Carbon nanodot-loaded anti-inflammatory and antibacterial hydrogel and preparation method and application thereof

Abstract

The present application relates to the technical field of active material preparation, and particularly relates to an anti-inflammatory and antibacterial hydrogel loaded with carbon nanodots and a preparation method and application thereof. The hydrogel loaded with carbon nanodots provided by the present application contains royal jelly carbon nanodots; and the raw materials of the hydrogel contain the following components in parts by weight: 4-8 parts of honey, 10-15 parts of esterified cassava starch, and 1-3 parts of tragacanth. The hydrogel loaded with carbon nanodots provided by the present application combines royal jelly carbon nanodots and composite hydrogel, has good antibacterial, anti-inflammatory, antioxidant and wound healing effects, and has excellent biocompatibility and mechanical properties, and is expected to become an effective substitute for antibiotics, and is used for the treatment of bacterial infection wounds and has a good application prospect in the preparation of wound dressings.

Description

Technical Field

[0001] This invention relates to the field of active material preparation technology, and in particular to an anti-inflammatory and antibacterial hydrogel loaded with carbon nanodots, its preparation method and application. Background Technology

[0002] Bacterial infections and antibiotic resistance pose serious challenges to human health. For example, Staphylococcus aureus has a significant ability to adhere to skin keratinocytes. Its colonization and invasive infection of skin wounds not only increase the level of reactive oxygen species (ROS) in keratinocytes but also exacerbate DNA damage, triggering persistent inflammation and becoming a key factor hindering wound healing. Furthermore, bacterial infections of wounds can also induce systemic damage. Antibiotics are commonly used to treat bacterial infections, but routine or excessive use of antibiotics after trauma or bacterial infection is a major factor promoting the development of antibiotic resistance. Existing studies have shown that antibiotics may inhibit the wound healing process and are accompanied by serious side effects. Therefore, developing natural, non-toxic, multifunctional active substances with antibacterial, antioxidant, and anti-inflammatory properties for wound healing treatment is considered one of the effective strategies to reduce antibiotic dependence.

[0003] Royal jelly possesses various pharmacological properties, such as anti-inflammatory, antibacterial, and antioxidant effects. However, royal jelly is rich in large-molecule proteins and lipids, resulting in low bioavailability and limiting its therapeutic efficiency. Carbon nanodots, also known as carbon dots, are a novel type of carbon-based nanomaterial with low toxicity, good biocompatibility, and stable physicochemical properties, and have been widely used in drug delivery systems. By controlling the retention of functional groups during the synthesis of carbon dots, carbon dot materials with various functional activities can be prepared. Therefore, the preparation of royal jelly carbon nanodots plays an important role in improving the utilization and bioactivity of royal jelly. However, carbon nanodots often suffer from poor wound adhesion, leading to ineffective treatment. Hydrogels can serve as carriers to improve this drawback of carbon nanodots. Therefore, developing hydrogels carrying carbon nanodots as an antibiotic alternative is of great significance for the treatment of bacterial wound infections. Summary of the Invention

[0004] This invention provides an anti-inflammatory and antibacterial hydrogel loaded with carbon nanodots, its preparation method, and its application.

[0005] This invention first developed a type of royal jelly carbon nanodots, which significantly improves the antioxidant, antibacterial, and anti-inflammatory properties compared to natural royal jelly. Furthermore, this invention optimized the raw materials for the hydrogel material carrying the royal jelly carbon nanodots and discovered that a composite hydrogel obtained by combining esterified cassava starch and tragali gum with honey, when used to carry the aforementioned carbon nanodots, not only exhibits good biocompatibility and gelling properties but also significantly enhances anti-inflammatory, antibacterial, and wound-healing abilities.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a hydrogel loaded with carbon nanodots, wherein the carbon nanodots are royal jelly carbon nanodots; the raw materials of the hydrogel contain the following components in parts by weight: 4-8 parts of honey, 10-15 parts of esterified cassava starch, and 1-3 parts of yarrow gum.

