Photoresponsive carbon monoxide releasing nanogel and preparation method and application thereof

Abstract

The application provides a photoresponsive carbon monoxide releasing nanogel, a preparation method and application thereof, and solves the technical problems of uncontrollable release, non-targeted release and low bioavailability of CO when CO is used as an antibacterial agent. The photoresponsive carbon monoxide releasing nanogel is formed by coupling of polyacrylhydrazine nanogel with a fluid dynamic average particle size of 130nm-160nm and a polydispersity coefficient less than 1 and a metal-free, photoresponsive carbon monoxide releasing molecule after carboxyl functionalization; under visible light, the carbon monoxide slow-releasing nanogel releases CO, and the release period is 1h-3h.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and materials technology, specifically relating to a photoresponsive carbon monoxide-releasing nanogel, its preparation method, and its application. Background Technology

[0002] Since their discovery, antibiotics have been used as a powerful weapon against bacterial infections. Undeniably, as a routine treatment for bacterial infections, antibiotics have saved countless lives. However, large-scale clinical dosage and long-term use of antibiotics have inevitably led to the emergence of drug-resistant bacteria, especially multidrug-resistant bacteria. It is predicted that by 2050, drug-resistant infections could cause 10 million deaths annually. Non-antibiotic therapies offer new strategies for combating drug-resistant infections.

[0003] In recent years, endogenous gaseous signaling molecules, such as carbon monoxide (CO), have been considered a novel strategy against drug-resistant infections and have attracted increasing attention. Compared with traditional antibiotic treatment, they have shown many unique advantages, such as no drug resistance and low toxicity. To deliver CO safely and conveniently, CO-releasing molecules that can release CO under specific conditions have been developed. Although metal carbonyl carbon monoxide-releasing molecules are a powerful tool for local CO delivery, their bactericidal mechanism remains unclear, and uncontrolled release cannot be avoided. In addition, CO-releasing molecules, as small molecules, suffer from poor solubility, poor photostability, and poor tissue accumulation. Therefore, current CO delivery strategies should mainly address two aspects: controlled release and improved bioavailability.

[0004] Therefore, there is an urgent need for a delivery scheme that can achieve controlled release of CO and effectively improve its bioavailability. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problems of uncontrollable release, untargetable release, and low bioavailability of CO when used as an antibacterial agent, and to provide a photoresponsive carbon monoxide-releasing nanogel, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution provided by this invention is:

[0007] First, a carrier capable of loading CO donors is provided, and then a photoresponsive carbon monoxide-releasing nanogel is further prepared based on this carrier.

[0008] The special feature of polyacrylhydrazide nanogels is that their molecular structure is as follows:

[0009]

[0010] x and y represent the number of repeating units of the two repeating units of the nanogel, respectively, and z represents the degree of crosslinking; x, y, and z are all positive numbers, and x:y is approximately equal to 4:1;

[0011] Its hydrodynamic average particle size is 130nm to 160nm (this particle size range is suitable; particles smaller than 100nm have poor enrichment at the infection site, while particles that are too large are kinetically unstable), and its polydispersity index is less than 1.

[0012] The above-mentioned method for preparing polyacrylhydrazine nanogels involves free radical emulsion polymerization of hydrophobic nanogel monomer AH-Boc with hydrophilic monomer polyethylene glycol methyl ether acrylate (PEGA), crosslinking agent MBA, and initiator APS in an oxygen-free environment to obtain polyacrylhydrazine nanogels; specifically, it includes the following steps:

[0013] A. Preparation of polyacrylhydrazine nanogel monomer tert-butyl 2-acryloylhydrazine-1-carboxylate (AH-Boc)

[0014] A1. Add acrylic acid to tert-butyl hydrazide and mix evenly, then continue to dissolve using a mixed solution of deionized water and tetrahydrofuran (THF) to obtain solution 1;

[0015] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC was dissolved in a mixed solution of deionized water and tetrahydrofuran (THF) to obtain solution 2;

[0016] Add solution 2 dropwise to solution 1 in three to five portions and carry out the reaction at room temperature;

[0017] A2. After extraction, washing, and drying of the reaction solution obtained in A1, the solvent was removed by suspension evaporation, and the product was purified by recrystallization to obtain polyacrylhydrazine nanogel monomer 2-acrylhydrazine-1-carboxylic acid tert-butyl ester AH-Boc.

[0018] B. Preparation of polyacrylhydrazide nanogels

[0019] B1. Add the AH-Boc obtained in A2, the hydrophilic monomer PEGA, the crosslinking agent MBA, and the surfactant SDS (surfactant used to stabilize the hydrophobic monomer) to a Schlenk flask and dissolve them in deionized water; purge with an inert gas (such as argon) for 30 minutes to remove oxygen and maintain an airtight environment.

[0020] B2. Heat the mixture obtained in B1 to 70-75°C. After the temperature stabilizes, add an aqueous initiator APS to initiate the reaction. Ensure that the reaction is carried out at 70-75°C for 4-6 hours and then stop by cooling to 0°C.

[0021] B3. Dialyze the solution after reaction B2 with deionized water for 2-3 days, changing the dialysis water two to three times a day to obtain polyacrylhydrazine nanogel.

[0022] C Preparation of polyacrylhydrazide nanogels

[0023] C1. Add hydrochloric acid to the poly(acrylamide hydrazide-carboxylic acid ester) nanogel obtained in B3, acidify the solution to make the hydrochloric acid concentration 1 mol / L and stir for 10-12 h to remove the protection of tert-butyl formate groups.

[0024] C2. Add NaOH solution to the solution obtained in C1 to neutralize it, and stir for 30-60 minutes;

[0025] C3. Dialyze the solution obtained in C2 with deionized water for 2-3 days, changing the dialysis water two to three times a day to obtain polyacrylhydrazine nanogel.

[0026] Furthermore, in A1, deionized water and tetrahydrofuran are mixed in a volume ratio of 2:1;

[0027] The molar ratio of tert-butyl hydrazine carbamate, acrylic acid, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1:1;

[0028] For every 1 mmol of tert-butyl hydrazine carbamate, add 2.5–3 mL of a mixed solution of deionized water and tetrahydrofuran; dissolve 1 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 0.5–1 mL of a mixed solution of deionized water and tetrahydrofuran.

