A preparation method of a selenium nanoparticle composite hydrogel based on quaternary ammonium chitosan stabilization

Abstract

This invention discloses a method for preparing and applying a quaternized chitosan-stabilized selenium nanoparticle composite hydrogel, comprising the following components: quaternized chitosan selenium nanoparticles, chitosan, collagen, and oxidized konjac glucomannan. The collagen solution has a concentration of 1% w / v, the chitosan solution has a concentration of 2% w / v, the oxidized konjac glucomannan solution has a concentration of 2.4% w / v, and the concentrations of quaternized chitosan selenium nanoparticles are 10, 20, and 50 μg / mL. The volume ratio of collagen, chitosan, and oxidized konjac glucomannan is 1:1:1. This antibacterial hydrogel combines chitosan, collagen, oxidized konjac glucomannan, and quaternized chitosan selenium nanoparticles, exhibiting excellent antibacterial properties. The hydrogel enables the slow and continuous release of selenium nanoparticles, prolonging the antibacterial action time and improving the antibacterial efficacy and duration. It also demonstrates excellent wound-healing capabilities and shows promising application prospects in the healing of open wounds.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a method for preparing and applying a quaternized chitosan-stabilized selenium nanoparticle composite hydrogel. Background Technology

[0002] The development trend of wound dressings is towards multifunctionality to facilitate convenient and long-term wound care. Compared with traditional antibiotic treatment, the antibacterial effect of selenium nanoparticles is not limited by bacterial resistance. Collagen (COL) and chitosan (CS) are both natural biomolecules and have been developed as ideal biomedical materials with excellent biocompatibility. Based on the theory of moist wound healing, a self-healing hydrogel was designed, composed of collagen (COL), chitosan (CS), and oxidized modified konjac glucomannan (OKGM). OKGM acts as a macromolecular crosslinking agent to construct dynamic Schiff base bonds. Quaternized chitosan selenium nanoparticles (QCS / SeNPs) were introduced as a functional material into the COL-CS-OKGM hydrogel matrix to develop a functional hydrogel with good antibacterial properties that can be widely used in open wounds. Summary of the Invention

[0003] To develop a functional hydrogel with good antibacterial properties that can be widely used in open wounds, the following technical solution was adopted:

[0004] The first aspect of the present invention provides an antibacterial hydrogel comprising the following components: collagen (COL), chitosan (CS) and oxidized modified konjac glucomannan (OKGM) copolymer, loaded with selenium nanoparticles (QCS / SeNPs) stabilized by quaternized chitosan as functional substances.

[0005] Furthermore, in the antibacterial hydrogel, the concentration of collagen is 1%, the concentration of chitosan is 2%, the concentration of oxidized konjac glucomannan is 2.4%, and the concentrations of quaternized chitosan selenium nanoparticles are 10, 20, and 50 micrograms / mL, respectively. The volume ratio of collagen, chitosan, and oxidized konjac glucomannan is 1:1:1.

[0006] A second aspect of the present invention provides a method for preparing any of the above-mentioned antibacterial hydrogels, comprising the following steps:

[0007] Preparation of S1 Oxidized Konjac Glucomannan: Konjac glucomannan was added to deionized water and stirred until completely dissolved. Then sodium periodate was added, and the mixture was stirred in the dark. Ethylene glycol was added to terminate the reaction, and the mixture was stirred again. The resulting solution was dialyzed in deionized water, and the resulting solution was freeze-dried to obtain oxidized konjac glucomannan.

[0008] Preparation of S2 antibacterial hydrogel: Collagen solution and chitosan solution were stirred at low temperature, and oxidized konjac glucomannan solution was added dropwise. Then, a suspension of quaternized chitosan selenium nanoparticles was added, and the pH of the solution was adjusted with sodium hydroxide to obtain a collagen-chitosan-oxidized konjac glucomannan-quaternized chitosan selenium nanoparticle composite hydrogel.

[0009] Furthermore, step S1 specifically includes:

[0010] S11: Add 5 g of konjac glucomannan to 500 ml of deionized water; add 1.32 g of sodium periodate and stir in the dark at 30°C for 12 h; add 10 mL of ethylene glycol to terminate the reaction and stir for another 2 h; the dialysis time should not be less than 72 h.

[0011] Furthermore, step S2 specifically includes:

[0012] S21: Collagen concentration is 1%, chitosan concentration is 2%, oxidized konjac glucomannan concentration is 2.4%, quaternized chitosan selenium nanoparticle concentration is 10, 20, and 50 μg / mL, and the volume ratio of collagen, chitosan, and oxidized konjac glucomannan is 1:1:1.

