CN112661740B - Light-responsive donor molecule capable of cooperatively releasing carbon monoxide and nitric oxide and derivatives, preparation method and application thereof - Google Patents

Abstract

The invention provides a donor molecule that releases carbon monoxide and nitric oxide synergistically in response to light, and has a structure represented by formula (I). The carbon monoxide and nitric oxide donor molecules shown in the structure of formula (I) provided by the present invention can sensitively and rapidly release carbon monoxide and nitric oxide under the response of low-intensity visible light, and can be further prepared into polymer prodrugs and assembled to obtain A high-performance polymer drug, and the molecule can efficiently kill Gram-positive bacteria. The structural molecule does not involve metal ions and other components that are harmful to the human body. It is completely dispersed and stable in aqueous solution, and has broad application prospects.

Description

Donor molecules and their derivatives for synergistic release of carbon monoxide and nitric oxide in response to light and preparation methods and applications

technical field

The invention relates to the field of signal molecule carrier preparation and polymer polyprodrug materials, in particular to a photoresponsive and coordinated release of carbon monoxide and nitric oxide donor molecules and their derivatives, as well as their preparation methods and applications.

Background technique

Carbon monoxide (CO) has always been considered a poisonous gas. People's perception of CO is that it competes with oxygen to combine with hemoglobin, causing hypoxia in the human body and eventually leading to suffocation and death. However, studies in recent years have found that carbon monoxide, similar to nitrogen monoxide and hydrogen sulfide, is also a gas signal molecule that can play an important role in signal transmission. Carbon monoxide produced in the human body is mainly produced by heme under the action of heme oxygenase to generate biliverdin, and release ferrous ions and carbon monoxide at the same time. Carbon monoxide generally acts as a messenger molecule and has many physiological functions, such as anti-inflammation, vasodilation, and wound repair. In addition, carbon monoxide at a slightly higher concentration (μM) can inhibit the growth of bacteria and kill bacteria at the same time, but due to the high toxicity of carbon monoxide, it is not suitable for treatment as a gas inhalation method, so various carbon monoxide releasing molecules have been developed. Capable of releasing carbon monoxide triggered by different stimuli. The existing carbon monoxide release donors include metal coordination compounds, cyclohexamethylenetetraamine carboxyborane and 3-hydroxyflavone derivatives, etc. The presence of metal ions in metal coordination compounds leads to poor biocompatibility, and the release is uncontrollable, and it is released immediately in the water environment. However, hexamethylenetetraminecarboxyborane will generate hydrogen gas while releasing carbon monoxide, which will have adverse effects on the release of CO by organisms. 3-Hydroxyflavone derivatives can be released under light irradiation, and have fluorescence properties that can be imaged at the cell level. However, 3-Hydroxyflavone derivatives also have disadvantages such as poor solubility. It is urgent to develop a new type of carbon monoxide donor. In order to achieve controlled release and good biocompatibility, it can be applied in the field of biomedicine.

Nitric oxide is the first gas signal molecule to be discovered. It is recognized as an important gas signal molecule in the cardiovascular and central nervous systems. It has many physiological functions, including platelet aggregation and adhesion, vasodilation, and wound repair. , immune response and carcinogenesis-related aspects. Studies have shown that low concentrations of nitric oxide can reverse the multidrug resistance of tumors or bacteria, and high concentrations of nitric oxide can directly kill cancer cells or bacteria. As a signaling molecule in the human body, NO can be produced endogenously in the human body. Nitric Oxide Synthase (NOS) exists in vascular endothelial cells, NOS is a protease family, mainly including neural nitric oxide synthase, inducible nitric oxide synthase and endothelial nitric oxide synthase . Endogenous NO is mainly derived from L-arginine. Under certain conditions, NOS can decompose L-arginine into NO and L-citrulline. Since NO is a gas at room temperature and is easily oxidized in the air, precise control of its concentration and dosage is not convenient for clinical operation, which greatly limits its clinical application. Currently, inhalation of NO to treat neonatal persistent pulmonary hypertension requires the use of special flow control equipment. Typical NO donors include nitro compounds (such as nitroglycerin), metal nitrosyl complexes (such as sodium nitroprusside), diazenium diols (NONOates) and sulfur nitrosyl compounds (RSNO), etc. These donor molecules usually have problems such as difficulty in synthesis and purification, insufficient stability in physiological environment, and uncontrollable release. For example, the commonly used diethylammoniumdiolate diazenium (DEA·NONOate) needs to be synthesized and prepared under high pressure (5-10atm) and strong alkali conditions for a long time (~3days), and it releases NO spontaneously under physiological conditions. The half-life is about 2.1min; the synthesis reaction of sulfur nitrosyl compounds (RSNO) will produce a large number of by-products, which are difficult to separate and purify; and NO can be released under the conditions of thiol, transition metal ion (Cu ⁺ ) and light. Therefore, it is of great significance to develop a new type of nitric oxide prodrug release donor that can release precisely and controllably and has good biocompatibility.

