A dual-functional hydrogel integrated with silver ions and hair extraction micro-particles, and a preparation method and application thereof

Abstract

The application discloses a kind of silver ion and hair extraction micro-particle integrated dual-function hydrogel and its preparation method and application, the hydrogel includes temperature-sensitive gel matrix, hair extraction micro-particle (HMP), silver ion antibacterial component;The hair extraction micro-particle (HMP) and silver ion antibacterial component are jointly loaded in temperature-sensitive hydrogel matrix;Wherein, the hair extraction micro-particle is spindle-shaped hair-derived micro-particle.The hydrogel of the application can reduce potential toxicity to normal cells while ensuring antibacterial effect, significantly improve the overall biological safety of dressing.In addition, HMP itself has inherent cell proliferation activity, can directly stimulate fibroblast, keratinocyte and endothelial cell proliferation, accelerate granulation tissue formation, re-epithelialization and angiogenesis, promote pressure ulcer healing from multiple targets.The hydrogel of the application is a body temperature responsive hydrogel, which is liquid at room temperature and becomes gel state after contacting skin temperature, convenient to carry and use.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a bifunctional hydrogel integrating silver ions and hair extraction microparticles, its preparation method, and its application. Background Technology

[0002] Pressure ulcers, also known as pressure injuries, are localized damage to the skin or subcutaneous tissue caused by prolonged or intense pressure and shear forces. Their pathological process is complex, involving persistent ischemia and hypoxia, reperfusion injury, bacterial colonization and infection, and excessive inflammatory responses. Among these, infection and the oxidative stress microenvironment are the core obstacles leading to persistent pressure ulcer healing. Activated neutrophils and macrophages within the wound produce large amounts of reactive oxygen species (ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. Under physiological conditions, ROS participate in signal transduction and host defense; however, in chronic pressure ulcers, the excess of ROS far exceeds the clearance capacity of the endogenous antioxidant system. This persistent oxidative stress directly damages lipids, proteins, and DNA, leading to apoptosis or necrosis; prolongs inflammation by activating pathways such as NF-κB to release large amounts of pro-inflammatory factors; inhibits fibroblast proliferation, migration, and collagen synthesis; and impairs endothelial cell function, hindering angiogenesis. Therefore, actively intervening in the oxidative stress microenvironment of pressure ulcers is one of the key strategies to break healing stagnation. On the other hand, silver-containing dressings are a first-line option for controlling wound infection and have been widely used in clinical practice. Silver ions exert a broad-spectrum antibacterial effect by disrupting bacterial cell membranes and interfering with DNA replication and enzyme activity. However, numerous clinical and basic studies have pointed out its inherent limitations: 1) Risk of cell function inhibition: At effective antibacterial concentrations, silver ions may be toxic to fibroblasts and keratinocytes, inhibiting their proliferation and migration; 2) Lack of microenvironment regulation: Existing dressings mainly play a passive antibacterial role and lack the ability to actively regulate excessive inflammation and reactive oxygen species (ROS) accumulation in wounds. Summary of the Invention

[0003] The present invention aims to provide a bifunctional hydrogel integrating silver ions and hair extraction microparticles, which has broad-spectrum and highly efficient ability to scavenge reactive oxygen species, inherent cell-promoting activity, and antibacterial properties; another objective of the present invention is to provide a method for preparing a bifunctional hydrogel integrating silver ions and hair extraction microparticles; yet another objective of the present invention is to provide an application of a bifunctional hydrogel integrating silver ions and hair extraction microparticles.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention provides a bifunctional hydrogel integrating silver ions and hair extract microparticles. The hydrogel includes a temperature-sensitive gel matrix, hair extract microparticles (HMP), and silver ion antibacterial components. The HMP hair extract microparticles and silver ion antibacterial components are co-loaded in the temperature-sensitive hydrogel matrix.

[0006] The hair extract microparticles are spindle-shaped hair-derived microparticles.

[0007] Preferably, the concentration of the temperature-sensitive gel matrix is ​​15-25% (w / v).

[0008] Preferably, the concentration of the hair-derived particles is 100-1000 μg / mL.

[0009] Preferably, the hair-derived particles have a particle size of 1-5 μm.

