A photoresponsive nitric oxide donor functional material and its preparation method and application

Abstract

The present invention belongs to the technical field of gaseous transmitter donor functional materials, and specifically relates to a photoresponsive nitric oxide donor functional material and a preparation method and application thereof. The raw material of the photoresponsive nitric oxide donor material includes a nitric oxide donor molecule, and the nitric oxide donor has a structure shown in formula (I): formula (I); wherein, m and n are selected from any integer between 1 and 3. The photoresponsive nitric oxide donor functional material can be stably present in an aqueous environment, and also has visible light responsiveness. Under 410nm light irradiation, it still has the release characteristics of nitric oxide, and has a significant bactericidal effect on Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli).

Description

Technical Field

[0001] The invention belongs to the technical field of gaseous transmitter donor functional materials, and in particular relates to a light-responsive nitric oxide donor functional material and a preparation method and application thereof. Background Art

[0002] Nitric oxide (NO), as a gaseous signaling molecule, can participate in many physiological and pathological processes, especially in the field of antibacterial infection treatment. NO has a broad spectrum of antibacterial activity and can prevent bacteria from developing drug resistance. Its development prospects as an antibacterial agent have received increasing attention.

[0003] However, as a gas molecule, NO is mainly troubled by the safety issues of NO donors, residual toxicity of components or complicated procedures. In addition, due to the low stability of NO, the application of exogenous gaseous NO in antibacterial therapy is relatively rare. It is necessary to develop systems containing NO donors and methods for producing NO to effectively deliver NO. How to achieve efficient loading and controllable release of NO molecules is the key to achieving its high antibacterial efficiency.

[0004] In past studies, exogenous nitric oxide donor compounds mainly include organic nitrates, nitro compounds (such as nitroglycerin), thionitrosothiols, diazepine diol derivatives (NONOates) and metal-nitroso complexes (such as sodium nitroprusside). These compounds all have a common shortcoming, which is poor stability, causing problems such as premature leakage of nitric oxide and physiological toxicity. Among them, diazepine diol derivatives need to be synthesized under high pressure conditions, thionitrosothiols can respond to a variety of substances under physiological conditions, such as temperature, reducing substances, etc., and metal-nitroso complexes are prone to physiological toxicity due to their inherent metal ions. Therefore, it is very necessary to synthesize nitric oxide donor molecules with high stability and achieve controlled release of nitric oxide under external stimulation conditions.

[0005] As an external stimulus, light has the advantage of being controllable in time and space. The controlled release of nitric oxide by light triggering has been widely studied. Some light-responsive NO donors have been developed, which have unique advantages: the dose, rate and location of NO release can be easily regulated by controlling the illumination time, illumination position and illumination intensity, thereby achieving the purpose of effectively regulating NO release and related physiological processes.

[0006] Currently, in order to achieve the release of nitric oxide in a controlled manner, a nitric oxide donor that can be stimulated by light to release nitric oxide has been developed. Among the light-responsive nitric oxide donor molecules, nitrobenzene derivatives require high irradiation intensity to release nitric oxide, but high irradiation intensity is harmful to organisms. Therefore, in order to reduce the irradiation intensity, a nitric oxide donor has been developed. N -nitrosamine derivatives, which achieve light-triggered release of nitric oxide through homolytic cleavage of the N-N bond.N -The photosensitivity of nitrosamine derivatives is not ideal, and they are toxic, which greatly limits the practical application of photoresponsive nitric oxide donor molecules in biomedicine.

[0007] Therefore, it is of great significance to provide a photoresponsive nitric oxide donor that is weakly irradiated and has low toxicity. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides a photoresponsive nitric oxide donor functional material and its preparation method and application. The material is a nanoparticle functional material that can stably exist in an aqueous environment and also has visible light responsiveness.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] A photoresponsive nitric oxide donor material, wherein the raw material of the photoresponsive nitric oxide donor material comprises a nitric oxide donor molecule, and the nitric oxide donor has a structure shown in formula (I):

[0011] Formula (I);

[0012] Wherein, m and n are both selected from any integer between 1 and 3.

