Preparation method of CO2 antibacterial dressing with pH indication function

Abstract

The present invention relates to the technical field of biomedical materials, and discloses a method for preparing a CO2 antibacterial dressing with a pH indication function. First, a phenol red (PR)-grafted PCN-SF composite material PR-PCN-SF is prepared, followed by preparing a mesoporous silica MSN@FeCO loaded with carbonyl iron (FeCO), and finally loading MSN@FeCO onto the PR-PCN-SF composite material to obtain a composite aerogel MSN@FeCO-PR-PCN-SF. The present invention indicates the wound infection condition by the color change of the loaded phenol red indicator, controls the temperature of the near-infrared (NIR) light source on / off control system, adjusts the release amount of CO2, realizes the CO2 antibacterial efficacy started on demand, and then achieves an efficient and controllable wound treatment effect. The present invention is simple to prepare, safe and controllable, and is not prone to bacterial resistance, and has potential application prospects in the field of biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a method for preparing a CO2 antibacterial dressing with a pH indicator function. Background Art

[0002] Microorganisms such as viruses, bacteria, and fungi are generally pathogenic to humans and animals and cause a variety of serious diseases, resulting in extremely high morbidity and mortality. Common diseases include upper respiratory tract infections (URTI), skin and soft tissue infections (SSI), surgical wound infections (SWI), etc. Since the discovery of penicillin, pharmaceutical companies have produced many antimicrobial drugs containing antibiotics, which have improved sanitary conditions. However, the widespread use of antibiotics in recent years has caused bacteria to develop drug resistance, and many new drug-resistant strains have been found in patients around the world (Hamley, Small Bioactive Peptides for Biomaterials Design and Therapeutics, Chemical Reviews, 2017, 117(24), 14015-14041). Therefore, studying antimicrobial strategies that are less likely to induce bacterial resistance is an important direction for antimicrobial therapy.

[0003] Antimicrobial wound dressings have garnered increasing attention in recent years. Most commercial suppliers currently offer silver-coated or nanoparticle-impregnated dressings. While the broad-spectrum antimicrobial efficacy of these dressings has been demonstrated, some studies have also reported silver cytotoxicity to mammalian cells and negative effects on wound healing. Furthermore, silver-based wound dressings are expensive, and there are ongoing concerns about nanoparticle absorption and nanotoxicity. Studies have demonstrated that wound fluid pH plays a crucial role in the wound healing cascade. Upon injury, microvascular leakage lowers the wound pH, approaching the physiological pH that favors bacterial infection. During wound healing, various factors, including hypoxia and increased lactate production, contribute to a decrease in the wound microenvironment's pH (N. Ninan, A. Forget, V. Prasad Shastri, et al., ACS Applied Materials & Interfaces, 2016, 8, 28511-28521). Therefore, wound pH can be used to indicate wound healing progress and ensure the safety and effectiveness of the treatment process.

[0004] Gas therapy, as a new treatment modality, can use gas molecules to treat diseases. Based on the many advantages of this treatment method, it has received increasing attention in recent years. (1) Gas molecules are less likely to develop drug resistance and can increase the sensitivity of drug-resistant cells to chemotherapy drugs. (2) Some gas molecules, as important endogenous signaling molecules, play an important role in various physiological processes. In recent years, researchers have studied and developed five typical gas molecules (hydrogen, nitric oxide, carbon monoxide, hydrogen sulfide and sulfur dioxide) for the diagnosis and treatment of various diseases. Among them, CO has the advantage of being relatively stable and has a variety of potential therapeutic uses under physiological metabolic and pathological conditions (cell protection, anti-apoptosis, anti-cancer and anti-inflammatory, etc.). Summary of the Invention

[0005] Purpose of the Invention: To address the challenges of the prior art, the present invention provides a method for preparing a light-controlled CO2 antimicrobial wound dressing with pH indication. The color change of a loaded phenol red indicator indicates wound infection. By controlling the on / off function of a near-infrared (NIR) light source to regulate the system temperature and adjust the CO2 release, the dressing achieves on-demand CO2 antimicrobial efficacy, thereby achieving efficient and controllable wound treatment. This method is simple to prepare, safe, controllable, and unlikely to induce bacterial resistance, offering potential applications in the biomedical field.

