A thermosensitive antibacterial hydrogel based on ultra-small copper nanoparticles and its preparation method

Abstract

This invention discloses a thermosensitive antibacterial hydrogel based on ultra-small diameter copper nanoparticles and its preparation method, relating to the field of biomedical materials technology. The preparation method of the thermosensitive antibacterial hydrogel includes the following steps: uniformly mixing a gel matrix, a stabilizer, a pH buffer, and ultra-small diameter copper nanoparticles to obtain the thermosensitive antibacterial hydrogel; the preparation method of the ultra-small diameter copper nanoparticles includes the following steps: dissolving lactoferrin in water, adding copper sulfate pentahydrate, adjusting the pH of the solution to 8-14 with an alkaline solution, adding a reducing agent to carry out the reaction, and then dialysis and drying to obtain the ultra-small diameter copper nanoparticles. This hydrogel has good injectability, can release copper nanoparticles under specific conditions, and rapidly kills bacteria. Furthermore, the hydrogel is easy to synthesize, simple to operate, has good biocompatibility, and exhibits low cytotoxicity to cells, showing great application potential in the treatment of skin wound infections.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a thermosensitive antibacterial hydrogel based on ultra-small copper nanoparticles and its preparation method. Background Technology

[0002] Skin wound healing is a complex and highly coordinated process, comprising four phases: hemostasis, inflammation, proliferation, and remodeling. Skin wounds are susceptible to bacterial infection during healing, leading to delayed healing and scarring. Chronic wounds, such as those from diabetic patients, are particularly vulnerable due to persistent inflammation, dysregulation of proteases and their inhibitors, and hyperglycemia, resulting in an extremely high risk of bacterial infection. Severe infections in diabetic patients can lead to amputation and even death. Therefore, there is an urgent need to develop novel antibacterial wound dressings to address the growing problem of bacterial infection.

[0003] Antibiotics are the primary treatment for bacterial infections; however, overuse of antibiotics can easily lead to antibiotic resistance. Metal nanoparticles, due to their broad-spectrum antibacterial properties, persistence, and lack of resistance development, exhibit excellent bactericidal effects and represent a promising solution to address antibiotic resistance. Copper, in particular, demonstrates significant inhibitory effects against a variety of bacteria and viruses. It exhibits excellent antibacterial activity against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus vulgaris, making it a promising antibacterial agent. However, current methods for preparing copper nanoparticles primarily involve dispersion using surfactants, which suffers from drawbacks such as large particle size, poor stability, complex preparation processes, poor biosafety, and inability to be metabolized in vivo. Therefore, there is an urgent need to develop novel methods to prepare ultra-small copper nanoparticles to enhance their antibacterial activity and in vivo metabolism, while reducing the amount of copper required.

[0004] Traditional wound dressings, such as gauze and bandages, are characterized by low cost, easy availability, and good breathability and moisture absorption. Their main function is to cover and protect the wound from external contamination. However, they have disadvantages such as the need for frequent changes, which may increase the risk of wound infection, and their tendency to adhere to the wound, potentially causing secondary damage. Furthermore, they are not suitable for wounds with large exudates, and may require additional fixation measures to prevent dressing slippage. Hydrogel dressings can maintain a moist environment and are suitable for wounds with significant exudate. Hydrogels can keep the wound moist, promote the growth of new tissue, accelerate wound healing, and reduce scar formation. However, due to the current lack of technology for preparing ultra-small particle size copper nanoparticles, there is a lack of antibacterial hydrogel wound dressings based on copper nanoparticles. Therefore, the preparation of ultra-small particle size copper nanoparticle antibacterial hydrogels is crucial for improving the antibacterial efficacy and biosafety of hydrogel wound dressings. Summary of the Invention

