Nitric oxide controlled release system and use thereof

Abstract

The application discloses a controllable release system of nitric oxide and application thereof. The controllable release system of nitric oxide comprises a polymeric macromolecular acid and a nitric oxide donor nitrite. By adjusting the structure and proportion of a crosslinking agent in the polymeric macromolecular acid, a spatial structure with different crosslinking densities can be formed, the reaction between protons provided by the polymeric macromolecular acid and nitrite ions in the nitric oxide donor component is slowed down by using a shielding effect, and the diffusion rate of generated NO is controlled, so that the controllable adjustment of a maximum concentration (C max ) and a half-life (T 1 / 2 ) of NO release is realized. Meanwhile, by changing the dosage form of the nitric oxide donor, the distribution rate of nitrite in the system during the reaction is controlled, and the half-life (T 1 / 2 ) of nitric oxide release of the system is further adjusted. The controllable release system of nitric oxide has an anti-pathogenic microorganism activity, and the application thereof in the preparation of an anti-pathogenic microorganism product.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a controlled-release nitric oxide system. This system exhibits antimicrobial activity and its application in the preparation of antimicrobial products. Background Technology

[0002] Nitric oxide (NO) has physiological functions such as vasodilation and immune regulation, and exhibits broad-spectrum antibacterial, antiviral, and wound-healing activities. However, the pharmacological activity of NO is highly dependent on its duration of action and concentration. Therefore, achieving controlled release of NO is of great clinical significance.

[0003] NO can be generated through the chemical reaction of inorganic nitrites with small molecule acids, such as citric acid, ascorbic acid, or inorganic acids. Sanotize developed NORS™, a nasal spray for combating COVID-19, which works by releasing NO through the reaction of sodium nitrite with citric acid. However, in this system, NO originates from the instantaneous reaction between sodium nitrite and a small molecule acid, resulting in a short half-life (T0). 1 / 2 =0.06 hours), making it difficult to achieve sustained NO release and thus failing to meet the needs of long-acting therapy. (WO 2022 / 013614A1) Previous studies have used microencapsulation technology to encapsulate sodium nitrite and citric acid to achieve sustained NO release; however, the nitrite in this microencapsulation system needs to be prepared and stored under anhydrous conditions, which brings inconvenience to clinical applications. (CN103622917A) NOVAN has developed ZELSUVMI™, a nitric oxide-releasing gel, by introducing diol diazepinenium functional groups onto polysiloxane macromolecules as nitric oxide donor materials. This gel releases NO through reaction with acidic aqueous solutions, addressing the issue of short NO release half-life (half-life T0). 1 / 2 It can release NO in up to 1.98 hours, achieving controlled release. (CN102711729B; WO 2013 / 029009A1) However, this material also needs to be prepared and stored under anhydrous conditions, which limits the convenience of its clinical application.

[0004] NOVAN's NO-emission material structure and NO release diagram Summary of the Invention

[0005] The purpose of this invention is to provide a controlled release system for nitric oxide. This system comprises a polymeric macromolecular acid component and a nitric oxide donor nitrite component. By adjusting the structure and ratio of the crosslinking agent in the polymeric macromolecular acid, spatial structures with different crosslinking densities are formed. The cage effect is utilized to slow down the reaction between the protons donated by the polymeric macromolecular acid and the nitrite ions in the nitric oxide component, and to control the diffusion rate of NO generation, thereby achieving different maximum NO release concentrations (C0). max ) and half-life (T) 1 / 2 The controllable adjustment of nitric oxide (NO) can be achieved by changing the formulation of the nitric oxide donor component and controlling the distribution rate of nitrite in the system during the reaction, thereby further regulating the NO release half-life (T0). 1 / 2 This enables the controlled release of nitric oxide.

[0006] The controlled release system of nitric oxide of the present invention includes a polymeric macromolecular acid component and a nitric oxide donor component. The polymeric macromolecular acid has a cross-linked structure. The two components in the system are stored independently. Before use, the polymeric macromolecular acid component and the nitric oxide donor component are mixed evenly in a volume ratio of 1:0.3 to 1:3.

[0007] The general structural formula of the polymeric macromolecular acid is as follows: m: Number of acrylic monomer units; n: Number of crosslinking agent units; x: Number of methacrylic acid monomer units; R originates from a crosslinking agent structural fragment: .

[0008] In some embodiments of the present invention, the nitric oxide donor is selected from one or more of sodium nitrite, calcium nitrite, and potassium nitrite, preferably sodium nitrite.