[0013] Preferably, the structural formula of the nitric oxide donor molecule is selected from any one of formula (I-1), formula (I-2) and formula (I-3),

[0014]

[0015] Formula (I-1),

[0016]

[0017] Formula (I-2),

[0018]

[0019] Formula (I-3).

[0020] Preferably, the nitric oxide donor molecule releases nitric oxide upon illumination at 400-420 nm.

[0021] Preferably, the method for preparing the nitric oxide donor molecule comprises the following steps:

[0022] Compound 1 and N , N -Carbonyldiimidazole reacts in solvent A to obtain a structure shown in formula (I), wherein the structure of compound 1 is shown below:

[0023] .

[0024] Further preferably, the compound 1 and N , N The molar ratio of '-carbonyldiimidazole is 1:3-5, the solvent A is anhydrous tetrahydrofuran, and the reaction time is 2-3 h.

[0025] The above-mentioned nitric oxide donor molecule is a small organic molecule, which is insoluble in aqueous solution, and its photoresponse effect in aqueous solution cannot be tested; a series of functional nanomaterials are prepared and derived from this, which on the one hand utilize the photoresponse of nitric oxide donor molecules, and on the other hand improve their water solubility so that they can be stably used in aqueous solution.

[0026] Preferably, the raw material of the photoresponsive nitric oxide donor material further comprises an amphiphilic polymer, and the amphiphilic polymer has a structure shown in formula (II):

[0027] Formula (II);

[0028] Wherein, x is selected from any integer between 5 and 10.

[0029] Further preferably, the molecular weight of the amphiphilic polymer is 3000-6000 Da.

[0030] More preferably, the structure of the amphiphilic polymer is the following formula (II-1):

[0031] Formula (II-1).

[0032] Further preferably, the mass ratio of the nitric oxide donor molecule to the amphiphilic polymer is 1:4-6.

[0033] The present invention also relates to a method for preparing the above-mentioned light-responsive nitric oxide donor material, comprising the following steps:

[0034] (1) mixing a nitric oxide donor molecule, an amphiphilic polymer and a solvent B to obtain a mixed solution 1;

[0035] (2) Adding Zn(NO3)2 aqueous solution to the mixed solution 1, stirring, and dialyzing to obtain the light-responsive nitric oxide donor material.

[0036] Preferably, the solvent B in step (1) is N , N -dimethylformamide, the mass volume ratio of the nitric oxide donor molecule to the solvent B in the mixed solution 1 is 1g:450-550 mL.

[0037] Preferably, the mass concentration of the Zn(NO3)2 aqueous solution in step (2) is 4-5 mg / mL, and the mass ratio of the Zn(NO3)2•6H2O to the nitric oxide donor molecule is 0.8-1:1.

[0038] Preferably, the stirring time in step (2) is 5.5-6.5 h, the stirring speed is 700-1500 rpm, and the dialysis molecular weight cutoff is 13000-15000 Da.

[0039] The present invention also relates to the use of the above-mentioned light-responsive nitric oxide donor material or the light-responsive nitric oxide donor material prepared by the above-mentioned preparation method in the preparation of antibacterial drugs.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The nitric oxide donor molecule in the present invention has a symmetrical structure and is used N , N '-Carbonyldiimidazole covalently modified nitric oxide donor molecules N , N '-Dinitroso-p-phenylenediamine derivatives were used to obtain the imidazole-terminated nitric oxide donor molecule NORM-CDI. The prepared donor molecule has visible light responsiveness and can release nitric oxide under 410 nm light irradiation;

[0042] (2) The nanoparticle functional material prepared by the present invention can stably exist in an aqueous environment and also has visible light responsiveness. Under 410 nm light irradiation, it still has the characteristic of releasing nitric oxide and has no significant effect on Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ) have significant bactericidal effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the NORM-CDI donor molecule in Example 1 of the present invention;