[0005] The purpose of this invention is to provide a thermosensitive antibacterial hydrogel based on ultra-small copper nanoparticles and its preparation method, thereby solving the problems existing in the prior art. This hydrogel has good injectability, can release copper nanoparticles under specific conditions for rapid sterilization, and is convenient to synthesize, simple to operate, has good biocompatibility, and exhibits low cytotoxicity to cells, showing great application potential in the treatment of skin wound infections.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing ultra-small diameter copper nanoparticles, comprising the following steps:

[0008] Lactoferrin was dissolved in water, copper sulfate pentahydrate was added, the pH was adjusted to 8-14 to form a complex, a reducing agent was added to carry out the reaction, and then the mixture was dialyzed and dried to obtain the ultra-small copper nanoparticles.

[0009] Furthermore, the mass ratio of the lactoferrin to the copper sulfate pentahydrate is 20:(1-10).

[0010] Furthermore, the concentration of the lactoferrin is 20 mg / mL.

[0011] Furthermore, the molar ratio of the reducing agent to copper in the reaction system is 1:(1-10).

[0012] Furthermore, the reducing agent is at least one selected from hydrazine hydrate, ascorbic acid, sodium citrate, sodium borohydride, and sodium cyanoborohydride.

[0013] The present invention also provides an ultra-small copper nanoparticle prepared according to the above preparation method.

[0014] The present invention also provides a method for preparing a thermosensitive antibacterial hydrogel based on ultra-small diameter copper nanoparticles, comprising the step of mixing a gel matrix, a stabilizer, a pH buffer solution and the aforementioned ultra-small diameter copper nanoparticles uniformly to prepare the thermosensitive antibacterial hydrogel.

[0015] Furthermore, the gel matrix comprises component 1 and component 2;

[0016] Component 1 is Pluronic F127 or hydroxypropyl methylcellulose;

[0017] Component 2 is carbomer.

[0018] Furthermore, the stabilizer is at least one of ascorbic acid and sodium citrate.

[0019] The present invention also provides a thermosensitive antibacterial hydrogel prepared according to the above preparation method.

[0020] The present invention also provides the application of the above-mentioned thermosensitive antibacterial hydrogel in the preparation of wound dressing products.

[0021] The present invention discloses the following technical effects:

[0022] The copper nanoparticles provided by this invention have a simple preparation process, high stability, good dispersibility, high solubility, and exhibit ultra-small particle size and excellent antibacterial properties. Lactoferrin is a protein with antibacterial activity extracted from milk, possessing ultra-small particle size and an internal cavity structure. Using lactoferrin as a dispersant provides excellent dispersibility and biocompatibility, resulting in copper nanoparticles with good dispersibility and high stability. This overcomes the problems of large particle size and poor solubility of current copper nanoparticles. Ultra-small particle size copper nanoparticles have many advantages: large specific surface area, increased contact with bacteria, and strong antibacterial ability; they can cross the glomerular capillary wall of the kidneys, exhibiting rapid renal clearance; they reduce the dosage of copper used, decrease cytotoxicity, and improve biosafety.

[0023] This invention discloses a temperature-sensitive antibacterial hydrogel based on ultra-small copper nanoparticles. This hydrogel is temperature-sensitive, rapidly forming a hydrogel at a physiological temperature of 37°C and remaining in a solution state below 37°C. It also exhibits good injectability, releasing copper nanoparticles under specific conditions for rapid sterilization. The hydrogel is easy to synthesize and operate, possesses good biocompatibility, and demonstrates low cytotoxicity, showing significant application potential in the treatment of skin wound infections. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Particle size distributions of lactoferrin and LF-Cu are shown below; (a) shows the particle size distribution of lactoferrin; (b) shows the particle size distribution of LF-Cu.

[0026] Figure 2 Transmission electron micrographs of lactoferrin and LF-Cu are shown; (a) is a transmission electron micrograph of lactoferrin; (b) is a transmission electron micrograph of LF-Cu.