[0009] In some embodiments of the present invention, the polymeric macromolecular acid is selected from one or more of the following: polyacrylic acid and its derivatives, cross-linked polyacrylic acid and its derivatives, polylactic acid, and alginate, preferably cross-linked polyacrylic acid; the cross-linked polyacrylic acid is formed by copolymerization of acrylic acid or its derivative monomers and a cross-linking agent to form a cross-linked network structure. The precipitation polymerization reaction system includes monomers, cross-linking agents, solvents, dispersants, and free radical initiators; wherein the solvent is preferably a cyclohexane / ethyl acetate mixed solvent, the dispersant is preferably Hypermer B246, and the free radical initiator is preferably dodecyl peroxide. The specific preparation process includes the following steps: Add 4 wt%–10 wt% monomer, 50 wt%–55 wt% cyclohexane, 40 wt%–45 wt% ethyl acetate, and the required initiator, dispersant, and crosslinking agent to the reaction vessel. Bubble with nitrogen for 30–60 min, then heat at 65 °C. o C~75 o The reaction was carried out at C for 4-10 h until the monomer was completely produced, at which point the reaction was terminated. The resulting white powder was obtained by filtration and vacuum drying.

[0010] In some embodiments of the present invention, the monomer in the polymeric macromolecular acid is selected from one or more of the following: acrylic acid, methacrylic acid; the monomer accounts for 95% to 99.95% of the total mass of the crosslinked polyacrylic acid. In some embodiments of the present invention, the crosslinking agent of the polymeric macromolecular acid contains multiple olefin functional groups within a single molecule and is selected from one or more of the following: ethylene glycol diallyl ether, propylene glycol diallyl ether, butanediol diallyl ether, glycerol-α,α'-diallyl ether, glycerol triallyl ether, pentaerythritol tetraallyl ether, pentaerythritol triallyl ether, allyl sucrose ether; preferably pentaerythritol triallyl ether; the crosslinking agent accounts for 0.05% to 5% of the monomer by mass.

[0011] In some embodiments of the present invention, the polymeric macromolecular acid component comprises 0.04% to 20% by mass. In some embodiments of the present invention, the mass fraction of nitrite in the nitric oxide donor component is 0.02% to 10%.

[0012] In some embodiments of the present invention, the nitric oxide release half-life of the system is adjustable in the range of 0.15 h to 10.9 h, and the maximum instantaneous concentration of nitric oxide release is adjustable in the range of 0.5 ppm to 380 ppm.

[0013] In some embodiments of the present invention, the nitric oxide controlled release system is characterized in that the nitric oxide donor component is a liquid formulation, a gel, or a solid formulation; the nitric oxide liquid formulation, gel, or solid formulation is composed of a nitric oxide donor component and an excipient; the polymeric macromolecular acid component is a liquid formulation or a gel; the polymeric macromolecular acid liquid formulation or gel is composed of a polymeric macromolecular acid component and an excipient.

[0014] In some embodiments of the present invention, the excipients of the polymeric macromolecular acid components in the controlled release system of nitric oxide are selected from one or more of the following: hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methylcellulose, carboxymethyl cellulose or sodium carboxymethyl cellulose, carbomer, hyaluronic acid, poloxamer, sodium alginate, xanthan gum or chitosan.

[0015] In some embodiments of the present invention, the nitric oxide component excipient of the controlled release system is selected from one or more of the following: hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methylcellulose, carboxymethyl cellulose or sodium carboxymethyl cellulose, carbomer, hyaluronic acid, poloxamer, sodium alginate, xanthan gum or chitosan, mannitol, trehalose, gelatin, and lactose.

[0016] In some embodiments of the present invention, the acidity and cross-linking structure of the polymeric macromolecular acid component in the controlled release system of nitric oxide are provided by cross-linked polyacrylic acid or cross-linked polymethacrylic acid.

[0017] In some embodiments of the present invention, the internal cross-linking structure of the polymeric macromolecular acid is adjusted by changing the proportion of the cross-linking agent during polymerization, resulting in polymeric macromolecular acids with different cross-linking densities. Different cross-linking densities exhibit different NO release behaviors, thereby meeting the NO concentration requirements of different therapeutic scenarios. In specific embodiments, the mass fraction of the cross-linking agent is 0.05% to 5% of the monomer.

[0018] In some embodiments of the present invention, NO release behavior with extended half-life is achieved by changing the formulation of nitric oxide.

[0019] In some embodiments of the present invention, the controlled release system of nitric oxide is prepared into an antimicrobial product, wherein the pathogenic microorganisms include fungi, bacteria and viruses; the fungi are Candida albicans, the bacteria are Propionibacterium acnes, Staphylococcus aureus and Escherichia coli, and the virus is herpes simplex virus.