[0013] S22: Stir and mix at a low temperature maintained by an ice bath, with the temperature being 0°C, for a stirring time and rate of 500 r / min for 10 mins.

[0014] S23: Adjust pH value: Adjust the pH value of the solution to approximately 6-7 using 1 mol / L sodium hydroxide.

[0015] The beneficial effects of this invention are:

[0016] 1. When the amount of quaternized chitosan selenium nanoparticles added is 20 micrograms / ml, the hydrogel exhibits good antioxidant and antibacterial properties.

[0017] 2. The composite hydrogel has a good hemostatic effect, and its healing efficiency for open wounds reaches 91.87% in 14 days, which far exceeds the healing efficiency of currently commercially available gels.

[0018] 3. The preparation process is simple, the raw materials are readily available, and it is easy to achieve industrial production, providing a new and effective treatment method for clinical use, with significant social benefits. Attached Figure Description

[0019] Figure 1 Morphology and particle size characterization of quaternized chitosan selenium nanoparticles

[0020] Figure 2Morphological characterization of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel

[0021] Figure 3 Antioxidant properties of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel

[0022] Figure 4 Antibacterial properties of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel

[0023] Figure 5 Healing properties of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0025] (I) Implementation Examples

[0026] Preparation method of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticles: 5 ml of collagen (Col) solution (1%, w / v) and 5 ml of chitosan (CS) solution (2%, w / v) were mixed at 500 r / min for 10 min under low temperature maintained in an ice bath. A certain amount of OKGM solution (2.4%, w / v) was added dropwise while stirring. Next, a certain amount of concentrated QCS / SeNPs-2 suspension was added to achieve selenium nanoparticle concentrations of 10, 20, and 50 μg / mL in the final hydrogel. Subsequently, the pH of the solution was adjusted to approximately 6-7 with 1 mol / L sodium hydroxide to obtain a collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel.

[0027] (II) Experimental Examples

[0028] 2.1 Test Materials

[0029] 2.1.1 Reagents and Animal Models

[0030] 1) The antibacterial hydrogel of the present invention is prepared according to the above preparation method.

[0031] 2) Relevant reagents: Commercial hydrogel rhEGF Gel, DPPH, ABTS

[0032] 3) Animal model: Female KM mice weighing 20–30 g

[0033] 2.2 Experimental Methods and Results

[0034] 2.2.1 Morphology and particle size characterization of quaternized chitosan selenium nanoparticles

[0035] Experimental methods:

[0036] Microscopic images of quaternized chitosan selenium nanoparticles were obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM); particle size and potential were measured by a ZS90 Malvern nanoparticle size analyzer.

[0037] Experimental results:

[0038] Both atomic force microscopy and transmission electron microscopy revealed that the quaternized chitosan selenium nanoparticles exhibited a spherical shape with a stable diameter around 200 nm, reaching the nanoscale, and possessed a larger specific surface area, better loading capacity, and epidermal permeability. The particle size measurements were consistent with the microscopic results. Potential measurements demonstrated the stability of the quaternized chitosan selenium nanoparticles. The selenium nanoparticles showed a positive potential of approximately 25 mV, indicating that the selenium nanoparticles prepared using modified chitosan exhibited better stability and were more conducive to the long-term stability of their biological activity.

[0039] 2.2.2 Morphological characterization of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel

[0040] Experimental methods:

[0041] Microscopic images of the composite hydrogel were obtained using atomic force microscopy (AFM).

[0042] Experimental results:

[0043] As can be seen, collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticles form a dense network cross-linked structure, and the height map shows that the gel surface has a porous structure. The height morphology map (where the horizontal axis represents the scanning range in μm and the vertical axis represents the surface height in nm) reveals the microscopic three-dimensional morphology of the hydrogel surface. The curve fluctuates dramatically within ±3 nm, indicating that the hydrogel surface is not ideally smooth, exhibiting nanoscale undulations and textures, a typical characteristic of polymer hydrogels. Two prominent sharp peaks are observed at approximately 1.2 μm and 2.9 μm, possibly representing areas of aggregation micro-regions, particles, or cross-linking points on the hydrogel surface; while deep valleys correspond to softer areas with higher swelling degrees or porous structures. The nanoscale undulations directly reflect the non-uniformity of cross-linking density within the hydrogel—areas with high cross-linking density are "harder," appearing as protrusions in AFM scanning; areas with low cross-linking density and sufficient swelling are "softer," appearing as depressions. Significant roughness may affect biocompatibility characteristics such as cell adhesion and protein adsorption. The scale of peaks and valleys (nm level) is also closely related to the mechanical properties and swelling behavior of hydrogels.