Light response has excellent characteristics as a trigger release condition, a molecule of flavonoid derivatives can release a molecule of carbon monoxide under light; nitrogen nitrosamines can undergo N-N bond homolysis under low light intensity irradiation, releasing a molecule of nitric oxide free radicals. Based on the above, the release of nitric oxide and carbon monoxide is controllable. Therefore, it is of great significance to develop a fluorescent donor that can release nitric oxide and carbon monoxide synergistically in response to light.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a photoresponse synergistic release of carbon monoxide and nitric oxide donor molecule and its derivatives as well as its preparation method and application, the obtained flavone derivatives can The synergistic release of carbon monoxide and nitric oxide is accompanied by obvious fluorescence changes, which can be used for biological imaging and monitoring of release in vivo.

In order to achieve the above purpose, the present invention provides a donor molecule that releases carbon monoxide and nitric oxide synergistically in response to light, which has a structure shown in formula (I):

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The R ₂ is a light-responsive protection element.

Preferably in the present invention, the R ₁ is selected from any structure in formula (R ₁ -1) to formula (R ₁ -14):

in, Indicates that it is connected with nitrogen, and * indicates that it is connected with methylene.

Preferably in the present invention, the R ₂ is selected from any structure in (R ₂ -a), formula (R ₂ -b), (R ₂ -c), (R ₂ -d):

Preferably in the present invention, the donor molecule has any structure in formula (I-1) to formula (I-7):

The present invention provides a method for preparing the above-mentioned donor molecule, comprising the following steps:

React compound II with R ₁ -NH ₂ to form a Schiff base, through reduction and nitrosation steps, and then react with methacryloyl chloride to obtain the structure shown in formula (I),

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The R ₂ is a light-responsive protection element.

The present invention provides an amphiphilic prodrug polymer with fluorescent properties that releases carbon monoxide and nitric oxide synergistically in response to light, and has a structure represented by formula (III):

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The _R2 is a light-responsive protection element;

The n is 1-30;

Said _n1 is 30-70.

The present invention provides a preparation method of the above-mentioned amphiphilic prodrug polymer with fluorescent properties that releases carbon monoxide and nitric oxide synergistically in response to light, comprising the following steps:

Mixing the compound of formula (IV) and the photoresponse of formula (I) with fluorescent properties to synergistically release monomer molecules of carbon monoxide and nitric oxide, reacting in the presence of the initiator AIBN to obtain the structure shown in formula (III) Amphiphilic carbon monoxide and nitric oxide prodrug polymers;

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The R ₂ is a light-responsive protection element.

The n is 1-30;

Said _n1 is 30-70.

The present invention provides an assembly, which is composed of the above-mentioned amphiphilic and fluorescent photoresponse synergistically releasing carbon monoxide and nitric oxide prodrug polymer or the amphiphilic and fluorescent photoresponse synergistically releasing carbon monoxide prepared by the above preparation method Prodrug polymers with nitric oxide, obtained by self-assembly.

The invention provides the application of the above-mentioned assembly as a signal molecule gas carrier in the preparation of polymer medicine.

The invention provides the application of the above-mentioned assembly as a signal molecule gas carrier in the preparation of a drug for killing Gram-positive bacteria.

Compared with the prior art, the present invention provides a donor molecule that releases carbon monoxide and nitric oxide synergistically in response to light, and has a structure represented by formula (I). The carbon monoxide and nitric oxide donor molecules shown in the structure of formula (I) provided by the present invention can sensitively and rapidly release carbon monoxide and nitric oxide under the response of low-intensity visible light, and can be further prepared into polymer prodrugs and assembled to obtain A high-performance polymer drug, and the molecule can efficiently kill Gram-positive bacteria. The structural molecule does not involve metal ions and other components that are harmful to the human body. It is completely dispersed and stable in aqueous solution, and has broad application prospects.