[0020] In some embodiments of the present invention, the nitric oxide release system of the present invention maintains a long-lasting antibacterial effect by continuously releasing NO even after 24 hours of mixing, which is superior to the reaction system with citric acid and sodium nitrite.

[0021] In one embodiment of the present invention, the antimicrobial product includes a drug or medical device for wound healing, hemorrhoids, acne or pathogenic microbial infection.

[0022] This invention discloses a controlled nitric oxide release system. This system generates NO through the reaction of a polymeric macromolecular acid with nitrite. Utilizing the cage effect created by the spatial structure of the polymeric macromolecular acid, the movement of protons it provides is restricted, and the diffusion rate of the generated NO is controlled, thereby achieving controlled NO release. Simultaneously, the NO release half-life can be adjusted by changing the formulation of the nitric oxide component. Compared with existing nitric oxide release systems, the controlled nitric oxide release system provided by this invention has significant advantages in terms of water preparation and storage stability and controlled NO release. By adjusting the spatial structure of the polymeric macromolecular acid and the NO donor formulation, the maximum concentration of nitric oxide released (C0) can be controlled. max ) and half-life (T) 1 / 2 This allows for controlled release of NO based on the required NO levels for different treatment scenarios. The controlled release system for nitric oxide of this invention exhibits antimicrobial activity and its application in the preparation of antimicrobial products. Attached Figure Description

[0023] Figure 1 A: NO release results of polymeric macromolecular acids ZD-1~ZD-4 and ZD-7 synthesized with crosslinking agents of different structures; B: NO release results of ZD-5, ZD-16~ZD-19 synthesized with crosslinking agents of different proportions; C: NO release results of mixing sodium nitrite aqueous solution of different concentrations with ZD-5 aqueous solution; D: NO release results of reacting citric acid aqueous solution and ZD-5 aqueous solution with sodium nitrite aqueous solution respectively.

[0024] Figure 2 The infrared spectrum and potentiometric titration results of the polymeric macromolecular acid ZD-1 synthesized in this invention.

[0025] Figure 3 Scanning electron microscope image of the polymeric macromolecular acid synthesized in this invention.

[0026] Figure 4 The swelling curves and swelling ratios of the polymeric macromolecular acids synthesized in this invention.

[0027] Figure 5 The antibacterial activity results of the controlled release system of nitric oxide described in this invention.

[0028] Figure 6 Results of a mouse wound healing model of Staphylococcus aureus infection treated with the controlled release system of nitric oxide described in this invention. Detailed Implementation

[0029] The present invention will be further described in conjunction with the accompanying drawings and embodiments. These embodiments are provided for illustrative purposes only and are not intended to be restrictive, unless otherwise specified.

[0030] Examples 1-22 describe the synthesis of polymeric macromolecular acids ZD-1-ZD-22 by reacting acrylic acid monomers or methacrylic acid monomers with crosslinking agents of different structures and proportions; Example 23 describes the structural characterization of the polymeric macromolecular acid provided by this invention; Examples 24-26 describe the preparation of NO donor components in different dosage forms. Example 1: Synthesis and structural characterization of the polymeric macromolecular acid ZD-1 Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 15 mg of ethylene glycol diallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble under nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-1.

[0031] The chemical structure of the synthesized polymeric macromolecular acid ZD-1 was characterized by infrared spectroscopy, and the carboxyl content was quantified by potentiometric titration.

[0032] 1. Infrared spectrum of ZD-1 The synthesized crosslinked polyacrylic acid was prepared by potassium bromide tableting at 4000-400 cm⁻¹. -1 Its infrared spectrum was measured at 3159 cm⁻¹. -1 The broad peak at 2958 cm⁻¹ corresponds to the stretching vibration peak of the hydroxyl group; -1 The peak corresponds to the stretching vibrations of the methyl and methylene groups; 1710 cm⁻¹ -1 The peak corresponds to the stretching vibration of the carbonyl group in the polyacrylic acid backbone; 1454 cm⁻¹ -1 With 1417 cm -1 The peak corresponds to the deformation vibration and bending vibration of the methylene group; 1247 cm⁻¹ -1 With 1171 cm -1 The peak corresponds to the stretching vibration of the ether bond; this absorption peak originates from the allyl ether structure of the crosslinking agent. Infrared spectrum (cm²) -1 ): 3126, 2960, 1710, 1454, 1418, 1247, 1171, Figure 2 This is the infrared spectrum of ZD-1.