[0006] Technical solution: The present invention provides a method for preparing a CO2 antibacterial dressing with a pH indicator function, comprising the following steps:

[0007] S1. Grind urea and dicyandiamide and calcine in a muffle furnace to produce porous g-C3N4.

[0008] S2. Dicyclohexylcarbodiimide was used as a dehydrating agent and lutidine as a catalyst. The mixture containing silk fibroin SF and porous g-C3N4 was added and stirred to mix evenly. After a period of reaction, PCN-SF was obtained.

[0009] S3 dicyclohexylcarbodiimide as a dehydrating agent, lutidine as a catalyst, was added to a mixed solution containing phenol red PR and PCN-SF, stirred and mixed, and the reaction was continued for a period of time to obtain a PR-PCN-SF solution;

[0010] S4. MSN was used as a carrier and dissolved in chloroform, frozen and degassed under liquid nitrogen. A photothermal responsive CO donor was added. The mixture was protected from light and stirred under nitrogen. The mixture was centrifuged, washed, and dried to obtain MSN@FeCO nanoparticles.

[0011] S5. Aqueous solutions of sodium N-lauroylsarcosinate, MSN@FeCO, and PR-PCN-SF were mixed, ultrasonically treated, placed in a silica gel mold, and completely frozen with liquid nitrogen before drying to obtain a composite aerogel.

[0012] Furthermore, in S1, the mass ratio of urea to dicyandiamide is 3:1;

[0013] And / or, in S2, the mass ratio of the porous g-C3N4, silk fibroin SF, dicyclohexylcarbodiimide and lutidine is 1:1:2:0.05;

[0014] And / or, in S3, the mass ratio of phenol red PR, PCN-SF, dicyclohexylcarbodiimide and lutidine is 1:1:2:0.05;

[0015] And / or, in S4, the mass ratio of the MSN, CO donor and chloroform is 5-20: 3-10: 2.5-10.

[0016] Furthermore, in S1, the calcination temperature is 500-540° C., and the calcination time is 4-5 h.

[0017] Furthermore, in S2, the reaction temperature is room temperature and the reaction time is 12 to 24 h;

[0018] And / or, in S3, the reaction temperature is room temperature and the reaction time is 12 to 24 hours.

[0019] Furthermore, in S2, the solvent used in the mixed solution of the silk fibroin SF and porous g-C3N4 is any one of the CaCl2-H2O-CH3CH2OH ternary system and the LiBr-H2O binary system, and the bath ratio is 1: 10-30;

[0020] And / or, in S3, the solvent used in the mixed solution of phenol red PR and PCN-SF is any one of H2O, CH3CH2OH, dichloromethane, chloroform, acetone or cyclohexane, and the bath ratio is 1:10~30.

[0021] Furthermore, in S4, the stirring time is 24-30 h, the drying temperature is 50-60° C., and the drying time is 12-14 h.

[0022] Furthermore, in S4, the CO donor is Fe(CO)5 or Fe3(CO) 12 Any of .

[0023] Furthermore, in S5, the concentration of the aqueous solution of MSN@FeCO and PR-PCN-SF is 10-50 mg / mL.

[0024] Furthermore, in S5, the concentration of sodium N-lauroyl sarcosinate is 1-4 mg / mL.