[0008] In the process of optimizing the hydrogel formulation, this invention discovered that, compared with other colloidal or starch-based raw material blends, when esterified cassava starch and tragali gum are blended within the aforementioned ratio range, the long chains of tragali gum can better form a denser three-dimensional network with the linear / branched structures of esterified cassava starch through hydrogen bonds and hydrophobic interactions, thereby significantly enhancing the elasticity of the hydrogel. Combined with honey, this further improves the viscosity and water retention of the hydrogel. Simultaneously, the phenolic compounds in honey can enhance the antibacterial properties of the hydrogel. Controlling the ratio of the three components within the aforementioned range can improve the antibacterial properties of the hydrogel while ensuring good mechanical strength and gelation characteristics. Furthermore, this invention found that royal jelly carbon nanodots exhibit significantly enhanced antibacterial, anti-inflammatory, and antioxidant activities compared to natural royal jelly.

[0009] Preferably, the raw materials of the hydrogel contain the following components in parts by weight: 4-5 parts honey, 10-11 parts esterified tapioca starch, and 1.5-2.5 parts tragacanth gum.

[0010] In this invention, the esterified cassava starch is preferably acetate-esterified cassava starch. The combination of acetate-esterified cassava starch and tragacanth gum can form a denser three-dimensional network, resulting in superior gel properties such as elasticity of the hydrogel.

[0011] The raw materials for the hydrogel described above also include water. The amount of water used is in a mass ratio of (3-5):1 to the total mass of esterified cassava starch and tragacanth gum.

[0012] Preferably, the raw materials of the hydrogel contain the following components in parts by weight: 4-8 parts honey, 10-15 parts esterified tapioca starch, 1-3 parts tragacanth gum, and 45-75 parts water.

[0013] More preferably, the raw material of the hydrogel comprises the following components in parts by weight: 4-5 parts honey, 10-11 parts esterified tapioca starch, 1.5-2.5 parts tragacanth gum, and 46-50 parts water. Preferably, the degree of substitution (DS) of the acetate esterified tapioca starch is 0.01-0.1. More preferably, it is 0.04-0.06.

[0014] The royal jelly carbon nanodots described above are preferably synthesized using an ultrasound-assisted hydrothermal method.

[0015] Preferably, the method for preparing the royal jelly carbon nanodots includes: mixing royal jelly with water and then subjecting it to ultrasonic treatment to obtain a royal jelly solution; and reacting the solution at 180-220°C.

[0016] Preferably, the mass ratio of royal jelly to water is 1:(4-8). After mixing, the mixture is stirred (preferably for 10-40 minutes) and then subjected to ultrasonic treatment.

[0017] Preferably, the frequency of the ultrasonic treatment is 35-45 kHz and the power is 180-220 W.

[0018] Preferably, the ultrasonic treatment time is 20-50 minutes.

[0019] Preferably, the reaction time is 6-15 hours.

[0020] Preferably, after the reaction, the supernatant is separated, purified, and freeze-dried to obtain powdered royal jelly carbon nanoparticles. The supernatant can be separated by centrifugation. The purification includes membrane filtration and dialysis (preferably using a regenerated cellulose dialysis membrane with a molecular weight cutoff of 1 kDa in water). After dialysis, the liquid inside the dialysis membrane is collected and freeze-dried.

[0021] In some embodiments of the present invention, the ultrasonic-assisted hydrothermal synthesis includes: mixing royal jelly with water at a mass ratio of 1:(4-8), stirring magnetically for 20-30 minutes, and then ultrasonically treating for 30-40 minutes at a frequency of 38-42 kHz and a power of 190-210 W; placing the resulting solution in a high-temperature reactor and reacting at 180-220°C for 10-15 hours; after the reaction, centrifuging the reaction solution at 8000-10000 rpm for 20-30 minutes, and filtering the supernatant through a 0.22-micron filter membrane; placing the filtrate into a regenerated cellulose dialysis membrane with a molecular weight cutoff of 1 kDa, and dialyzing it in pure water for 24-48 hours until the dialysis fluid is colorless; collecting the liquid inside the dialysis membrane and freeze-drying it under vacuum for 12-24 hours to obtain powdered royal jelly carbon nanoparticles.

[0022] Preferably, the preparation method of the hydrogel includes: mixing esterified cassava starch and tragali gum in water and reacting them, and then mixing them with honey.

[0023] Preferably, the mass ratio of the royal jelly carbon nanodots to the total mass of the hydrogel raw materials is 1:50-1:100.

[0024] Secondly, the present invention provides a method for preparing the above-mentioned hydrogel loaded with carbon nanodots, the method comprising: mixing esterified cassava starch and yarrow gum in water to react, mixing the reaction product with honey, and then mixing it with royal jelly carbon nanodots.