[0044] 2.2.3 Antioxidant properties of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel

[0045] Experimental methods:

[0046] (1) Determination of DPPH free radical scavenging

[0047] Composite hydrogels prepared with different amounts of selenium nanoparticles were mixed with 2 mL of DPPH-ethanol (0.2 mmol / L). After reacting in the dark for 30 minutes, the absorbance at 517 nm was measured using a UV spectrophotometer. Each group was tested in triplicate. The scavenging activity of DPPH free radicals was calculated as follows:

[0048] DPPH free radical scavenging capacity (%) = [1 - (A1 - A2) / A0] × 100%

[0049] Where A0 is the absorbance after the reaction of water with DPPH, A1 is the absorbance after the reaction of the composite hydrogel with DPPH, and A2 is the absorbance after the reaction of water with the composite hydrogel.

[0050] (2) Determination of ABTS free radical scavenging

[0051] An ABTS radical solution was prepared by mixing equal volumes of 7 mM ABTS solution and 2.45 mM potassium persulfate solution and incubating in the dark for 12–16 h. The ABTS radical solution was diluted with 10 mM PBS to an absorbance of 0.7 ± 0.02 at 734 nm. 400 μL of a composite hydrogel with different amounts of selenium nanoparticles was mixed with 400 μL of the ABTS radical solution and reacted in the dark for 6 mins. The absorbance was measured at 734 nm using a UV-Vis spectrophotometer. A mixture of 400 μL anhydrous ethanol and 400 μL of ABTS solution served as a control group. The ABTS scavenging rate was calculated using the following equation.

[0052] ABTS radical scavenging rate (%) = (As – Ac) / As × 100%

[0053] Where As is the absorbance value of the control group and Ac is the absorbance value of the experimental group.

[0054] Experimental results:

[0055] In the ABTS⁺ experiment, there was no significant difference in free radical scavenging rates among the three groups of samples with different amounts of SeNPs, all of which were significantly higher than the group without SeNPs and the control group. In the DPPH experiment, there were significant differences in DPPH free radical scavenging rates among the three groups of samples with different amounts of SeNPs. The CCOSe20 group showed the highest DPPH scavenging rate (approximately 98%), significantly better than CCOSe50 (approximately 96%) and CCOSe10 (approximately 90%), and significantly higher than the group without SeNPs and the control group. These results indicate that the addition of SeNPs can significantly enhance the antioxidant activity of the gel material. In conclusion, the addition of SeNPs can significantly enhance the antioxidant activity of the gel material alone, with CCOSe20 showing the best performance in the DPPH system. All groups with added SeNPs exhibited stable and efficient scavenging activity in the ABTS⁺ system.

[0056] 2.2.4 Antibacterial properties of collagen-chitosan-oxidized konjac glucomannan-selenium nanoparticle composite hydrogel

[0057] Gram-positive bacteria (Staphylococcus aureus ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 27195) were selected to test the antibacterial activity of the composite hydrogel. Briefly, Muller-Hinton broth (MHB) was incubated at 37°C until an absorbance value of 0.5 was obtained, corresponding to 1 × 10⁻⁶. 8 CFU / mL. Dilute the inoculum in MHB medium to approximately 1 × 10⁻⁶ CFU / mL. 4The final concentration of CFU / mL was determined. The lyophilized composite hydrogel was dissolved in deionized water to a concentration of 2 mg / mL and sterilized by UV irradiation for 30 mins. Different concentrations of samples (2–2000 μg / mL) were analyzed by serial 2-fold dilutions in 96-well plates. The absorbance at 260 nm and 280 nm was determined by plating the plates onto plate counting agar after incubation at 37°C (24 h).

[0058] Experimental results:

[0059] The addition of selenium nanoparticles significantly improved the antibacterial properties of the hydrogel material. In the control group, both E. coli and S. aureus grew in large numbers, but after treatment with the hydrogel material, the colony counts of E. coli and S. aureus decreased significantly, while the antibacterial efficiency of the hydrogel with added selenium nanoparticles exceeded 99%.

[0060] The antibacterial mechanism of the hydrogel was detected by absorbance measurement at 260nm / 280nm. As shown in the figure, the leakage of bacterial nucleic acid and protein increased significantly after treatment with CCOSe material, and was negatively correlated with the colony count, indicating that cell membrane disruption is its core antibacterial mechanism.