[0025] Preferably, in S1, the washing is first washing with dilute nitric acid having a concentration of 0.1 mol / L, and then washing with deionized water;

[0026] And / or, in S5, the ultrasonic treatment is specifically water bath ultrasonic treatment for 1 hour, followed by probe ultrasonic treatment for 20 to 40 minutes;

[0027] And / or, in S5, the drying is specifically drying in a freeze dryer for 2 to 3 days. Beneficial effects

[0028] Compared with the prior art, the present invention uses silk fibroin (SF) with high biocompatibility as the wound dressing substrate, and uses a chemical grafting method to firmly connect the pH color indicator phenol red (PR) and the photothermal reagent (porous g-C3N4, PCN) to SF to obtain a PR-PCN-SF composite material. The photothermal responsive CO donor (MSN@FeCO) is further loaded onto PR-PCN-SF by freeze drying to obtain an MSN@FeCO-PR-PCN-SF composite hydrogel dressing. Among them, PR can respond to the weakly acidic environment of bacterial infection, and then indicate the inflammation of the wound through a color change visible to the naked eye. Then, according to the color change of the hydrogel dressing, the irradiation conditions of near-infrared light (NIR) are controlled to regulate the temperature of the photothermal reagent PCN, thereby triggering the thermosensitive CO donor (FeCO) in the hydrogel system to produce CO. By controlling the on / off, illumination power, and illumination time of the NIR light source, the release efficiency and antibacterial efficacy of CO can be adjusted to achieve on-demand wound treatment efficacy, thereby improving the safety and controllability of CO in the field of antibacterial and wound treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a scanning electron microscope morphology of the MSN@FeCO-PR-PCN-SF composite aerogel prepared in the present invention;

[0030] Figure 2 Graph showing the photothermal experimental data of the MSN@FeCO-PR-PCN-SF composite aerogel prepared in embodiments 1-3 and the SF aerogel prepared in the comparative example;

[0031] Figure 3 Graph showing the CO release kinetics of the MSN@FeCO-PR-PCN-SF composite aerogels prepared in embodiments 1-3 and the SF aerogels prepared in the comparative example;

[0032] Figure 4 Schematic diagram of the color change of the MSN@FeCO-PR-PCN-SF composite aerogel prepared in the present invention in a weak acid environment. DETAILED DESCRIPTION

[0033] The present invention is described in detail below with reference to the embodiments.

[0034] Implementation method 1:

[0035] This embodiment provides a method for preparing a light-controlled CO2 antibacterial wound dressing with a pH indicator function, and the specific steps are as follows:

[0036] Step 1: Preparation of porous g-C3N4 (PCN): Urea and dicyandiamide were mixed in a 3:1 mass ratio and manually ground for 4 hours to obtain an ultrafine powder. The ground powder was then calcined in a muffle furnace at 530°C for 4 hours to obtain a yellow product. The yellow product was washed in 0.1 mol / L dilute nitric acid and deionized water, respectively, to remove any residue on the PCN surface.

[0037] Step 2. Preparation of PCN-loaded silk fibroin composite material (PCN-SF): Using dicyclohexylcarbodiimide (DCC) as a dehydrating agent and dimethylpyridine (DMAP) as a catalyst, SF and PCN were added to an organic solvent and reacted at room temperature for 12 to 24 hours in a volume ratio of PCN, SF, DCC and DMAP of 1:1:2:0.05, so that the amino groups rich in the edges of the PCN pores reacted with the carboxyl groups of SF to obtain a PCN-loaded silk fibroin composite material (PCN-SF).

[0038] Step 3. Preparation of phenol red grafted PCN-SF composite material (PR-PCN-SF): Using dicyclohexylcarbodiimide (DCC) as a dehydrating agent and catalyzed by dimethylpyridine (DMAP), phenol red and PCN-SF were added to an organic solvent and reacted at room temperature for 12 to 24 hours in a volume ratio of PR:PCN-SF:DCC:DMAP of 1:1:2:0.05 to allow the hydroxyl groups on phenol red to react with the amino groups on silk fibroin to obtain PR-PCN-SF.