It should be noted that in the current prior art, there is no donor that can synergistically release carbon monoxide and nitric oxide in the field of releasing gas signal molecules, and the donor can simultaneously release carbon monoxide and nitric oxide under the irradiation of a low-intensity light source. nitrogen oxides. The donor molecule of the present invention also has multi-functionality, can release two kinds of signal molecules synergistically and have fluorescence changes at the same time, produce strong red fluorescence under light, and the red fluorescence disappears after the gas molecules are released. The donor molecule can prepare a polymer capable of releasing carbon monoxide and nitric oxide under the trigger of visible light through a controlled polymerization reaction, which improves the stability of NO and avoids safety problems caused by its premature release.

Description of drawings

Fig. 1 is the proton nuclear magnetic spectrum and the carbon nuclear magnetic spectrum of the monomer NCORM that the embodiment of the present invention 1 obtains;

Fig. 2 is the high-resolution mass spectrogram and the high performance liquid chromatogram of the monomer NCORM obtained in Example 1 of the present invention;

Fig. 3 is the NMR and gel chromatogram curves of the amphiphilic block polymer BP (PEG ₄₅ -b-PCNORM ₉ ) obtained in Example 1 of the present invention;

4 is a TEM photograph of micellar nanoparticles self-assembled by the BP (PEG ₄₅ -b-PCNORM ₉ ) polymer of Example 1 of the present invention;

Fig. 5 is the fluorescence change curve of nanoparticles prepared from the BP (PEG ₄₅ -b-PCNORM ₉ ) polymer provided in Example 1 of the present invention under 410nm illumination;

Fig. 6 is the curve of quantitative analysis of carbon monoxide release in pure water and phosphate buffer solution of BP (PEG ₄₅ -b-PCNORM ₉ ) polymer micelle nanoparticles in Example 3 of the present invention under 410nm light;

Fig. 7 is the curve of quantitative analysis of nitric oxide released by BP (PEG ₄₅ -b-PCNORM ₉ ) polymer micelle nanoparticles in Example 4 of the present invention under 410nm light;

Fig. 8 is the detection and quantification of nitric oxide and carbon monoxide released by BP (PEG ₄₅ -b-PCNORM ₉ ) polymer micellar nanoparticles in Example 2 of the present invention under different light conditions;

Fig. 9 is a bar graph showing the killing of Gram-positive bacteria Staphylococcus aureus by BP (PEG ₄₅ -b-PCNORM ₉ ) polymer micellar nanoparticles according to Example 2 of the present invention under 410 nm light.

Detailed ways

The present invention provides a donor molecule that releases carbon monoxide and nitric oxide synergistically in response to light, which has the structure shown in formula (I):

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The R ₂ is a light-responsive protection element.

According to the present invention, the X is preferably oxygen or nitrogen; the Y is preferably oxygen or sulfur.

According to the present invention, the R ₁ is selected from any structure in formula (R ₁ -1) to formula (R ₁ -14):

in, Indicates that it is connected with nitrogen, and * indicates that it is connected with methylene.

In the present invention, and * indicate the attachment position; a single bond indicates a methyl group.

According to the present invention, the R ₂ is more specifically selected from any structure in (R ₂ -a), formula (R ₂ -b), (R ₂ -c), (R ₂ -d):

In the present invention, the photoresponse with fluorescent properties cooperatively releases carbon monoxide and nitric oxide donor molecules having any structure in formula (I-1) to formula (I-7):

The present invention provides a method for preparing the above-mentioned photoresponsive coordinated release of carbon monoxide and nitric oxide donor molecules with fluorescent properties, comprising the following steps:

React compound II with R ₁ -NH ₂ to form a Schiff base, through reduction and nitrosation steps, and then react with methacryloyl chloride to obtain the structure shown in formula (I),

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The R ₂ is a light-responsive protection element.

The ranges of X, Y, R ₁ , and R ₂ are the same as above, and will not be repeated here.

In the present invention, there is no special limitation on the conditions of the above reaction, and the reaction can be carried out simply by mixing, and those skilled in the art can select a suitable preparation process according to the common knowledge in the field.

The present invention also provides an amphiphilic prodrug polymer with fluorescent properties that releases carbon monoxide and nitric oxide synergistically in response to light, which has the structure shown in formula (III):

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The _R2 is a light-responsive protection element;

The n is 1-30;

Said _n1 is 30-70.