[0033] 2. Determination of carboxyl content in ZD-1 by potentiometric titration The carboxyl content of ZD-1 was determined by potentiometric titration. 0.4 g of ZD-1 powder was accurately weighed and dissolved in 400 mL of deionized water, stirred until uniformly dispersed. Titration was performed using standard sodium hydroxide titrant (0.25 M). Near the endpoint, a stable reading was maintained for at least 2 minutes after each addition of titrant. The relative potential was measured and recorded using a pH meter during titration. After titration, a curve was plotted, and the first derivative was taken. The lowest point of the first derivative was determined as the titration endpoint. The carboxyl content of ZD-1 was 56.0%.

[0034] Example 2: Synthesis of polymeric macromolecular acid ZD-2.

[0035] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 15 mg of propylene glycol diallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and purge with nitrogen gas for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-2. Infrared spectrum (cm²) -1 ZD-2 contains 3144, 2958, 1726, 1453, 1416, 1249, and 1172 carboxyl groups. The carboxyl content of ZD-2 is 57.4%.

[0036] Example 3: Synthesis of polymeric macromolecular acid ZD-3.

[0037] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 15 mg of butanediol diallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-3. Infrared spectrum (cm²) -1 ZD-3 contains 3104, 2965, 1727, 1453, 1416, 1249, and 1172 carboxyl groups. The carboxyl content of ZD-3 is 56.8%.

[0038] Example 4: Synthesis of polymeric macromolecular acid ZD-4.

[0039] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 15 mg of glycerol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-4. Infrared spectrum (cm²) -1 ZD-4 contains 3134, 2961, 1710, 1454, 1416, 1248, and 1171 carboxyl groups. The carboxyl content of ZD-4 is 57.1%.

[0040] Example 5: Synthesis of polymeric macromolecular acid ZD-5.

[0041] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 15 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-5. Infrared spectrum (cm²) -1 ZD-5 contains 3134, 2958, 1725, 1454, 1416, 1247, and 1172 carboxyl groups. The carboxyl content of ZD-5 is 61.8%.

[0042] Example 6: Synthesis of polymeric macromolecular acid ZD-6.

[0043] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 15 mg of pentaerythritol tetraallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, cooled to room temperature, nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-6.

[0044] Example 7: Synthesis of polymeric macromolecular acid ZD-7.

[0045] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, and 15 mg of glycerol to a three-necked flask. α,α Diallyl ether, 20 mg Hypermer B246, and 10 mg dodecyl peroxide were dissolved by stirring and bubbling under nitrogen for 30 min to remove oxygen. The mixture was then heated to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-7.

[0046] Example 8: Synthesis of polymeric macromolecular acid ZD-8.

[0047] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 3 mg of glycerol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-8.

[0048] Example 9: Synthesis of polymeric macromolecular acid ZD-9.

[0049] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 6 mg of glycerol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-9.

[0050] Example 10: Synthesis of polymeric macromolecular acid ZD-10.

[0051] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 9 mg of glycerol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-10.

[0052] Example 11: Synthesis of polymeric macromolecular acid ZD-11.

[0053] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 12.5 mg of glycerol triallyl ether, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and purge with nitrogen gas for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-11.

[0054] Example 12: Synthesis of polymeric macromolecular acid ZD-12.

[0055] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 25 mg of glycerol triallyl ether, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-12.

[0056] Example 13: Synthesis of polymeric macromolecular acid ZD-13.

[0057] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 37.5 mg of glycerol triallyl ether, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. oThe reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-13. Infrared spectrum (cm²) -1 ): 3145, 2959, 1724, 1454, 1414, 1247, 1173.

[0058] Example 14: Synthesis of polymeric macromolecular acid ZD-14.

[0059] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 2.5 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and purge with nitrogen gas for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-14.

[0060] Example 15: Synthesis of polymeric macromolecular acid ZD-15.

[0061] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 5 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, cooled to room temperature, nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-15.

[0062] Example 16: Synthesis of polymeric macromolecular acid ZD-16.

[0063] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 20 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. oThe reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-16. Infrared spectrum (cm²) -1 (): 3160, 2958, 1710, 1454, 1417, 1248, 1171. The carboxyl content of ZD-16 is 62.1%.

[0064] Example 17: Synthesis of polymeric macromolecular acid ZD-17.

[0065] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 30 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-17. Infrared spectrum (cm²) -1 ): 3127, 2958, 1711, 1454, 1416, 1249, 1172. The carboxyl content of ZD-17 is 60.9%.

[0066] Example 18: Synthesis of polymeric macromolecular acid ZD-18.

[0067] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 40 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-18. Infrared spectrum (cm²) -1 ): 3144, 2958, 1710, 1454, 1415, 1248, 1172. The carboxyl content of ZD-18 is 59.8%.

[0068] Example 19: Synthesis of polymeric macromolecular acid ZD-19.