[0039] Step 4: Synthesis of mesoporous silica (MSN) and carbonyl iron-loaded mesoporous silica (MSN@FeCO): 5 g of hexadecyltrimethylammonium chloride (CTAC) and 0.05 g of triethanolamine (TEA) were added to deionized water and mixed. The SiO₂ aqueous solution was then added and stirred. 0.5 mL of tetraethyl orthosilicate (TEOS) was also added, stirred, and washed by centrifugation. The resulting product was extracted multiple times with a methanolic NaCl solution and dried to obtain MSN. Next, 20 mg of MSN was dissolved in 10 mL of chloroform, frozen under liquid nitrogen, and degassed. FeCO₂ was added, and the mixture was stirred for 24 h under nitrogen in the dark. After centrifugation, the mixture was washed with chloroform and dried at 60°C to obtain MSN@FeCO₂ nanoparticles.

[0040] Step 5: Loading MSN@FeCO onto the PR-PCN-SF composite material to prepare a composite aerogel (MSN@FeCO-PR-PCN-SF): Sodium N-lauroylsarcosinate (SNS) was used as a surfactant. A 4 mg / mL SNS solution was prepared and added to a 20 mg / mL MSN@FeCO and PR-PCN-SF solution. The initial hydrogel dispersion of the composite material was obtained at 25°C. After waterbath sonication for 1 hour and probe sonication for 30 minutes, the sample was transferred to a silica gel mold and frozen in liquid nitrogen for 2 days. Once completely frozen, the sample was transferred to a freeze dryer and dried for 2–3 days to obtain the MSN@FeCO-PR-PCN-SF composite aerogel.

[0041] Implementation 2:

[0042] This embodiment is substantially the same as Embodiment 1, except that the concentration of the MSN@FeCO and PR-PCN-SF solutions in this embodiment is 30 mg / mL.

[0043] Apart from this, this embodiment is identical to Embodiment 1 and will not be described in detail here.

[0044] Implementation 3:

[0045] This embodiment is substantially the same as Embodiment 1, except that the concentration of the MSN@FeCO and PR-PCN-SF solutions in this embodiment is 40 mg / mL.

[0046] Apart from this, this embodiment is identical to Embodiment 1 and will not be described in detail here.

[0047] Comparative Example:

[0048] SF was dissolved in a CaCl2-H2O-CH3CH2OH ternary system with a bath ratio of 1:20 and dispersed evenly after ultrasonic treatment. It was then transferred into a silica gel mold and frozen using liquid nitrogen. After the sample was completely frozen, it was transferred to a freeze dryer and dried for 3 days to obtain SF aerogel.

[0049] Photothermal effect test:

[0050] In order to study the photothermal properties of the samples, Implementation Example 1, Implementation Example 2, Implementation Example 3 and the comparative example were irradiated with 808 nm NIR laser for 10 min, and the temperature changes of the samples were recorded every 2 min using a thermocouple thermometer.

[0051] CO release kinetics test:

[0052] The CO production of MSN@FeCO-PR-PCN-SF composite gel was detected by hemoglobin method. 1.5 mg of hemoglobin (Hb) was added to 5 mL of PBS solution, and then 18 mg of sodium dithionite was added. After thorough stirring, the sample was added and N2 was bubbled under vacuum for 15 minutes. Implementation Example 1, Implementation Example 2, Implementation Example 3 and the comparative example were irradiated with 808 nm NIR laser for 10 minutes. The absorption peak of Hb solution at 350-600 nm was tested every 2 minutes using a microplate reader. The conversion of Hb to HbCO was calculated using the strong absorption peaks of carboxyhemoglobin (HbCO) and Hb at 410 nm and 430 nm, and the concentration of CO released (C CO ) is calculated using the following formula:

[0053]

[0054] Among them, C CO is the concentration of released CO, C Hb is the initial Hb concentration, I 410nm and I 430nm Represent the absorption spectrum intensities at 410 nm and 430 nm, respectively.

[0055] In vitro antibacterial test:

[0056] In the in vitro antibacterial experiment, Pseudomonas aeruginosa and Staphylococcus aureus were used as experimental bacteria. The revived bacteria were placed in a liquid culture medium and shaken on a constant temperature shaker at 37°C overnight. Implementation 1, Implementation 2, Implementation 3 and the comparative example samples were placed in a 12-well plate, and 200 μL of bacterial solution was respectively added to the sample surface. After contact with the bacterial solution for 3 hours, the pH value of the gel sample surface was tested and its color change was observed. Then, the samples were irradiated with an 808 nm NIR laser for 10 minutes, and then 1 mL of PBS solution was added to each group of samples. After ultrasonic treatment, 50 μL of the bacterial solution was plated and placed on a constant temperature shaker at 37°C overnight to observe the number of bacterial colonies. The dark group samples were subjected to antibacterial experiments using the same method under light-proof conditions.

