CN111675778B - Azo reductase responsive near-infrared polymer fluorescent probe prepared based on PISA method and application thereof - Google Patents

Azo reductase responsive near-infrared polymer fluorescent probe prepared based on PISA method and application thereof Download PDF

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CN111675778B
CN111675778B CN202010544263.4A CN202010544263A CN111675778B CN 111675778 B CN111675778 B CN 111675778B CN 202010544263 A CN202010544263 A CN 202010544263A CN 111675778 B CN111675778 B CN 111675778B
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周年琛
王雨晴
李立山
王书媛
孙亚兰
张伟
张正彪
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Suzhou University
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Abstract

The invention relates to an Azo reductase responsive near-infrared polymer fluorescent probe prepared based on a PISA method and application thereof, and provides an Azo reductase responsive molecule PPEGMA-ADP-Azo-CDPA which can be used as a near-infrared macromolecular chain transfer agent in a PISA polymerization method, and further, benzyl methacrylate is efficiently synthesized into the Azo reductase responsive near-infrared polymer fluorescent probe under the action of an initiator and the infrared macromolecular chain transfer agent by the PISA method, polymer nanoparticles eliminate aggregation quenching effect fluorescence enhancement under the action of the Azo reductase, and the near-infrared polymer nanoparticles realize the functions of the fluorescent probe and a drug carrier by utilizing the properties.

Description

Azo reductase responsive near-infrared polymer fluorescent probe prepared based on PISA method and application thereof
Technical Field
The invention relates to the technical field of material preparation, in particular to a PISA (particle image sensing system) method-based azo reductase responsive near-infrared polymer fluorescent probe and application thereof.
Background
In recent years, the application of polymer nanomaterials in drug carriers, nano-drugs, drug delivery, bio-imaging, and the like has attracted extensive attention of researchers. Stimuli-responsive nanoparticles may undergo reversible or irreversible physical or chemical changes under factors such as light, pH, temperature, enzymes, redox, etc., and may be applied to drug delivery, diagnostic imaging, biosensors, bio-separations, etc. Traditional solution self-assembly is usually that amphiphilic block polymer is dissolved in selected solvent and poor solvent is added, and then dialysis is carried out to freeze segments to form polymer nanoparticle assembly, however, the preparation method can only be carried out at low concentration (< 1% w/w) and the steps are tedious, which limits the commercial application of the method. With the development of living radical polymerization, polymerization induced self-assembly (PISA) has become a hot spot of research in this field due to the advantages of simple and efficient preparation method and capability of preparing nanoparticles with controllable morphology at high concentration. The steps of polymerization-induced self-assembly are generally as follows: the soluble chain segment as the macromolecule RAFT reagent is completely dissolved in a solvent, then another proper monomer is added for chain extension, the solubility of the second-stage polymer is gradually reduced in the chain extension process, the amphiphilic polymer nano-assembly is driven to form in the system in order to keep the balance of the interaction force, and the polymer nano-particles with different shapes and particle sizes can be obtained by controlling the length of the hydrophobic chain segment, the proportion of the hydrophilic chain segment and the hydrophobic chain segment and the polymerization time in the polymerization-induced self-assembly system. For the reasons mentioned above, the preparation of stimuli-responsive polymer nanoparticles for controlled drug release by means of polymerization-induced self-assembly would be of great advantage (see: Zhang, W.J., Hong, C.Y., Pan, C.Y., Biomacromolecules2017,18, 1210-one 1217.).
In recent years, the construction of drug targeted delivery strategies in the colon has attracted a great deal of attention from researchers. A large number of anaerobic microorganisms exist in the colon region of the human body (10)10～1012Bacteria/g) can generate enzymes such as azoreductase, nitroreductase and the like, the drug-loaded polymer nanoparticles containing azo bonds can release drugs in the colon in a positioning way, and side effects are reduced while the colon diseases are treated, so that inspiration is provided for the system design of drug targeted delivery for treating the colon diseases. Fluorescent probeDue to the advantages of high efficiency, sensitivity and visualization, the method is widely applied to the fields of biological imaging, biological sensing and the like. Near-infrared fluorescence becomes a research hotspot of a fluorescent probe due to the advantages of high transmittance, low background noise, reduction of injury to human tissues and organs and the like during biological imaging. The fluorescent probe has the advantage of intuitively, efficiently and sensitively realizing the monitoring of the drug release in a drug delivery system, and becomes a reliable way for researching the drug distribution and monitoring the drug delivery process. The near-infrared fluorescent molecule Aza-BODIPY not only has the advantages of high molar absorption coefficient, high fluorescence quantum yield, high structural stability, low pH sensitivity and the like, but also is an ACQ type dye molecule, namely, the ACQ type dye molecule can quench fluorescence in an aggregation state and recover the fluorescence in a dissolution state, so that the amphiphilic polymer nanoparticle probe capable of simulating the response of azoreductase in a colon environment can be efficiently designed for drug release of near-infrared fluorescence monitoring by utilizing the strategy.
The currently reported near-infrared polymer fluorescent probe responsive to azoreductase is relatively less used for targeted delivery and controllable release of colon drugs, and the PISA strategy is utilized to prepare the near-infrared probe in-situ high-concentration drug-coated drug to realize drug targeted delivery and release of fluorescence monitoring, so that the PISA in-situ preparation of the near-infrared polymer drug-coated nanoparticle fluorescent probe responsive to azoreductase has important significance for further imaging and treatment of colon.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a PISA (particle image sensing system) method-based azo reductase responsive near-infrared polymer fluorescent probe and application thereof.
The first purpose of the invention is to provide a molecule PPEGMA-ADP-Azo-CDPA with Azo reductase responsiveness, the structural formula of which is shown in the formula (I):
Figure BDA0002540129360000021
wherein n is more than or equal to 11 and less than or equal to 25.