[0027] Figure 3 Here are the XPS spectra of LF-Cu; where (a) shows the chemical composition detection results; and (b) shows the electronic structure detection results.

[0028] Figure 4 The graph shows the detection results of the hydrogen peroxide scavenging rate of LF-Cu.

[0029] Figure 5 The figure shows the results of the rheological property testing of the nano-copper gel.

[0030] Figure 6 The live / dead staining fluorescence image after co-incubation of copper nanogel and cells for 24 h;

[0031] Figure 7 The figure shows the detection results of the antibacterial ability of the nano-copper gel against three types of bacteria. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Example 1

[0038] First, dissolve 100 mg of lactoferrin in 5 mL of deionized water, add 5 mg of copper sulfate pentahydrate, and stir for 20 min. After stirring evenly, adjust the pH to 11 with sodium hydroxide solution (or ammonia) to form a complex, and stir for another 20 min. Then, add hydrazine hydrate as a reducing agent (with a copper to reducing agent molar ratio of 1:5), and continue the reaction for 3 h to obtain a homogeneous copper nanoparticle solution. Dialyze the solution in deionized water using a dialysis bag semi-permeable membrane to remove all small molecule impurities, then freeze the solution and freeze-dry it to obtain copper nanoparticles (LF-Cu).

[0039] The size distribution of lactoferrin and LF-Cu was determined using a particle size analyzer, and the average size was calculated by fitting. Figure 1 The particle size distribution of lactoferrin in (a) is uniform, with a particle size of approximately 6 nm. Figure 1 Figure (b) shows that the particle size of LF-Cu is 7.4 nm after loading with copper nanoparticles.

[0040] The microstructure of copper nanoparticles was characterized using transmission electron microscopy (TEM). The accelerating voltage was adjusted (typically between 80-300 kV), and appropriate current and focusing conditions were selected. TEM results of LF-Cu are shown below. Figure 2 The results indicate that the copper nanoparticles are uniformly dispersed in LF, with spherical particles representing copper nanoparticles and a particle size of approximately 1–2 nm. Furthermore, multiple dispersed copper nanoparticles are encapsulated by a single lactoferrin protein. These results demonstrate that the synthesized copper nanoparticles possess ultra-small particle size and uniform dispersibility.

[0041] X-ray photoelectron spectroscopy (XPS) was used to detect the chemical composition and electronic structure. XPS spectra confirmed the presence of Cu, O, N, C, S, and Fe. Figure 3 XPS spectra show that Cu 3p 3 / 2 and Cu 2p 1 / 2 Two distinct binding energy peaks exist at 932.90 eV and 953.20 eV, respectively.

[0042] Hydrogen peroxide scavenging capacity was tested. H₂O₂ reacted with titanium sulfate to form a yellow precipitate of peroxide-titanium complex, which could be dissolved by H₂SO₄ and measured colorimetrically at 415 nm. Within a certain range, the color intensity showed a linear relationship with the H₂O₂ concentration. The scavenging capacity of different concentrations of LF-Cu for hydrogen peroxide was tested using a hydrogen peroxide content detection kit. The mixture was incubated in a shaker at 37℃ in the dark for 30 min. Finally, the absorbance of the solution at 415 nm was measured using a UV spectrophotometer. The scavenging capacity of LF-Cu at concentrations of 5, 10, 20, and 30 μg / mL for hydrogen peroxide was measured, and the results are shown in the table below. Figure 4Experimental results show that LF-Cu at a concentration of 30 μg / mL has a 100% scavenging capacity for hydrogen peroxide, indicating that low concentrations of LF-Cu have excellent scavenging capacity for hydrogen peroxide.