[0010] Preferably, the silver ion antibacterial component is at least one of silver nitrate, silver acetate, and silver citrate.

[0011] Preferably, the concentration of silver ions is 0.1-1.0 μg / mL.

[0012] This invention also provides a method for preparing a bifunctional hydrogel integrating silver ions and hair extraction microparticles, comprising the following steps:

[0013] (1) Dissolve the thermosensitive gel matrix in phosphate buffer solution and stir to dissolve to obtain matrix solution;

[0014] (2) The washed human hair was hydrolyzed in an alkaline solution, then centrifuged, washed and freeze-dried to obtain hair-derived microparticles; the hair-derived particles were dispersed in a portion of the matrix solution and sonicated to obtain a dispersion.

[0015] (3) Mix the silver nitrate solution with the dispersion, then adjust the volume of the remaining matrix solution to the target concentration, and stir evenly at 4°C to obtain the composite hydrogel precursor solution.

[0016] Preferably, in step (1), the temperature-sensitive gel matrix is ​​dissolved in phosphate buffer at 4-10℃.

[0017] Preferably, in step (2), the specific preparation process of the hair-derived particles is as follows: human hair is washed, degreased, and dried, and then hydrolyzed in a 1-2 M alkaline solution at 50-80 °C for 1-4 hours; after the reaction is completed, the mixture is centrifuged, washed, precipitated, and then freeze-dried.

[0018] This invention also provides the application of a bifunctional hydrogel integrating silver ions and hair extraction microparticles in the preparation of drugs or medical devices for pressure ulcers, wherein the pressure ulcers are bacterial infectious or oxidative stress pressure ulcers.

[0019] The design principle of this invention lies in utilizing human hair-derived microparticles (HMPs) as a functional carrier. On one hand, HMPs, by retaining natural melanin, possess a broad-spectrum and highly efficient ability to scavenge reactive oxygen species (ROS), neutralizing excess superoxide anions and hydroxyl radicals in pressure ulcer wounds, thus reducing oxidative stress damage to fibroblasts and endothelial cells. Simultaneously, their unique spindle-shaped structure (approximately 1 μm in length) makes them difficult to penetrate the skin barrier, ensuring biocompatibility while exerting their efficacy. On the other hand, HMPs indirectly antagonize silver ion-induced oxidative stress toxicity by scavenging ROS, broadening the therapeutic window of silver ions. Furthermore, HMPs themselves possess cell proliferation-promoting activity, directly stimulating the proliferation of repair cells and accelerating granulation tissue formation and vascularization.

[0020] Beneficial effects: Compared with the prior art, the present invention has significant advantages:

[0021] I. The hydrogel of this invention provides long-lasting and broad-spectrum antibacterial protection with silver ions, effectively killing common pathogens in pressure ulcer wounds (such as MRSA). HMP, by retaining natural melanin, can efficiently remove excess ROS from the wound, including superoxide anions, hydrogen peroxide, and hydroxyl radicals, directly reducing oxidative stress damage to host cells and creating a favorable microenvironment for tissue repair. The synergistic effect of these two components overcomes the limitations of traditional silver-containing dressings that focus on antibacterial activity while neglecting repair. HMP indirectly reduces silver ion-induced oxidative stress damage by clearing ROS, thereby broadening the therapeutic window of silver ions. While ensuring antibacterial efficacy, it reduces potential toxicity to normal cells, significantly improving the overall biocompatibility of the dressing. Furthermore, HMP itself has inherent cell proliferation-promoting activity, directly stimulating the proliferation of fibroblasts, keratinocytes, and endothelial cells, accelerating granulation tissue formation, re-epithelialization, and angiogenesis, promoting pressure ulcer healing from multiple targets. Based on the properties of the temperature-sensitive gel matrix, the hydrogel precursor solution of this invention can flow at low temperatures, making it easy to apply or inject onto irregular pressure ulcers; upon contact with body temperature, it rapidly gels in situ, closely adhering to the wound surface, forming a moist healing environment, and enabling the continuous release of active ingredients.