[0044] Figure 2 is the carbon NMR spectrum of the NORM-CDI donor molecule in Example 1 of the present invention;

[0045] Figure 3 is a high performance liquid chromatogram of the NORM-CDI donor molecule in Example 1 of the present invention;

[0046] Figure 4 is the mass spectrum of the NORM-CDI donor molecule in Example 1 of the present invention;

[0047] Figure 5 The amphiphilic polymer PEG containing imidazole in Example 1 of the present invention45 - b -H NMR spectrum of PMNZ8;

[0048] Figure 6 The amphiphilic polymer PEG containing imidazole in Example 1 of the present invention 45 - b -Size exclusion chromatogram of PMNZ8;

[0049] Figure 7 The particle size distribution diagram of the nanoparticle functional material MNZ-NORM that releases nitric oxide in response to visible light prepared in Example 1 of the present invention before and after irradiation with 410 nm light;

[0050] Figure 8 The transmission electron micrographs of the nanoparticle functional material MNZ-NORM that releases nitric oxide in response to visible light prepared in Example 1 of the present invention before and after irradiation at 410 nm;

[0051] Fig. 9 This is a graph showing changes in the ultraviolet absorption spectrum of the visible light responsive nitric oxide releasing nanoparticle functional material MNZ-NORM prepared in Example 1 of the present invention under 410 nm light as the light exposure time increases;

[0052] Fig.10 The electron paramagnetic resonance spectra of nitric oxide detected under different treatment conditions of the nanoparticle functional material MNZ-NORM for releasing nitric oxide in response to visible light prepared in Example 1 of the present invention;

[0053] Fig.11 This is a quantitative result diagram of nitric oxide detected by Griess reagent under 410 nm light for the visible light responsive nitric oxide releasing nanoparticle functional material MNZ-NORM prepared in Example 1 of the present invention;

[0054] Fig.12 Figure 2 shows the antibacterial results of the visible light responsive nitric oxide releasing nanoparticle functional material MNZ-NORM prepared in Example 1 of the present invention at different concentrations against Escherichia coli under 410 nm light. The data are expressed as mean ± standard deviation (n = 3), where ns means not significant and ** means p <0.01, **** indicates p <0.0001;

[0055] Fig.13Figure 2 shows the antibacterial results of the visible light responsive nitric oxide releasing nanoparticle functional material MNZ-NORM prepared in Example 1 of the present invention at different concentrations against Staphylococcus aureus under 410 nm light. The data are expressed as mean ± standard deviation (n = 3), where ns means not significant and *** means p <0.001, **** indicates p <0.0001. DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0057] Example 1

[0058] 1. The synthesis of the visible light responsive nitric oxide donor molecule NORM-CDI is as follows:

[0059]

[0060] The specific preparation process is as follows:

[0061] Compound 1 was prepared according to a previous report ( J. Mater. Chem. B , 2020, 8, 7009).

[0062] For the preparation of NORM-CDI, compound 1 (1.60 g, 3.43 mmol, 1.0 eq.) was dissolved in 100 mL of anhydrous THF, added to a round-bottom flask and stirred at room temperature under nitrogen protection. N,N-carbonyldiimidazole (CDI, 2.23 g, 13.76 mmol, 4.0 eq.) was dissolved in 50 mL of anhydrous THF and added dropwise to the above round-bottom flask using a constant pressure dropping funnel. After stirring the reaction for 2.5 h, the resulting mixture was concentrated and washed three times with anhydrous ether. After sufficient drying, 2.1 g of a light yellow solid product, NORM-CDI, was obtained.

[0063] The structure and purity of the obtained visible light responsive nitric oxide donor molecule NORM-CDI were characterized and verified by nuclear magnetic resonance hydrogen spectrum, carbon spectrum, mass spectrum and high performance liquid chromatography. The test results are as follows Figures 1 to 4 shown.