The second object of the present invention is to provide a method for preparing an azoreductase responsive molecule represented by formula (I), comprising the steps of:
(1) reacting a compound ADP and Azobr in a formula (1) in an organic solvent at 60-65 ℃ under the action of alkali metal salt and potassium iodide to obtain a compound ADP-Azo in a formula (2) after the reaction is completed;
(2) reacting a compound ADP-Azo of a formula (2) with bromopropyne in an organic solvent at 60-65 ℃ under the action of an alkali metal salt, and obtaining a compound Al-ADP-Azo of a formula (3) after the reaction is completed;
(3) reacting Al-ADP-Azo with a RAFT reagent CDPA in an organic solvent at 0-25 ℃ under the action of DMAP and DCC, performing column chromatography after the reaction is completed, and separating a compound Al-ADP-Azo-CDPA in the formula (4);
(4) Al-ADP-Azo-CDPA and the compound PPEGMA-N of the formula (5) are added under the protection of inert atmosphere3Under the action of cuprous bromide and PMDETA, reacting in an organic solvent at 60-65 ℃ to obtain an azo reductase responsive molecule shown in formula (I) after complete reaction; wherein the structural formulas of the formulas (1) to (5) and the formula of the Azobr are as follows in sequence:
Figure BDA0002540129360000031
in the step (1), the molar ratio of ADP, Azobr, alkali metal salt and potassium iodide is 1 (1.1-1.5) to 2: 0.1.
Further, in the step (1), the preparation method of AzoBr comprises the following steps:
preparing diazonium salt of p-aminobenzyl alcohol: dropwise adding an aqueous solution of nitrite into aminobenzyl alcohol at the temperature of-5-0 ℃ in the presence of concentrated hydrochloric acid, and reacting completely to obtain a diazonium salt solution of the aminobenzyl alcohol;
preparing Azo: dropwise adding a diazonium salt solution of p-aminobenzyl alcohol into an aqueous solution of alkali metal salt and phenol at 0-5 ℃, and reacting completely to obtain Azo;
preparation of Azobr: the Azo and 1, 6-dibromohexane react in an organic solvent at 65 ℃ under the action of alkali metal salt, and after the reaction is completed, the Azober is obtained by column chromatography.
In step (2), the molar ratio of ADP-Azo, bromopropyne and alkali metal salt is 1 (1.1-1.5): 2.
Further, in the step (1) and the step (2), the alkali metal salt is selected from potassium carbonate.
Preferably, in step (1) and step (2), the organic solvent is acetone, in step (3), the organic solvent is dichloromethane, and in step (4), the organic solvent is anhydrous toluene.
Further, in the step (3), the molar ratio of Al-ADP-Azo, CDPA, DMAP and DCC is 1 (1.1-1.3) to 0.5 (1.0-1.2); in step (4), Al-ADP-Azo-CDPA, PPEGMA-N3The molar ratio of cuprous bromide (CuBr) to PMDETA is (1.1-1.5) to (1-2) to (2-3).
Further, in the step (4), the compound of formula (5), PPEGMA-N3The preparation method comprises the following steps:
(S1) under the anaerobic condition, PEAMA, EBIB, PMDETA and cuprous bromide react in anisole at 90 ℃ for 1.5 hours to obtain PPEGMA, wherein the proportion of PEGMA, EBIB, PMDETA and cuprous bromide is (1.1-1.5): 1:2: 3;
(S2) under the protection of inert atmosphere, reacting PPEGMA with sodium azide in an organic solvent at 80-85 ℃ to obtain the compound PPEGMA-N of the formula (5) after complete reaction3。
Preferably, the number average molecular weight (M) of PPEGMAn,NMR) 6500 and 7500g/mol (determined by nuclear magnetic testing); or number average molecular weight (M)n,SEC) 6300 to 7900g/mol (measured by gel chromatography (SEC)).
Preferably, the azo reductase responsive molecule of formula (I) above is prepared as follows:
Figure BDA0002540129360000051
a third object of the present invention is to disclose the use of the azo reductase responsive molecule of formula (I) as a near infrared macromolecular chain transfer agent.
It is a fourth object of the present invention to provide a method for preparing an azoreductase responsive near-infrared polymer in situ by a PISA (polymerization induced self-assembly) method, comprising the steps of:
under the protection of inert atmosphere, taking Azo reductase responsive molecules shown in formula (I) as near-infrared macromolecular chain transfer agents, reacting benzyl methacrylate (BzMA) in an organic solvent at 68-73 ℃ under the action of an initiator and the near-infrared macromolecular chain transfer agents, and obtaining Azo reductase responsive near-infrared polymers (PPEGMA-ADP-Azo-PBzMA) shown in formula (II) after complete reactionx) Wherein formula (II) is as follows:
Figure BDA0002540129360000061
wherein n is more than or equal to 5 and less than or equal to 87, and x is more than or equal to 3 and less than or equal to 75. Preferably, 11. ltoreq. n.ltoreq.25, 3. ltoreq. x.ltoreq.40.
Furthermore, the molar ratio of BzMA, PPEGMA-ADP-Azo-CDPA and the initiator is 6:1 (0.2-0.4).
Further, the initiator is AIBN (azobisisobutyronitrile).
The fifth object of the present invention is to provide an azo reductase-responsive near infrared polymer represented by the formula (II) prepared by the above preparation method.
The near-infrared polymer responding to the azo reductase shown in the formula (II) prepared by the invention is an amphiphilic block polymer, the preparation process is carried out in poor solvent ethanol at a hydrophobic end, the solubility of the polymer is deteriorated in the polymerization process, and finally the polymer exists in an assembly form with different shapes.
The azo reductase responsive near-infrared polymer assembly obtained by the polymerization-induced self-assembly is dialyzed to prepare corresponding micelle PBS solution, and the corresponding micelle PBS solution is added with Na2S2O4The assembly is destroyed by the action, and the phenomenon that the fluorescence is gradually enhanced is accompanied.