[0022] II. The preparation method of this invention is simple to operate, requiring only steps such as dissolution, hydrolysis, ultrasonic mixing, and volume adjustment. No complex equipment is needed, making it suitable for large-scale production. All operations are carried out at low temperatures (4~10℃) or with gentle heating (50~80℃), avoiding damage to the active ingredients from high temperatures, high pressures, or organic solvents, thus ensuring the natural structure of HMP and the stability of silver ions. Furthermore, by adjusting parameters such as hydrolysis time, ultrasonic power, and component concentration, the particle size, dispersibility, and silver ion loading of HMP can be precisely controlled, thereby achieving flexible adjustment of product performance. In addition, the raw materials used in the hydrogel of this invention (human hair, F127, and silver salts) are widely available and cost-effective, and the preparation process is easy to scale up, showing good prospects for clinical application.

[0023] Third, the hydrogel of this invention can be used to prepare drugs or medical devices for treating bacterial or oxidative stress-induced pressure ulcers. It can simultaneously address the two core issues of pressure ulcer healing: bacterial infection and oxidative stress, filling the gap in the single-function dressings of existing treatments.

[0024] In vitro experiments have demonstrated that the hydrogel of this invention can completely kill high concentrations of MRSA (>10) within 1 hour at extremely low silver ion concentrations (0.4 μg / mL). 6 (CFU / mL); Antioxidant experiments confirmed that its scavenging rates of ABTS, superoxide anion, hydrogen peroxide, and hydroxyl radicals were significantly superior to the control; Cell scratch assays showed that HMP / Ag@Gel hydrogel could effectively antagonize the inhibitory effect of oxidative stress on cell migration and promote wound repair; Animal experiments showed that in an MRSA infectious pressure ulcer model, the wound healing speed of the hydrogel treatment group was significantly faster than that of the control group, with higher granulation tissue formation and vascularization. The HMP provided by this invention can be personalized for different groups, extracting HMP from individual hair, achieving individual-specific use and avoiding the side effects of immune rejection. Attached Figure Description

[0025] Figure 1 This is a scanning electron microscope (SEM) image of hair-derived microparticles.

[0026] Figure 2 Comparison of the gelation state of F127 hydrogels of different concentrations at 4℃ and 37℃;

[0027] Figure 3 The scavenging ability of the hydrogel prepared in Example 1 for different reactive oxygen species (ROS);

[0028] Figure 4 For different Ag + The bactericidal effect of high-concentration hydrogels on MRSA;

[0029] Figure 5The effect of HMP / Ag@Gel hydrogel on scratch healing of HACAT cells under oxidative stress;

[0030] Figure 6 Representative photographs of wound healing in rats of each group (days 0, 2, 4, 6, and 8). Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] Weigh 2.0 g of Pluronic F127 powder, add 8 mL of pre-cooled PBS buffer (pH=7.4), and stir magnetically overnight at 4°C to obtain a 20% (w / v) clear F127 stock solution.

[0034] Take 2 grams of healthy human hair, wash, degrease, and dry it, then place it in a round-bottom flask containing 50 mL of 1.5 M NaOH solution. Stir and react in a 50°C water bath for 2 hours. After the reaction is complete, centrifuge the mixture at 8000 rpm for 10 minutes and discard the supernatant. Collect the precipitate, wash repeatedly with ultrapure water until neutral, and freeze-dry to obtain the desired product. Figure 1 As shown, the microparticles are spindle-shaped, hair-derived particles, approximately 1 μm long and 200±50 nm wide.

[0035] Weigh 1.0 mg of the prepared hair-derived microparticles, add 1.0 mL of the above F127 stock solution, and sonicate in an ice-water bath for 5 minutes. The sonication power is 300 W, the working time is 2 s, and the interval time is 3 s to obtain a uniformly dispersed hair-derived microparticle suspension.

[0036] Take 10 μL of AgNO3 standard solution with a concentration of 40 μg / mL, using ultrapure water as the solvent, and add it to 1.0 mL of hair-derived microparticle suspension. Gently shake to mix.

[0037] Add 1.0 mL of F127 stock solution to the above hair-derived microparticle suspension and gently stir at 4°C for 30 minutes to obtain a homogeneous composite hydrogel precursor solution. In this solution, the final concentration of F127 is 20%, the final concentration of HMP is 500 μg / mL, and the Ag... +The final concentration was 0.4 μg / mL. The solution transformed into a non-flowing, transparent hydrogel within 3 seconds at human body surface temperature (31-34 °C).