[0025] Preferably, the esterified cassava starch and yarrow gum are mixed and reacted at 65-95°C (preferably 65-70°C) for 15-30 minutes.

[0026] Preferably, after mixing and reacting esterified cassava starch and yarrow gum at 65-95°C for 15-30 minutes, the mixture is cooled to 45-55°C, honey is added, and the mixture is reacted for 5-10 minutes. Then, it is mixed with royal jelly carbon nanodots for further reaction.

[0027] Thirdly, the present invention provides the application of the above-described hydrogels carrying carbon nanodots in antibacterial activity.

[0028] The bacteria mentioned include, but are not limited to, Staphylococcus aureus.

[0029] The antibacterial activity may be for purposes other than disease diagnosis and treatment.

[0030] Fourthly, the present invention provides the application of the above-described hydrogels carrying carbon nanodots in the preparation of products having one or more functions selected from antibacterial, anti-inflammatory, antioxidant, and wound-healing-promoting properties.

[0031] The products include dressings, disinfectants, and antibacterial or bacteriostatic agents.

[0032] Fifthly, the present invention provides the application of the above-described hydrogels carrying carbon nanodots in antibacterial, anti-inflammatory, antioxidant and / or wound healing promotion.

[0033] The beneficial effects of the present invention include at least the following: The hydrogel with carbon nanodots provided by the present invention combines royal jelly carbon nanodots and composite hydrogel, which not only has good antibacterial, anti-inflammatory, antioxidant and wound healing promoting effects, but also the hydrogel material has excellent gel properties such as biocompatibility and mechanical properties, and is expected to become an effective alternative to antibiotics for the treatment of bacterial infected wounds, and has good application prospects in wound dressings. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a transmission electron microscope (TEM) image of royal jelly carbon nanodots from Experiment Example 1 of this invention.

[0036] Figure 2 The graph shows the test results of the storage modulus (G') and dissipation modulus (G'') of the hydrogel in Experimental Example 1 of this invention.

[0037] Figure 3 This is a scanning electron microscope (SEM) image of the hydrogel in Experimental Example 1 of this invention.

[0038] Figure 4 The images show the viscosity test results of the hydrogels from Example 1 (with honey hydrogel) and Comparative Example 1 (without honey hydrogel) of the present invention.

[0039] Figure 5 The figures show the water retention test results of the hydrogels from Example 1 (with honey hydrogel) and Comparative Example 1 (without honey hydrogel) of the present invention.

[0040] Figure 6 This is a growth curve of Staphylococcus aureus treated with different concentrations of royal jelly carbon nanodots in Experimental Example 3 of the present invention.

[0041] Figure 7 This study illustrates the effect of different concentrations of royal jelly carbon nanodots on the activity of mouse macrophages (RAW 264.7) in Experimental Example 3 of this invention.

[0042] Figure 8 This study describes the effect of different concentrations of royal jelly carbon nanodots on nitric oxide (NO) release from mouse macrophages (RAW 264.7) in Experimental Example 3 of this invention.

[0043] Figure 9 This study illustrates the effect of different concentrations of royal jelly carbon nanodots on the release of reactive oxygen species (ROS) from mouse macrophages (RAW 264.7) in Experimental Example 3 of this invention.

[0044] Figure 10 This is a growth curve of Staphylococcus aureus treated with a composite hydrogel containing royal jelly carbon nanodots in Experimental Example 3 of the present invention.

[0045] Figure 11 This study describes the effect of a composite hydrogel containing royal jelly carbon nanodots on the activity of mouse macrophages (RAW264.7) in Experimental Example 3 of this invention.

[0046] Figure 12The diagram shows the antibacterial performance test results of hydrogels with and without honey in Experiment 4 of this invention. In the diagram, hydrogel without honey represents the hydrogel with royal jelly carbon nanoparticles in Comparative Example 1, hydrogel with 2 grams of honey represents the hydrogel with royal jelly carbon nanoparticles in Comparative Example 2, hydrogel with 4 grams of honey represents the hydrogel with royal jelly carbon nanoparticles in Example 3, and hydrogel with 5 grams of honey represents the hydrogel with royal jelly carbon nanoparticles in Example 1.

[0047] Figure 13 The effects of the composite hydrogel containing royal jelly carbon nanodots in Experimental Example 5 of this invention on the wound area, pro-inflammatory factor interleukin-6 (IL-6), anti-inflammatory factor interleukin-10 (IL-10), platelet endothelial cell adhesion molecule-31 (CD31), and α-smooth muscle actin (α-SMA) expression in mice. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0049] The degree of substitution of acetate-esterified tapioca starch used in the following examples and comparative examples is 0.05.