[0057] The scanning electron microscopy morphology of MSN@FeCO-PR-PCN-SF composite aerogel is shown in Figure 2. Figure 1 As shown in the figure, the gel material has a porous structure and nanoparticles are distributed on the surface of the material, proving that the composite material was successfully synthesized. At the same time, the porous structure of the gel material is conducive to the production and release of CO gas molecules, which can ensure the smooth progress of subsequent antibacterial experiments.

[0058] Photothermal experimental data such as Figure 2As shown in Figure 1, the temperature of the samples was observed over time under 808 nm NIR light irradiation. The results show that after 10 minutes of NIR irradiation, the temperature of the comparative example did not change much with the irradiation time, while the temperatures of Implementation 1, Implementation 2, and Implementation 3 gradually increased, and there was no significant difference in their photothermal temperatures. This proves that the doping level of MSN@FeCO has little effect on the photothermal temperature, and that the gel system achieves its photothermal effect primarily through the loading of PCN.

[0059] CO release kinetics data such as Figure 3 As shown in the figure, the CO production of the samples was observed under 808 nm NIR light irradiation. The results show that under NIR irradiation, the comparative example sample did not produce CO, while the CO production of Implementation 1, Implementation 2, and Implementation 3 gradually increased over time. Moreover, as the doping amount of MSN@FeCO increased, the amount of CO released increased. After 10 minutes of irradiation, the CO concentrations reached 0.185 mM, 0.255 mM, and 0.385 mM, respectively. This gel system mainly achieves controllable CO release through the loading of MSN@FeCO and NIR light.

[0060] The surface color changes of the gel samples after contact with PBS buffer solutions of different pH values (pH=5.0, 5.6, 6.2, 6.8, 7.4) for 20 min are shown in the figure. Figure 4 As shown, it is proved that the gel material can respond to a weakly acidic environment and indicate it through a color change visible to the naked eye.

[0061] Tables 1 and 2 show the plate colony counts from the in vitro antibacterial experiments. The results show that under NIR irradiation, Implementations 1, 2, and 3 all exhibited effective antibacterial effects against Pseudomonas aeruginosa and Staphylococcus aureus. With increasing MSN@FeCO doping levels, the antibacterial efficiency significantly increased, reaching as high as 99%. In comparison, the control group and the dark control group showed no significant antibacterial effect. These results demonstrate that the antibacterial effect of this gel system can be controlled by adjusting the MSN@FeCO doping level and NIR irradiation, ensuring a safe and controllable treatment process.

[0062] Table 1 Antibacterial test results obtained under 808 nm NIR light in comparative examples and embodiments 1 to 3 of the present invention

[0063] Comparative Example Implementation Method 1 Implementation Method 2 Implementation 3 Pseudomonas aeruginosa 7% 81% 93% 97% Staphylococcus aureus 9% 84% 96% 99%

[0064] Table 2 Antibacterial test results obtained in dark conditions for the comparative examples and embodiments 1 to 3 of the present invention

[0065] Comparative Example Implementation Method 1 Implementation Method 2 Implementation 3 Pseudomonas aeruginosa 9% 12% 13% 14% Staphylococcus aureus 8% 10% 11% 11%