[0064] 2. Amphiphilic polymer PEG containing imidazole 45 - b -PMNZ8 was synthesized as follows:

[0065]

[0066] (1) The specific preparation process of compound 3 is as follows:

[0067] Metronidazole (5.0 g, 29.2 mmol, 1.0 eq.) and isocyanoethyl methacrylate (9.065 g, 58.4 mmol, 2.0 eq.) were dissolved in a THF / DMF mixed solvent (120 mL, v / v=5:1) and added to a round-bottom flask. 0.06 mL of DBTL was added to the mixture, stirred at room temperature for 3 h, and then concentrated on a rotary evaporator. The concentrated solution was then precipitated into 300 mL of n-hexane three times. Subsequently, the filter cake was collected by vacuum filtration. Finally, the solid product was dried in a vacuum oven overnight to obtain 8.7 g of compound 3.

[0068] (2) Amphiphilic polymer PEG containing imidazole 45 - b -The specific preparation process of PMNZ8 is as follows:

[0069] Compound 3 (500 mg, 1.532 mmol, 10.0 eq.), PEG45-CTA (343.9 mg, 0.1532 mmol, 1.0 eq.) and AIBN (5.03 mg, 0.03064 mmol, 0.2 eq.) were added to a 3 mL sealed tube, and the raw materials were dissolved with 2 mL DMSO. The oxygen in the sealed tube was removed by freeze-degassing-thawing, and the sealed tube was sealed. After the polymerization reaction was carried out at 70 ° C and 850 rpm for 10 h, the sealed tube was opened and the mixture was precipitated into 40 mL of anhydrous ether. The operation was repeated four times and fully dried to obtain a viscous brown-yellow polymer PEG. 45 - b -PMNZ8230 mg.

[0070] The obtained amphiphilic polymer PEG 45 - b -PMNZ8 was characterized by H NMR and size exclusion chromatography. The test results are as follows Figure 5 and Figure 6 shown.

[0071] 3. The preparation process of visible light responsive nitric oxide nanoparticles MNZ-NORM is as follows:

[0072] Take NORM-CDI (5.0 mg) and dissolve it in 0.5 mL DMF and add it to 2 mL of polymer PEG 45 - b-PMNZ8 (25 mg) in DMF solution to fully dissolve the raw materials. Add 1 mL of Zn(NO3)2 (4.5 mg) aqueous solution at 25°C and 800 rpm, and continue stirring for 6 hours. Then, dialyze the obtained 3.5 mL reaction solution with a MW 14000 Da dialysis bag. After the dialysis is completed, add water to 8 g. Centrifuge the obtained sample at 25°C and 4000 rpm for 5 min, and take the supernatant for subsequent testing.

[0073] The particle size of the prepared visible light responsive nitric oxide nanoparticles MNZ-NORM before and after 410 nm irradiation was characterized by ALV dynamic light scattering and transmission electron microscopy. Figure 7 and Figure 8 shown.

[0074] Example 2

[0075] 1. The degradation process of MNZ-NORM under 410 nm light was studied by UV absorption spectroscopy. The specific process is as follows:

[0076] A MNZ-NORM nanoparticle aqueous solution with a concentration of 80 μM was prepared and the UV absorption spectrum was tested using a 2 mm pathlength UV cuvette. 2 ) Irradiate the sample and track the changes in the UV absorption spectrum as the illumination time increases. The test results are as follows: Fig. 9 shown.

[0077] like Fig. 9 As shown in the figure, with the increase of 410 nm illumination time, the characteristic absorption peak (315 nm) of the ultraviolet absorption spectrum of MNZ-NORM gradually decreased, indicating that light can change the molecular structure of MNZ-NORM.

[0078] 2. The electron paramagnetic resonance (EPR) method was used to test the nitric oxide release of MNZ-NORM under 410 nm light. The specific process is as follows:

[0079] MGD and FeSO4 in PBS (10 mM, pH 7.4) were added to the NORM-CDI solution in DMSO / H2O (v / v=6:4) to control the NO release element concentration to 50 μM, MGD concentration to 3 mM, and FeSO4 concentration to 0.75 mM. Control group: The same treatment was performed except that NORM-CDI molecules were not included and no light was applied. Then, the air was replaced with nitrogen for 30 min, and the light group was irradiated with 410 nm light for 15 min (28.1 mW / cm 2 ), the non-illumination group was kept in the dark for the same period of time and subjected to EPR test. The test results are as follows Fig.10shown.