So far, the preparation of azo reductase responsive near-infrared polymer fluorescent probes by the PISA strategy has been rarely reported. The invention discloses a method for preparing an azo reductase responsive near-infrared polymer fluorescent probe by a PISA strategy.
The invention also claims application of the azo reductase responsive near-infrared polymer shown in the formula (II) in preparation of azo reductase responsive fluorescent probes.
The invention further claims application of the azo reductase responsive near-infrared polymer shown in the formula (II) in preparation of azo reductase responsive drug carriers or biological imaging preparations.
Further, near infrared fluorescence can be used to monitor the drug release process of the drug carrier or the targeted site of the bioimaging agent.
Further, an in-situ drug loading mode is adopted to prepare the azoreductase response near-infrared polymer drug carrier, and the method comprises the following steps:
the PPEGMA-ADP-Azo-CDPA shown in the formula (I), the initiator, BzMA and the hydrophobic drug are reacted in an organic solvent at 70 ℃ for 24 hours, and the amphiphilic block polymer drug-loaded micelle responding to the Azo reductase is obtained by a polymerization induced self-assembly (PISA) method.
Preferably, the hydrophobic drug is Doxorubicin (DOX), and other types of hydrophobic drugs with other properties can be selected and contained in the azo reductase responsive polymer micelle.
The prepared drug-loaded micelle can realize the controllable release of the drug only by simulating the colon environment in the presence of azoreductase and is accompanied with the activation and enhancement phenomenon of near-infrared fluorescence. Under the environment of simulating human colon, the azo bond in the azoreductase specificity reducing polymer changes the hydrophilic-hydrophobic proportion of the micelle to destroy the assembly to release the drug, and along with the release of the drug, the near infrared fluorescent molecule is no longer in an aggregation quenching state but is better dispersed in a PBS buffer solution along with the hydrophilic end, so that the fluorescence reduction is continuously enhanced. As azo reductase mainly exists in the colon of a human body, the drug-loaded micelle prepared by adopting the PISA strategy has potential application in the fields of preparation for treating colon diseases, drug monitoring and biological imaging preparation.
By the scheme, the invention at least has the following advantages:
(1) the invention synthesizes an Azo reductase response molecule PPEGMA-ADP-Azo-CDPA, which introduces near infrared fluorescent group at the hydrophilic chain end and can be used as macromolecular chain transfer agent with near infrared characteristic. (2) Chain extension is carried out on BzMA by using Azo reductase responsive near-infrared macromolecular chain transfer agent PPEGMA-ADP-Azo-CDPA, and PISA strategy is utilized to synthesize Azo reductase responsive near-infrared amphiphilic polymer PPEGMA-ADP-Azo-PBzMAxThe hydrophobic chain segment is continuously increased in the polymerization process, and the amphiphilic polymer forms a nano assembly. (3) The performance of the near-infrared fluorescent molecule ACQ is skillfully utilized to quench the near-infrared fluorescence in the process of forming an assembly, and Na is utilized2S2O4Or the azo reductase turns on fluorescence to realize the probe performance of the assembly. (4) The polymer hydrophilic end introduces near infrared fluorescence, and has higher signal-to-noise ratio and less human body injury when in biological imaging compared with fluorescence imaging in an ultraviolet visible light region.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
FIG. 1 shows the reaction of Azo in DMSO-d according to the invention6Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 2 shows the reaction of Azobr in DMSO-d according to the present invention6Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 3 shows the reaction of Al-ADP-Azo in DMSO-d according to the invention6Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 4 shows the reaction of Al-ADP-Azo-CDPA in DMSO-d according to the invention6Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 5 shows PPEGMA-N of the present invention3In CDCl3Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 6 shows PPEGMA-ADP-Azo-CDPA in DMSO-d according to the present invention6Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 7 shows PPEGMA-ADP-Azo-PBzMA of the present invention5In CDCl3Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 8 shows the Al-ADP-Azo-CDPA and the polymer PPEGMA-ADP-Azo-PBzMA obtained by polymerization-induced self-assembly in the present inventionxGPC outflow curve of (a);
FIG. 9 shows PPEGMA-ADP-Azo-PBzMA prepared by PISA according to the present inventionxA TEM pattern of (A);
FIG. 10 shows the in situ preparation of assemblies PPEGMA-ADP-Azo-PBzMA of PISA according to the inventionxIn PBS (0.3mg/mL) of Na2S2O4Ultraviolet-visible spectrums before and after reduction;
FIG. 11 Assembly PPEGMA-ADP-Azo-PBzMA prepared in situ from PISA according to the inventionxIn PBS (0.3mg/mL) of Na2S2O4Fluorescence spectra before and after reduction (excitation wavelength 650 nm);
FIG. 12 shows the in situ preparation of assemblies PPEGMA-ADP-Azo-PBzMA of PISA according to the inventionxIn PBS (0.3mg/mL) of Na2S2O4TEM image of the reduced sample;
FIG. 13 shows the in situ preparation of assemblies PPEGMA-ADP-Azo-PBzMA of PISA according to the inventionxIn PBS (0.1mg/mL) of Na2S2O4Corresponding dynamic particle size scattering (DLS) plots before and after reduction;
FIG. 14 shows PPEGMA-ADP-Azo-PBzMA of the present invention5@ DOX in CDCl3Nuclear magnetic hydrogen spectrum of1H NMR chart;
FIG. 15 shows PPEGMA-ADP-Azo-PBzMA of the present invention5The GPC outflow curve of @ DOX;
FIG. 16 shows DOX-entrapped micellar solution PPEGMA-ADP-Azo-PBzMA5@DOX(0.3mg/mL) UV-visible spectra at different times of enzymatic hydrolysis;
FIG. 17 shows the results of drug release over time of DOX-loaded micelle solution (0.3mg/mL) by azoreductase;
FIG. 18 shows DOX-entrapped micellar solution (0.3mg/mL) PPEGMA-ADP-Azo-PBzMA5@ DOX fluorescence spectrum (excitation wavelength 650nm) with time under the action of azo reductase;
FIG. 19 is a drug-loaded micelle PPEGMA-ADP-Azo-PBzMA5@ DOXPBS solution (0.3mg/mL) (a) Transmission Electron Microscopy (TEM) image before enzymatic hydrolysis (b) after enzymatic hydrolysis for 24 h;
FIG. 20 is a dynamic particle size scattering (DLS) plot of drug-loaded micelle PBS solution (0.1mg/mL) before and after 24h of enzymatic hydrolysis;
FIG. 21 is a schematic diagram of the synthesis of azo reductase responsive drug loaded micelles and the process of drug release.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The invention discloses a strategy for in-situ preparation of an azo reductase responsive amphiphilic block polymer micelle by PISA (platelet-activating antigen), which comprises the following steps:
(1) carrying out diazo coupling reaction on p-aminobenzyl alcohol and phenol to prepare azobenzene Azo with functional groups of phenolic hydroxyl and alcoholic hydroxyl respectively:
Figure BDA0002540129360000091
azo through nucleophilic substitution reaction, synthesizing Azo Br:
Figure BDA0002540129360000092
and (3) reacting near-infrared fluorescent molecules ADP with the Azobr to prepare ADP-Azo through nucleophilic substitution:
Figure BDA0002540129360000093
and (2) synthesizing Al-ADP-Azo with alkynyl and Azo bonds by using ADP-Azo and bromopropyne through an esterification reaction:
Figure BDA0002540129360000094
Al-ADP-Azo and a micromolecular RAFT reagent CDPA are synthesized into Al-ADP-Azo-CDPA through an esterification reaction.
