Preparation method and application of in-situ electrospinning photodynamic nanofiber dressing for promoting healing of diabetic wounds
Optical nano -fiber dressing prepared through the in -situ static spinning technology, combined with silk protein, brilliant, polylastic acid and photoretinomic agent TPIS to solve the problem of difficulty healing of diabetic foot ulcer wounds and multiple drug resistance bacteria, realizing efficient efficiency The effect of sterilization and hemostasis has promoted wound healing.
Patent Information
- Application Number
- CN202411125323.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-16
- Publication Date
- 2025-01-24
AI Technical Summary
It is difficult to heal the wound of diabetic foot ulcers. Long-term use of traditional antibiotics can easily lead to the emergence of multidrug-resistant bacteria, and the use of photosensitizers directly at the wound has difficulty in aggregation and recycling.
The in -situ static spinning technology combines silk protein, glue, polymapilic acid and photoretinomic agent TPIS to prepare optical nano -fiberic dressing to achieve one -stop sterilization and hemostatic effect.
This dressing has excellent antibacterial properties, can effectively inhibit bacterial growth, promote wound healing, and at the same time has good fit and safety, avoiding multiple drug resistance problems of traditional treatment methods.
Smart Images
Figure CN119345435A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedical materials, and in particular to a preparation method and application of an in-situ electrospun photodynamic nanofiber dressing for promoting diabetic wound healing. Background Art
[0002] As one of the most common diseases in the world, diabetes has a high mortality rate and complex complications. Among them, diabetic foot ulcers are a typical chronic skin trauma with difficult wound healing. Wounds associated with diabetic foot ulcers are difficult to repair, which is closely related to factors such as wound infection, changes in inflammatory response, lack of extracellular matrix, and failure of angiogenesis. Studies have shown that methicillin-resistant Staphylococcus aureus is one of the main sources of infection in diabetic patients. Traditional antibiotics are the most common treatment for preventing bacterial infection of diabetic wounds. However, the healing cycle of diabetic foot ulcers is long, and long-term use of antibiotics increases the possibility of the emergence of multidrug-resistant bacteria.
[0003] Antimicrobial photodynamic therapy is a method in which photosensitizers produce highly destructive reactive oxygen species under light, which damage bacterial membrane structures, enzymes, proteins, nucleic acids, etc., causing bacterial death, and have the same killing effect on multidrug-resistant bacteria. Photosensitizers can be combined with light exposure time and light exposure position to achieve the controllability of treatment. Compared with antibiotics, they have a broad-spectrum bactericidal effect, have less adverse reactions and damage to host tissue cells, and can be used repeatedly and safely. However, directly using powdered or granular photosensitizers on diabetic infected wounds is prone to aggregation and difficult to recover, and cannot achieve the effect of hemostasis. Loading photosensitizers on electrospun nanofiber dressings can obtain the unique ultrafine diameter, high porosity and high specific surface area of nanofibers while retaining the original properties of the photosensitizer. The high specific surface area and porosity of nanofiber dressings ensure the free exchange of gas, steam and liquid molecules. Nanofibers with random mesh structures similar to extracellular matrix have been shown to accelerate the process of cell adhesion and proliferation. In addition, in situ electrospinning at the wound site will not cause secondary damage to the wound, and the nanofiber dressing can fit well with the skin wound.
[0004] Therefore, the present invention combines silk fibroin, gelatin, polylactic acid and photosensitizer TPIS, and adopts the method of in-situ electrostatic spinning to prepare photodynamic nanofiber dressing to achieve one-stop sterilization and hemostasis. In addition, the method of in-situ electrostatic spinning can directly deposit on the wound surface to form a nanofiber dressing, which can be well fitted with the skin wound by the effect of electrostatic force, so that it can effectively inhibit bleeding. The dressing preparation method is simple and low in cost. With the addition of photosensitizer TPIS, the photodynamic nanofiber dressing has excellent antibacterial properties and meets the requirements of biomedical dressings. Summary of the invention
[0005] In view of the above technical problems, the purpose of the present invention is to provide a photodynamic nanofiber dressing for promoting diabetic wound healing and a preparation method thereof. The dressing has excellent antibacterial properties and is beneficial to diabetic wound healing. The dressing comprises the following steps:
[0006] (1) Extraction of silk fibroin: degummed silk was dissolved in a ternary dissolution system of calcium chloride, anhydrous ethanol and deionized water, excess impurities were removed by dialysis, and the resulting solution was freeze-dried;
[0007] (2) Preparation of nanofiber dressing spinning solution: dissolving silk fibroin, gelatin and polylactic acid in a mixed solution of hexafluoroisopropanol and formic acid, and stirring until the polymer is completely dissolved to obtain a nanofiber dressing spinning solution;
[0008] (3) Preparation of photosensitizer TPIS: The photosensitizer has a structure shown in Formula I, and its preparation method has been mentioned in the article Small2022, 18, 2203825;
[0009]
[0010] (4) Preparation of photodynamic nanofiber dressing spinning solution: mixing the photosensitizer TPIS with the spinning solution of step (2) to prepare a photodynamic nanofiber dressing spinning solution;
[0011] (5) Preparation of photodynamic nanofiber dressing: placing the photodynamic nanofiber dressing spinning solution of step (4) in a syringe of an electrospinning device, applying a high-voltage electrostatic field between the spinneret of the syringe and a grounded receiving device, the polymer spinning solution generates a jet under the action of electrostatics, and a photodynamic nanofiber dressing consisting of parallelly arranged polymer electrospun nanofibers is obtained on the roller receiving device;
[0012] Wherein, in step (1), the reaction dissolution temperature is 78-82°C, and the molar ratio of calcium chloride, anhydrous ethanol and deionized water is 1:(1-3):(9-7). Preferably, the molar ratio of the three is 1:2:8.