The ranges of X, Y, R ₁ , and R ₂ are the same as above, and will not be repeated here.

The n is preferably 5-20, more preferably 5-15.

The n ₁ is preferably 45-60, more preferably 50-55.

The present invention provides a preparation method of the above-mentioned amphiphilic prodrug polymer with fluorescent properties that releases carbon monoxide and nitric oxide synergistically in response to light, comprising the following steps:

Mixing the compound of formula (IV) and the photoresponse of formula (I) with fluorescent properties to synergistically release monomer molecules of carbon monoxide and nitric oxide, reacting in the presence of the initiator AIBN to obtain the structure shown in formula (III) Amphiphilic carbon monoxide and nitric oxide prodrug polymers;

Wherein, the X is oxygen element or nitrogen element;

The Y is oxygen element or sulfur element;

The R ₁ is a substituted or unsubstituted phenylene group, a coumarinyl group, a benzofurazanyl group;

The substituents of the phenylene, coumarinyl, and benzofurazanyl groups are selected from one or more of nitro and trifluoromethyl;

The R ₂ is a light-responsive protection element.

The n is 1-30;

Said _n1 is 30-70.

The ranges of X, Y, R ₁ , R ₂ , n, and n ₁ are the same as above, and will not be repeated here.

The preparation method of the above-mentioned amphiphilic prodrug polymer with fluorescent properties that releases carbon monoxide and nitric oxide synergistically in response to light is not limited to reversible addition-fragmentation chain transfer polymerization (RAFT), and other suitable methods known to those skilled in the art can also be used. Synthetic method to obtain the structure described in the present invention.

Further, the amphiphilic block polymer can form a nano-assembly, and the polymer assembly can release nitric oxide and nitric oxide under the irradiation of 410nm visible light. Compared with small molecules, the stability of NO is improved. To avoid safety issues caused by its premature release, it will not be metabolized in a short period of time.

Based on this, the present invention provides an assembly, which is a prodrug polymer that releases carbon monoxide and nitric oxide synergistically from the above-mentioned amphiphilic photoresponse with fluorescent properties or the amphiphilic photoresponse with fluorescent properties prepared by the above preparation method A prodrug polymer that releases carbon monoxide and nitric oxide synergistically, obtained by self-assembly.

The method of the self-assembly is not particularly limited in the present invention, and may be a self-assembly method well known to those skilled in the art. The embodiment of the present invention uses a co-solvent, and then slowly adds water to assemble. The co-solvent includes but not limited to dimethyl sulfoxide, N,N-dimethylformamide, 1,4-dioxane, tetrahydrofuran, etc. One or more of them can be used as co-solvents to obtain stable nanoscale polymer assemblies.

Specifically, the amphiphilic block polymer is dissolved in an organic solvent as a co-solvent, added to purified water (such as ultrapure water) at a certain temperature and under stirring conditions, and then dialysis is performed at a certain temperature Removing the organic solvent; specifically, placing the obtained polymer nanoparticles in a dialysis bag at a certain temperature to remove the organic solvent by dialysis, thereby obtaining the polymer nanoparticle aqueous dispersion.

In the present invention, the amphiphilic polymer with fluorescent properties and photoresponse synergistically releasing carbon monoxide and nitric oxide obtained by selecting appropriate structures and functional groups can control and accurately release carbon monoxide and nitric oxide in the water phase. The specific implementation method is, after obtaining a stable nanoparticle dispersion, use a detection instrument or detection reagent to test the quantitative results of nitric oxide and carbon monoxide gas molecules released by the nanoparticle dispersion of the assembly under light at 410 nm. In addition, under 410nm light, the fluorescence changes of the test samples are sampled at different times, and the macroscopic fluorescence changes of the nanoparticle dispersion can be observed at the same time.

The assembly obtained by the self-assembly of the present invention can be used as a drug carrier and has a good effect as a drug for killing Gram-positive bacteria.

Based on this, the present invention provides the application of the above assembly as a signal molecule gas carrier in the preparation of polymer drugs.

The present invention also provides the application of the above-mentioned assembly as a signal molecule gas carrier in the preparation of a drug for killing Gram-positive bacteria.

Next, the technical solutions in the embodiments of the present invention will be clearly and completely described. Apparently, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In order to further understand the photoresponsive synergistic release of carbon monoxide and nitric oxide donor molecules and their derivatives as well as their preparation methods and applications described in the present invention, they will be described in detail in combination with examples.