[0038] Example 2

[0039] Weigh 2.0 g of Pluronic F127 powder, add 8 mL of pre-cooled PBS buffer (pH=7.4), and stir magnetically overnight at 4°C to obtain a 15% (w / v) clear F127 stock solution.

[0040] Take 2 grams of healthy human hair, wash, degrease, and dry it, then place it in a round-bottom flask containing 50 mL of 1.5 M NaOH solution. Stir and react at a constant temperature of 50°C for 2 hours. After the reaction is complete, centrifuge the mixture at 8000 rpm for 10 minutes and discard the supernatant. Collect the precipitate, wash repeatedly with ultrapure water until neutral, and freeze-dry to obtain hair-derived microparticles.

[0041] Weigh 1.0 mg of the prepared hair-derived microparticles, add 1.0 mL of the above F127 stock solution, and sonicate in an ice-water bath for 5 minutes. The sonication power is 300 W, the working time is 2 s, and the interval time is 3 s to obtain a uniformly dispersed hair-derived microparticle suspension.

[0042] Take 10 μL of AgNO3 standard solution with a concentration of 40 μg / mL, using ultrapure water as the solvent, and add it to 1.0 mL of hair-derived microparticle suspension. Gently shake to mix.

[0043] Add 1.0 mL of F127 stock solution to the above hair-derived microparticle suspension and gently stir at 4°C for 30 minutes to obtain a homogeneous composite hydrogel precursor solution. In this solution, the final concentration of F127 is 15%, the final concentration of HMP is 100 μg / mL, and the Ag... + The final concentration was 0.1 μg / mL. The solution transformed into a non-flowing, transparent hydrogel within 3 seconds at human body surface temperature (31-34 °C).

[0044] Example 3

[0045] Weigh 2.0 g of Pluronic F127 powder, add 8 mL of pre-cooled PBS buffer (pH=7.4), and stir magnetically overnight at 4°C to obtain a 25% (w / v) clear F127 stock solution.

[0046] Take 2 grams of healthy human hair, wash, degrease, and dry it, then place it in a round-bottom flask containing 50 mL of 1.5 M NaOH solution. Stir and react at a constant temperature of 50°C for 2 hours. After the reaction is complete, centrifuge the mixture at 8000 rpm for 10 minutes and discard the supernatant. Collect the precipitate, wash repeatedly with ultrapure water until neutral, and freeze-dry to obtain hair-derived microparticles.

[0047] Weigh 1.0 mg of the prepared hair-derived microparticles, add 1.0 mL of the above F127 stock solution, and sonicate in an ice-water bath for 5 minutes. The sonication power is 300 W, the working time is 2 s, and the interval time is 3 s to obtain a uniformly dispersed hair-derived microparticle suspension.

[0048] Take 10 μL of AgNO3 standard solution with a concentration of 40 μg / mL, using ultrapure water as the solvent, and add it to 1.0 mL of hair-derived microparticle suspension. Gently shake to mix.

[0049] Add 1.0 mL of F127 stock solution to the above hair-derived microparticle suspension and gently stir at 4°C for 30 minutes to obtain a homogeneous composite hydrogel precursor solution. In this solution, the final concentration of F127 is 25%, the final concentration of HMP is 1000 μg / mL, and the Ag... + The final concentration was 1.0 μg / mL. The solution transformed into a non-flowing, transparent hydrogel within 3 seconds at human body surface temperature (31-34 °C).

[0050] Comparative Example 1

[0051] Preparation of pure F127 hydrogel (20%, w / v)

[0052] Weigh 2.0 g of Pluronic F127 powder and add 8 mL of pre-cooled phosphate buffer (PBS, pH=7.4). Stir magnetically overnight at 4°C to obtain a 20% (w / v) clear F127 stock solution. Take 2.0 mL of the above F127 stock solution and gently stir at 4°C for 30 minutes to obtain a pure F127 hydrogel precursor solution.