[0061] 2.2.5 Healing Properties of Collagen-Chitosan-Oxidized Konjac Glucomannan-Selenium Nanoparticle Composite Hydrogel

[0062] Female KM mice weighing 20–30 g were randomly divided into a control group and groups with different amounts of selenium nanoparticles, with 5 mice in each group (n = 5). After anesthesia, the mice's dorsal hair was removed, and a wound model was created using a 1.5 cm diameter punch. The wounds were then covered with composite hydrogels loaded with different amounts of selenium nanoparticles. The control group received no treatment, while the control group's wounds were cleaned with PBS. The wounds were photographed at 0, 3, 7, 14, and 21 days. Mice were sacrificed on day 21, and the burn area was calculated and statistically analyzed.

[0063] Experimental results:

[0064] As shown in the figure, CCOSe50 was compared with commercially available rhEGF gel to evaluate its effectiveness in actual wound healing. It can be seen that both the rhEGF gel group and the CCOSe50 group significantly accelerated wound healing in mice compared to the control group (p<0.05). The CCOSe50 group showed a better healing effect, with the wound area shrinking to 68.47% by day 3, significantly better than the rhEGF gel group (72.27%). By day 14, the wound area was only 8.13%, indicating near-healing, while the wound area in the rhEGF gel group remained at 15.63%, and the unhealed wound area in the control group reached 38.67%. These results indicate that the CCOSe50 hydrogel, rich in SeNPs, can significantly accelerate wound contraction and epidermal repair, improving wound healing efficiency, and its healing efficiency is superior to that of clinically used rhEGF gel.

Claims

1. A method for preparing selenium nanoparticle composite hydrogels based on quaternized chitosan-stabilized nanoparticles, characterized in that, The process includes the following steps: mixing collagen solution and chitosan solution under low temperature maintained by an ice bath, adding oxidized konjac glucomannan dropwise, and then adding a suspension of quaternized chitosan-based selenium nanoparticles to obtain a collagen-chitosan-oxidized konjac glucomannan-quaternized chitosan selenium nanoparticle composite hydrogel.

2. The method according to claim 1, characterized in that, The collagen solution has a concentration of 1% w / v, and the chitosan solution has a concentration of 2% w / v.

3. The method according to claim 1, characterized in that, The mixture was stirred and mixed at a low temperature maintained in an ice bath (0°C) for 10 min at a stirring speed of 500 r / min.

4. The method according to claim 1, characterized in that, Oxidized konjac glucomannan was obtained by modifying konjac glucomannan. The specific preparation method was as follows: 5 g of konjac glucomannan was added to 500 ml of deionized water and stirred until completely dissolved. Then, 1.32 g of sodium periodate was added, and the mixture was stirred in the dark at 30°C for 12 h. The reaction was terminated by adding 10 mL of ethylene glycol, and the mixture was stirred for another 2 h. The resulting solution was dialyzed against deionized water for 72 h. After centrifugation (3000 rpm, 20 min), the supernatant was collected, and the resulting solution was freeze-dried to obtain oxidized konjac glucomannan.

5. The method according to claim 1, characterized in that, The quaternized chitosan-based selenium nanoparticles were prepared as follows: 0.032 g of quaternized chitosan was dissolved in 10 mL of 4% acetic acid solution, followed by the addition of 10 mL of 0.1 M sodium selenite and 10 mL of 0.1 M vitamin C. The reaction system was stirred at room temperature for 30 minutes. After centrifugation at 10,000 rpm for 10 minutes at room temperature, the supernatant was removed, and the remaining red precipitate was the quaternized chitosan selenium nanoparticles.

6. The method according to claim 1, characterized in that, The concentrations of quaternized chitosan-based selenium nanoparticles added were 10, 20, and 50 μg / mL, respectively.

7. The method according to claim 1, characterized in that, The pH of the solution was adjusted to approximately 6-7 using 1 mol / L sodium hydroxide to obtain a collagen-chitosan-oxidized konjac glucomannan-quaternized chitosan selenium nanoparticle composite hydrogel.

8. The method according to claim 1, characterized in that, The volume ratio of the original protein, chitosan, and oxidized konjac glucomannan is 1:1:

1.

9. The use of the composite hydrogel according to any one of claims 1 to 8 in the preparation of drugs for antibacterial diseases.

10. The application according to claim 9, characterized in that, The antimicrobial-related disease is open wound healing.

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