(2) Selecting PEGMA as monomer, selecting anisole as solvent under the action of EBIB, CuBr and PMDETA, carrying out ATRP polymerization to obtain PPEGMA, adding sodium azide into the polymer PPEGMA to synthesize PPEGMA-N with azide as end group3：
Figure BDA0002540129360000095
PPEGMA-N with azide as end group3And a RAFT reagent Al-ADP-Azo-CDPA connected with near infrared fluorescent molecules is reacted with the RAFT reagent Al-ADP-Azo-CDPA to synthesize a macromolecular chain transfer agent PPEGMA-ADP-Azo-CDPA by click chemistry CuAAC:
Figure BDA0002540129360000101
(3) PPEGMA-ADP-Azo-CDPA is taken as a macromolecular chain transfer agent, BzMA is taken as a monomer, ethanol is taken as a solvent for polymerization induced self-assembly, and the amphiphilic block polymer micelle responding to Azo reductase is prepared, wherein the corresponding amphiphilic block polymer is PPEGMA-ADP-Azo-PBzMA:
Figure BDA0002540129360000102
the performance test method in the specific embodiment is as follows:
1. nuclear magnetic hydrogen spectrum (1H NMR) was measured by a Bruker 300MHz NMR spectrometer with Tetramethylsilane (TMS) as an internal standardThe sample is CDCl3Testing after the solvent is dissolved;
2. number average molecular weight (M) of the polymern) Weight average molecular weight (M)w) And molecular weight distribution index (M)w/Mn) Measuring on a TOSOH HLC-8320 gel chromatograph (SEC) equipped with a differential refraction detector and an ultraviolet detector, connecting two TSKgel Super Mutipore HZ-N (3 mu m beads size) columns in series, wherein the molecular weight range is 500-190,000 g/mol, chromatographic pure THF is used as a mobile phase, the flow rate is 0.35mL/min, the test is carried out at 40 ℃, and the molecular weight of the polymer is corrected by using polymethyl methacrylate (narrow distribution) as a standard sample;
3. the fluorescence emission spectrum is obtained by testing a Hitachi F-4600 type fluorescence photometer;
4. UV-visible absorption Spectroscopy measurements were carried out on a UV-2600 UV-visible spectrometer (Shimadzu, Nakagyo-ku, Kyoto, Japan) at 25 ℃;
5. a Transmission Electron Microscope (TEM) adopts HITACHI HT7700TEM, and the working acceleration voltage is 120 kV;
6. dynamic Light Scattering (DLS) Using a Malvern Zetasizer Nano-ZS90, test angle 90 ° and test temperature 25 ℃.
Example one
The preparation of azo reductase responsive amphiphilic block polymer micelle by PISA comprises the following steps:
1. synthesis of Azo
4.0g (32.4mmol) of p-aminobenzyl alcohol was added to a 100mL beaker, 6.8mL of concentrated hydrochloric acid was added, and 20mL of water was added and the mixture was stirred in an ice-water bath. Dissolving 3.3g (34.0mmol) of sodium nitrite in 10mL of water, dropwise adding the prepared aqueous solution of the sodium nitrite into a beaker, continuously reacting for half an hour after the dropwise addition is finished, and keeping the temperature at-5-0 ℃ all the time to completely react to obtain the diazonium salt of the p-aminobenzyl alcohol. Adding 3.2g (34.0mmol) of phenol and 6.3g (45.4mmol) of potassium carbonate into a 250mL beaker, adding 100mL of ice water to dissolve the phenol and the potassium carbonate, placing the mixture in an ice water bath, adding diazonium salt of p-aminobenzyl alcohol into the ice water bath, slowly dropwise adding the diazonium salt at 0-5 ℃, and continuing the reaction after the dropwise adding is finished. After the reaction is completed, the mixture is filtered by suction and washed by 20mL of ice waterAnd collecting and drying a filter cake for three times to obtain an orange solid with the yield of 91 percent. FIG. 1 is Azo1H NMR spectrum.