[0053] Comparative Example 2

[0054] Preparation of low-concentration F127 hydrogel (10%, w / v)

[0055] Weigh 1.0 g of Pluronic F127 powder and add 9 mL of pre-cooled phosphate buffer (PBS, pH=7.4). Stir magnetically overnight at 4°C to obtain a 10% (w / v) clear F127 stock solution. Take 2.0 mL of this stock solution and gently stir at 4°C for 30 minutes to obtain a low-concentration F127 hydrogel precursor solution.

[0056] Comparative Example 3

[0057] Preparation of high-concentration F127 hydrogel (30%, w / v)

[0058] Weigh 3.0 g of Pluronic F127 powder and add 7 mL of pre-cooled phosphate buffer (PBS, pH=7.4). Stir magnetically overnight at 4°C to obtain a 30% (w / v) clear F127 stock solution. Take 2.0 mL of this stock solution and gently stir at 4°C for 30 minutes to obtain a high-concentration F127 hydrogel precursor solution.

[0059] Comparative Example 4

[0060] Low concentration of Ag + Preparation of hydrogels.

[0061] The preparation steps were the same as in Example 1, except that AgNO3 with a concentration of 5 μg / mL was used, while all other conditions remained unchanged. This yielded the HMP / Ag@Gel hydrogel precursor solution. The Ag in this solution... + The final concentration was 0.05 μg / mL.

[0062] Comparative Example 5

[0063] High concentration of Ag + Preparation of hydrogels.

[0064] The preparation steps were the same as in Example 1, except that AgNO3 with a concentration of 200 μg / mL was used, while all other conditions remained unchanged. This yielded the HMP / Ag@Gel hydrogel precursor solution. The Ag in this solution... + The final concentration was 2.0 μg / mL.

[0065] Comparative Example 6

[0066] Preparation of high-concentration hair-derived microparticle hydrogels.

[0067] The preparation steps were the same as in Example 1, except that 3.0 mg of hair-derived microparticles (HMP) were used, while all other conditions remained unchanged. This yielded an HMP / Ag@Gel hydrogel precursor solution. The final HMP concentration in this solution was 1500 μg / mL. The precursor solution was placed in a 37°C incubator and observed to transform into a non-flowing, transparent hydrogel within 3 seconds, consistent with the gelation behavior in Example 1. However, after standing for 24 hours, a small amount of particle precipitation was visible on the gel surface, indicating a slight decrease in the dispersion stability of high-concentration HMP.

[0068] Experimental Example 2

[0069] To verify the temperature-sensitive properties of the hydrogel precursor solution of the present invention and to demonstrate the critical significance of the F127 concentration range, the hydrogel precursor solutions prepared in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were placed at 4°C (storage temperature) and 37°C (simulated human body temperature) to observe the changes in their physical state.

[0070] like Figure 2 As shown, at 4°C (storage state), the hydrogel precursor solution prepared in Example 1 was clear and transparent, and flowed freely; the hydrogel precursor solution prepared in Comparative Example 1 was clear and transparent, and flowed freely; the hydrogel precursor solution prepared in Comparative Example 2 was clear and transparent, with excellent flowability; and the hydrogel precursor solution prepared in Comparative Example 3 was extremely viscous and had poor flowability. At 37°C (use state), the hydrogel precursor solution prepared in Example 1 completely transformed into a non-flowing solid gel; the hydrogel precursor solution prepared in Comparative Example 1 completely transformed into a non-flowing solid gel; the hydrogel precursor solution prepared in Comparative Example 2 remained in a flowing state and could not form a stable gel; the hydrogel precursor solution prepared in Comparative Example 3 formed a gel, but due to its excessive viscosity at low temperature, its clinical operability was poor.

[0071] Therefore, it can be seen that, compared with Comparative Example 1, the gelation behavior of Example 1 and Comparative Example 1 is completely identical at 4℃ and 37℃, indicating that the addition of HMP and Ag... + The temperature-sensitive gelling properties of F127 are not affected; Comparative Example 2 cannot gel at 37°C, indicating that F127 concentration below 15% cannot be used as a molding dressing; Comparative Example 3 can gel, but the viscosity is too high at 4°C, resulting in poor clinical operability and making it difficult to apply.