[0050] Example 1 This embodiment provides a hydrogel loaded with royal jelly carbon nanodots, wherein the raw materials of the hydrogel contain the following components in parts by weight: 5 parts honey, 10 parts acetate-esterified tapioca starch, and 2 parts tragacanth gum.

[0051] The mass ratio of royal jelly carbon nanodots to the raw materials of the hydrogel (honey, acetic acid transcribed cassava starch and yarrow gum) is 1:100.

[0052] The preparation method of the above-mentioned hydrogel loaded with royal jelly carbon nanodots includes the following steps: Preparation of carbon nanodots: Royal jelly was dissolved in pure water at a mass ratio of 1:6 and stirred magnetically for 20 minutes at room temperature. Then, it was sonicated at 40 kHz and 200 W for 30 minutes at room temperature to ensure complete dissolution. The resulting solution was placed in a high-temperature reactor and reacted at 180℃ for 15 hours. After the reaction, the reaction solution was centrifuged at 8000 rpm for 30 minutes at room temperature. The supernatant was filtered through a 0.22-micron filter membrane. The filtrate was placed in a regenerated cellulose dialysis membrane with a molecular weight cutoff of 1 kDa and dialyzed in pure water at room temperature for 24 hours until the dialysate was colorless. The liquid inside the dialysis membrane was collected and freeze-dried under vacuum for 24 hours to obtain powdered royal jelly carbon nanodots.

[0053] Hydrogel preparation and royal jelly carbon nanodot loading: Acetate-esterified cassava starch and tragali gum were weighed out according to the mass ratio and dissolved in water at a material-to-liquid ratio of 1:4. The mixture was stirred and reacted at 65°C for 30 minutes. After cooling to 45°C, honey was added and the mixture was stirred and reacted for 10 minutes to obtain a hydrogel. Royal jelly carbon nanodots were added to the hydrogel solution and incubated at room temperature for 30 minutes to obtain a hydrogel loaded with royal jelly carbon nanodots.

[0054] Example 2 This embodiment provides a hydrogel loaded with royal jelly carbon nanodots, wherein the raw materials of the hydrogel contain the following components in parts by weight: 7 parts honey, 15 parts acetic acid esterified tapioca starch, and 3 parts tragacanth gum.

[0055] The mass ratio of royal jelly carbon nanodots to the raw materials of the hydrogel (honey, acetic acid cassava starch and yarrow gum) is 1:50.

[0056] The preparation method of the above-mentioned hydrogel loaded with royal jelly carbon nanodots includes the following steps: Preparation of carbon nanodots: Royal jelly was dissolved in pure water at a mass ratio of 1:6 and stirred magnetically for 30 minutes at room temperature. Then, it was sonicated at 40 kHz and 200 W for 40 minutes at room temperature to ensure complete dissolution. The resulting solution was placed in a high-temperature reactor and reacted at 220℃ for 10 hours. After the reaction, the reaction solution was centrifuged at 10,000 rpm for 20 minutes at room temperature. The supernatant was filtered through a 0.22-micron filter membrane. The filtrate was placed in a regenerated cellulose dialysis membrane with a molecular weight cutoff of 1 kDa and dialyzed in pure water at room temperature for 48 hours until the dialysate was colorless. The liquid inside the dialysis membrane was collected and freeze-dried under vacuum for 12 hours to obtain powdered royal jelly carbon nanodots.

[0057] Hydrogel preparation and royal jelly carbon nanodot loading: Acetate-esterified cassava starch and tragali gum were weighed out according to the mass ratio and dissolved in water at a material-to-liquid ratio of 1:4. The mixture was stirred and reacted at 65°C for 30 minutes. After cooling to 45°C, honey was added and the mixture was stirred and reacted for 10 minutes to obtain a hydrogel. Royal jelly carbon nanodots were added to the hydrogel solution and incubated at room temperature for 30 minutes to obtain a hydrogel loaded with royal jelly carbon nanodots.

[0058] Example 3 This embodiment provides a hydrogel loaded with royal jelly carbon nanodots, wherein the raw materials of the hydrogel contain the following components in parts by weight: 4 parts honey, 10 parts acetate-esterified tapioca starch, and 2 parts tragacanth gum.