2. Synthesis of Azobr
2.0g (8.8mmol) of Azo, 2.1g (10.5mmol) of 1, 6-dibromohexane and 4.8g (17.6mmol) of potassium carbonate were charged into a 250mL round-bottomed flask, 100mL of acetone was added thereto, and the reaction was refluxed at 65 ℃ for 12 hours. After the reaction is finished, ethyl acetate is added for extraction, water washing is carried out for three times, the organic layer is taken and added with anhydrous sodium sulfate for drying, rotary evaporation is carried out, and then the organic layer is placed in a vacuum oven for drying and column chromatography (eluent: V)Petroleum ether/V Ethyl acetate4/1) gave a yellow solid in 64.4% yield.
FIG. 2 is of Azobr1H NMR spectrum.
3. Synthesis of ADP-Azo
0.7g (1.3mmol) of ADP, 0.5g (1.3mmol) of Azobr and 0.3g (2.6mmol) of potassium carbonate and 21.6mg (0.13mmol) of potassium iodide were charged into a 50mL round-bottomed flask, 20mL of acetone was added, and the reaction was refluxed at 65 ℃ for 24 hours. And after the reaction is finished, cooling to room temperature, adding ethyl acetate for extraction, washing for three times, taking an organic layer, adding anhydrous sodium sulfate for drying, carrying out rotary evaporation, placing in a vacuum oven for drying, and putting a crude product ADP-Azo into the next reaction.
4. Synthesis of Al-ADP-Azo
Adding 0.6g (0.7mmol) of ADP-Azo, 99.6mg (0.8mmol) of bromopropyne and 98.4mg (0.72mmol) of potassium carbonate into a 50mL round-bottom flask, adding 20mL of acetone, refluxing at 65 ℃ for 24 hours, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, extracting with ethyl acetate, washing with water for three times, taking an organic layer, adding anhydrous sodium sulfate, drying, rotary steaming, drying in a vacuum oven, and performing column chromatography (eluent: V)Petroleum ether/V Ethyl acetate2/1) gave Al-ADP-Azo as a dark green solid in 45.4% yield. FIG. 3 shows Al-ADP-Azo1H NMR spectrum.
5. Synthesis of Al-ADP-Azo-CDPA
0.5g (0.6mmol) of Al-ADP-Azo, 276.1mg (0.7mmol) of CDPA, 8.5mg (0.3mmol) of DMAP were placed in a 50mL three-necked flask under an inert gas argon atmosphere and dissolved by adding 10mL of dichloromethane. Will be provided withAdding 141.3mg (0.7mmol) of DCC dissolved in 10mL of dichloromethane into a constant-pressure dropping funnel, slowly dropping the DCC in the constant-pressure dropping funnel into a three-neck flask under ice bath, continuing to react for 15 minutes under ice bath after the dropping of the DCC is finished, and removing the ice water bath and stirring at 25 ℃ for reacting for 36 hours. After the reaction was complete, the mixture was filtered twice, rotary evaporated to dryness and dried under vacuum at 20 ℃. The product was a dark green solid in 56.7% yield. FIG. 4 shows Al-ADP-Azo-CDPA1H NMR spectrum.
6. Synthesis of PPEGMA-N3
1.2g (2.4mmol) of PEGMA, 48.5mg (0.3mmol) of PMDETA, 27.3mg (0.15mmol) of EBIB and 20.1mg (0.15mmol) of CuBr are weighed into a 5mL ampoule, 2.5mL of anisole are added, oxygen is removed by three cycles of freeze-pump-thaw-gas filling, and the mixture is transferred to 90 ℃ for reaction for 2 h. After the reaction is finished, breaking the tube, and settling in normal hexane to obtain the viscous polymer PPEGMA.
1.0g (0.14mmol) of PPEGMA and 0.14g (2.15mmol) of sodium azide were weighed, 10mL of anhydrous DMF was added, and the reaction was carried out at 80 ℃ for 24 hours. After the reaction is finished, adding dichloromethane and saturated saline water for extraction for 8 times, collecting a water layer dissolved with sodium azide for centralized treatment, simultaneously collecting an organic layer, adding anhydrous sodium sulfate for drying, performing suction filtration, collecting filtrate, performing rotary evaporation and concentration, and settling in normal hexane to obtain PPEGMA-N3The yield was 85.2%. FIG. 5 is PPEGMA-N3Is/are as follows1H NMR spectrum.
7. Synthesis of hydrophilic macromolecular chain transfer agent PPEGMA-ADP-Azo-CDPA
231.6mg (0.36mmol) of Al-ADP-Azo-CDPA and PPEGMA-N3(molecular weight is 7000g/mol, 2.1g,0.30mmol), 43.03mg (0.3mmol) cuprous bromide and 104.0mg (0.6mmol) PMDETA are added into a 25mL schlenk tube, 8mL of anhydrous toluene is added for dissolution, oxygen is removed through three cycles of freezing-air extraction-unfreezing-air filling, the tube is placed in an oil bath kettle at 60 ℃ for reaction for 24 hours, the reaction is cooled to room temperature after the reaction is finished, the tube is placed in a dialysis bag (MWCO3500), unreacted Al-ADP-Azo-CDPA and small molecular weight polymers and byproducts and the like are removed through dialysis in anhydrous ethanol, the tube is subjected to rotary steaming after dialysis, and the tube is placed in a vacuum oven at 25 ℃ for drying, thus obtaining PPEGMA-ADP-Azo-CDPA, wherein the yield is 87.6%.FIG. 6 is a representation of PPEGMA-ADP-Azo-CDPA1H NMR spectrum.
Wherein the number n of repeating units of the hydrophilic segment is determined by the ratio of PPEGMA-ADP-Azo-CDPA1The H NMR (FIG. 6) was calculated using the following formula (1):
m＝(I4.01-4.16/2)/(I7.84-7.96/8) formula (1)
I4.01-4.16: 4.01-4.16ppm of spectrogram corresponding to-OCH in hydrophilic chain segment PPEGMA2CH2Proton peak of O-fragment (a);
I7.84-7.96: the proton peaks (g, h) on the benzene ring of the near infrared fluorophore and azobenzene are corresponding to 7.84-7.96ppm in the spectra.