[0069] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 50 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, and the solid is collected by vacuum filtration. The filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding a white powder, ZD-19. Infrared spectrum (cm²) -1 ): 3134, 2959, 1710, 1454, 1415, 1249, 1172. The carboxyl content of ZD-19 is 58.6%.

[0070] Example 20: Synthesis of polymeric macromolecular acid ZD-20.

[0071] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 250 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and purge with nitrogen gas for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-20.

[0072] Example 21: Synthesis of polymeric macromolecular acid ZD-21.

[0073] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 4 g of acrylic acid monomer, 1 g of methacrylic acid monomer, 40 mg of pentaerythritol triallyl ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask. Stir to dissolve, and bubble with nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 hours, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-21.

[0074] Example 22: Synthesis of polymeric macromolecular acid ZD-22.

[0075] Add 23 mL of cyclohexane, 17 mL of ethyl acetate, 5 g of acrylic acid monomer, 15 mg of allyl sucrose ether, 20 mg of Hypermer B246, and 10 mg of dodecyl peroxide to a three-necked flask, stir to dissolve, and bubble under nitrogen for 30 min to remove oxygen. Heat to 73°C under nitrogen protection. o The reaction begins at C, and a white precipitate gradually appears in the system. The reaction is stopped after 8 h, the temperature is lowered to room temperature, the nitrogen protection is removed, the solid is collected by vacuum filtration, the filter cake is washed with ethyl acetate (30 mL × 3), and the solid is dried under vacuum to remove the solvent, yielding white powder ZD-22.

[0076] Example 23: Scanning electron microscope image and swelling experiment of polymerized macromolecular acid.

[0077] The internal cross-section of polymeric macromolecular acids was observed using scanning electron microscopy to analyze their microstructure. Figure 3 The images show cross-sectional electron micrographs of Examples 1-5 and Examples 16-19. The highly cross-linked polyacrylic acid backbone forms a tight network structure through the cross-linking agent. The spatial structures of the polymeric macroacids ZD-1-ZD-3 synthesized with the two-arm cross-linking agent are significantly sparse, with larger internal voids. In contrast, the polymeric macroacids ZD-4 and ZD-5, which increase the number of cross-linking sites and introduce hydroxyl groups as hydrogen bonding sites, exhibit a tighter network structure. As the proportion of cross-linking agent in Examples 16-19 increases, their network structure gradually becomes tighter, eventually exhibiting a film-like structure. This result demonstrates that the spatial structure of the polymeric macroacids of the present invention can be adjusted by changing the structure and proportion of the cross-linking agent, further enabling the controlled release of NO.

[0078] This embodiment measures the swelling rate of the polymeric macromolecular acid polymers in Examples 5 and 16-19. The polymeric macromolecular acid powder was held at 15 MPa for 10 min to form a 13 mm diameter disc. After weighing and recording the weight, the disc was placed in a sieve and immersed in deionized water. At fixed time points, the sieve and the polymeric macromolecular acid powder were removed, and the resulting gel was carefully removed from the sieve surface and weighed. The swelling rate was calculated based on the change in gel weight. Figure 4 The swelling curves and equilibrium swelling rates of ZD-5, ZD-16~ZD-19 were obtained. Equilibrium was reached after 24 h. With the increase of the crosslinking agent ratio, the equilibrium swelling rate of the polymeric macromolecular acid decreased, which indicates a tighter internal spatial structure and a greater crosslinking density, which can enhance the cage effect of the macromolecular acid and facilitate the slow release of NO.

[0079] Example 24: Preparation of aqueous solution of NO donor component.

[0080] Sodium nitrite (0.02 g to 10 g) was dissolved in 50 mL of deionized water and then diluted to 100 mL with deionized water. The solution was stirred and mixed thoroughly to obtain an aqueous solution of NO donor components with a concentration ranging from 0.02% to 10%.

[0081] Example 25: Preparation of NO donor component hydrogel.

[0082] Sodium nitrite (0.02 g to 10 g) and hydroxyethyl cellulose (HEC 250HX, 0.7 g) were dissolved in 50 mL of deionized water and then diluted to 100 mL with deionized water. The mixture was stirred until homogeneous to obtain a NO donor component hydrogel with a concentration ranging from 0.02% to 10%.

[0083] Example 26: Preparation of NO donor component lyophilizing agent.

[0084] 10 mg of sodium nitrite and 30 mg of mannitol were dissolved in 3 mL of deionized water and then freeze-dried under vacuum to obtain the NO donor component lyophilized agent.

[0085] Example 27: NO release results from the reaction of polymerized macromolecular acid with NO donor components.