[0029] The reaction time is 5–6 hours;

[0030] A2 is specifically prepared by extracting the reaction solution obtained from A1 multiple times with ethyl acetate in a separatory funnel, then washing it twice with a 0.05-0.1 mol / L sodium carbonate aqueous solution, and finally washing it twice with ultrapure water; drying it with anhydrous sodium sulfate for 20 min, removing the solvent by evaporation, and then recrystallizing and purifying it to obtain the product polyacrylhydrazine nanogel monomer 2-acrylhydrazine-1-carboxylic acid tert-butyl ester.

[0031] In B1, the molar ratio of AH-Boc, PEGA, MBA, SDS, and APS in the nanogel solution is 4:1:0.4:0.06:0.09; and 8 mL of deionized water is added for every 0.4 mmol of AH-Boc; the concentration of MBA used is 0.1 mol / L, the concentration of SDS is 0.06 mol / L, and the concentration of APS aqueous solution is 0.006 mol / L; ensuring that the concentration of crosslinking agent MBA in the nanogel solution is 0.04 mmol / L and the concentration of initiator APS is 0.009 mmol / L;

[0032] In B2, the reaction time is 4 hours;

[0033] In C2, the concentration of NaOH solution added is 5 mol / L.

[0034] The above-mentioned polyacrylhydrazine nanogel is used as a drug carrier.

[0035] A type of photoresponsive carbon monoxide-releasing nanogel (CORNs) is unique in that:

[0036] It is formed by coupling of polyacrylhydrazine nanogel with a hydrodynamic average particle size of 130nm to 160nm and a polydispersity index of less than 1 with a carboxyl-functionalized metal-free, photoresponsive carbon monoxide-releasing molecule.

[0037] Under visible light, carbon monoxide is slowly released from the nanogel, which then releases CO over a period of 1 to 3 hours.

[0038] Its structural formula is as follows:

[0039]

[0040] x and y represent the number of repeating units of the two types of repeating units in the nanogel, respectively; z represents the degree of crosslinking; and xn represents the number of repeating units loaded with CORM. x, y, z, and n are all positive numbers, with x:y approximately equal to 4:1 and x:n approximately equal to 30 to 40:1.

[0041] The present invention also provides a method for preparing the above-mentioned photoresponsive carbon monoxide-releasing nanogel CORN.

[0042] The substances used in the preparation process include: 3-hydroxy-2-naphthoic acid, lithium methyl (LiMe), hydrogen peroxide (H2O2), sodium hydroxide (NaOH), 4-aldehyde benzoic acid, acrylic acid (AA), tert-butyl hydrazide carbazate, N,N'-methylenebisacrylamide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N,N'-methylenebisacrylamide (MBA), and ammonium persulfate (APS). The monomer, polyethylene glycol methyl ether acrylate (PEGA) with an average Mn of 480, was purified in a solvent purification system (Pure Solv™) before the reaction by passing it through a column packed with basic alumina to remove polymerization inhibitors, sodium hydroxide (NaOH), tetrahydrofuran (THF), dimethylformamide (DMF), ethyl acetate (EA), petroleum ether (PE), anhydrous ethanol (Ethanol), hydrochloric acid, and deionized water (Milli-Q water).

[0043] Includes the following steps:

[0044] 1) Preparation of metal-free, photoresponsive carbon monoxide-releasing molecule CORM

[0045] The preparation method of the metal-free, photoresponsive carbon monoxide-releasing molecule CORM (3-Hydroxy-2-(4-carboxy)phenyl-benzo[g]chromen-4-one) is as follows:

[0046] 3-hydroxy-2-acetylnaphthalene and 4-aldehyde benzoic acid were dissolved in anhydrous ethanol and subjected to a cyclization reaction catalyzed by hydrogen peroxide and sodium hydroxide to obtain a carboxyl-functionalized metal-free, photoresponsive carbon monoxide-releasing molecule CORM.

[0047] 2) Preparation of photoresponsive carbon monoxide-releasing nanogels CORN

[0048] The above-mentioned polyacrylhydrazine nanogel (prepared using the same method as described above, with no strict order in the preparation of the carbon monoxide-releasing molecule CORM and the polyacrylhydrazine nanogel) was deprotected by the tert-butyl formate group. This was then dissolved with the metal-free, photoresponsive carbon monoxide-releasing molecule CORM obtained in step 1) in a mixed solution of dimethylformamide and water. The metal-free, photoresponsive carbon monoxide-releasing nanogel CORN was obtained by catalytic reaction using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The catalytic system composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) effectively catalyzes the reaction of the carboxyl groups, improving the coupling efficiency.

[0049] Furthermore, in step 1), the specific steps for preparing the metal-free, photoresponsive carbon monoxide-releasing molecule CORM are as follows:

[0050] S1 Preparation of carbon monoxide-releasing molecular precursor 3-hydroxy-2-acetylnaphthalene-1-(3-hydroxynaphthalen-2-yl)ethan-1-one

[0051] S1.1 At room temperature, 3-hydroxy-2-naphthoic acid is added to tetrahydrofuran (THF) in the solvent purification system. After stirring until dissolved, an inert gas (such as argon) is introduced for at least 30 minutes to maintain an airtight environment (to ensure that the subsequent reaction is carried out in an oxygen-free inert environment). Then, the reaction temperature is lowered to 0°C using an ice-water bath (since methyllithium is exothermic, it needs to be lowered to 0°C in advance to avoid excessive exothermic reaction), and methyllithium is added dropwise to carry out the reaction.

[0052] S1.2 Add 0.5 mol / L to 1 mol / L hydrochloric acid to the solution after the reaction in S1.1 until no more bubbles are generated, ensuring that the unreacted methyllithium reacts completely; remove tetrahydrofuran (THF) under vacuum and continue to add 0.5 mol / L to 1 mol / L hydrochloric acid to adjust the pH of the solution to neutral; extract the acidified solution multiple times with dichloromethane and collect the lower organic layer; add anhydrous sodium sulfate to the organic layer for drying (at least 20 min), filter to remove sodium sulfate, and suspend in the solution to increase the concentration and ensure that no crystals precipitate; hydrochloric acid is used here mainly because it has no oxidizing properties and will not affect the reaction products;

[0053] S1.3 uses pure ethyl acetate and petroleum ether as eluents and silica gel as the stationary phase to remove impurities from the solution obtained in S1.2 by rapid column chromatography; then anhydrous sodium sulfate solution is added to dry the solution, the sodium sulfate is removed by filtration, the solvent is removed by suspension evaporation, and the solution is dried to obtain the carbon monoxide release molecule precursor 3-hydroxy-2-acetylnaphthalene.