According to the integral area ratio of proton signal peaks at 4.01-4.16ppm and 7.84-7.96ppm, the-OCH of PPEGMA-ADP-Azo-CDPA is calculated by the formula (1)2CH2The number of repeating units of the O-segment was 14. Molecular weight and molecular weight distribution of PPEGMA-ADP-Azo-CDPA were characterized by GPC, and the molecular weight (M) is shown by the plot macro-RAFT in FIG. 8n) 10500g/mol, molecular weight distribution index (M)w/Mn) Is 1.12.
8. Polymerization induced self-assembly
Preparing Azo reductase responsive near-infrared nano particles by using a polymerization-induced self-assembly strategy to prepare a nano probe PPEGMA-ADP-Azo-PBzMA5For example, a typical procedure for the polymerization-induced self-assembly is as follows: 120.0mg (0.014mmol) of PPEGMA-ADP-Azo-CDPA, 0.8mg (0.003mmol) of AIBN and 28.0mg (0.084mmol) of BzMA were added to the ampoule in a molar ratio [ BzMA [)]0/[PPEGMA-ADP-Azo-CDPA]0/[AIBN]030/5/1. Dissolving the mixture by using 1440.0mg of absolute ethyl alcohol, wherein the solid content is 10 percent, stirring the mixture until the mixture is completely dissolved, removing oxygen in the system by three cycles of freezing, air extraction, unfreezing and air inflation, sealing the tube, placing the ampoule bottle at 70 ℃ for reacting for 24 hours, and then breaking the tube. Settling a part of micelle solution in n-hexane to obtain a green viscous solid, removing n-hexane, vacuum drying (30 deg.C) to obtain dark green viscous solid containing amphiphilic block polymer PPEGMA-ADP-Azo-PBzMA5By using a coreThe product was determined by magnetic, GPC and found to be about 91% yield, Mn＝12400g/mol，Mw/Mn＝1.15。
And taking out part of the polymer assembly ethanol solution, placing the part of the polymer assembly ethanol solution in a dialysis bag (MWCO8000) and dialyzing the part of the polymer assembly ethanol solution in a PBS buffer solution (pH is 7.4) for 24 hours to remove dead chains and ethanol solution generated in the polymerization process, and replacing water every 4 hours during the dialysis, thereby finally obtaining the near-infrared polymer nanoparticle PBS solution responding to the azo reductase.
PPEGMA-ADP-Azo-PBzMA preparation methodxExcept that the molar ratio of PPEGMA-ADP-Azo-CDPA and BzMA was varied.
FIG. 7 shows PPEGMA-ADP-Azo-PBzMA5Is/are as follows1H NMR spectrum, FIG. 8 shows PPEGMA-ADP-Azo-CDPA (curve macro-RAFT) and PPEGMA-ADP-Azo-PBzMA in the present inventionxGPC elution curve of (1). Wherein the number (x) of repeating units of the hydrophobic segment can be calculated by the following formula (2):
x＝(I4.80-4.97/2)/(I7.84-7.96/8) formula (2)
I4.80-4.97: 4.80-4.97ppm of spectrogram corresponds to-OCH in a hydrophilic chain segment PBzMA2Proton peak of fragment (d);
I7.84-7.96: the proton peaks (g, h) on the benzene ring of the near infrared fluorophore and azobenzene are corresponding to 7.84-7.96ppm in the spectra.
Such as PPEGMA-ADP-Azo-PBzMA5For example, the average degree of polymerization of the BzMA repeating unit was calculated to be 5.1 by the formula (2) from the integrated areas of the characteristic signal peaks 4.80 to 4.97 and 7.84 to 7.96. PPEGMA-ADP-Azo-PBzMA5The GPC curve of (A) is shown in FIG. 8, and the molecular weight (M) is shown by the corresponding curven) 12400g/mol, molecular weight distribution (M)w/Mn) Is 1.15. In FIG. 8, PPEGMA-ADP-Azo-PBzMA9Molecular weight (M) ofn) 14000g/mol, molecular weight distribution (M)w/Mn) Is 1.19; PPEGMA-ADP-Azo-PBzMA18Molecular weight (M) ofn) 15800g/mol, molecular weight distribution (M)w/Mn) Is 1.17; PPEGMA-ADP-Azo-PBzMA35Molecular weight (M) ofn) 17600g/mol, molecular weight distribution (M)w/Mn) Is 1.20.
9. Preparation and characterization of micellar solutions
Diluting 100 μ L of polymer assembly ethanol solution to 5mg/mL, transferring the micelle solution into a dialysis bag (MWCO8000), and dialyzing with ultrapure water for 24 hr to remove macromolecular chain transfer agent and ethanol which do not participate in polymerization. After the dialysis was completed, the volume was adjusted to 1mg/mL with a PBS solution (pH 7.4).
The micelle solution was diluted to 0.2mg/mL with PBS buffer, 15. mu.L of the micelle solution was dropped on a pure carbon film, left for 30 seconds, and then blotted dry with filter paper. Then 15. mu.L of a phosphotungstic acid solution having a concentration of 1 wt% was dropped thereon, left for 20 seconds, blotted with filter paper, dried, and then tested by a Transmission Electron Microscope (TEM). Meanwhile, the size thereof was characterized by Dynamic Light Scattering (DLS) to confirm the results of the TEM test. FIG. 9 shows the assembly PPEGMA-ADP-Azo-PBzMA of the present inventionxTEM spectrum of (A) and (D) in the sequence of PPEGMA-ADP-Azo-PBzMA5、PPEGMA-ADP-Azo-PBzMA8、PPEGMA-ADP-Azo-PBzMA18、PPEGMA-ADP-Azo-PBzMA35TEM spectrum of micellar solution.