[0059] The mass ratio of royal jelly carbon nanodots to the raw materials of the hydrogel (honey, acetic acid transcribed cassava starch and yarrow gum) is 1:100.

[0060] The preparation method of the hydrogel loaded with royal jelly carbon nanodots is the same as that in Example 1.

[0061] Comparative Example 1 This comparative example provides a hydrogel loaded with royal jelly carbon nanodots, which differs from the hydrogel loaded with royal jelly carbon nanodots in Example 1 only in that the honey is removed.

[0062] The only difference between the above-mentioned method for preparing hydrogels loaded with royal jelly carbon nanodots and the method in Example 1 is that the honey addition step is omitted.

[0063] Comparative Example 2 This comparative example provides a hydrogel loaded with royal jelly carbon nanodots, which differs from the hydrogel loaded with royal jelly carbon nanodots in Example 1 only in that the raw materials of the hydrogel contain the following components in parts by weight: 2 parts honey, 10 parts acetate-esterified tapioca starch, and 2 parts yarrow gum.

[0064] The preparation method of the hydrogel loaded with royal jelly carbon nanodots is the same as that in Example 1.

[0065] Comparative Example 3 This comparative example provides a hydrogel loaded with royal jelly freeze-dried powder, which differs from the hydrogel loaded with royal jelly carbon nanodots in Example 1 only in that the royal jelly carbon nanodots are replaced with an equal mass of royal jelly freeze-dried powder.

[0066] The only difference between the above-mentioned method for preparing hydrogels loaded with royal jelly freeze-dried powder and the method in Example 1 is that the royal jelly carbon nanodot preparation step is removed and the royal jelly carbon nanodots are replaced with royal jelly freeze-dried powder.

[0067] Comparative Example 4 This comparative example provides a hydrogel loaded with royal jelly carbon nanodots, which differs from the hydrogel loaded with royal jelly carbon nanodots in Example 1 only in that the acetate-esterified tapioca starch is replaced with an equal amount of tapioca starch.

[0068] Comparative Example 5 This comparative example provides a hydrogel loaded with royal jelly carbon nanodots, which differs from the hydrogel loaded with royal jelly carbon nanodots in Example 1 only in that: yarrow gum is replaced with an equal amount of xanthan gum.

[0069] Example 1: Physicochemical characterization of hydrogels loaded with royal jelly carbon nanodots The physicochemical properties of the royal jelly carbon nanodots and the hydrogels loaded with royal jelly carbon nanodots prepared in the above embodiments and comparative examples were characterized, and the specific methods and results are described below.

[0070] 1.1 Experimental Methods The morphology of royal jelly carbon nanodots was detected using transmission electron microscopy (TEM, Hitachi HT7700). The storage modulus (G') and dissipation modulus (G'') of the hydrogel were characterized using a rheometer (MCR301, Austria) at dynamic strain frequencies of γ = 0.1–100% and dynamic scanning frequencies of 0.1–100 rad / s. The surface morphology of the hydrogel was detected using scanning electron microscopy (SEM, Hitachi S-4800).

[0071] 1.2 Experimental Results The test results of royal jelly carbon nanodots and hydrogels loaded with royal jelly carbon nanodots in Example 1 are as follows: Figure 1 , Figure 2 and Figure 3 As shown. TEM analysis revealed ( Figure 1 The royal jelly carbon nanoparticles exhibited well-dispersed spherical particles without obvious aggregates. Rheological analysis revealed that... Figure 2 At dynamic strain frequencies ranging from 0.1% to 100%, the storage modulus (G') of the gel was consistently higher than its dissipation modulus (G''), and this was further demonstrated by the consistently higher storage modulus (G') at dynamic scan frequencies gradually increasing from 0.1% to 100 rad / s. This indicates that the hydrogel possesses good mechanical strength. SEM analysis revealed... Figure 3 The hydrogel has an interconnected porous structure in its cross-section, which increases the specific surface area of ​​the hydrogel, thereby ensuring the stable release of royal jelly carbon nanodots and the absorption of tissue fluid.

[0072] Experiment Example 2: Determination of Gel Properties of Hydrogels Loaded with Royal Jelly Carbon Nanodots The gelation properties of the hydrogels containing royal jelly carbon nanodots prepared in the above embodiments and comparative examples were measured, and the specific methods and results are as follows.