[0054] S2 Preparation of Metal-Free, Photoresponsive Carbon Monoxide-Releasing Molecule CORM(3-Hydroxy-2-(4-carboxy)phenyl-benzo[g]chromen-4-one)

[0055] S2.1 Add the carbon monoxide-releasing precursor 3-hydroxy-2-acetnaphthalene obtained in S1.3 to a light-protected reaction vessel (to protect the target product generated during the reaction). First, add anhydrous ethanol and stir until homogeneous. Then, add a 5 mol / L NaOH aqueous solution (a high concentration of NaOH is used because CORM is insoluble in water, and adding too low a concentration of NaOH will affect the solubility). Stir at room temperature for 30-60 minutes, then add 4-aldehyde benzoic acid, and then stir under light-protected conditions for 5-6 hours. Use an ice-water bath to lower the solution to 0°C (to inhibit the decomposition of the subsequently added H2O2), and add 30 wt% H2O2 dropwise (for easy quantitative addition, it is best to use commercially available H2O2).

[0056] S2.2 The reaction solution of S2.1 was stirred in an ice-water bath for 10-12 hours, and then gradually brought to room temperature. Under light-protected conditions, 0.5 mol / L to 1 mol / L hydrochloric acid aqueous solution was added to adjust the pH to 6.5. The solution was filtered under light-protected conditions and washed with ice-cold anhydrous ethanol. Then it was dried in a vacuum drying oven to obtain metal-free, photoresponsive carbon monoxide-releasing molecules CORM.

[0057] Furthermore, step 2) specifically involves:

[0058] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and the metal-free, photoresponsive carbon monoxide-releasing molecule CORM prepared in step 1) were dissolved in dimethylformamide. Polyacrylamide nanogel solution was added to the mixed solution. After reacting at room temperature for 12-14 hours, the suspension turned orange-red. Dialysis with deionized water was performed for 2-3 days, changing the dialysis water two to three times a day to remove dimethylformamide and unreacted reactants. The suspension was filtered to obtain the photoresponsive carbon monoxide-releasing nanogel CORN.

[0059] Further, in S1.1, for every 1 mmol of 3-hydroxy-2-naphthoic acid, 4-5 mL of tetrahydrofuran and 1.9-2.0 mL of 1.6 mol / L methyllithium are added;

[0060] Maintain an airtight environment for 30 minutes; reaction time is 4-5 hours.

[0061] In S1.3, the ratio of ethyl acetate to petroleum ether is 1:1.5 to 3;

[0062] In S2.2, for every 1 mmol of 3-hydroxy-2-acetylnaphthalene, add 2-3 mL of anhydrous ethanol, 1 mL of 5 mol / L NaOH aqueous solution, and 0.9-1 mL of 30 wt% H2O2; wherein the molar ratio of 3-hydroxy-2-acetylnaphthalene to 4-aldehyde benzoic acid is 1:1; the light-protected reaction vessel refers to a brown reaction vessel wrapped with tin foil; use a No. F sintered glass funnel for suction filtration.

[0063] Further, in step 2, the molar ratio of carbon monoxide releasing molecule CORM, polyacrylhydrazine nanogel, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 12-16:1:60-80:60-80; and 4-5 mL of DMF is added for every 0.1 mmol of carbon monoxide releasing molecule CORM.

[0064] The present invention also provides the application of the above-mentioned photoresponsive carbon monoxide-releasing nanogel in the preparation of antibacterial agents, and the antibacterial agent product thereof.

[0065] The advantages of this invention are:

[0066] 1. This invention utilizes metal-free, photoresponsive CO-releasing molecules, avoiding the potential toxicity issues of transition metal ions while achieving controlled CO release (i.e., photoresponsive release). Metal-free CO-releasing molecules avoid the uncontrolled release of metal carbonyl CO-releasing molecules in the human body and the potential toxicity issues of transition metal ions. Furthermore, to more intelligently control CO release behavior for in vivo applications, ensuring sufficient stability of the polymer CO-releasing system during in vivo circulation, and specifically releasing CO at the infection site, the core design goal is to provide stimulus-responsive nanocarriers that offer an ideal solution for regulating CO release. Light is an ideal exogenous stimulus, capable of precisely regulating the release time and dosage of CO with minimal impact on important physiological parameters (achieving locally controlled release through localized light irradiation of the wound). Additionally, physiologically friendly visible light overcomes the side effects of ultraviolet light on the human body, making it more advantageous in regulating CO release.

[0067] 2. This invention uses polyacrylhydrazine nanogel as a nanocarrier to load CO-releasing molecules, improving the solubility, photostability, and bioavailability of CO-releasing molecules, and extending the release cycle. Nanogels are intramolecularly cross-linked polymer gels existing in the form of nanoparticles (particle size 1nm-1000nm), with a typical network structure, capable of dispersing into nano-sized hydrogel particles in aqueous solution. Compared to other gels, nanogels have several unique advantages: small size, easy phagocytosis by cells; easy penetration of various protective membranes in the human body, enabling deep drug delivery; and high drug loading efficiency. In this invention, the hydrazine functional group of the nanogel can rapidly react with a series of other functional groups under mild conditions to achieve CORM loading. Furthermore, the protonated hydrazine group under acidic conditions possesses bacterial targeting properties. Due to the negative charge of bacterial cell membranes, the microenvironment of bacterial infection is slightly acidic. The adhesion between this negatively charged bacterial cell wall and the positively charged nanogel promotes CO release at close range. To balance targeting capability and stealth properties, PEGA is used, thereby extending pharmacokinetics and improving particle biodistribution. The CORM release cycle of this nanogel is about 1h to 3h, and there is no release in the dark, which effectively prolongs the reaction time of CO, improves photostability, and realizes the controllable release of CO.