[0072] Experimental Example 3

[0073] Antioxidant function test of hydrogel

[0074] To verify the antioxidant function of the hydrogel of the present invention, the hydrogel prepared in Example 1 was tested and compared with phosphate-buffered saline (PBS) as a blank control.

[0075] (1) Sample preparation

[0076] The HMP / Ag@Gel hydrogel precursor solution (F127 20%, HMP 500 μg / mL, Ag) prepared according to the method in Example 1 + 0.4 μg / mL);

[0077] Control group: Phosphate-buffered saline (PBS, pH=7.4).

[0078] (2) Total antioxidant capacity (ABTS free radical scavenging)

[0079] Prepare ABTS free radical cation working solution. Take 100 μL of each sample solution and mix it with 2.0 mL of ABTS working solution. React at room temperature in the dark for 6 minutes, and measure the absorbance at a wavelength of 734 nm to calculate the scavenging rate.

[0080] (3) Superoxide anion (O2) - •) Clearance ability

[0081] A superoxide dismutase (SOD) assay kit was used. 20 μL of sample, 160 μL of WST-8 working solution, and 20 μL of reaction start-up solution were added sequentially to each well of a 96-well plate. The plate was incubated at 37°C for 30 minutes, and the absorbance was measured at 450 nm. The effect on O2 was calculated. - • The inhibition rate of generation.

[0082] (4) Hydrogen peroxide (H2O2) scavenging ability

[0083] 100 μL of sample was mixed with 100 μL of 1 mM H2O2 solution and reacted at room temperature for 9 hours. 17.5 μL of the reaction mixture was then added to 132.5 μL of Ti(SO4)2 solution and reacted at room temperature for 30 minutes. The absorbance was measured at 415 nm to calculate the H2O2 scavenging rate.

[0084] (5) Hydroxyl radical (·OH) scavenging ability

[0085] 100 μL of sample, 100 μL of 2 mM FeSO4, and 100 μL of 1 mM H2O2 were mixed sequentially and incubated at room temperature for 30 minutes. 50 μL of 4 mM salicylic acid was added to capture ·OH, and the absorbance was measured at 510 nm to calculate the ·OH scavenging rate.

[0086] (6) Experimental results

[0087] like Figure 3 As shown, the hydrogel prepared in Example 1 exhibited significant scavenging abilities against ABTS free radicals, superoxide anions, hydrogen peroxide, and hydroxyl free radicals, with scavenging rates of 5%, 28%, 38%, and 21%, respectively. This indicates that the HMP / Ag@Gel hydrogel prepared in this invention possesses broad-spectrum and highly efficient antioxidant activity.

[0088] Test Example 4

[0089] In vitro rapid contact sterilization performance test

[0090] (1) Sample preparation

[0091] The hydrogel precursor solution (F127 20%, HMP 500 μg / mL, Ag) prepared according to the method in Example 1 + 0.4 μg / mL);

[0092] The hydrogel precursor solution (F127 15%, HMP 100 μg / mL, Ag) prepared according to the method in Example 2 + 0.1 μg / mL);

[0093] The hydrogel precursor solution (F127 25%, HMP 1000 μg / mL, Ag) prepared according to the method in Example 3 + 1.0 μg / mL);

[0094] A pure F127 hydrogel precursor solution (20%, w / v) was prepared according to the method of Comparative Example 1.

[0095] The low Ag prepared according to the method of Comparative Example 4 + Concentration hydrogel precursor solution (Ag) + 0.05 μg / mL);

[0096] High Ag prepared according to the method of Comparative Example 5 + Concentration hydrogel precursor solution (Ag) + 2.0 μg / mL);

[0097] (2) Experimental methods

[0098] This study simulates the contact scenario between hydrogel dressings and bacteria when applied to warm, infected wounds. The hydrogel was first equilibrated at physiological temperature (37°C) to reach its application state before bacteria were introduced, thus evaluating the material's rapid bactericidal ability under simulated real-world conditions.