[0086] NO release behavior was detected using a Sievers NOA 280i nitric oxide analyzer. A standard curve was established using the NO signal generated by adding a certain concentration of sodium nitrate standard to vanadium trichloride. The polymeric macromolecular acid component and the NO donor component were mixed in a 1:1 ratio and injected into the reaction chamber. N2 was used as the carrier gas at a flow rate of 200 mL / min to carry the real-time generated NO to the detector. During the detection period, NO reacted with O3 to generate a light signal, which was used to quantify the real-time NO concentration. The NO release curve was recorded to calculate the NO release data.

[0087] Table 1. NO release from the reaction of polymeric macromolecular acids with NO donor components Table 1 shows the NO release results of the polymeric macromolecular acid provided in this invention reacting with NO donor components of different formulations in Examples 24-26. By changing the structure (number of crosslinking sites and introduction of hydroxyl groups) and ratio of the crosslinking agent, polymeric macromolecular acids with different density spatial structures were obtained. The compact spatial structure can enhance the cage effect of the polymer and slow down the generation and diffusion of NO. The synthesized polymeric macromolecular acid reacted with NO donor components of different formulations, providing the highest NO release concentration (C0.05) in the range of 0.5 ~ 380 PPM. max ) and a half-life of 0.15–10.9 h (T 1 / 2This method achieves controlled NO release, significantly outperforming existing nitric oxide release systems and extending the NO release half-life by up to 10.9 h. Furthermore, the AUC of NO release increases proportionally with increasing sodium nitrite and polymeric macromolecular acid content, exhibiting a good linear relationship.

[0088] The NO release results of the reactions of equimolar amounts of the polymeric macromolecular acid ZD-5 aqueous solution and the traditional small-molecule acid citric acid aqueous solution with 0.2% sodium nitrite aqueous solution were compared. Under the same molar amounts, the NO release half-life of the reaction of 0.2% sodium nitrite solution with citric acid was 0.06 h. ZD-5, through its tight network structure, slowed down the reaction of nitrite ions and the diffusion of NO within the system. With a similar total release amount, its half-life was extended to 1.03 h, effectively avoiding the burst release effect of NO reacting with the small-molecule acid and achieving controlled NO release.

[0089] Example 28: Results of the antimicrobial activity of a NO controlled release system composed of ZD-5 aqueous solution and NaNO2 aqueous solution.

[0090] 1. Antibacterial activity of the NO controlled release system composed of ZD-5 aqueous solution and NaNO2 aqueous solution. In Example 5, 2 g each of a 2.5% aqueous solution of ZD-5 and a 0.2% aqueous solution of sodium nitrite were mixed thoroughly and then reacted with the bacterial solution for 2 minutes. Samples were taken to detect the number of surviving colonies, and the antibacterial rate was calculated. The results are shown in Table 2 (each test bacterium was repeated three times). As shown in Table 2, the NO controlled-release system composed of 2.5% ZD-5 aqueous solution and 0.2% NaNO2 aqueous solution exhibits broad-spectrum antibacterial activity.

[0091] Table 2. Results of the antimicrobial activity of the NO controlled release system composed of 2.5% ZD-5 aqueous solution and 0.2% NaNO2 aqueous solution. This invention also determined the anti-Staphylococcus aureus and anti-Candida albicans activities of 2.5% ZD-5 aqueous solution or 2.5% ZD-13 aqueous solution and 0.2% sodium nitrite aqueous solution, respectively, used alone and in combination, with PBS as the control group. See Table 3 for details. Figure 5 , 6As shown, the colony counts obtained after using 2.5% ZD-5 aqueous solution, 2.5% ZD-13 aqueous solution, or 0.2% sodium nitrite aqueous solution alone were not significantly different from those in the Control group, indicating that the individual components had no antibacterial activity. Furthermore, when 2.5% ZD-5 aqueous solution or 2.5% ZD-13 aqueous solution was mixed with 0.2% sodium nitrite aqueous solution and then reacted with the bacterial solution, no colonies were produced. This indicates that the antibacterial activity of the nitric oxide release system provided by this invention does not originate from the individual polymeric macromolecular acid or nitrite, but rather from the NO produced by the reaction of the two.