[0043] Example 2

[0044] First, 100 mg of lactoferrin was dissolved in 5 mL of deionized water, and 20 mg of copper sulfate pentahydrate was added. The mixture was stirred for 20 min. After thorough mixing, the pH was adjusted to 8 with sodium hydroxide solution to form a complex, and the mixture was stirred for another 20 min. Then, hydrazine hydrate (with a copper to reducing agent molar ratio of 1:1) was added as a reducing agent, and the reaction was continued for 6 h to obtain a homogeneous copper nanoparticle solution. Dialysis was performed in deionized water using a dialysis bag semi-permeable membrane to remove all small molecule impurities. After freezing, the solution was lyophilized to obtain LF-Cu.

[0045] Example 3

[0046] First, 100 mg of lactoferrin was dissolved in 5 mL of deionized water, and 50 mg of copper sulfate pentahydrate was added. The mixture was stirred for 20 min. After thorough mixing, the pH was adjusted to 14 with sodium hydroxide solution to form a complex, and the mixture was stirred for another 20 min. Then, hydrazine hydrate (with a copper to reducing agent molar ratio of 1:10) was added as a reducing agent, and the reaction was continued for 1 h to obtain a homogeneous copper nanoparticle solution. Dialysis was performed in deionized water using a dialysis bag semi-permeable membrane to remove all small molecule impurities. After freezing, the solution was lyophilized to obtain LF-Cu.

[0047] Example 4

[0048] First, dissolve 100 mg of lactoferrin in 5 mL of deionized water, add 5 mg of copper sulfate pentahydrate, and stir for 20 min. After stirring evenly, adjust the pH to 11 with sodium hydroxide solution (or ammonia) to form a complex, and stir for another 20 min. Then, add ascorbic acid as a reducing agent (with a copper to reducing agent molar ratio of 1:5), and continue the reaction for 3 h to obtain a homogeneous copper nanoparticle solution. Dialyze the solution in deionized water using a dialysis bag semi-permeable membrane to remove all small molecule impurities, then freeze the solution and freeze-dry it to obtain copper nanoparticles (LF-Cu).

[0049] Example 5

[0050] First, dissolve 100 mg of lactoferrin in 5 mL of deionized water, add 5 mg of copper sulfate pentahydrate, and stir for 20 min. After stirring evenly, adjust the pH to 11 with sodium hydroxide solution (or ammonia) to form a complex, and stir for another 20 min. Then, add sodium citrate as a reducing agent (the molar ratio of copper to reducing agent is 1:5), and continue the reaction for 3 h to obtain a homogeneous copper nanoparticle solution. Dialyze the solution in deionized water using a dialysis bag semi-permeable membrane to remove all small molecule impurities. After freezing, freeze the solution using a freeze dryer to obtain copper nanoparticles (LF-Cu).

[0051] Example 6

[0052] First, dissolve 100 mg of lactoferrin in 5 mL of deionized water, add 5 mg of copper sulfate pentahydrate, and stir for 20 min. After stirring evenly, adjust the pH to 11 with sodium hydroxide solution (or ammonia) to form a complex, and stir for 20 min. Then, add sodium borohydride as a reducing agent (the molar ratio of copper to reducing agent is 1:5), and continue the reaction for 3 h to obtain a homogeneous copper nanoparticle solution. Dialyze the solution in deionized water using a dialysis bag semi-permeable membrane to remove all small molecule impurities, then freeze the solution and freeze-dry it to obtain copper nanoparticles (LF-Cu).

[0053] Example 7

[0054] First, dissolve 100 mg of lactoferrin in 5 mL of deionized water, add 5 mg of copper sulfate pentahydrate, and stir for 20 min. After stirring evenly, adjust the pH to 11 with sodium hydroxide solution (or ammonia) to form a complex, and stir for 20 min. Then, add sodium cyanoborohydride as a reducing agent (the molar ratio of copper to reducing agent is 1:5), and continue the reaction for 3 h to obtain a homogeneous copper nanoparticle solution. Dialyze the solution in deionized water using a dialysis bag semi-permeable membrane to remove all small molecule impurities, then freeze the solution and freeze-dry it to obtain copper nanoparticles (LF-Cu).