Example two
The reduction of the micellar solution corresponds to the activation of fluorescence, as follows:
simulating colon environment, selecting sodium hydrosulfite Na2S2O4The reductive release behavior of several micelles obtained in example one was investigated as reducing agents mimicking azoreductase. First, 3.0mL of a micellar solution having a concentration of 0.3mg/mL was transferred to a cuvette, deoxygenated by introducing argon gas for 5 minutes, and 5mg of Na was added2S2O4And the mixture is placed in a water bath at 37 ℃ and stirred in a closed manner. During the period, the reduction response behavior of the near-infrared polymer micelle probe is monitored by fluorescence spectrum, and the excitation wavelength is 650 nm.
Meanwhile, the morphology and the size of the sample before and after reduction and the change of a characteristic absorption peak are characterized by TEM, DLS and UV-vis.
By observing the UV-vis UV-Vis spectrum (FIG. 10), in Na2S2O4Has a polymerization degree of 5, 9, 18 (FIGS. 10a, b, c)The absorption peak of azo at 360nm of the polymer nanoprobe disappeared, confirming the cleavage of the azo bond. Since the polymer chain is broken due to the breakage of the azo bond, the assembly cannot be maintained in an original stable state, and the polymer assembly is broken. While the nano probe with the vesicle morphology with the solvophobic chain segment of 35 repeating units is in Na2S2O4Shows a certain stability in the presence of (figure 10 d). Due to the gradual elimination of the ACQ function, the fluorescence spectrum shows that (figure 11) the fluorescence intensity of the polymer nano-particles with polymerization degrees of 5, 9 and 18 (figures 11a, b and c) in the near-infrared region of 700-750 nm is gradually increased, and the fluorescence enhancement phenomenon does not occur in the near-infrared region of the polymer nano-probe with the shape of the vesicle. In this reduction reaction, the assembly with a degree of polymerization of 35 and a vesicle morphology showed resistance to reduction (fig. 11d), probably because the azo moiety was packed tightly as a hydrophobic segment in the membrane of the vesicle, and the active site of the reaction was tightly embedded with the difficult-to-reduce azo bond.
Morphology and particle size changes before and after micelle reduction were tested by TEM and DLS. Fig. 12 and 13 show that the assembly with the polymerization degree of 5 (fig. 12a and 13a), the polymerization degree of 9 (fig. 12b and 13b) and the polymerization degree of 18 (fig. 12c and 13c) is broken into irregular assembly from the original regular assembly morphology due to the breaking of the azo bond, and at the same time, the DLS map confirms that the particle size of the assembly is enlarged, indicating that the assembly is broken under the reduction action. While the morphology and particle size of the polymersome with a polymerization degree of 35 (fig. 12d, 13d) are not changed significantly, a certain reduction resistance and stability are shown.
EXAMPLE III
The preparation method of the azo reductase responsive amphiphilic block polymer drug-loaded micelle by PISA comprises the following steps:
1. entrapment of Doxorubicin (DOX) by polymerization-induced self-assembly
120.0mg (0.014mmol) of PPEGMA-ADP-Azo-CDPA, 0.8mg (0.002mmol) of AIBN and 20mg of BzMA (0.06mmol) were added to an ampoule in a molar ratio [ BzMA ]]0/[PPEGMA-ADP-Azo-CDPA]0/[AIBN]0At the same time, 1mg of the compound was added thereto at 30:5:1Doxorubicin (DOX). Dissolved in 1400mg of absolute ethanol, the solid content being 10%. Stirring until the mixture is completely dissolved, removing oxygen in the system through three cycles of freezing, air extraction, unfreezing and air inflation, reacting at 70 ℃ for 24 hours, and then breaking the tube. After the polymerization was completed, a part of the polymer was taken out and settled in n-hexane to obtain a dark green polymer, which was named PPEGMA-ADP-Azo-PBzMA5@ DOX, Nuclear magnetic testing of the product (yield about 90%, M)n＝11500g/mol，Mw/Mn1.17). FIG. 14 is PPEGMA-ADP-Azo-PBzMA5Of @ DOX coated micelles1H NMR spectrum, FIG. 15 shows PPEGMA-ADP-Azo-PBzMA5@ DOX GPC elution Profile of coated micelles. And taking out the other part of the polymeric micelle solution, adding the polymeric micelle solution into a dialysis bag (MWCO8000) to perform dialysis in ultrapure water for 48 hours to remove the non-entrapped DOX, the macromolecular chain transfer agent which does not participate in the polymerization or is terminated in advance and ethanol, and fixing the volume of the polymeric micelle coated with the drug to 1mg/mL by using PBS buffer solution after the dialysis is finished to be reserved for the subsequent tests of the encapsulation rate, the entrapment rate and the reduction corresponding behavior.
2. Doxorubicin entrapped (DOX) content test
Taking 1mL of the drug-coated micelle solution prepared by PISA, freeze-drying the drug-coated micelle solution to remove the aqueous solution, and adding 3mLDMSO to completely dissolve the drug-coated micelle and destroy the micelle to release DOX. The content of DOX was tested by fluorescence spectroscopy (Hitachi F-4600): under the excitation wavelength of 480nm, measuring the fluorescence emission intensity of DOX at 590nm, measuring the fluorescence intensity of DOX/DMSO solutions with different concentrations to obtain a DOX concentration and fluorescence standard curve, obtaining the DOX concentration by comparing the fluorescence standard curve, and further calculating to obtain the encapsulation amount and encapsulation rate of the DOX encapsulated by the drug-encapsulated micelle.
The Drug Loading (DLC) and encapsulation efficiency (DLE) were obtained according to the following equations:
drug loading (wt%) × 100% (weight drug loaded/weight polymer nanoparticles)
Encapsulation efficiency (wt%) (weight of loaded drug/total drug input) × 100%
According to the formula of the drug loading rate (DLC) and the encapsulation efficiency (DLE), the concentration of the polymer micelle solution is 1mg/mL, the theoretical drug loading rate is 0.47 wt%, the actual drug loading rate is 0.22-0.34 wt%, and the encapsulation efficiency is 46.2-0.72%.