[0073] 2.1 Experimental Methods The viscosity of the hydrogel was tested using a rheometer (MCR301, Austria); and the water retention of the hydrogel was evaluated by testing the water loss of the hydrogel through freeze-drying tests (lasting 4 hours, 8 hours, and 12 hours).

[0074] 2.2 Experimental Results Comparing the viscosity data of the hydrogels containing royal jelly carbon nanodots in Example 1 and Comparative Example 1, the results show that the hydrogel containing honey (Example 1) has significantly improved viscosity compared to the hydrogel without honey (Comparative Example 1). Figure 4 As shown), water retention is significantly improved (e.g. Figure 5 (As shown). However, when acetate-esterified tapioca starch was replaced with an equal amount of tapioca starch (i.e., Comparative Example 4), the viscosity of the hydrogel decreased by approximately 43%; when tragali gum was replaced with an equal amount of xanthan gum (i.e., Comparative Example 5), the water retention of the hydrogel decreased significantly by approximately 38%. Therefore, the hydrogel formulation of the present invention significantly improves the adhesion and water retention of the gel, thereby enhancing its adhesive and moisturizing properties when used as a skin dressing.

[0075] Experiment Example 3: Evaluation of the bioactivity of royal jelly carbon nanodots and hydrogels loaded with royal jelly carbon nanodots The bioactivity of the royal jelly carbon nanodots and the hydrogels loaded with royal jelly carbon nanodots prepared in the above embodiments and comparative examples was evaluated, with the royal jelly freeze-dried powder used in Comparative Example 3 as a control. The specific methods and results are as follows.

[0076] 3.1 Experimental Methods 3.1.1 Antibacterial test Staphylococcus aureus growing in the logarithmic phase (1×10⁻⁶) 7 Royal jelly carbon nanodots and hydrogels loaded with royal jelly carbon nanodots were added to solutions containing CFU / mL, with concentrations ranging from 2-50 μg / mL. The solutions were incubated at 37°C. Every 4 hours, 200 μL of the bacterial suspension was collected, and the OD values ​​were measured using a microplate reader. 600 Absorbance value, based on OD 600 The absorbance values ​​were used to plot the growth curve of Staphylococcus aureus, thereby analyzing its antibacterial ability.

[0077] 3.1.2 Cytotoxicity assay Mouse macrophages (RAW 264.7) were cultured in a 37°C CO2 incubator. Cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 μg / mL penicillin, and 100 μg / mL streptomycin. RAW264.7 cells were cultured at 10... 5 The cells were seeded at a density of 80% in 96-well plates. Different concentrations of royal jelly carbon nanodots and extracts of hydrogels containing royal jelly carbon nanodots (the extract obtained by soaking the hydrogel containing royal jelly carbon nanodots in water for 6 hours, i.e., dissolving the royal jelly carbon nanodots from the hydrogel) were added for pretreatment. After incubation for 24 h, 10 μL of CCK-8 reagent was added to each well, and after another 2 h of incubation, the OD value was measured at 450 nm using a microplate reader to determine the effect of royal jelly carbon nanodots and hydrogels containing carbon nanodots on the viability of RAW 264.7 cells.

[0078] 3.1.3 Anti-inflammatory capacity test Royal jelly carbon nanodots were added to RAW264.7 cell culture medium and incubated for 1 h. Then, the cells were stimulated with 20 μg / mL of inactivated Staphylococcus aureus for 24 h. The cell culture medium was collected, and the NO content in the cell culture medium was detected using a nitric oxide (NO) detection kit (Shanghai Beyotime Technology Co., Ltd., China).

[0079] 3.1.4 Antioxidant capacity test Royal jelly carbon nanodots were added to RAW264.7 cell culture medium and incubated for 1 h. Then, the cells were stimulated with 20 μg / mL of inactivated Staphylococcus aureus for 24 h. After removing the culture medium, 10 μM reactive oxygen species fluorescent probe (DCFH-DA) was added and incubated for 20 min. After washing with DMEM, the cells were collected and the cell fluorescence was detected by flow cytometry (BD FACSVerse, USA). The excitation and emission wavelengths were set to 488 nm and 525 nm, respectively. The results were analyzed using FlowJo software. The ratio of fluorescence intensity to total cell number is the relative ROS level.