[0055] Example 8

[0056] Equal volumes of 1 wt% Prönnicke F127 and 1 wt% Carbomer were mixed, and ascorbic acid (stabilizer) was added at a concentration of 0.5 wt%. The pH was adjusted to 7.4 using phosphate buffer, and then 25 wt% LF-Cu (prepared in Example 1) was added. The mixture was stirred until homogeneous to obtain a precursor solution for the nano-copper gel. The nano-copper gel was obtained by heating (when the temperature rises to 37°C, the precursor solution system undergoes a sol-gel transition).

[0057] The elastic modulus (G′) and loss modulus (G″) of the nano-copper gel were measured using a rotational rheometer at different temperatures with a strain of 1% and a frequency range of 0.1–1.2 Hz. Figure 5 The rheological properties of the nano-copper gel were tested, showing that the rheological characteristics of the temperature-sensitive hydrogel are highly dependent on temperature changes. Experimental results indicate that when the temperature is 3°C below its phase transition temperature, the system exhibits a sol state with low viscosity. From 5°C to 35°C, the viscosity remains essentially unchanged with increasing temperature. As the temperature rises above the phase transition temperature of 35°C, the viscosity increases significantly. When the physiological temperature of 37°C is reached, the aqueous solution system transforms into a gel state.

[0058] Figure 6 The images show the live / dead cell fluorescence images after 24 hours of co-incubation of copper nanogel and cells. The live / dead cell staining method was used to qualitatively evaluate the effect of the hydrogel extract on the activity of L929 mouse fibroblasts. Fluorescence images stained with AO / PI (acridin orange / propidium iodide) after 24 hours of culture are shown below. Figure 6 As shown, compared with the control group, the hydrogel group showed more green fluorescence (live cells) and almost no red fluorescence (dead cells), indicating that the nano-copper gel has good cell compatibility.

[0059] Example 9

[0060] After mixing 1 wt% Prönnicke F127 and 10 wt% Carbomer in equal volumes, ascorbic acid (stabilizer) was added at a concentration of 0.1 wt%. The pH was adjusted to 7.4 using phosphate buffer, and then 1 wt% LF-Cu (prepared in Example 1) was added. The mixture was stirred until homogeneous to obtain a precursor solution for the nano-copper gel. The nano-copper gel was obtained by heating (when the temperature rises to 37°C, the precursor solution system will undergo a sol-gel transition).

[0061] Example 10

[0062] After mixing 20 wt% Prönnicke F127 and 1 wt% carbomer, ascorbic acid (stabilizer) was added at a concentration of 1 wt%. The pH was adjusted to 7.4 using phosphate buffer, and then 50 wt% LF-Cu (prepared in Example 2) was added. The mixture was stirred until homogeneous to obtain a precursor solution for the nano-copper gel. The nano-copper gel was obtained by heating (when the temperature rises to 37°C, the precursor solution system will undergo a sol-gel transition).

[0063] Example 11

[0064] Equal volumes of 1 wt% hydroxypropyl methylcellulose and 1 wt% carbomer were mixed, and sodium citrate (stabilizer) was added, with the amount of sodium citrate added being 0.5 wt%. The pH was adjusted to 7.4 using phosphate buffer, and then 25 wt% LF-Cu (prepared in Example 3) was added. The mixture was stirred evenly to obtain a precursor solution for the nano-copper gel. The nano-copper gel was obtained by heating (when the temperature rises to 37°C, the precursor solution system will undergo a sol-gel transition).

[0065] Example 12

[0066] Equal volumes of 1 wt% hydroxypropyl methylcellulose and 10 wt% carbomer were mixed, and sodium citrate (stabilizer) was added at a concentration of 0.1 wt%. The pH was adjusted to 7.4 using phosphate buffer, and then 1 wt% LF-Cu (prepared in Example 4) was added. The mixture was stirred until homogeneous to obtain a precursor solution for the nano-copper gel. The nano-copper gel was obtained by heating (when the temperature rises to 37°C, the precursor solution system undergoes a sol-gel transition).

[0067] Example 13

[0068] After mixing 10 wt% hydroxypropyl methylcellulose and 1 wt% carbomer in equal volumes, sodium citrate (stabilizer) was added, with the amount of sodium citrate added being 1 wt%. The pH was adjusted to 7.4 using phosphate buffer, and then 50 wt% LF-Cu (prepared in Example 5) was added. The mixture was stirred evenly to obtain a precursor solution for the nano-copper gel. The nano-copper gel was obtained by heating (when the temperature rises to 37°C, the precursor solution system will undergo a sol-gel transition).

[0069] Comparative Example 1

[0070] Same as Example 8, except that LF-Cu is replaced with 20nm nano-copper (PVP-Cu).

[0071] Example 14

[0072] The antibacterial properties of the nano-copper gel prepared in Example 8 were studied.

[0073] Take a sterilized 5mL glass vial and add 100μL of test bacterial solution (concentration of 10). 5 Add 900 μL of liquid culture medium ( / mL), then add 100 μL of the nano-copper gel prepared in Example 8 or Comparative Example 1. Gently pipette to mix evenly and incubate at 37°C for 12 hours. After dilution, spread on plates and calculate the inhibition rate by colony counting.

[0074] Figure 7The antibacterial effect of the nano-copper gel was shown in the figure. The antibacterial effect of the nano-copper gel against *Escherichia coli*, *Staphylococcus aureus*, and methicillin-resistant *Staphylococcus aureus* (MRSA) was statistically analyzed using the colony plate counting method. No colonies grew in any of the three groups, indicating that the nano-copper gel achieved a 100% inhibition rate against *E. coli*, *S. aureus*, and MRSA. The experimental results demonstrate that the nano-copper gel exhibits excellent antibacterial effects against *E. coli*, *S. aureus*, and MRSA. Furthermore, at the same concentration, the smaller the size of the prepared nano-copper particles, the better the antibacterial effect.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing ultra-small diameter copper nanoparticles, characterized in that, Includes the following steps: Lactoferrin was dissolved in water, copper sulfate pentahydrate was added, the pH was adjusted to 8-14 to form a complex, a reducing agent was added to carry out the reaction, and then the mixture was dialyzed and dried to obtain the ultra-small copper nanoparticles.

2. The preparation method according to claim 1, characterized in that, The mass ratio of lactoferrin to copper sulfate pentahydrate is 20:(1-10).

3. The preparation method according to claim 1, characterized in that, The molar ratio of the reducing agent to copper in the reaction system is (1-10):

1.

4. The preparation method according to claim 1, characterized in that, The reducing agent is at least one of hydrazine hydrate, ascorbic acid, sodium citrate, sodium borohydride, and sodium cyanoborohydride.

5. Ultra-small copper nanoparticles prepared by the preparation method according to any one of claims 1-4.

6. A method for preparing a thermosensitive antibacterial hydrogel based on ultra-small diameter copper nanoparticles, characterized in that, The method includes the step of uniformly mixing a gel matrix, a stabilizer, a pH buffer, and the ultra-small copper nanoparticles as described in claim 5 to prepare the temperature-sensitive antibacterial hydrogel.

7. The preparation method according to claim 6, characterized in that, The gel matrix comprises component 1 and component 2; Component 1 is Pluronic F127 or hydroxypropyl methylcellulose; Component 2 is carbomer.

8. The preparation method according to claim 6, characterized in that, The stabilizer is at least one of ascorbic acid and sodium citrate.

9. A thermosensitive antibacterial hydrogel prepared by the preparation method according to any one of claims 6-8.

10. The application of the thermosensitive antibacterial hydrogel as described in claim 9 in the preparation of wound dressing products.

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