Example 1

1-1) Synthetic light-responsive synergistic release of nitric oxide and carbon monoxide donor molecules (R ₁ is phenylene, R ₂ is o-nitrobenzyl, X is O, Y is O, and the left side is a naphthalene ring structure)

Preparation method: Weigh 1 (1g, 5.37mmol), add 15mL of ethanol to form a suspension, add aqueous sodium hydroxide solution (5.4mL, 5M), stir at room temperature for 30min, add p-hydroxymethylbenzaldehyde (748mg, 5.5mmol) In the reaction bottle, stir at room temperature for 5 hours. After the reaction, cool to 0°C, add 2mL of 30% hydrogen peroxide dropwise and react overnight, acidify the pH to 6.5 with 0.5M HCl, form a yellow precipitate, and obtain the product by suction filtration; weigh the product (288mg, 1mmol), potassium carbonate (140mg, 1.0mmol), o-nitrobenzyl bromide (258mg, 1.2mmol) were dissolved in 10mL DMF, and the mixture was stirred at room temperature for 12h. After the reaction, it precipitated into water, and the product was obtained by suction filtration; The product from the previous step (453mg, 1mmol) and pyridinium chlorochromate (620mg, 1.2mmol) were dissolved and clarified by adding 55mL of anhydrous dichloromethane, reacted overnight at room temperature, washed with suction three times, and recrystallized to obtain a yellow product; Add 20 mL of THF and 15 mL of ethanol to the obtained aldehyde-based product (450 mg, 1 mmol) to dissolve and clarify, add p-aminobenzyl alcohol (563 mg, 3.68 mmol), and react at room temperature overnight, and a solid precipitates out. In dichloromethane, add 266 mg of sodium cyanoborohydride, react for 2 hours, remove the solvent by rotary evaporation of the reaction solution, add 10 mL of THF to dissolve, add 5 mL of glacial acetic acid, then add 55 mg of sodium nitrite, react overnight, and obtain a yellow product by column chromatography . Weigh the precursor product (293mg, 0.5mmol) and dissolve it in 30mL of anhydrous dichloromethane, add anhydrous triethylamine (100mg, 1mmol), add methacryloyl chloride (105mg, 1mmol), react at room temperature for 6h, wash the column with water Chromatography gave 325 mg of a yellow solid.

The structure of the obtained donor molecule NCORM, which releases nitric oxide and carbon monoxide synergistically in response to light, was characterized by H NMR and C NMR spectra. The results are shown in Figure 1. At the same time, the obtained donor molecule NCORM was further characterized, and the results are shown in Figure 2. In Figure 2, the upper figure is the high-resolution mass spectrum, and the lower figure is the high-performance liquid chromatography. Fig. 1 and Fig. 2 are the proton nuclear magnetic spectrum, carbon spectrogram, high-resolution mass spectrogram and high-performance liquid chromatogram of the photoacoustic synergistic release of nitric oxide and carbon monoxide monomer molecules obtained in Example 1 of the present invention. It can be seen from the results that the obtained monomer molecular structure and purity are in line with the expected design.

1-2) Synthesis of prodrug block polymer BP with fluorescent properties and synergistic release of nitric oxide and carbon monoxide in response to light

Reaction characteristics: By adding PEO macroRAFT agent and the above-mentioned monomers with fluorescent properties that can release nitric oxide and carbon monoxide in photoresponse synergistically, the photoresponsive synergistic release of nitric oxide is obtained by reversible addition-fragmentation chain transfer polymerization (RAFT) Prodrug block polymer PCNORM with carbon monoxide. The polymerization degree n of the hydrophobic segment can be effectively changed by changing polymerization parameters and conditions, preferably n=5-20. Those skilled in the art understand that the degree of polymerization n is not critical to the present invention as long as it has no detrimental effect on the present invention. In order to help understanding more clearly, the polymer with n=9 is used as an example for illustration below.

Preparation method: Add 300mg NCORM monomer (0.4575mmol), 103mg PEO macroRAFT agent ((0.1mmol; refer to Macromolecules, 2008, 41, 12) for the synthesis method) and 16.53mg AIBN (0.045mmol) into a sealed tube, dissolve in 2mL In DMSO. Fully degas, seal the tube after degassing for the last time, and place it in an oil bath at 70°C. After 10 hours of polymerization, stop the reaction, add a small amount of dichloromethane to the obtained polymer solution, and precipitate in anhydrous ether , repeated the precipitation three times, and dried in a vacuum oven to obtain 217 mg of a yellowish polymer BP (yield: 53.8%).