[0066] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a CO2 antibacterial dressing with pH indication, characterized in that: The following steps are involved: S1. Grind urea and dicyandiamide and calcine in a muffle furnace to produce porous g-C3N4. S2. Dicyclohexylcarbodiimide was used as a dehydrating agent and lutidine as a catalyst. The mixture containing silk fibroin SF and porous g-C3N4 was added and stirred to mix evenly. After a period of reaction, PCN-SF was obtained. S3 dicyclohexylcarbodiimide as a dehydrating agent, lutidine as a catalyst, was added to a mixed solution containing phenol red PR and PCN-SF, stirred and mixed, and the reaction was continued for a period of time to obtain a PR-PCN-SF solution; S4. MSN was used as a carrier and dissolved in chloroform, frozen and degassed under liquid nitrogen. A photothermal responsive CO donor was added. The mixture was stirred in the dark under nitrogen, centrifuged, washed, and dried to obtain MSN@FeCO nanoparticles. S5. An aqueous solution of sodium N-lauroylsarcosinate, MSN@FeCO, and PR-PCN-SF was mixed, ultrasonically treated, placed in a silica gel mold, completely frozen in liquid nitrogen, and then dried to obtain a composite aerogel. The CO2 antibacterial dressing with pH indication regulates the temperature of the photothermal reagent PCN by controlling the irradiation conditions of near-infrared light, thereby triggering the thermosensitive CO2 donor FeCO2 in the hydrogel system to produce CO2.

2. The method for preparing the CO2 antibacterial dressing with pH indication according to claim 1, characterized in that: In S1, the mass ratio of urea to dicyandiamide is 3:1; And / or, in S2, the mass ratio of the porous g-C3N4, silk fibroin SF, dicyclohexylcarbodiimide and lutidine is 1:1:2:0.05; And / or, in S3, the mass ratio of phenol red PR, PCN-SF, dicyclohexylcarbodiimide and lutidine is 1:1:2:0.05; And / or, in S4, the mass ratio of the MSN, CO donor and chloroform is 5-20: 3-10: 2.5-10.

3. The method for preparing the CO2 antibacterial dressing with pH indicator according to claim 1, characterized in that: In S1, the calcination temperature is 500-540° C., and the calcination time is 4-5 h.

4. The method for preparing the CO2 antibacterial dressing with pH indicator according to claim 1, characterized in that: In S2, the reaction temperature is room temperature and the reaction time is 12–24 h; And / or, in S3, the reaction temperature is room temperature and the reaction time is 12 to 24 hours.

5. The method for preparing the CO2 antibacterial dressing with pH indication according to claim 1, characterized in that: In S2, the solvent used in the mixed solution of silk fibroin SF and porous g-C3N4 is any one of the CaCl2-H2O-CH3CH2OH ternary system and the LiBr-H2O binary system, and the bath ratio is 1: 10-30; And / or, in S3, the solvent used in the mixed solution of phenol red PR and PCN-SF is any one of H2O, CH3CH2OH, dichloromethane, chloroform, acetone or cyclohexane, and the bath ratio is 1:10~30.

6. The method for preparing the CO2 antibacterial dressing with pH indicator according to claim 1, characterized in that: In S4, the stirring time is 24-30 h, the drying temperature is 50-60° C., and the drying time is 12-14 h.

7. The method for preparing the CO2 antibacterial dressing with pH indication according to claim 1, characterized in that: In S4, the CO donor is either Fe(CO)5 or Fe3(CO)12.

8. The method for preparing the CO2 antibacterial dressing with pH indicator function according to claim 1, characterized in that: In S5, the concentration of the aqueous solution of MSN@FeCO and PR-PCN-SF is 10-50 mg / mL.

9. The method for preparing the CO2 antibacterial dressing with pH indicator function according to claim 1, characterized in that: In S5, the concentration of sodium N-lauroyl sarcosinate is 1-4 mg / mL.

10. The method for preparing a CO2 antibacterial dressing with pH indication according to any one of claims 1 to 9, characterized in that: In S1, the washing is specifically washing with dilute nitric acid having a concentration of 0.1 mol / L, and then washing with deionized water; And / or, in S5, the ultrasonic treatment is specifically water bath ultrasonic treatment for 1 hour, followed by probe ultrasonic treatment for 20 to 40 minutes; And / or, in S5, the drying is specifically drying in a freeze dryer for 2 to 3 days.

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