[0068] 3. The nanogel of this invention has undergone meticulous process optimization, resulting in a uniform particle size distribution, controllable morphology, low hemolysis rate, and good biocompatibility. This invention designed and optimized the effects of factors such as the ratio of the two monomers, the concentration of the crosslinking agent, the concentration of the initiator, and reaction time, exploring the optimal nanogel formulation (in the nanogel solution, hydrophobic monomer: hydrophilic monomer = 4:1; crosslinking agent concentration: 0.04 mmol / L; initiator concentration: 0.009 mmol / L; reaction time: 4 h), thus obtaining a monodisperse nanogel with a particle size of approximately 160 nm and a polydispersity index <1. Experiments confirmed that this CORN achieved a bactericidal rate of over 99% against MRSA and ER after light irradiation. Simultaneously, blood compatibility testing using rabbit erythrocytes showed no significant difference between the nanogel and the negative control, demonstrating good blood compatibility and showing significant application prospects in biomedical materials. Attached Figure Description

[0069] Figure 1 A simplified process flow diagram for preparing polyacrylhydrazide nanogels and CORN provided by the present invention, and a diagram showing the bactericidal effect of releasing CO under visible light.

[0070] Figure 2 The products of each step involved in this invention 11H NMR spectra; (a) CORM; (b) CORN; (c) Polyacrylhydrazide nanogel; (d) Poly(acrylhydrazide-carboxylic acid ester) nanogel;

[0071] Figure 3 The formulation optimization process for preparing nanogels in this invention is shown in (a) for the kinetic diameter and PDI of nanogels with different formulations; (b) for the SEM image of poly(acrylhydrazine-carboxylic acid ester) nanogels; and (c) for the SEM image of polyacrylhydrazine nanogels.

[0072] Figure 4 The DLS test results of the polyacrylhydrazine nanogel, CORN, and CORN after irradiation with 410nm light for 1 hour involved in the embodiments of the present invention are as follows:

[0073] Figure 5 The diagram shows the antibacterial effect of CORN provided by the present invention; wherein, (a) CORN's bactericidal effect on MRSA and ER under dark and light conditions; and (b) CORN's bactericidal rate on MRSA and ER under dark and light conditions.

[0074] Figure 6 These are blood compatibility test images of polyacrylhydrazide nanogel and CORN under no light conditions and under light conditions in this invention. Detailed Implementation

[0075] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0076] Example 1:

[0077] Product preparation

[0078] A method for preparing photoresponsive carbon monoxide-releasing nanogels, such as... Figure 1 As shown, the entire synthesis process, in sequence, involves the synthesis of polyacrylhydrazine nanogels and the coupling of the polyacrylhydrazine nanogels with the carbon monoxide-releasing molecule CORM. Specifically, it includes the following steps:

[0079] 1) Preparation of carbon monoxide-releasing molecule precursor 3-hydroxy-2-acetylnaphthalene (1-(3-hydroxynaphthalen-2-yl)ethan-1-one)

[0080] 3-Hydroxy-2-naphthoic acid (0.940 g, 5 mmol) was dissolved in dry tetrahydrofuran (20 mL), and the mixture was stirred at 0 °C under argon atmosphere for 30 min. A solution of methyl lithium in diethoxymethane (9.4 mL, 1.6 mol / L, 15.04 mmol) was added dropwise to the mixture using a syringe. The resulting mixture was stirred at 0 °C for 5 h. The solution was acidified with 0.5 mol / L HCl until no bubbles were generated. The solvent was then removed under reduced pressure, and the residue was added to 0.5 mol / L HCl (30 mL). The organic phase of the suspension was extracted three times with dichloromethane. The solvent in the extract was removed under reduced pressure. The crude product was then purified by flash column chromatography (using ethyl acetate as eluent) to give the yellow target product (yield 0.655 g, 69%).

[0081] 2) Preparation of carbon monoxide-releasing molecules CORM

[0082] Sodium hydroxide (1 mL, 5 mol / L, 5 mmol) was added to a suspension of 3-hydroxy-2-acetylnaphthalene (0.186 g, 1 mmol) in ethanol (3 mL), and the mixture was stirred at room temperature for 30 min. Then, 4-aldehyde benzoic acid (0.15 g, 1 mmol) was added to the mixture, and the reaction was stirred at room temperature for 5 h. The resulting mixture was cooled to 0 °C, and hydrogen peroxide (2.00 mL, 30%) was added dropwise. The reaction mixture was then stirred overnight and gradually heated to room temperature. Subsequently, 0.5 mol / L hydrochloric acid was added to the solution until pH = 6.5. The resulting yellow precipitate was separated by filtration and washed with cold ethanol to give the product (yield 0.212 g, 64%).

[0083] 3) Preparation of nanogel monomer AH-Boc

[0084] 1.586 g (12 mmol) of tert-butyl hydrazinoformate and 0.7 mL (10 mmol) of acrylic acid were dissolved in a mixture of THF and water (1:2, 25 mL). EDC (2.108 g, 11 mmol) was dissolved in the mixed solvent (5 mL) and added to the reaction mixture in portions. The mixture was stirred at 25 °C for 5 h, then extracted with ethyl acetate (30 mL × 3), and washed with sodium carbonate solution (0.1 M, 30 mL × 3) and deionized water (30 mL × 3). The product was dried over anhydrous sodium sulfate and condensed under reduced pressure to form a crude white solid. The target product was obtained by recrystallization from ethyl acetate (yield 1.165 g, 31.2%).

[0085] 4) Preparation of polyacrylhydrazide nanogels (optimal formulation and synthesis route)

[0086] In a Schlenk flask, AH-Boc (0.075 g, 0.4 mmol), PEGA (0.045 mL, 0.1 mmol), MBA (0.4 mL, 0.1 mol / L, 0.04 mmol), and SDS (0.1 mL, 0.06 mol / L, 0.006 mmol) were dissolved in deionized water (8 mL) and purged with Ar gas for 30 min to remove oxygen. The mixture was then heated to 70 °C, and an aqueous solution of APS (1.5 mL, 0.006 mol / L, 0.009 mmol) was added to initiate the reaction. The reaction was allowed to proceed at 70 °C for 4 h and stopped by cooling to 0 °C to obtain a poly(acryloylhydrazine-carboxylic acid ester) nanogel solution. 10 mL of the poly(acryloylhydrazine-carboxylic acid ester) nanogel was acidified with hydrochloric acid to 1 mol / L and stirred overnight to remove the tert-butyl formate protecting the hydrazine group. NaOH solution was added to neutralize the mixture and recover the amino group. After stirring for one hour, the solution was dialyzed with deionized water for two days, with the dialysate being changed twice a day.