[0080] 3.2 Experimental Results The growth curve of Staphylococcus aureus was monitored at 4-hour intervals, and the results showed that ( Figure 6 Under treatment with different concentrations of royal jelly carbon nanodots (prepared using the carbon nanodot preparation method in Example 1), the growth and reproduction of Staphylococcus aureus were significantly inhibited. Furthermore, as the concentration of carbon nanodots increased, the slope of the growth curve of Staphylococcus aureus gradually decreased, indicating that its growth and reproduction time was continuously delayed until it stopped growing. This demonstrates that royal jelly carbon nanodots exhibit good antibacterial ability.

[0081] The effect of royal jelly carbon nanodots (prepared using the carbon nanodot preparation method in Example 1) on the viability of RAW 264.7 cells was determined using the CCK-8 assay. The results showed that ( Figure 7 The carbon nanodots maintained over 85% viability in RAW 264.7 cells at concentrations ranging from 2-50 μg / mL, indicating good biocompatibility. Inactivation of Staphylococcus aureus induced an inflammatory response in RAW 264.7 cells, leading to increased NO and ROS levels. Figure 8 As shown, royal jelly carbon nanodots (prepared using the carbon nanodot preparation method in Example 1) significantly reduced NO release and alleviated cellular inflammation within a concentration range of 5-50 μg / mL. Royal jelly carbon nanodots achieved an inhibition rate of 68.65% on cellular NO release at a concentration of only 5 μg / mL, and an inhibition rate exceeding 90% at a concentration of 20 μg / mL. Furthermore, as... Figure 9 As shown, both 10 and 20 μg / mL of royal jelly carbon nanodots (prepared using the carbon nanodot preparation method in Example 1) significantly inhibited the generation of cellular ROS. This indicates that royal jelly carbon nanodots possess good biocompatibility, anti-inflammatory activity, and antioxidant capacity.

[0082] Staphylococcus aureus was treated with the hydrogel containing royal jelly carbon nanodots from Example 1, and the amount of royal jelly carbon nanodots (i.e., the mass ratio of carbon nanodots to the total mass of honey, acetic acid cassava starch, and tragali gum) was varied. The dynamic changes in the growth curve of Staphylococcus aureus were observed. Figure 10 When the amount of royal jelly carbon nanodots loaded was 1 mg / g, 5 mg / g, and 10 mg / g, the gels all achieved significant antibacterial effects. Furthermore, the effect of the hydrogel loaded with royal jelly carbon nanodots on the viability of RAW264.7 cells was investigated. Figure 11 The viability of RAW264.7 cells remained above 90%, indicating that the hydrogel carrying royal jelly carbon nanodots has good biocompatibility.

[0083] Furthermore, natural royal jelly freeze-dried powder showed no significant antibacterial, anti-inflammatory, or antioxidant properties at the same concentration (i.e., within the concentration range of 2-50 μg / mL). This indicates that compared with royal jelly freeze-dried powder, the antibacterial, anti-inflammatory, and antioxidant activities of royal jelly carbon nanodots were significantly enhanced.

[0084] Example 4: Evaluation of the effect of honey on the antibacterial activity of hydrogels The antibacterial activity of the hydrogels (without royal jelly carbon nanodots) in Examples 1 and 3 and Comparative Examples 1 and 2 was determined, and the specific methods and results are as follows.

[0085] 4.1 Experimental Methods Use a sterilized punch to make holes (6 mm in diameter) in a test plate coated with Staphylococcus aureus. Place a hydrogel sample matching the hole size into the hole and incubate for a period of time to determine the size of the inhibition zone.

[0086] 4.2 Experimental Results like Figure 12 As shown, compared with the hydrogels of Comparative Examples 1 and 2, the addition of the hydrogels of Examples 1 and 3 resulted in the formation of a clear antibacterial zone, indicating a significant improvement in their antibacterial properties.

[0087] Experiment Example 5: Hydrogels loaded with royal jelly carbon nanodots were used to treat bacterial wound infections in mice. Animal experiments were conducted on the hydrogels containing royal jelly carbon nanodots prepared in the above embodiments and comparative examples to evaluate their efficacy in promoting wound healing. The specific methods and results are described below.