3. Azo reductase responsive drug-coated micelle drug release behavior research
Removing oxygen from 1mL of drug-coated micelle solution (0.6mg/mL) by three cycles of freezing, air suction, unfreezing and inflation, transferring the deoxygenated micelle solution into a 2.0mL reaction tube under the protection of inert gas, then respectively adding 0.20mg of azoreductase (DT-diaphorase Human) and 1.0mg of coenzyme (NADPH), simultaneously continuing to perform constant volume by using PBS buffer solution after bubbling deoxygenation, completely deoxygenating the closed reaction tube, placing the closed reaction tube in a constant temperature oscillator at 37 ℃ and oscillating for 1, 3, 6, 9, 12, 18 and 24 hours in a dark place, then respectively taking the tube to monitor the drug release and the fluorescence enhancement effect, and respectively monitoring the near infrared fluorescence change and the drug release effect under the excitation wavelengths of 650nm and 480nm through fluorescence spectroscopy. And testing the change of the characteristic absorption intensity of azobenzene around 360nm before and after enzymolysis through ultraviolet absorption spectrum to observe the breaking condition of azobenzene. And simultaneously, the changes of the appearance, the size and the particle size distribution of the micelle before and after reduction are represented by TEM and DLS.
As shown in FIG. 16, the azo bond is continuously cleaved into aniline fragments by the action of the azo reductase, as it can be seen from this UV-visible spectrum that the absorption intensity of the azo bond is continuously decreased with the increase of the reduction time. With the action of the azoreductase, as can be seen from fig. 17, DOX is released continuously and shows a trend of increasing continuously, the release amount can reach 82% at the time of reduction for 24 hours, meanwhile, near infrared fluorescence is monitored, as can be clearly seen from fig. 18, the near infrared fluorescence is gradually enhanced along with the increase of the reduction time, and the near infrared polymer nanoparticle fluorescent probe responding to the azoreductase is confirmed to realize the drug targeted release effect monitored by fluorescence.
The morphology and particle size variation of DOX-entrapped micelles were tested by TEM and DLS. Observing the morphology of the micelles before (fig. 19a) and after (fig. 19b) the reduction of fig. 19 shows that after the action of the azoreductase, the spherical micelles are greatly reduced, and irregular aggregates with larger sizes are generated. This is because the spherical micelle is dissociated by the breakage of the azo bond. The dynamic particle size scattering (DLS) plots (fig. 20) of the drug-loaded micelles before and after 24 hours of enzymatic hydrolysis are well documented.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims (10)

1. An azoreductase responsive molecule having the structural formula shown in formula (I):
Figure FDA0002540129350000011
wherein n is more than or equal to 11 and less than or equal to 25.
2. A method of preparing an azoreductase responsive molecule of claim 1 comprising the steps of:
(1) reacting a compound ADP and Azobr in a formula (1) in an organic solvent at 60-65 ℃ under the action of alkali metal salt and potassium iodide to obtain a compound ADP-Azo in a formula (2) after the reaction is completed;
(2) reacting a compound ADP-Azo of a formula (2) with bromopropyne in an organic solvent at 60-65 ℃ under the action of an alkali metal salt, and obtaining a compound Al-ADP-Azo of a formula (3) after the reaction is completed;
(3) reacting Al-ADP-Azo with a RAFT reagent CDPA in an organic solvent at 0-25 ℃ under the action of DMAP and DCC, and separating a compound Al-ADP-Azo-CDPA of the formula (4) after the reaction is completed;
(4) Al-ADP-Azo-CDPA and the compound PPEGMA-N of the formula (5) are added under the protection of inert atmosphere3Under the action of cuprous bromide and PMDETA, reacting in an organic solvent at 60-65 ℃ to obtain an azo reductase responsive molecule shown in formula (I) after complete reaction; wherein the structural formulas of the formulas (1) to (5) and the formula of the Azobr are as follows in sequence:
Figure FDA0002540129350000012
Figure FDA0002540129350000021
3. the method of claim 2, wherein: in the step (1), the molar ratio of ADP, Azobr, alkali metal salt and potassium iodide is 1 (1.1-1.5) to 2: 0.1.
4. The method of claim 2, wherein: in the step (2), the mole ratio of ADP-Azo, bromopropyne and alkali metal salt is 1 (1.1-1.5): 2.
5. The method of claim 2, wherein: in the step (3), the molar ratio of Al-ADP-Azo, CDPA, DMAP and DCC is 1 (1.1-1.3) to 0.5 (1.0-1.2); in step (4), the Al-ADP-Azo-CDPA, PPEGMA-N3The molar ratio of the cuprous bromide to the PMDETA is (1.1-1.5) to (1-2) to (2-3).
6. Use of the azo reductase-responsive molecule of claim 1 as a near infrared macromolecular chain transfer agent.
7. A method for preparing an azo reductase responsive near-infrared polymer in situ by a PISA process, comprising the steps of:
under the protection of inert atmosphere, taking the azo reductase responsive molecule of claim 1 as a near-infrared macromolecular chain transfer agent, and reacting benzyl methacrylate in an organic solvent at 68-73 ℃ under the action of an initiator and the near-infrared macromolecular chain transfer agent to obtain the azo reductase responsive near-infrared polymer shown in formula (II), wherein the formula (II) is as follows:
Figure FDA0002540129350000031
wherein n is more than or equal to 11 and less than or equal to 25, and x is more than or equal to 3 and less than or equal to 40.
8. An azo reductase-responsive near-infrared polymer represented by the formula (II) prepared by the preparation method according to claim 7.
9. Use of the azoreductase-responsive near-infrared polymer of claim 8 in the preparation of an azoreductase-responsive fluorescent probe.
10. Use of the azoreductase-responsive near infrared polymer of claim 8 in the preparation of an azoreductase-responsive pharmaceutical carrier or a bioimaging formulation.
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