[0087] 5) Synthesize CORN

[0088] EDC (0.155 g, 1 mmol), NHS (0.115 g, 1 mmol), and CORM (0.066 g, 0.2 mmol) were dissolved in DMF (5 ml). Ultrafiltration-centrifuged polyacrylhydrazine nanogel (14.25 mg, 0.0125 mmol) was added to the mixture. After reacting at room temperature for one day, the suspension turned orange-red. DMF and unreacted reactants were removed by dialyzing with deionized water for 3 days. Excess CORM precipitated from CORN was removed by filtration to obtain the final product.

[0089] To obtain a product with uniform particle size, the process for polyacrylhydrazine nanogel (step 4 above) was optimized in this invention as follows: Figure 3 As shown:

[0090] Figure 3 The images show the effects of the formulation used in the preparation of polyacrylhydrazine nanogels in this invention during the specific optimization process. (a) shows the process optimization of polyacrylhydrazine nanogels (the hydrodynamic diameter and PDI of the gel were measured by DLS); (b) shows the SEM image of poly(acrylhydrazine-carboxylic acid ester) nanogels; and (c) shows the SEM image of polyacrylhydrazine nanogels.

[0091] Polyacrylhydrazide nanogels are composed of monomers AH-Boc and PEGA, using MBA as a crosslinking agent and APS as an initiator. They are readily soluble in water under all pH conditions, thus contributing to improved cytocompatibility and solubility of CORM. The carbonyl hydrazide functional group in AH-Boc can rapidly react with a range of other functional groups under mild conditions, enabling CORM loading. Furthermore, the protonated hydrazide group under acidic conditions possesses antibacterial properties. However, it also facilitates drug adhesion, recognition, and clearance. To balance targeting capability and stealth properties, this invention utilizes PEGA. The stealth function prolongs particle pharmacokinetics and improves biodistribution.

[0092] Polyacrylhydrazide nanogels were synthesized via emulsion free radical polymerization, employing a rapid and simple synthetic route. To better control the partial size and molecular weight distribution, the effects of varying the ratio of the two monomers, the concentration of the crosslinking agent, the concentration of the initiator, and the reaction time were investigated. The hydrodynamic diameter of the polyacrylhydrazide nanogels was determined using DLS.

[0093] AH-Boc, containing a hydrazide group, is hydrophobic, while PEGA is hydrophilic. This invention investigated the ratio of these two monomers to determine the amount of hydrophobic monomer, thereby obtaining the optimal nanogel distribution and the highest level of active functionality. First, polyacrylamide hydrazide nanogels were prepared with different molar ratios of AH-Boc and PEGA at fixed concentrations of crosslinking agent, initiator, and reaction time. When the proportion of AH-Boc monomer was low, the diameter and PDI value of the polyacrylamide hydrazide nanogels became very large. As the proportion of AH-Boc monomer increased, the diameter and PDI value tended to stabilize. When the ratio of AH-Boc to PEGA was 4:1, the PDI value was 0.088, below 0.1, indicating a monodisperse size distribution. With further increases in the proportion of AH-Boc, the PDI value also increased slightly to 0.098. In terms of particle size and monodispersity, the optimal molar ratio of AH-Boc to PEGA was 4:1. Furthermore, the results show that the addition of AH-Boc can effectively optimize the performance of the polyacrylamide hydrazide nanogels.

[0094] After determining the monomer ratio, the effect of APS concentration on the size and molecular weight distribution was investigated. APS, as an initiator, determines the monomer conversion rate; a high conversion rate leads to stable dispersion polymerization kinetics. While keeping the monomer ratio, crosslinking agent concentration, and reaction time constant, polyacrylhydrazine nanogels were first prepared at different concentrations of APS. DLS results showed that when the APS concentration was below 0.9 mM, the monomer was not completely converted, resulting in smaller particle sizes. At 0.9 mM, the PDI value reached its minimum, with an average particle size of approximately 161.3 nm.

[0095] Since the crosslinking agent MBA has a significant impact on the dispersibility of nanogels, the concentration of MBA was further investigated under fixed monomer ratios, crosslinking agent concentrations, and reaction times. The results of the size and PDI values ​​of the nanogels synthesized at different MBA concentrations showed that the effect of MBA on the nanogel size was not as significant as the previous two variables. The optimal PDI value for the nanogel was achieved at an MBA concentration of 4 mM.

[0096] To optimize the synthesis process of nanogels, this invention also investigated the effect of reaction time on the nanogels. This invention used DLS to track the relationship between particle growth and time. Since the nanogels are synthesized via free radical polymerization, the polymerization is unstable at the beginning, and the measured size and PDI value are highly uncertain. As time increases, the polymerization reaction tends to end, and the size of the nanogels tends to stabilize. Optimal monodispersity was obtained at a reaction time of 4 hours.

[0097] Based on the research discussion, a monomer ratio of 4:1, 0.9 mM APS, 4 mM MBA, and a reaction time of 4 hours can be used for further experiments because it provides suitable particle size and optimal monodispersity. Advances in nanogel formulation optimization facilitate controlled drug release.

[0098] from Figure 3 As can be seen from (a), the particle size changes with the ratio of hydrophilic to hydrophobic monomers, MBA concentration, APS concentration, and reaction time; at the same time, observations (b) and (c) show that the deprotection process has no significant effect on the morphology of the gel.