[0099] 100 μL of the hydrogel precursor solutions prepared in Examples 1-3, Comparative Examples 1, and Comparative Examples 4 and 5 were added to 96-well plates. The plates were incubated at 37°C for 10 minutes to allow them to completely solidify into a gel, simulating the actual application of dressings on wounds. 100 μL of a 1×10⁻⁶ hydrogel precursor solution was added to each well. 6 A CFU / mL suspension of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was prepared, ensuring the bacterial suspension covered the gel. The plate was incubated at 37°C in the dark for another 60 minutes. Immediately after incubation, samples were taken from each well, serially diluted, and plated onto LB agar plates. After incubation at 37°C for 24 hours, colony counting was performed.

[0100] (3) Experimental results

[0101] like Figure 4 As shown, in Comparative Example 1, the antibacterial activity was close to 0%, indicating that the vector F127 itself had no antibacterial contribution; in Comparative Example 4 (Ag +The bactericidal rate of the hydrogel prepared at 0.05 μg / mL was close to that of the blank control (<10%), indicating that silver ions had almost no antibacterial effect at this concentration; the bactericidal rates of the hydrogels prepared in Examples 1 and 3 both reached over 99.9%. The bactericidal rate of the hydrogel prepared in Example 2 was approximately 96.8%.

[0102] This shows that the antibacterial efficacy is related to Ag. + The concentrations are positively correlated, and in Ag + A concentration of 0.4 μg / mL is sufficient to completely kill MRSA after 1 hour of contact.

[0103] Experimental Example 5

[0104] Effects of the HMP / Ag@Gel hydrogel prepared in this invention on cell migration ability under oxidative stress conditions

[0105] (1) Sample preparation

[0106] Control group: Low serum DMEM medium (containing 2% fetal bovine serum)

[0107] Hydrogen peroxide group: low serum DMEM medium + 200 μM H2O2 (simulating oxidative stress environment)

[0108] Treatment group: low serum DMEM medium + 200 μM H2O2 + HMP / Ag@Gel hydrogel (added via Transwell chamber).

[0109] The gel used in the treatment group was the HMP / Ag@Gel hydrogel precursor solution prepared in Example 1. The gel was placed in the upper chamber of a Transwell chamber, and 100 μL of the gel precursor solution was added to each well. The chamber was incubated at 37°C for 10 minutes to allow it to gel. The chamber was then placed in a cell culture plate to allow the active ingredients released by the gel to permeate through the membrane and act on the cells in the lower chamber.

[0110] (2) Experimental methods

[0111] Human immortalized keratinocytes (HACAT) in the logarithmic growth phase were harvested at a density of 1 × 10⁻⁶ cells per well. 6 Cells were seeded into 6-well plates. After confluence, the culture medium was discarded. Low-serum DMEM was added to the Control group, and low-serum DMEM containing 200 μM H2O2 was added to the Hydrogen Peroxide group and the Treatment group. The cells were incubated for another 6 hours to establish an oxidative stress model.

[0112] Six hours later, each group was replaced with fresh low-serum DMEM, and straight scratches were made on the monolayer of cells using a 200 μL sterile pipette tip. The cells were then washed with PBS to remove detached cells. In the treatment group, Transwell chambers containing gel were simultaneously placed in well plates (the gel did not directly contact the scratched surface). Cells from each group were incubated at 37°C in a 5% CO2 incubator, and photographed under a microscope at 0, 12, 24, and 48 hours.

[0113] (3) Experimental results

[0114] like Figure 5 As shown, at 0 hours: the scratch widths were consistent across all groups; at 12 hours: the scratch widths in the Control and Treatment groups began to shrink, while the scratch widths in the hydrogen peroxide group showed no significant change; at 24 hours: the scratch widths in the Control and Treatment groups further decreased, and their healing degrees were similar; the scratches in the hydrogen peroxide group still showed no significant healing; at 48 hours: the scratches in the Control and Treatment groups were basically healed, while the scratches in the hydrogen peroxide group still had obvious gaps.

[0115] Therefore, it can be seen that under oxidative stress conditions, the HMP / Ag@Gel hydrogel prepared in this invention can significantly promote cell migration, and its effect is comparable to that of control without oxidative stress, while the cell migration ability of hydrogen peroxide group is severely impaired.

[0116] Application Example 6

[0117] To evaluate the in vivo therapeutic effect of the hydrogel of this invention on infectious pressure ulcers, a rat model was established and the following group intervention study was conducted:

[0118] I. Animal grouping and model establishment

[0119] 1. Uninfected group: A simple pressure injury model was established, with full-thickness skin defects and ischemic pressure ulcers on the back, without bacterial inoculation.