[0088] 5.1 Experimental Methods Mouse experiments: Six-week-old female C57BL / 6 mice weighing 16-20 grams were selected and housed in an animal room meeting specific pathogen-free (SPF) standards, with a 12-hour alternating light and dark cycle. The ambient temperature was maintained at 20-24℃, and the relative humidity was controlled at 50±5%. Mice were provided with standard AIN-93 feed, sterile drinking water, and filtered pathogen-free air daily. To establish a mouse wound model of Staphylococcus aureus infection, a full-thickness circular wound with a diameter of 10 mm was made on the back of each mouse. Then, 10 µL of Staphylococcus aureus was evenly applied to the surface of the mouse wound. At predetermined time points (i.e., days 0, 1, 3, and 5), a hydrogel containing royal jelly carbon nanodots was fixed to the mouse back wound and left for 6 hours. The hydrogel was then removed, and the wound was covered with a sterile dressing. The wound condition was recorded using a camera on days 0, 4, 7, and 12, and the wound area was measured. At the end of the experiment (i.e., day 12), skin tissue samples from each group were collected for immunohistochemical analysis to detect the expression levels of pro-inflammatory cytokines IL-6, anti-inflammatory cytokines IL-10, α-smooth muscle actin (α-SMA), and platelet endothelial cell adhesion molecule-31 (CD31).

[0089] 5.2 Experimental Results A mouse model of Staphylococcus aureus-infected wounds was used to evaluate the wound-healing effect of a hydrogel loaded with royal jelly carbon nanodots. The experimental results of the hydrogel loaded with royal jelly carbon nanodots in Example 1 are as follows: The wound healing process in mice was recorded by photography, and the results showed ( Figure 13On day 7, the average wound area of ​​mice treated with the hydrogel containing royal jelly carbon nanodots was 19.9%, significantly lower than that of the control group (38.4%) without hydrogel treatment. Furthermore, immunohistochemical results showed that the expression level of the pro-inflammatory cytokine interleukin-6 (IL-6) in the hydrogel-treated group was significantly lower than that in the control group, while the expression level of the anti-inflammatory cytokine interleukin-10 (IL-10) was significantly higher, indicating that the hydrogel has a good anti-inflammatory effect. Moreover, the expression levels of platelet endothelial cell adhesion molecule-31 (CD31) and α-smooth muscle actin (α-SMA) in the hydrogel-treated group were significantly higher than those in the control group, indicating that the hydrogel can promote the proliferation of myofibroblasts and angiogenesis in mouse wound tissue, thereby promoting wound healing.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hydrogel loaded with carbon nanodots, characterized in that, The carbon nanodots are royal jelly carbon nanodots; the raw materials of the hydrogel contain the following components in parts by weight: 4-8 parts honey, 10-15 parts esterified cassava starch, and 1-3 parts yarrow gum.

2. The hydrogel carrying carbon nanodots according to claim 1, characterized in that, The esterified cassava starch is acetate-esterified cassava starch.

3. The hydrogel carrying carbon nanodots according to claim 1 or 2, characterized in that, The raw materials for the hydrogel also include water, and the amount of water used is in a mass ratio of (3-5):1 to the total mass of esterified cassava starch and yarrow gum.

4. The hydrogel carrying carbon nanodots according to any one of claims 1 to 3, characterized in that, The royal jelly carbon nanodots were synthesized using an ultrasound-assisted hydrothermal method. Preferably, the method for preparing the royal jelly carbon nanodots includes: mixing royal jelly with water and then subjecting it to ultrasonic treatment to obtain a royal jelly solution; and reacting the solution at 180-220°C.

5. The hydrogel with carbon nanodots according to claim 4, characterized in that, The reaction time is 6-15 hours; And / or, the frequency of the ultrasonic treatment is 35-45 kHz and the power is 180-220 W.

6. The hydrogel carrying carbon nanodots according to claim 4 or 5, characterized in that, After the reaction was completed, the supernatant was separated, purified and freeze-dried to obtain powdered royal jelly carbon nanodots.

7. The hydrogel carrying carbon nanodots according to any one of claims 1 to 6, characterized in that, The mass ratio of the royal jelly carbon nanodots to the total mass of honey, acetic acid cassava starch, and yarrow gum is 1:50-1:

100.

8. The method for preparing the hydrogel with carbon nanodots according to any one of claims 1 to 7, characterized in that, The method includes: mixing esterified cassava starch and yarrow gum in water to react, mixing the reaction product with honey, and then mixing it with royal jelly carbon nanodots.

9. The preparation method according to claim 8, characterized in that, The mixing reaction is carried out at 65-95°C for 15-30 minutes.

10. The use of the hydrogel with carbon nanodots according to any one of claims 1 to 7 in the preparation of products having one or more functions selected from antibacterial, anti-inflammatory, antioxidant, and wound healing promoting properties.

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