[0099] Product confirmation:

[0100] The present invention performed proton nuclear magnetic resonance (NMR) spectroscopy tests on CORM, CORN, poly(acrylamide hydrazide-carboxylic acid ester) nanogels and polyacrylamide hydrazide nanogels involved in the embodiments. Figure 2 The above are the proton NMR spectra of the products and intermediates involved in this invention, wherein, (a) is the NMR spectrum of CORM dissolved in DMSO. 1 (a) 1H NMR spectrum; (b) Measured CORN dissolved in DMSO. 1 (c) ¹H NMR spectrum; (c) shows the determination of polyacrylhydrazine nanogel dissolved in DMSO. 1 1H NMR spectrum; (d) shows the determination of poly(acrylamide hydrazide-carboxylic acid ester) nanogel dissolved in DMSO. 1 H NMR spectrum. Figure 2 (d) confirms the successful synthesis of poly(acryloylhydrazide-carboxylic acid ester) nanogels (e.g., the characteristic peak at 1.38 ppm is a hydrogen atom on tert-butyl formate). Comparison Figure 2In (c) and (d), it can be observed that the tert-butyl formate group at 1.38 ppm, a characteristic peak, has been effectively removed, confirming that the protection of the tert-butyl formate group in the acidified poly(acrylamide hydrazine-carboxylic acid ester) nanogel has been effectively removed, yielding polyacrylamide hydrazine nanogel. Comparing (a) and (b), it can be observed that the characteristic peak in the CORM spectrum between 7 ppm and 9 ppm is present in both sets of NMR spectra, indicating that the polyacrylamide hydrazine nanogel has been successfully coupled with CORM.

[0101] Subsequently, DLS tests were performed on the polyacrylhydrazine nanogel, CORN, and CORN after irradiation with 410nm light for 1 hour involved in the embodiments of the present invention. Figure 4 As shown, compared to nanogels, the average diameter of CORN increased from 161 nm to 420 nm, and the PDI value also increased slightly. After 1 hour of irradiation at 410 nm, the nanostructure of CORN was preserved, and the average diameter decreased slightly from 420 nm to 409 nm, proving that CORM in CORN had undergone photo-oxidation and released CO.

[0102] Antibacterial effect test:

[0103] Figure 5 The diagram shows the performance of CORN provided by this invention; (a) the bactericidal effect of CORN on methicillin-resistant Staphylococcus aureus (MRSA) and tetracycline-resistant Escherichia coli (ER) under dark and light conditions; (b) the bactericidal rate of CORN on MRSA and ER under dark and light conditions. The specific experimental steps are as follows:

[0104] To verify the bactericidal effect of CORN, a bacterial experiment was conducted. Methicillin-resistant Staphylococcus aureus (MRSA) and tetracycline-resistant Escherichia coli (ER) were incubated with PBS and CORN for 20 min each. Then, one group each of PBS and CORN was irradiated with 410 nm light for 30 min. These four groups (PBS-no light, PBS-light, CORN-no light, CORN-light) were then incubated again and spread onto culture media, followed by overnight incubation at a greenhouse. Figure 5 (a) The results showed that CORN had a significant killing effect on MRSA and ER-resistant organisms after light exposure.

[0105] To further verify its bactericidal effect, the bactericidal rate of CORN against two drug-resistant bacteria after light exposure was calculated using a bacterial spot test. Antibacterial activity was studied using the standard colony-forming unit (CFU) counting method, with ER and MRSA used as representative bacterial strains. Specifically, different concentrations of CORN were co-incubated with bacterial PBS suspensions for 20 min, followed by irradiation under 410 nm light for 30 min. Afterwards, 20 μL of the bacterial dispersion was plated onto a gel-like TSB agar plate to allow colony formation. The bactericidal efficiency was calculated by counting the number of bacterial colonies. Figure 5 (b) shows that CORN achieved a bactericidal rate of over 99% against both drug-resistant bacteria after light exposure.

[0106] Blood compatibility:

[0107] Figure 6 These are blood compatibility test images of polyacrylhydrazide nanogel and CORN under no-light and light-light conditions, respectively, as used in this invention. The specific experimental steps are as follows:

[0108] The hemolysis assay used rabbit red blood cells. First, the red blood cell dispersion was centrifuged at 2000 rpm for 10 minutes, and the cells were resuspended three times in PBS to remove hemoglobin. 1% Triton X-100 in PBS was used as a positive control, and pure PBS as a negative control. Equal volumes of red blood cell suspension and 6 g / L CORN were incubated at 37°C for 1 hour. After centrifugation at 2000 rpm for 10 minutes, the supernatant (100 μL) was transferred to a 96-well plate, and the absorbance at 576 nm was collected. The hemolysis percentage was calculated using the following formula: Hemolysis % = (A... 576，实验组 -A 576，空白 ) / (A 576，对照组 -A 576，空白 ()×100%) from Figure 6 It can be seen that both the nanogel and CORN have good blood compatibility.

[0109] Example 2:

[0110] Product preparation

[0111] A method for preparing photoresponsive carbon monoxide-releasing nanogels specifically includes the following steps:

[0112] 1) Preparation of carbon monoxide-releasing molecule precursor 3-hydroxy-2-acetylnaphthalene (1-(3-hydroxynaphthalen-2-yl)ethan-1-one)

[0113] 3-Hydroxy-2-naphthoic acid (0.940 g, 5 mmol) was dissolved in dry tetrahydrofuran (20 mL), and the mixture was stirred at 0 °C under argon atmosphere for 30 min. A solution of methyl lithium in diethoxymethane (9.4 mL, 1.6 mol / L, 15.04 mmol) was added dropwise to the mixture using a syringe. The resulting mixture was stirred at 0 °C for 5 h. The solution was acidified with 0.5 mol / L HCl until no bubbles were generated. The solvent was then removed under reduced pressure, and the residue was added to 0.5 mol / L HCl (30 mL). The organic phase of the suspension was extracted three times with dichloromethane. The solvent in the extract was removed under reduced pressure. The crude product was then purified by flash column chromatography (using ethyl acetate as eluent) to give the yellow target product (yield 0.655 g, 69%).

[0114] 2) Preparation of carbon monoxide-releasing molecules CORM

[0115] Sodium hydroxide (1 mL, 5 mol / L, 5 mmol) was added to a suspension of 3-hydroxy-2-acetylnaphthalene (0.186 g, 1 mmol) in ethanol (3 mL), and the mixture was stirred at room temperature for 30 min. Then, 4-aldehyde benzoic acid (0.15 g, 1 mmol) was added to the mixture, and the reaction was stirred at room temperature for 5 h. The resulting mixture was cooled to 0 °C, and hydrogen peroxide (2.00 mL, 30%) was added dropwise. The reaction mixture was then stirred overnight and gradually heated to room temperature. Subsequently, 0.5 mol / L hydrochloric acid was added to the solution until pH = 6.5. The resulting yellow precipitate was separated by filtration and washed with cold ethanol to give the product (yield 0.212 g, 64%).