[0092] Table 3. Antibacterial activity of 2.5% ZD-5 aqueous solution or 2.5% ZD-13 aqueous solution and 0.2% sodium nitrite aqueous solution, used alone and in combination. 2. Antiviral activity of the controlled NO release system composed of ZD-5 aqueous solution and NaNO2 aqueous solution. Anti-HSV-1 plaque tropism assay was performed using a 2.5% ZD-5 solution and 0.2% sodium nitrite. The specific experimental method is as follows: VERO cells were seeded in 24-well plates at a rate of 2 × 10⁶ cells per well. 5 37 cells o Culture at C for 24 h for later use. Mix 2.5% ZD-5 aqueous solution with 0.2% sodium nitrite aqueous solution thoroughly, and add 3×10 8 After vortexing the HSV-1 virus suspension in PFU until homogeneous, let it stand for 10 min, then add the sample to a 24-well plate at 37°C. o After co-incubating at C for 1 h, 0.5 mL of methylcellulose medium solution was added to each well of the 24-well plate, and the mixture was incubated at 37°C. o Incubate at C for 48 h, then observe under a microscope after crystal violet staining and record the number of viral plaques in the well plate.

[0093] The experimental results are shown in Table 4. The results show that the inhibition rate of HSV-1 by mixing 2.5% ZD-5 aqueous solution with 0.2% sodium nitrite aqueous solution reached 100%, indicating that the nitric oxide release system of the present invention has good anti-HSV-1 virus activity.

[0094] Table 4. Antiviral activity results of the NO controlled release system composed of 2.5% ZD-5 aqueous solution and 0.2% NaNO2 aqueous solution. Example 29: Long-term antibacterial test of a NO controlled release system composed of ZD-5 aqueous solution and NaNO2 aqueous solution.

[0095] To verify the advantages of the controlled nitric oxide release system provided by this invention compared to the nitric oxide release system based on the reaction of small molecule acids with sodium nitrite, this embodiment uses 2.5% ZD-5 solution and 1.5% citric acid solution, respectively, to react with 0.2% sodium nitrite as comparative examples. Samples were taken 10 min and 24 h after mixing the two components and reacted with Candida albicans, and the number of colonies was counted using the plate method.

[0096] Table 5. Antibacterial duration of NO generated by the reaction of ZD-5 aqueous solution and small molecule citric acid aqueous solution with sodium nitrite aqueous solution. The antibacterial effects of the two groups at each time point are shown in Table 5. After 10 min of mixing the two components, the antibacterial rate against Candida albicans was close to 100%. However, the antibacterial results at 24 h after mixing showed that the system with citric acid as an acid reacting with sodium nitrite had only a 20% antibacterial rate, while the system with the polymeric macromolecular acid of the present invention reacting with sodium nitrite still maintained a 96% antibacterial rate. This proves that the nitric oxide release system composed of the polymeric macromolecular acid and sodium nitrite provided by the present invention has a long-lasting effect and is significantly better than the existing nitric oxide release system composed of citric acid and sodium nitrite.

[0097] Example 30: Effect of a controlled NO release system composed of ZD-13 aqueous solution and sodium nitrite hydrogel on wound healing in mice infected with Staphylococcus aureus.

[0098] The effect of 2.5% ZD-13 aqueous solution and 0.2% sodium nitrite hydrogel on wound healing in mice infected with Staphylococcus aureus was evaluated. Control group, positive control group, and drug administration group were established. The control group was not administered drugs during modeling, the positive control group received oxacillin (10 mg / day, dissolved in 0.7% HEC), and the drug administration group received a mixture of 0.2% NaNO2 hydrogel and 2.5% ZD-13 aqueous solution before administration.

[0099] Four-week-old male ICR mice were used in the experiment. After anesthesia, the mice had their dorsal hair removed, and the full-thickness skin was excised to create a circular wound with a diameter of 8 mm. 50 µL of 10 6 CFU / mL Staphylococcus aureus bacterial suspension was used to colonize the wound with gauze for 24 hours, creating a wound model of Staphylococcus aureus infection. Treatment began on the second day after bacterial infection, with administration once daily in different groups. 0.1 g each of ZD-13 aqueous solution and sodium nitrite hydrogel were gently mixed at the wound site, spread evenly, and covered with gauze. In the positive group, oxacillin was dissolved in the matrix and applied directly to the wound, followed by gauze coverage.

[0100] Starting from day 0 of the drug administration, the wound volume was recorded by taking photos daily. ImageJ was used to calculate the wound area and healing rate to observe the wound healing process. Figure 6 Photos of wounds and healing rate curves for each group on days 0, 2, 4, 6, 8, and 10.

[0101] Table 6 shows the wound healing rates for each group on days 3, 6, and 10 (n=3). The wound healing rate was calculated using the following formula: Wound healing rate = (Wound area on day 0 - Wound area on day 10) / Wound area on day 0 * 100%.

[0102] The data comparison in Table 6 shows that the wound healing rate of the treatment group was significantly better than that of the positive and control groups, and the effect was more pronounced in the early stage of wound healing. This indicates that the nitric oxide release system of the present invention has good antibacterial ability and wound healing promotion effect.

