluorescence is activated and gradually increases.Therefore, this polymer can not only be used Make pharmaceutical carrier, while there is fluorescent probe function, can effectively monitor the process of drug release, to realize that drug is being tied Controllable and Targeting delivery, is a kind of potential colon specific drug controlled release carrier in intestines.

Claims (10)

1. a kind of Amphipathilic block polymer near infrared fluorescent probe of azo reductase response, the azo reductase response The general structure of Amphipathilic block polymer near infrared fluorescent probe is as follows:
In formula, 50≤m≤200,40≤n≤400.
2. the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response according to claim 1, feature It is, the preparation method of the Amphipathilic block polymer near infrared fluorescent probe of the azo reductase response includes following step It is rapid:
(1) compound A is reacted to obtain compound B with al6Azo compound；
(2) compound B is reacted with propine compound, obtains alAB6Azo compound；
(3) cause lactide ring-opening polymerization using alAB6Azo compound, obtain alAB6Azo-PLA；
(4) by alAB6Azo-PLA and mPEG-N3Reaction obtains the Amphipathilic block polymer near-infrared of azo reductase response Fluorescence probe.
3. the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response according to claim 2, feature It is:
In step (1), the molar ratio of compound A and al6Azo compound is 1: 1；The reaction is in the presence of potassium carbonate, DMF It carries out；The temperature of the reaction is 60 DEG C, and the time is 24 hours；
In step (2), the molar ratio of compound B and propine compound is 1: 4；It is described reaction in the presence of potassium carbonate, DMF into Row；The temperature of the reaction is 50 DEG C, and the time is 4 hours；
In step (3), ring-opening polymerization carries out in nitrogen, in the presence of DMAP；AlAB6Azo compound, DMAP, lactide Molar ratio be 0.05: 0.20: (2.5~10)；The temperature of ring-opening polymerization is 40 DEG C, and the time is 24~48 hours；
In step (4), reacts in the presence of toluene, CuBr and PMDETA, carried out in nitrogen；The temperature of reaction is 65 DEG C, the time It is 24 hours；alAB6Azo-PLA,mPEG-N3, CuBr, PMDETA molar ratio be (1.0~1.2): 1.0: 7.5: 15.
4. the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response according to claim 2, feature It is: carries out coupling reaction in ice water with p-aminophenyl methanol diazonium salt and phenol and obtain alAzo compound；With alAzoization Object is closed to react to obtain al6Azo compound with 6- bromohexane；It is azido group by the end group modification of poly glycol monomethyl ether, obtains mPEG-N3。
5. the Amphipathilic block polymer near infrared fluorescent probe of azo reductase response according to claim 4, feature It is:
Coupling reaction is carried out in ice water with p-aminophenyl methanol diazonium salt and phenol and obtains alAzo compound specifically: will be contained There is the mixed solution of concentrated hydrochloric acid, p-aminophenyl methanol, ice water to be added dropwise in sodium nitrite in aqueous solution, is then added dropwise and contains with stirring There is the aqueous solution of phenol, potassium carbonate, continue to stir, then adjusting pH is 4~5, alAzo compound is obtained, then by alAzoization It closing object, 6- bromohexane, potassium carbonate to be dissolved in acetone, be cooled to room temperature after reaction, reaction solution is extracted by ethyl acetate, is washed, Column is crossed after obtained organic layer is dry to purify to obtain al6Azo compound；
It is azido group by the end group modification of poly glycol monomethyl ether, obtains mPEG-N3Specifically: under the conditions of ice-water bath, it will contain Poly glycol monomethyl ether, triethylamine dichloromethane solution be added dropwise to containing in the dichloromethane solution to tosylate chloride, instead The PEG-OTs that should obtain paratoluensulfonyl chloride sealing end, then by PEG-OTs, NaN3It is added in DMF, reaction obtains mPEG-N3。
6. polymer micelle, which is characterized in that the preparation method of the polymer micelle the following steps are included:
(1) compound A is reacted to obtain compound B with al6Azo compound；
(2) compound B is reacted with propine compound, obtains alAB6Azo compound；
(3) cause lactide ring-opening polymerization using alAB6Azo compound, obtain alAB6Azo-PLA；
(4) by alAB6Azo-PLA and mPEG-N3Reaction obtains the Amphipathilic block polymer of azo reductase response；
(5) under stirring, buffer is added dropwise in the Amphipathilic block polymer solution of azo reductase response, is mixed Liquid；Continue to stir after being added dropwise to complete, finally dialysis obtains polymer micelle.
7. polymer micelle according to claim 6, it is characterised in that: with p-aminophenyl methanol diazonium salt and phenol in ice water Middle progress coupling reaction obtains alAzo compound；It is reacted to obtain al6Azo compound with 6- bromohexane with alAzo compound；It will The end group modification of poly glycol monomethyl ether is azido group, obtains mPEG-N3；The propine compound is propargyl bromide；Buffer For PBS buffer solution；In the Amphipathilic block polymer solution of the azo reductase response, solvent DMF；The mixed liquor In, water content 80wt%.
8. polymer micelle according to claim 7, it is characterised in that:
Coupling reaction is carried out in ice water with p-aminophenyl methanol diazonium salt and phenol and obtains alAzo compound specifically: will be contained There is the mixed solution of concentrated hydrochloric acid, p-aminophenyl methanol, ice water to be added dropwise in sodium nitrite in aqueous solution, is then added dropwise and contains with stirring There is the aqueous solution of phenol, potassium carbonate, continue to stir, then adjusting pH is 4~5, alAzo compound is obtained, then by alAzoization It closing object, 6- bromohexane, potassium carbonate to be dissolved in acetone, be cooled to room temperature after reaction, reaction solution is extracted by ethyl acetate, is washed, Column is crossed after obtained organic layer is dry to purify to obtain al6Azo compound；
It is azido group by the end group modification of poly glycol monomethyl ether, obtains mPEG-N3Specifically: under the conditions of ice-water bath, it will contain Poly glycol monomethyl ether, triethylamine dichloromethane solution be added dropwise to containing in the dichloromethane solution to tosylate chloride, instead The PEG-OTs that should obtain paratoluensulfonyl chloride sealing end, then by PEG-OTs, NaN3It is added in DMF, reaction obtains mPEG-N3。
9. the Amphipathilic block polymer near infrared fluorescent probe of the response of azo reductase described in claim 1 is preparing near-infrared Application in fluorescence probe and/or anticancer drug.
10. the Amphipathilic block polymer near infrared fluorescent probe of the response of azo reductase described in claim 1 is preparing azo Application in the pharmaceutical carrier of reductase response.
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