[0116] 3) Preparation of nanogel monomer AH-Boc

[0117] 1.586 g (12 mmol) of tert-butyl hydrazinoformate and 0.7 mL (10 mmol) of acrylic acid were dissolved in a mixture of THF and water (1:2, 25 mL). EDC (2.108 g, 11 mmol) was dissolved in the mixed solvent (5 mL) and added to the reaction mixture in portions. The mixture was stirred at 25 °C for 5 h, then extracted with ethyl acetate (30 mL × 3), and washed with sodium carbonate solution (0.1 M, 30 mL × 3) and deionized water (30 mL × 3). The product was dried over anhydrous sodium sulfate and condensed under reduced pressure to form a crude white solid. The target product was obtained by recrystallization from ethyl acetate (yield 1.165 g, 31.2%).

[0118] 4) Preparation of polyacrylhydrazide nanogels (optimal formulation and synthesis route)

[0119] In a Schlenk flask, AH-Boc (0.075 g, 0.4 mmol), PEGA (0.045 mL, 0.1 mmol), MBA (0.4 mL, 0.1 mol / L, 0.04 mmol), and SDS (0.1 mL, 0.06 mol / L, 0.006 mmol) were dissolved in deionized water (8 mL) and purged with Ar gas for 30 min to remove oxygen. The mixture was then heated to 70 °C, and an aqueous solution of APS (1.5 mL, 0.006 mol / L, 0.009 mmol) was added to initiate the reaction. The reaction was allowed to proceed at 70 °C for 4 h and stopped by cooling to 0 °C to obtain a poly(acryloylhydrazine-carboxylic acid ester) nanogel solution. 10 mL of the poly(acryloylhydrazine-carboxylic acid ester) nanogel was acidified with hydrochloric acid to 1 mol / L and stirred overnight to remove the tert-butyl formate protecting the hydrazine group. NaOH solution was added to neutralize the mixture and recover the amino group. After stirring for one hour, the solution was dialyzed with deionized water for 3 days, with the dialysate changed twice a day.

[0120] 5) Synthesize CORN

[0121] EDC (0.1116 g, 0.75 mmol), NHS (0.086 g, 0.75 mmol), and CORM (0.050 g, 0.15 mmol) were dissolved in DMF (4 ml). Ultrafiltration-centrifuged polyacrylhydrazine nanogel (14.25 mg, 0.0125 mmol) was added to the mixture. After reacting at room temperature for one day, the suspension turned orange-red. DMF and unreacted reactants were removed by dialyzing with deionized water for 3 days. Excess CORM precipitated from CORN was removed by filtration to obtain the final product.

[0122] Based on the above verification of the product's performance and applications, this photoresponsive carbon monoxide-releasing nanomaterial can be used to kill bacteria and to prepare antibacterial agents.

[0123] The above description is only the preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the scope of the technology disclosed in the present invention, and such modifications or substitutions should be covered within the scope of protection of the present invention.

Claims

1. A polyacrylhydrazine nanogel for loading carbon monoxide-releasing molecules, characterized in that, The structural formula is as follows: x and y represent the number of repeating units in the two types of repeating units of the nanogel, respectively, and z represents the degree of crosslinking; x, y, and z are all positive numbers. Its average hydrodynamic particle size is 130nm to 160nm and its polydispersity index is less than 1.

2. The method for preparing the polyacrylhydrazide nanogel according to claim 1, characterized in that, Includes the following steps: A. Preparation of polyacrylhydrazide nanogel monomer 2-acrylhydrazide-1-carboxylic acid tert-butyl ester AH-Boc A1. Add acrylic acid to tert-butyl hydrazine carbamate and mix well. Then, continue to dissolve the mixture using a mixture of deionized water and tetrahydrofuran to obtain solution 1. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was dissolved in a mixed solution of deionized water and tetrahydrofuran to obtain solution 2; Add solution 2 dropwise to solution 1 in three to five portions and carry out the reaction at room temperature; A2. After extraction, washing, and drying of the reaction solution obtained in A1, the solvent was removed by suspension evaporation, and the product was purified by recrystallization to obtain polyacrylhydrazine nanogel monomer 2-acrylhydrazine-1-carboxylic acid tert-butyl ester AH-Boc. B. Preparation of poly(acrylhydrazide-carboxylic acid ester) nanogels B1. Add the AH-Boc obtained in A2, along with the hydrophilic monomer polyethylene glycol methyl ether acrylate (PEGA), the crosslinking agent N,N'-methylenebisacrylamide (MBA), and the surfactant sodium dodecyl sulfonate (SDS), to a Schlenk flask and dissolve them in deionized water; purge with an inert gas to remove oxygen and maintain an airtight environment; B2. Heat the mixture obtained in B1 to 70-75°C. After the temperature stabilizes, add an aqueous initiator APS to initiate the reaction. Ensure that the reaction is carried out at 70-75°C for 4-6 hours and then stop by cooling to 0°C. B3. Dialyze the solution after reaction B2 with deionized water for 2-3 days, changing the dialysis water two to three times a day to obtain poly(acrylamide hydrazide-carboxylic acid ester) nanogel. C Preparation of polyacrylhydrazide nanogels C1. Add hydrochloric acid to the poly(acrylamide hydrazide-carboxylic acid ester) nanogel obtained in B3, acidify the solution to make the hydrochloric acid concentration 1 mol / L and stir for 10-12 h to remove the protection of tert-butyl formate groups. C2. Add NaOH solution to the solution obtained in C1 to neutralize it, and stir for 30-60 minutes; C3. Dialyze the solution obtained in C2 with deionized water for 2-3 days, changing the dialysis water two to three times a day to obtain polyacrylhydrazine nanogel.