[0120] 2. Infection control group: A methicillin-resistant Staphylococcus aureus (MRSA) infectious pressure ulcer model was established. After modeling, the wound was inoculated with 10 7 CFU suspension of methicillin-resistant Staphylococcus aureus (MRSA).

[0121] 3. Carrier control group (infection + F127): An MRSA-infected pressure ulcer model was established, and pure F127 hydrogel (20%, w / v) was applied to the wound daily.

[0122] 4. Treatment group (infection + HMP / Ag@Gel): An MRSA-infected pressure ulcer model was established, and the wound was treated daily with the hydrogel (HMP / Ag@Gel) prepared in Example 1 of this invention.

[0123] II. Intervention Program and Observation Indicators

[0124] Treatment began on day 1 post-infection. Animals in each group received the corresponding drug once daily for 8 consecutive days. Wounds were photographed on days 0, 2, 4, 6, and 8 of treatment to assess healing progress.

[0125] III. Experimental Results

[0126] Wound healing status of rats in each group as follows Figure 6 As shown. Uninfected group: wound basically closed on day 8; Infected control group: wound not closed on day 8, necrotic tissue visible; Carrier control group (infected + F127): wound not closed on day 8; Treatment group (infected + HMP / Ag@Gel): wound basically closed on day 8, healing status close to the uninfected group, no obvious inflammation observed.

Claims

1. A bifunctional hydrogel integrating silver ions and hair extraction microparticles, characterized in that, The hydrogel comprises a thermosensitive gel matrix, HMP hair extract microparticles, and silver ion antibacterial components; the HMP hair extract microparticles and silver ion antibacterial components are jointly loaded in the thermosensitive hydrogel matrix. The hair extract microparticles are spindle-shaped hair-derived microparticles.

2. The bifunctional hydrogel integrating silver ions and hair extraction microparticles according to claim 1, characterized in that, The concentration of the thermosensitive gel matrix is ​​15-25% (w / v).

3. The bifunctional hydrogel integrating silver ions and hair extraction microparticles according to claim 1, characterized in that, The concentration of the hair-derived particles is 100-1000 μg / mL.

4. The bifunctional hydrogel integrating silver ions and hair extraction microparticles according to claim 1, characterized in that, The hair-derived microparticles have a particle size of 1-5 μm.

5. The bifunctional hydrogel integrating silver ions and hair extraction microparticles according to claim 1, characterized in that, The silver ion antibacterial component is one of silver nitrate, silver acetate, and silver citrate.

6. The bifunctional hydrogel integrating silver ions and hair extraction microparticles according to claim 1, characterized in that, The concentration of silver ions is 0.1-1.0 μg / mL.

7. The method for preparing the hydrogel according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Dissolve the thermosensitive gel matrix in phosphate buffer solution and stir to dissolve to obtain matrix solution; (2) The washed human hair was hydrolyzed in an alkaline solution, then centrifuged, washed and freeze-dried to obtain hair-derived particles; the hair-derived particles were dispersed in a portion of the matrix solution and sonicated to obtain a dispersion. (3) Mix the silver ion antibacterial component solution with the dispersion, then adjust the remaining matrix solution to the target concentration, and stir evenly at 4°C to obtain the composite hydrogel precursor solution.

8. The method for preparing the hydrogel according to claim 7, characterized in that, In step (1), the thermosensitive gel matrix is ​​dissolved in phosphate buffer at 4-10℃.

9. The method for preparing the hydrogel according to claim 7, characterized in that, In step (2), the specific preparation process of the hair-derived particles is as follows: human hair is washed, degreased, and dried, and then hydrolyzed in a 1-2 M alkaline solution at 50-80℃ for 1-4 hours; after the reaction is completed, the mixture is centrifuged, washed, precipitated, and then freeze-dried.

10. The use of the hydrogel according to any one of claims 1-9 in the preparation of pharmaceuticals or medical devices for pressure ulcers, characterized in that, The pressure ulcers are either bacterially infected or caused by oxidative stress.

Images