[0080] like Fig.10 As shown, only in the presence of MNZ-NORM, 410 nm light illumination can detect the triplet signal in the electron paramagnetic resonance spectrum using the mixed detection reagent of MGD and FeSO4, that is, the signal of nitric oxide is detected.

[0081] 3. Use Griess reagent to quantitatively detect the nitric oxide release of MNZ-NORM under 410 nm light. The specific process is as follows:

[0082] Preparation of Griess reagent: First prepare 0.1% N-(1-naphthyl)ethylenediamine hydrochloride (NED) and 1% p-aminobenzenesulfonic acid separately.

[0083] An aqueous solution of MNZ-NORM nanoparticles with a concentration of 80 μM was prepared, and first mixed with an equal volume of 1% p-aminobenzenesulfonic acid at 37°C for 5 min, and then an equal volume of 0.1% NED solution was added and cultured at 37°C for 10 min, and the UV absorption spectrum of the mixed solution at 400-700 nm was tested; in addition, aqueous sodium nitrite solutions with gradient concentrations were prepared: 0 μM, 12.5 μM, 25 μM, 50 μM, 100 μM and 200 μM, and co-cultured with the prepared Griess reagent according to the above method, and the UV absorption spectrum was recorded. The standard curve of UV absorption intensity-sample concentration at 523 nm was fitted for NO quantification. The nitric oxide quantification results are shown in Figure 2. Fig.11 shown.

[0084] like Fig.11 As shown in the quantitative results, with the increase of 410 nm illumination time, NO2 - The content gradually increased and tended to be stable, and the release rate was faster in the first 20 min.

[0085] Example 3

[0086] Antibacterial activity testing of visible light-responsive nitric oxide-releasing nanoparticles MNZ-NORM.

[0087] After the MNZ-NORM material was prepared, the antibacterial effects of samples of different concentrations on Escherichia coli and Staphylococcus aureus were tested before and after 410 nm light irradiation. The specific antibacterial process is as follows:

[0088] Gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus) were used for antibacterial performance studies. Bacterial culture: Use a sterile inoculation loop to take 3 colonies from a bacterial plate stored at 4°C and place them in 2.5 mL of sterile TSB medium. The bacterial solution is cultured in a 37°C shaking incubator for 18 h until it reaches the stable phase. Take another sterile inoculation tube, add 4 mL of sterile TSB medium, take 40 μL of the bacterial solution cultured to the stable phase, shake it, and continue to culture it in a 37°C shaking incubator for 1-2 h, controlling the OD600 at 0.6. Centrifuge the bacterial solution at 4°C and 10,000 rpm for 5 min, discard the supernatant, wash the bacteria twice with PBS, and finally disperse it in PBS for use.

[0089] Antibacterial experiment: PBS dispersion of MNZ-NORM nanoparticles (100 μL) and bacterial culture (50 μL) were added to a 96-well plate, and the final bacterial culture concentration was controlled to be 5×10 5 The bacterial solution was mixed by pipetting and co-cultured for 10 min, and then illuminated with 410 nm light (28.1 mW / cm 2 ) irradiation( E. coli , 20 min; S. aureus , 30 min). After the bacterial solution was irradiated with 410 nm light for 30 min in the dark, it was diluted 10 times twice in a row, 20 μL was taken from each well and coated on the plate, and the plate was counted after being cultured overnight in a 37°C incubator until it was visible to the naked eye. For the non-illuminated group, the sample and bacterial solution were mixed and co-cultured in the dark, and finally diluted and coated on the plate together with the experimental group. The blank group was a mixture of pure PBS and bacterial solution, and each group was paralleled to three groups.