3. The method for preparing polyacrylhydrazide nanogel according to claim 2, characterized in that: In A1, deionized water and tetrahydrofuran are mixed in a volume ratio of 2:

1. The molar ratio of tert-butyl hydrazine carbamate, acrylic acid, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1:1; For every 1 mmol of tert-butyl hydrazine carbamate, add 2.5–3 mL of a mixed solution of deionized water and tetrahydrofuran; dissolve 1 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 0.5–1 mL of a mixed solution of deionized water and tetrahydrofuran. The reaction time is 5–6 hours; A2 is specifically prepared by extracting the reaction solution obtained from A1 multiple times with ethyl acetate in a separatory funnel, then washing it twice with a 0.05-0.1 mol / L sodium carbonate aqueous solution, and finally washing it twice with ultrapure water; drying it with anhydrous sodium sulfate for 20 min, removing the solvent by evaporation, and then recrystallizing and purifying it to obtain the product polyacrylhydrazine nanogel monomer 2-acrylhydrazine-1-carboxylic acid tert-butyl ester. In B1, the molar ratio of AH-Boc, PEGA, MBA, SDS, and APS is 4:1:0.4:0.06:0.09; and 8 mL of deionized water is added for every 0.4 mmol of AH-Boc; the concentration of MBA used is 0.1 mol / L, the concentration of SDS is 0.06 mol / L, and the concentration of APS aqueous solution is 0.006 mol / L. In B2, the reaction time is 4 hours; In C2, the concentration of NaOH solution added is 5 mol / L.

4. A photoresponsive carbon monoxide-releasing nanogel CORN, characterized in that: It is formed by coupling the polyacrylhydrazine nanogel of claim 1 with a carboxyl-functionalized metal-free, photoresponsive carbon monoxide-releasing molecule; Under visible light, carbon monoxide-releasing nanogels release CO. Its structural formula is as follows: x and y represent the number of repeating units of the two types of repeating units in the nanogel, respectively; z represents the degree of crosslinking; and xn represents the number of repeating units loaded with CORM. x, y, z, and n are all positive numbers, with x:y equal to 4:1 and x:n equal to 30 to 40:

1.

5. The method for preparing the photoresponsive carbon monoxide-releasing nanogel CORN according to claim 4, characterized in that, Includes the following steps: 1) Preparation of metal-free, photoresponsive carbon monoxide-releasing molecule CORM 3-hydroxy-2-acetylnaphthalene and 4-aldehyde benzoic acid were dissolved in anhydrous ethanol and reacted under the co-catalysis of hydrogen peroxide and sodium hydroxide to obtain carboxyl-functionalized metal-free, photoresponsive carbon monoxide-releasing molecule CORM. 2) Preparation of photoresponsive carbon monoxide-releasing nanogels CORN The polyacrylhydrazine nanogel of claim 1 and the metal-free, photoresponsive carbon monoxide-releasing molecule CORM obtained in step 1) were dissolved in a mixed solution of dimethylformamide and water, and the photoresponsive carbon monoxide-releasing nanogel CORN was obtained by catalytic reaction using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

6. The method for preparing photoresponsive carbon monoxide-releasing nanogel CORN according to claim 5, characterized in that, Step 1) specifically involves: S1 Preparation of carbon monoxide-releasing molecular precursor 3-hydroxy-2-acetylnaphthalene S1.1 At room temperature, 3-hydroxy-2-naphthoic acid was added to tetrahydrofuran, stirred and dissolved, and then an inert gas was introduced to maintain an airtight environment. Subsequently, the reaction temperature was lowered to 0°C, and methyllithium was added dropwise to carry out the reaction. S1.2 Add 0.5 mol / L to 1 mol / L hydrochloric acid to the solution after the reaction of S1.1 until no more bubbles are produced; remove tetrahydrofuran under vacuum and then add 0.5 mol / L to 1 mol / L hydrochloric acid to adjust the pH of the solution to neutral. The acidified solution was extracted with dichloromethane multiple times, and the organic layer was collected. Anhydrous sodium sulfate was added to the organic layer for drying, the sodium sulfate was removed by filtration, and the solution concentration was increased by suspension evaporation to ensure that no crystals precipitate. S1.3 uses pure ethyl acetate and petroleum ether as eluents to remove impurities from the solution obtained in S1.2 by rapid column chromatography; then anhydrous sodium sulfate solution is added to dry the solution, the sodium sulfate is removed by filtration, the solvent is removed by suspension, and the solution is dried to obtain the carbon monoxide release molecule precursor 3-hydroxy-2-acetylnaphthalene. S2 Preparation of Metal-Free, Photoresponsive Carbon Monoxide-Releasing Molecule CORM S2.1 The carbon monoxide-releasing precursor 3-hydroxy-2-acetnaphthalene obtained in S1.3 was added to a light-protected reaction vessel. Anhydrous ethanol was added first and stirred until homogeneous, followed by the addition of 5 mol / L NaOH aqueous solution. The mixture was stirred at room temperature for 30 to 60 minutes, followed by the addition of 4-aldehyde benzoic acid. The mixture was then stirred under light-protected conditions for 5 to 6 hours. The temperature of the reaction solution was lowered to 0°C, and H2O2 was added dropwise. S2.2 The reaction solution of S2.1 was stirred in an ice-water bath for 10-12 hours, and then gradually allowed to return to room temperature. Under light-protected conditions, 0.5 mol / L to 1 mol / L hydrochloric acid aqueous solution was added to adjust the pH to 6.

5. The solution was filtered under light-protected conditions, washed with ice-cold anhydrous ethanol, and then dried to obtain metal-free, photoresponsive carbon monoxide-releasing molecules CORM.

7. The method for preparing photoresponsive carbon monoxide-releasing nanogel CORN according to claim 6, characterized in that, Step 2) specifically involves: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and the metal-free, photoresponsive carbon monoxide-releasing molecule CORM prepared in step 1) were dissolved in dimethylformamide. Polyacrylamide nanogel solution was added to the mixed solution. After reacting at room temperature for 12-14 hours, the suspension turned orange-red. Dialysis with deionized water was performed for 2-3 days, changing the dialysis water two to three times a day to remove dimethylformamide and unreacted reactants. The suspension was filtered to obtain the photoresponsive carbon monoxide-releasing nanogel CORN.

8. The application of the photoresponsive carbon monoxide-releasing nanogel according to claim 4 in the preparation of antibacterial agents.

9. An antibacterial agent, characterized in that: Its active ingredient is the photoresponsive carbon monoxide-releasing nanogel as described in claim 4.

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