The structure of the obtained polymer BP was characterized by proton nuclear magnetic spectrum and GPC, and the results are shown in Figure 3, in which the upper figure is the hydrogen nuclear magnetic spectrum and the lower figure is the gel chromatographic curve. It can be seen from the figure that the synthesized polymer of the present invention is as expected.

Example 2

Self-assembly to prepare nanomaterials, the specific description is as follows:

Dissolve 1 mg of BP polymer in 1 mL of N,N-dimethylformamide co-solvent, add 9 mL of ultrapure water to it at a rate of 0.9 mL/h under stirring at 500 r/min at room temperature, and dissolve the obtained white suspension The solution was placed in a MW14000 dialysis bag, dialyzed in ultrapure water, and the organic solvent was removed after dialysis for 12 hours. to obtain nanoassemblies.

The obtained nanomaterial assembly was tested, and the test results are shown in FIG. 4 . FIG. 4 is a TEM image of the nanomaterial obtained in Example 2. It can be seen from the figure that the obtained nanomaterial is a micelle with a diameter of about 200 nm.

Application example 1

Monitoring of Fluorescent Properties of Assemblies Under 410nm Illumination

Take 2mL of the assembly aqueous solution in a fluorescence cuvette with an optical path of 1cm, take samples at different times under 410nm illumination, and monitor the fluorescence changes of nanomaterials. The excitation wavelength is 405nm, and the fluorescence in the wavelength range of 415nm to 800nm is collected. At the same time, the changes in the fluorescent properties of the assembly can be seen with the naked eye. The test results are shown in Figure 5, in which the left picture is the change of fluorescence emission 5s before illumination, and the right figure is the change of fluorescence emission after 5s of illumination.

From Figure 5, it can be observed that the fluorescence properties change obviously. At 610nm, the red fluorescence increases rapidly at the initial stage of illumination. At the same time, the structure of flavonoids changes and carbon monoxide is released, and the red fluorescence disappears. This phenomenon is because the light-responsive protective group o-nitrobenzaldehyde falls off, exposing the hydroxyl group, which makes the flavonoids undergo ESIPT and produce strong red fluorescence. When a molecule of CO is released under the action of oxygen, the flavonoid structure changes, the ESIPT effect disappears, and the red fluorescence gradually disappears.

Application example 2

light releases carbon monoxide

A Deger gas detector was used to detect the release of the nanoparticles provided in Example 2 in different solutions under quantitative 410 nm light conditions. Specifically, 10 mL of the assembly water dispersion solution and pH 7.4 phosphate buffer dispersion solution were taken, and the carbon monoxide release was quantified in a closed environment under the conditions of stirring and 410 nm LED light (28.1 mW/cm ² ). The quantitative results are shown in Figure 6. It can be observed that under dark conditions, there are only fluctuations in the readings of the instrument, and there is almost no leakage of carbon monoxide, and the final release concentration is above 200nmol/mg.

Application example 3

nitric oxide released by light

Nitric oxide release was quantified with Griess reagent. The nanoassemblies obtained in Example 2 were irradiated with 410nm LED lamps (28.1mW/cm ² ). Specifically, the assemblies exposed to light for different times were mixed with an equal volume of Griess reagent, incubated in the dark for 10 minutes, and tested for ultraviolet absorption spectrum, and the content of nitric oxide released was quantified by the absorbance at 550 nm.

The results are shown in Fig. 7. Fig. 7 is the curve of quantitative analysis of nitric oxide released by micellar nanoparticles obtained by polymer BP self-assembly under the condition of visible light; Nitrogen release tended to be stable, and the assembly released 32.8% of nitric oxide.

Application example 4

Comparison of release of nitric oxide and carbon monoxide under different light intensities

Using 410nm light conditions, by changing the light intensity from weak to strong, the quantification of nitric oxide and carbon monoxide released from the quantitative assembly nanoparticle solution was detected. The quantitative results are shown in Figure 8, in which the left figure is the change of carbon monoxide release with light intensity, and the right figure is the change of nitric oxide release with light intensity. It can be seen from the figure that the release of nitric oxide and carbon monoxide also increases with the increase of light intensity, indicating that the release behavior of the two gas molecules is intensity-dependent. The more photon energy received, the more thorough the photochemical reaction occurs. So more is released.