[0103] Table 6. Effects of the controlled NO release system composed of ZD-13 aqueous solution and sodium nitrite hydrogel on a mouse wound healing model of Staphylococcus aureus infection.

Claims

1. A controlled release system for nitric oxide, comprising a polymeric macromolecular acid component and a nitric oxide donor component, characterized in that, The polymeric macromolecular acid is selected from one or more of the following: polyacrylic acid and its derivatives, cross-linked polyacrylic acid and its derivatives, polylactic acid, and alginate; the nitric oxide donor is selected from one or more of the following: sodium nitrite, calcium nitrite, and potassium nitrite; the two components in the system are stored independently and mixed evenly with a volume ratio of polymeric macromolecular acid component to nitric oxide donor component of 1:0.3 to 1:3 before use.

2. The controlled release system of nitric oxide according to claim 1, characterized in that, The polymeric macromolecular acid is selected as cross-linked polyacrylic acid, wherein the cross-linked polyacrylic acid is composed of acrylic acid or methacrylic acid as monomers, which copolymerize with the double bonds of a cross-linking agent to form a cross-linked network structure; the general formula of the polymeric macromolecular acid is as follows: ， Where: m: number of acrylic monomer units; n: number of crosslinking agent units; x: number of methacrylic acid monomer units; R originates from a crosslinking agent structural fragment: 。 3. The controlled release system of nitric oxide according to any one of claims 1 to 2, characterized in that, The crosslinking monomer is selected from one or more of the following: acrylic acid, methacrylic acid; the crosslinking monomer accounts for 95% to 99.95% of the total mass of crosslinked polyacrylic acid and its derivatives.

4. The nitric oxide releasable system according to any one of claims 1 to 2, characterized in that, The crosslinking agent contains multiple olefin functional groups within a single molecule and is selected from one or more of the following: ethylene glycol diallyl ether, propylene glycol diallyl ether, butanediol diallyl ether, glycerol-α,α'-diallyl ether, glycerol triallyl ether, pentaerythritol tetraallyl ether, pentaerythritol triallyl ether, and allyl sucrose ether; the crosslinking agent accounts for 0.05% to 5% of the monomer by mass.

5. The controlled release system of nitric oxide according to any one of claims 1 to 2, characterized in that, The polymeric macromolecular acid component comprises a mass fraction of 0.04% to 20%.

6. The controlled release system of nitric oxide according to claim 1, characterized in that, Sodium nitrite is preferred as the nitric oxide donor.

7. The controlled release system of nitric oxide according to claim 1, characterized in that, The mass fraction of nitrite in the nitric oxide donor component is 0.02% to 10%.

8. The controlled release system of nitric oxide according to any one of claims 1 to 7, characterized in that, The half-life of nitric oxide release in the system is in the range of 0.15 h to 10.9 h, and the maximum instantaneous concentration of nitric oxide release is in the range of 0.5 ppm to 380 ppm.

9. The controlled release system of nitric oxide according to any one of claims 1 to 7, characterized in that, The polymeric macromolecular acid component is a liquid formulation or a gelling agent; the polymeric macromolecular acid liquid formulation or gelling agent is composed of the polymeric macromolecular acid component and excipients. The nitric oxide donor component is a liquid formulation, a gelling agent, or a solid formulation; the nitric oxide liquid formulation, a gelling agent, or a solid formulation is composed of the nitric oxide donor component and excipients.

10. The controlled release system of nitric oxide according to any one of claims 1 to 4, characterized in that, The acidity and cross-linking structure of the polymeric macromolecular acid component are provided by cross-linked polyacrylic acid or cross-linked polymethacrylic acid.

11. The application of the controlled release system of nitric oxide according to any one of claims 1 to 10 in the preparation of antimicrobial products, characterized in that, The pathogenic microorganisms include fungi, bacteria, and viruses; the fungi are Candida albicans, the bacteria are Propionibacterium acnes, Staphylococcus aureus, and Escherichia coli, and the virus is herpes simplex virus; the anti-pathogenic microorganism products include drugs or medical devices for wound healing, hemorrhoids, acne, or pathogenic microorganism infections; the controlled release system of nitric oxide forms spatial structures with different cross-linking densities by adjusting the structure and ratio of the cross-linking agent in the polymeric macromolecular acid, utilizing the cage effect to slow down the reaction between the protons provided by the polymeric macromolecular acid and the nitrite ions in the nitric oxide donor component, and controls the diffusion rate of the generated nitric oxide; at the same time, by changing the dosage form of the nitric oxide donor component, the distribution rate of nitrite ions in the system during the reaction is controlled, further regulating the release of nitric oxide, and achieving controlled release of nitric oxide.

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