阅读说明：本技术 基于pisa方法制备的偶氮还原酶响应性的近红外聚合物荧光探针及其应用 (Azo reductase responsive near-infrared polymer fluorescent probe prepared based on PISA method and application thereof ) 是由 周年琛 王雨晴 李立山 王书媛 孙亚兰 张伟 张正彪 于 2020-06-15 设计创作，主要内容包括：本发明涉及一种基于PISA方法制备的偶氮还原酶响应性的近红外聚合物荧光探针及其应用,本发明提供了一种偶氮还原酶响应的分子PPEGMA-ADP-Azo-CDPA,其可作为PISA聚合法中的近红外大分子链转移剂,并进一步通过PISA方法,将甲基丙烯酸苄基酯在引发剂和红外大分子链转移剂的作用下,高效合成偶氮还原酶响应的近红外聚合物荧光探针,聚合物纳米粒子在偶氮还原酶作用下消除聚集猝灭作用荧光增强,利用上述性质该近红外聚合物纳米粒子实现荧光探针和药物载体功能。(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.)

1. An azoreductase responsive molecule having the structural formula shown in formula (I):

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:

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:

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.

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 probes are widely applied to the fields of biological imaging, biological sensing and the like due to the advantages of high efficiency, sensitivity and visualization. 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):

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:

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.

In 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) (ii) a 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 PPEGMA_n,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:

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 reaction_x) Wherein formula (II) is as follows:

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-PBzMA_xThe hydrophobic chain segment is continuously increased in the polymerization process, and the amphiphilic polymer forms a nano assembly. (3) Skillfully utilizes the ACQ performance of the near-infrared fluorescent molecules to form an assemblyQuenching near infrared fluorescence in the process using Na₂S₂O₄Or 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 invention_xGPC outflow curve of (a);

FIG. 9 shows PPEGMA-ADP-Azo-PBzMA prepared by PISA according to the present invention_xA TEM pattern of (A);

FIG. 10 shows the in situ preparation of assemblies PPEGMA-ADP-Azo-PBzMA of PISA according to the invention_xIn 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 invention_xIn 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 invention_xIn 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 invention_xIn 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) ultraviolet-visible spectra at different times of enzymolysis;

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:

azo through nucleophilic substitution reaction, synthesizing Azo Br:

and (3) reacting near-infrared fluorescent molecules ADP with the Azobr to prepare ADP-Azo through nucleophilic substitution:

and (2) synthesizing Al-ADP-Azo with alkynyl and Azo bonds by using ADP-Azo and bromopropyne through an esterification reaction:

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：

PPEGMA-N with azide as end group₃Synthesizing macromolecular chain transfer agent PPEGMA-AD by reacting with RAFT reagent Al-ADP-Azo-CDPA connected with near-infrared fluorescent molecules through click chemistry CuAAC reactionP-Azo-CDPA：

(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: