CN118178311A - Preparation method of thermosensitive hydrogel for promoting healing of bacterial infection wound of type I diabetes patient - Google Patents

Abstract

The present invention discloses a method for preparing a thermosensitive hydrogel that promotes wound healing of bacterial infections in patients with type 1 diabetes. The hydrogel is a GOx@Cu‑ZIF‑8/Alnu‑FS (GCZ/AFS) thermosensitive hydrogel. GCZ/AFS hydrogel is a novel material that immobilizes GOx@Cu‑ZIF‑8 and alnu ketone micelles in a hydrogel network composed of F127 and sodium alginate, and the MIC value for MRSA is 32 μg/mL. In addition, GCZ/AFS hydrogel can quickly promote the degree of healing of diabetic mouse skin wounds after MRSA infection. On the 9th day of MRSA infection in the mouse skin wound, the wound was almost completely healed, proving the excellent healing and anti-infection effects of the hydrogel. In addition, the present invention can also be used as a medical dressing for type 1 diabetic patients with skin wounds infected with MRSA. The present invention prepares GCZ/AFS healing-promoting hydrogels by a simple and quick method, providing a new method and new idea for the synergistic treatment of wound infections in diabetic patients.

Description

Preparation method of thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes

Technical Field

The invention belongs to the technical field of biomedical materials and relates to a preparation method and a treatment method of a thermosensitive hydrogel that promotes wound healing caused by bacterial infection in patients with type I diabetes.

Background technique

Diabetes is a common metabolic disease that can lead to various complications in patients, such as diabetic ulcers, due to systemic neuropathy and vasculopathy caused by high blood sugar. Chronic inflammation in diabetic ulcers hinders the skin regeneration process. At the same time, high sugar levels are a breeding ground for bacterial infection. The treatment outcomes of clinically used antibiotics and hypoglycemic drugs are highly affected by fluctuations in blood sugar levels and multidrug-resistant bacteria. Therefore, there is an urgent need to develop an antibacterial and anti-inflammatory strategy for diabetic wounds.

Glucose oxidase (GOx) can catalyze D-glucose to produce hydrogen peroxide and gluconic acid, consume excess glucose, reconstruct an acidic microenvironment, and simultaneously form reactive oxygen species (ROS) to kill bacteria. However, GOx itself is not an ideal antibacterial agent for treating diabetic infections because the generated H ₂ O ₂ concentration is low, which to some extent reduces the bactericidal activity. Practical strategies to improve treatment outcomes may rely on the generation of ROS with stronger oxidative properties, such as hydroxyl radicals (·OH), through cascade reactions.

Alnus ketone is a diphenylheptane compound, mainly found in plants such as cardamom and alder flower. In our previous study, we found that alnus ketone can significantly reduce the virulence of MRSA by inhibiting the synthesis of staphyloxanthin, but due to the extremely poor water solubility of alnus ketone, the biological activity and availability of alnus ketone are greatly affected. Using F127 to prepare alnus ketone into micelles and fix them in the three-dimensional network of hydrogel, alnus ketone can be slowly released to the target site with little side effects on normal tissues and organs. In particular, hydrogel wound dressings have the advantages of local targeting and minimally invasive drug delivery, showing great prospects for personalized treatment.

Therefore, the present invention fixes alnus ketone micelles and GOx@Cu-ZIF-8 in the network of hydrogel to obtain GOx@Cu-ZIF-8/Alnu-FS (GCZ/AFS) thermosensitive hydrogel, which can produce Fenton-like reaction and produce ·OH with stronger bactericidal effect while reducing the virulence of MRSA strain. This thermosensitive hydrogel is simple and convenient to use, can effectively treat wound infection of diabetic patients caused by MRSA, and can promote wound healing, providing a new idea for treating diabetic wound infection.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides a method for preparing a thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes, so as to solve the problem that wound infection in patients with diabetes is difficult to heal, and significantly alleviate the phenomenon that wounds in patients with diabetes cannot heal.

In order to achieve the above purpose, the technical solution adopted by the present invention is to form a hydrogel by immobilizing alder ketone micelles and GOx@Cu-ZIF-8, which reduces the toxicity of MRSA and produces ·OH to kill bacteria. In addition, the thermosensitive hydrogel not only gels quickly on the skin, but also has the effect of promoting wound healing. The present invention is achieved through the following technical solutions:

The present invention provides a method for preparing a thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes. The type 1 diabetes model is a diabetes model of healthy ICR mice induced by streptozotocin, and the blood glucose concentration is ≥16.7mmol/mL; the bacteria are methicillin-resistant Staphylococcus aureus; the hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes can heal the wound on the ninth day of infection.

Accordingly, a method for preparing a thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes comprises the following steps:

(1) Preparation of GOx@Cu-ZIF-8: Specific contents of GOx and 2-methylimidazole were dissolved in 7 mL of water to form solution A. Specific contents of Zn(NO ₃ ) ₂ ·6H ₂ O and Cu(NO ₃ ) ₂ ·3H ₂ O were dissolved in 3 mL of water to form solution B. Solution B was then added to solution A, and the mixed solution was stirred at room temperature for 30 min. Finally, the product was separated by centrifugation (10000 rpm, 10 min), washed three times with deionized water, and then freeze-dried for further use.

(2) Preparation of alder ketone micelles: Alder ketone and F127 were dissolved in tetrahydrofuran, respectively. Alder ketone solution was added to the F127 solution, and then the volume was fixed with tetrahydrofuran. Next, the above solution was added to deionized water for ultrasonication, and then placed in a fume hood to completely evaporate the tetrahydrofuran. The alder ketone micelles were dialyzed in a dialysis bag (3500MwCO), and the external deionized water was replaced several times during the process.

(3) Preparation of thermosensitive hydrogels for promoting wound healing of bacterial infection in patients with type 1 diabetes: GOx@Cu-ZIF-8 was added to deionized water and dissolved by ultrasonication, and then _CaCl2 was added and stirred (at room temperature) to make it uniform. F127 was added to the above solution and stirred in an ice bath until it was completely dissolved. Then the prepared alnus ketone micelles were added to the above system and stirred uniformly. Subsequently, sodium alginate was added to the above mixed solution and stirred in an ice bath for 1 hour.

Preferably, the mass ratio of the 2-methylimidazole to Zn(NO ₃ ) ₂ ·6H ₂ O is 11:5.

Preferably, the solid loading amount of GOx@Cu-ZIF-8 is 20 mg/mL.

Preferably, the immobilization amount of alnus ketone is 240 μg/mL.

Preferably, the content of _CaCl2 is 0.5% (w/v), the content of F127 is 18% (w/v), and the content of sodium alginate is 4% (w/v).

The GOx@Cu-ZIF-8 prepared in the present invention is a two-dimensional leaf-shaped MOFs material, which has a stronger bactericidal activity against MRSA than the traditional three-dimensional material (MIC value is 64μg/mL). The MIC value of the hydrogel containing GOx@Cu-ZIF-8 and alnus ketone micelles against MRSA is 32μg/mL, which indicates that the hydrogel can play the role of alnus ketone in reducing the virulence of MRSA and the bactericidal activity of GOx@Cu-ZIF-8, so that it can play a synergistic bactericidal role.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

Figure 1 is a scanning electron microscope image of GOx@Cu-ZIF-8

Figure 2 is the Fourier infrared spectrum of GOx@Cu-ZIF-8

Figure 3 is the X-ray diffraction pattern of GOx@Cu-ZIF-8

Figure 4 shows the MIC diagram of GOx@Cu-ZIF-8 and alder ketone synergistic bactericidal

Figure 5 is the antibacterial scanning electron microscopy image of GOx@Cu-ZIF-8 and alder ketone and GOx@Cu-ZIF-8 against MRSA

Figure 6 is the scanning electron microscopy and mapping image of GCZ/AFS hydrogel

Figure 7 is the Fourier infrared spectrum of GCZ/AFS hydrogel

Figure 8 is the X-ray diffraction pattern of GCZ/AFS hydrogel

Figure 9 shows the gelation temperature and time of GCZ/AFS hydrogel

Figure 10 is the rheological test graph of GCZ/AFS hydrogel

Figure 11 is the MIC graph of GCZ/AFS hydrogel sterilization

Figure 12 is a scanning electron microscopy image of the antibacterial effect of GCZ/AFS hydrogel on MRSA

Figure 13 shows GCZ/AFS hydrogel promoting wound healing on the back of diabetic mice

Detailed ways

In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined purpose, the specific operation mode and effect results of the present invention are described in detail below in combination with the accompanying drawings and embodiments.

Example 1

Step 1: Preparation of GOx@Cu-ZIF-8

5 mg of GOx and 550 mg of 2-methylimidazole were dissolved in 7 mL of water to form solution A. 150 mg of Zn(NO ₃ ) ₂ ·6H ₂ O and 63 mg of Cu(NO ₃ ) 2·3H ₂ O were dissolved in 3 mL of water to form solution B. Solution B was then added to solution A, and the mixed solution was stirred at room temperature for 30 minutes. Finally, the product was separated by centrifugation (10000 rpm, 10 min), washed 3 times with deionized water, and then lyophilized for further use. When used, phosphate buffered saline (PBS) was prepared into the desired concentration gradient by serial dilution method.

Step 2: Structural characterization of GOx@Cu-ZIF-8

The morphology of GOx@Cu-ZIF-8 was observed by scanning electron microscopy; the functional groups of GOx@Cu-ZIF-8 were determined by Fourier transform infrared spectrometer; and the crystal structure of GOx@Cu-ZIF-8 was further determined by X-ray diffractometer.

According to the scanning electron microscope image of Figure 1, it can be seen that GOx@Cu-ZIF-8 is a uniform two-dimensional leaf-like structure. The Fourier transform infrared spectrum analysis of Figure 2 shows that the characteristic peak at 1680cm ^-1 indicates the presence of the carboxyl stretching band of the amide function, which confirms the successful immobilization of glucose oxidase; the characteristic peaks of Zn-N, Zn-O and CN bonds appear at 422cm ^-1 , 750cm ^-1 and 1142cm ^-1 , respectively. The X-ray diffraction analysis of Figure 3 shows that each group of materials exhibits strong diffraction peaks at 10.34°, 12.68°, 14.66°, 16.50° and 18.00°, corresponding to the crystal planes of (002), (112), (022), (013) and (222), respectively. These results further show that the present invention successfully prepared GOx@Cu-ZIF-8.

Example 2

Step 1: Determination of the MIC value of the synergistic antibacterial effect of GOx@Cu-ZIF-8 and alnus ketone on MRSA

Different concentrations of ZIF-8, GOx@ZIF-8, Cu@ZIF-8 and GOx@Cu-ZIF-8 were ultrasonically dissolved in PBS and tested by 2-fold dilution method. Glucose and reduced glutathione were added to the culture medium to a concentration of 10mM. Briefly, 100μL of MRSA with a concentration of 1×10 ⁷ CFU/mL and different concentrations of MOFs materials were added to a 96-well plate, cultured at 37°C for 16-24h, and then 10μL of 6.25mg/mL resazurin solution was added. After standing for 1h, the color change was observed (the well turned red to indicate the presence of MRSA), and the concentration of alnus ketone was 8μg/mL.

According to the results in Figure 4, when the concentration of GOx@Cu-ZIF-8 is ≥200μg/mL, the growth of MRSA can be inhibited; when alder ketone is used in combination with GOx@Cu-ZIF-8, the growth of MRSA can be inhibited at a concentration of 50μg/mL. In addition, ZIF-8, GOx@ZIF-8, and Cu@ZIF-8 have almost no inhibitory effect on MRSA. This shows that GOx@Cu-ZIF-8 has an excellent inhibitory effect on MRSA, and the effect is better when used in combination with alder ketone.

Step 2: Microstructure observation of GOx@Cu-ZIF-8 and alder ketone after treatment of MRSA

Glucose and reduced glutathione were added to 3 mL of MRSA with a concentration of 1×10 ⁹ CFU/mL to make the concentration 10 mM. Alder ketone and GOx@Cu-ZIF-8 were then added to MRSA, cultured at 37°C for 4 h, and the morphology of MRSA was photographed using a scanning electron microscope (Hitachi SU8100, Japan).

The results in Figure 5 show that the bacteria in the blank control group and the alnus ketone treatment group did not show any rupture; the MRSA bacteria treated with GOx@Cu-ZIF-8 (200 μg/mL) and GOx@Cu-ZIF-8 (50 μg/mL) and alnus ketone (8 μg/mL) showed rupture, which indicates that the MRSA died after being treated with GOx@Cu-ZIF-8.

Example 3

Step 1: Morphology and element distribution of GCZ/AFS hydrogel

Add 1g GOx@Cu-ZIF-8 to 50mL deionized water and dissolve it by ultrasonic. Then add 0.5% _CaCl2 and stir evenly. Add 18% (w/v) F127 to the above solution and stir until completely dissolved (ice bath). Then add 5mL of alnus ketone micelles to the above system and stir evenly (ice bath). Subsequently, add sodium alginate with a concentration of 4% (w/v) to the above mixed solution and stir for 1 hour (ice bath). Then freeze-dry the prepared sample, and the morphology and element distribution of the dried hydrogel powder are photographed using a scanning electron microscope.

The results in Figure 6 show that there is a clear leaf-like morphology of GOx@Cu-ZIF-8 in the GCZ/AFS hydrogel, which indicates that GOx@Cu-ZIF-8 has been immobilized in the pores of the hydrogel. In addition, the EDS image of the GCZ/AFS hydrogel shows that C, N, O, Cu, and Zn elements are evenly distributed, which also indicates that GOx@Cu-ZIF-8 is evenly immobilized in the hydrogel.

Example 4

Step 1: Structural characterization of GCZ/AFS hydrogel

Fourier infrared spectrometer and X-ray diffractometer were used to detect the freeze-dried hydrogel powder in Example 3. The FSC hydrogel is a hydrogel composed of 18% F127, 4% sodium alginate and 0.5% _CaCl2 .

The FT-IR results in Figure 7 show that the main absorption peaks of F127 appear in GCZ/AFS hydrogel at 2885cm ^-1 (CH stretching vibration), 1472cm ^-1 (-methyl group PPO part), 1352cm ^-1 (OH bond) and 1110cm ^-1 (COC stretching vibration); the characteristic peaks of sodium alginate appear at 1297cm ^-1 (CO stretching) and 1600cm ^-1 (C＝O stretching vibration); the characteristic peaks of GOx@Cu-ZIF-8 appear at 420cm ^-1 (Zn-N) and 767cm ^-1 (Zn-O). The XRD results in Figure 8 show that GCZ/AFS hydrogel has the characteristic peaks of F127 and sodium alginate.

Example 5

Step 1: Thermosensitive gelation properties of GCZ/AFS hydrogel

After the GCZ/AFS hydrogel in Example 3 was prepared, it was placed in a refrigerator at 4° C. for temperature sensitivity testing.

The results in Figure 9 show that FSC hydrogel (left) and GCZ/AFS hydrogel do not gel at 20°C; the gelation time at 25°C is 300 and 276 s, respectively; and the gelation time at 37°C is 30 s and 25 s, respectively. This shows that the hydrogel prepared using F127, sodium alginate and _CaCl2 is temperature-sensitive and can gel quickly at 37°C, which is conducive to its application as a wound dressing.

Step 2: Rheological properties of GCZ/AFS hydrogels

After the GCZ/AFS hydrogel in Example 3 was prepared, it was placed in a refrigerator at 4° C. for rheological testing.

The results in Figure 10 show that when the temperature is between 20 and 22 °C, the state of the samples of FSC hydrogel and GCZ/AFS hydrogel changes. When the temperature is higher than this, the storage modulus (G’) of the hydrogel is greater than the loss modulus (G”), indicating that a hydrogel is formed at this time. In addition, as the temperature rises, the viscosity of the hydrogel also increases, which also shows that the GCZ/AFS hydrogel is a temperature-sensitive hydrogel.

Example 6

Step 1: Antibacterial effect of GCZ/AFS hydrogel on MRSA

The hydrogel was diluted with TSB medium to a concentration of 2048 μg/ml. Among them, FSCAG hydrogel is a hydrogel composed of 18% F127, 4% sodium alginate, 0.5% CaCl ₂ , 5mL alder ketone micelles and 10mg glucose oxidase. Glucose and reduced glutathione were added to the culture medium to a concentration of 10mM. In brief, 100μL of MRSA with a concentration of 1×10 ⁷ CFU/mL and MOFs materials of different concentrations were added to a 96-well plate, cultured at 37°C for 16-24h, and then 10μL of 6.25mg/mL resazurin solution was added. After standing for 1h, its color change was observed.

The results in Figure 11 show that FSC hydrogel has no inhibitory effect on MRSA; FSCAG hydrogel has an antibacterial effect on MRSA at high concentrations (≥1024μg/mL); and the MIC value of GCZ/AFS hydrogel against MRSA is only 32μg/mL, which indicates that GCZ/AFS hydrogel has excellent antibacterial activity against MRSA.

Step 2: Microscopic image of GCZ/AFS hydrogel after treating MRSA

Glucose and reduced glutathione were added to 3 mL of MRSA with a concentration of 1×10 ⁹ CFU/mL to make the concentration 10 mM. Then FSC hydrogel and GCZ/AFS hydrogel were added to the bacterial solution (concentration 32 μg/mL), cultured for 4 hours, and then the morphology of MRSA was photographed using a scanning electron microscope.

The results in Figure 12 show that the MRSA bacteria did not appear to be damaged after FSC hydrogel treatment, which was no different from the blank control group; while the bacteria appeared to be ruptured after GCZ/AFS hydrogel treatment, indicating that it has an antibacterial effect on MRSA.

Example 6

Step 1: GCZ/AFS hydrogel can promote skin wound healing in diabetic mice infected with MRSA

Construction of diabetic mouse model. The mice were intraperitoneally injected with 100 mg/kg of streptozotocin and then fed a high-sugar, high-fat diet for 7 days. The vital signs of the mice were closely observed within 6 hours after the injection to ensure adequate food and water. After 7 days, blood was collected from the tail of the mice and fasting blood sugar was measured. If the blood sugar was higher than 16.7 mmol/L, the type 1 diabetes model was considered to be successful. If it was unsuccessful, the same dose of streptozotocin was injected again and the blood sugar concentration was measured again after 7 days.

After the diabetic mouse model was successfully established, the hair on the back of the mice was removed, and a circular wound with a diameter of about 8 mm was formed on the back of each mouse with a hole punch. Subsequently, 50 μL of MRSA suspension (about 10 ⁸ CFU/mL) was spread on the wound surface to induce infection. On the first and third days after infection, 200 μL of saline, FSC hydrogel, and GCZ/AFS hydrogel were dispersed on the wound surface, respectively. Body weight and wound appearance were measured every other day. All mice were killed on the 9th day after infection, and the infected wound tissue was collected.

The results in Figure 13 show that the wounds of the mice in the blank control group and the model group still could not heal after 9 days of treatment with normal saline, and the wound area of the mice in the model group was larger, which indicates that wound healing after MRSA infection is more difficult. After FSC hydrogel treatment, most of the wounds of the mice healed; and after GCZ/AFS hydrogel treatment, the back wounds of the mice almost completely healed. The above shows that GCZ/AFS hydrogel can have an excellent effect in promoting wound healing and can be further used as wound dressings for diabetic patients.

Claims (6)

1. A thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes, characterized in that: the bacteria are methicillin-resistant Staphylococcus aureus; the type 1 diabetes model used is laboratory-induced diabetic ICR mice, with fasting blood glucose ≥ 16.7 mmol/L; the hydrogel is a thermosensitive hydrogel formed by self-assembly of GOx@Cu-ZIF-8, alder ketone micelles, sodium alginate, Pluronic F-127 and _CaCl2 , which can completely heal the wound on the 9th day. 2. The thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes according to claim 1 is characterized in that: the alnus ketone micelles contained in the hydrogel can inhibit the formation of staphyloxanthin in MRSA strains, making them more susceptible to killing by reactive oxygen; GOx@Cu-ZIF-8 can react with glucose to produce H ₂ O ₂ , and the copper ions in the material can decompose H ₂ O ₂ into ·OH with stronger bactericidal effect through a Fenton-like reaction; F127, the hydrogel network formed by sodium alginate and calcium ions can enable the alnus ketone micelles and GOx@Cu-ZIF-8 to exert a stronger synergistic bactericidal effect, with a MIC value of 32 μg/mL. 3. The thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes according to claim 1, characterized in that GOx@Cu-ZIF-8 can retain 85-90% of the activity of glucose oxidase. 4. The method for preparing a thermosensitive hydrogel for promoting wound healing of bacterial infection in patients with type 1 diabetes according to claim 1, characterized in that it comprises the following steps: Preparation of alder ketone micelles: Alder ketone and F127 were prepared into tetrahydrofuran solutions of 100 mg/mL and 50 mg/mL, respectively; Alder ketone solution was added to the F127 solution, and then the volume was adjusted to 1 mL with tetrahydrofuran; then, the above solution was added to deionized water for ultrasonication, and then placed in a fume hood; Alder ketone micelles were dialyzed in a dialysis bag, and the external deionized water was replaced several times during the process; GOx@Cu-ZIF-8 was added to deionized water and ultrasonically dissolved, and then _CaCl2 was added and stirred evenly; F127 was added to the above solution and stirred in an ice bath until completely dissolved; then the prepared alnus ketone micelles were added to the above system and stirred evenly; subsequently, sodium alginate was added to the above mixed solution and stirred evenly in an ice bath. 5. The use of the hydrogel according to claim 2 in wound healing, characterized in that the method for treating wound healing is to spray the low-temperature stored liquid hydrogel on the wound of mice infected with MRSA to directly form a gel film to sterilize and promote wound healing. 6. The hydrogel according to claim 3 is characterized in that it is temperature-sensitive, the hydrogel is liquid at a temperature below 25°C, and can quickly form a gel at a temperature above 25°C, and the higher the temperature, the faster the gelation speed, and the gelation time is about 30 seconds in an environment of 37°C.