[0090] The statistical results of the antibacterial activity of MNZ-NORM against Escherichia coli and Staphylococcus aureus are as follows: Fig.12 and Fig.13 shown.

[0091] like Fig.12 and Fig.13 As shown in the figure, under 410 nm light, with the increase of MNZ-NORM sample concentration, the survival rate of Escherichia coli and Staphylococcus aureus showed a decreasing trend. In addition, when the concentration of MNZ-NORM sample was 0.128 mg / mL, the survival rate of Escherichia coli was less than 5%; when the concentration of MNZ-NORM sample was 0.032 mg / mL, the survival rate of Staphylococcus aureus was less than 5%.

[0092] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A photoresponsive nitric oxide donor material, characterized in that: The method for preparing the photoresponsive nitric oxide donor material comprises the following steps: (1) mixing a nitric oxide donor molecule, an amphiphilic polymer and a solvent B to obtain a mixed solution 1; the solvent B is N , N - dimethylformamide; (2) adding Zn(NO3)2 aqueous solution to the mixed solution 1, stirring, and dialyzing to obtain the light-responsive nitric oxide donor material; Wherein, the nitric oxide donor molecule has a structure shown in formula (I): Formula (I); Wherein, m and n are selected from any integer between 1 and 3; The amphiphilic polymer has a structure shown in formula (II): Formula (II); Wherein, x is selected from any integer between 5 and 10.

2. The photoresponsive nitric oxide donor material according to claim 1, characterized in that: The structural formula of the nitric oxide donor molecule is selected from any one of formula (I-1), formula (I-2) and formula (I-3), Formula (I-1), Formula (I-2), Formula (I-3).

3. The photoresponsive nitric oxide donor material according to any one of claims 1 to 2, characterized in that: Nitric oxide donor molecules release nitric oxide upon irradiation with light at 400-420 nm.

4. The photoresponsive nitric oxide donor material according to any one of claims 1 to 2, characterized in that: The method for preparing the nitric oxide donor molecule comprises the following steps: Compound 1 and N , N -Carbonyldiimidazole reacts in solvent A to obtain a structure shown in formula (I), wherein the structure of compound 1 is shown below: 。 5. The photoresponsive nitric oxide donor material according to claim 4, characterized in that: The compound 1 and N , N The molar ratio of '-carbonyldiimidazole is 1:3-5, the solvent A is anhydrous tetrahydrofuran, and the reaction time is 2-3 h.

6. The photoresponsive nitric oxide donor material according to claim 1, characterized in that: The structure of the amphiphilic polymer is the following formula (II-1): Formula (II-1).

7. The photoresponsive nitric oxide donor material according to any one of claims 1 to 2, characterized in that: The mass ratio of the nitric oxide donor molecule to the amphiphilic polymer is 1:4-6, and the molecular weight of the amphiphilic polymer is 3000-6000Da.

8. A method for preparing the light-responsive nitric oxide donor material according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) mixing a nitric oxide donor molecule, an amphiphilic polymer and a solvent B to obtain a mixed solution 1; (2) adding Zn (NO3) 2 aqueous solution to the mixed solution 1, stirring, and dialyzing to obtain the light-responsive nitric oxide donor material; The solvent B in step (1) is N , N -Dimethylformamide.

9. The preparation method according to claim 8, characterized in that: The mass volume ratio of the nitric oxide donor molecule to the solvent B in the mixed solution 1 in step (1) is 1 g:450-550 mL; the mass concentration of the Zn(NO3)2 aqueous solution in step (2) is 4-5 mg / mL, the mass ratio of the Zn(NO3)2 to the nitric oxide donor molecule is 0.8-1:1, the stirring time is 5.5-6.5 h, the stirring speed is 700-1500 rpm, and the dialysis molecular weight cutoff is 13000-15000 Da.

10. Use of the photoresponsive nitric oxide donor material according to any one of claims 1 to 7 or the photoresponsive nitric oxide donor material prepared by the preparation method according to any one of claims 8 to 9 in the preparation of drugs against Escherichia coli and Staphylococcus aureus.

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