Application example 5

Assemblies kill Gram-positive bacteria—Staphylococcus aureus under light conditions

The specific operation is to use 2.5mL sterilized TSB liquid medium to cultivate the colonies on the solid agar plate for 16-18 hours. To the logarithmic growth phase, the final required bacterial concentration of 5*10 ⁵ CFU/mL was obtained by centrifugation, washing and dilution. Add 100 μL of assembly solutions of different concentrations and 50 μL of bacterial solution in a 96-well plate, and incubate at 37°C for 30 minutes after irradiating at 410 nm (28.1mW/cm ² , 15min), then dilute 100 times and take 20 μL to spread on the plate, and incubate at 37°C Colony counting was carried out at 15h, and the histogram of the bactericidal effect was obtained.

The experimental results are shown in Figure 9. It can be seen that 81% of bacteria can be killed at 6.25 μg/mL, and more than 99% of bacteria can be killed at 25 μg/mL. It can be seen from the results that this fluorescent polymer nanoparticle assembly solution that can release carbon monoxide and nitric oxide synergistically in response to light can effectively kill Gram-positive bacteria at a very low concentration. It shows that the polymer has broad application prospects in antibacterial aspects, and at the same time, killing bacteria by gas signal molecules can prevent bacteria from developing drug resistance, which provides a reliable and effective reference for the future application of gas signal molecules for sterilization.

The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (9)

1. A donor molecule that is light responsive to co-release of carbon monoxide and nitric oxide, having the structure of formula (i):

wherein, X is oxygen element;

y is oxygen;

the R is ₁ Is phenylene;

the R is ₂ Is of the formula (R) ₂ -a) a structure shown in:

2. the donor molecule of claim 1, wherein R ₁ Selected from the formula (R) ₁ -1) to formula (R) ₁ -any one of the structures of 3):

wherein ,represents bonding to nitrogen, represents bonding to methylene。

3. The donor molecule of claim 1, having any of the structures of formulae (I-1) to (I-3):

4. a method of preparing a donor molecule according to any of claims 1 to 3, comprising the steps of:

combining compound II with R ₁ -NH ₂ Reacting to generate Schiff base, reacting with methacryloyl chloride after reduction and nitrosation steps to obtain a structure shown in formula (I),

wherein, X is oxygen element;

y is oxygen;

the R is ₁ Is phenylene;

the R is ₂ Is of the formula (R) ₂ -a) a structure shown in:

5. an amphiphilic light-responsive prodrug polymer capable of cooperatively releasing carbon monoxide and nitric oxide with fluorescent property, which has a structure shown in a formula (III):

wherein, X is oxygen element;

y is oxygen;

the R is ₁ Is phenylene;

the R is ₂ Is of the formula (R) ₂ -a) a structure shown in:

n is 1-30;

said n ₁ 30 to 70.

6. The method for preparing the amphiphilic prodrug polymer with fluorescent property and capable of cooperatively releasing carbon monoxide and nitric oxide through light response as claimed in claim 5, which comprises the following steps:

mixing a compound with a structure shown in a formula (IV) and a monomer molecule with a fluorescent property, wherein the monomer molecule is used for cooperatively releasing carbon monoxide and nitric oxide through a light response with a fluorescent property, and reacting in the presence of an initiator AIBN to obtain an amphiphilic carbon monoxide and nitric oxide prodrug polymer with a structure shown in a formula (III);

wherein, X is oxygen element;

y is oxygen;

the R is ₁ Is phenylene;

the R is ₂ Is of the formula (R) ₂ -a) a structure shown in:

n is 1-30;

said n ₁ 30 to 70.

7. An assembly obtained by self-assembly from the amphiphilic prodrug polymer with fluorescence property for cooperatively releasing carbon monoxide and nitric oxide prepared by the preparation method of claim 5 or the amphiphilic prodrug polymer with fluorescence property for cooperatively releasing carbon monoxide and nitric oxide prepared by the preparation method of claim 6.

8. The use of the assembly of claim 7 as a carrier for signal molecule gases in the preparation of polymeric drugs.

9. Use of the assembly of claim 7 as a carrier for a signal molecule gas for the preparation of a medicament for killing gram positive bacteria.