[0013] Wherein, in step (2), the mass ratio of silk fibroin, gelatin and polylactic acid is 9:(2-0):(1-3), and the mass ratio of hexafluoroisopropanol and formic acid is (3-7):(7-3). Preferably, the mass ratio of silk fibroin, gelatin and polylactic acid is 9:1:2, and the mass ratio of hexafluoroisopropanol and formic acid is 7:3.
[0014] Wherein, in step (4), the mass concentration of the added photosensitizer TPIS is 0.6%-1.8%. Preferably, the mass concentration of the added photosensitizer TPIS is 1.8%.
[0015] Wherein, in step (5), the electrospinning conditions used in the electrospinning process are at room temperature, the high-voltage electrostatic field is 10-20KV, the flow rate is 8-24μL / min, the distance between the spinneret and the receiving device is 12-18cm, the deposition time is 120-360min, the receiving device is a roller, and the receiving speed of the roller is 60-100r / min. Preferably, the high-voltage electrostatic field is 20KV, the flow rate is 16μL / min, the distance between the spinneret and the receiving device is 15cm, the deposition time is 240min, and the receiving speed of the roller is 80r / min.
[0016] In the present invention, the photodynamic nanofiber dressing for diabetic wound healing can be directly deposited in situ on the wound to promote wound healing.
[0017] An in-situ electrospun photodynamic nanofiber dressing for promoting diabetic wound healing using the following steps:
[0018] Diabetic rats were used to establish a diabetic chronic wound model infected with methicillin-resistant Staphylococcus aureus. The SD rats were randomly divided into 4 groups: blank control group, ND group, TPIS@ND group without light, and 6mW / cm 2 TPIS@ND group treated with white light. These rats were fed with a normal diet and had free access to water. A wound with a diameter of 1.2 cm was cut on the back of each rat, and the wound was inoculated with a solution of methicillin-resistant Staphylococcus aureus (100 μL per wound, 1 × 10 8 CFU / mL) to establish a diabetic chronic wound model infected with methicillin-resistant Staphylococcus aureus. The wounds in the experimental group were covered with in situ electrospun nanofiber dressings, while the control group was not treated and covered with gauze. The macroscopic images of the wounds in each group were observed and recorded using a mobile phone camera on days 0, 3, 6, 9, and 12, and the wound area was measured by wound boundary using Image J to evaluate the wound healing process. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 A-1B are the normalized UV absorption and fluorescence emission spectra of the photodynamic nanofiber dressing in dimethyl sulfoxide solution, respectively. Figure 1 C is the TEM image of a single fiber in ND and TPIS@ND, which confirms that the photosensitizer TPIS is successfully loaded on the nanofiber dressing.
[0020] Figure 2 The generation of reactive oxygen species by photodynamic nanofiber dressing.
[0021] Figure 3 To study the blood compatibility and cell compatibility of photodynamic nanofiber dressing.
[0022] Figure 4 To test the antibacterial properties of photodynamic nanofiber dressing.
[0023] Figure 5 A diagram showing how photodynamic nanofiber dressing promotes wound healing in diabetic SD rats. DETAILED DESCRIPTION
[0024] In order to clearly illustrate the technical features of the present invention, the present invention is further described in detail in conjunction with the following specific embodiments and drawings.
[0025] Embodiment 1:
[0026] A preparation method of photodynamic nanofiber dressing:
[0027] Step 1: Weigh 3.48 g of calcium chloride and dissolve it in a mixed solution of 1.80 mL of anhydrous ethanol and 5.08 mL of deionized water, then dissolve 1 g of degummed silk in the mixed solution at 80°C, place the dissolved solution in a dialysis bag after cooling, dialyze with distilled water for three days, and obtain silk fibroin after freeze-drying;
[0028] Step 2: Weigh 0.9 g of silk fibroin and 0.3 g of polylactic acid, dissolve all of them in a mixed solution of 2.28 mL of hexafluoroisopropanol and 6.96 mL of formic acid, then add TPIS with a mass concentration of 0.6% (7.2 mg) to the mixed solution, stir thoroughly at room temperature until completely dissolved, and prepare a spinning precursor solution;
[0029] Step 3: Add the spinning precursor solution prepared in step 2 into a 5 mL syringe (needle diameter 0.7 mm), and install it on a portable electrospinning device. Hold the device with your hand, turn on the power switch, set the high-voltage electrostatic field to 10 KV, the flow rate to 8 μL / min, adjust the distance between the spinneret and the receiving device to 12 cm, and perform spinning. Deposit the nanofiber dressing TPIS@ND directly on the drum receiving device with a receiving speed of 60 r / min, and the deposition time is 120 min.
[0030] Embodiment 2:
[0031] A preparation method of a photodynamic nanofiber dressing:
[0032] Step 1: Weigh 3.20 g of calcium chloride and dissolve it in a mixed solution of 3.32 mL of anhydrous ethanol and 4.15 mL of deionized water, then dissolve 1 g of degummed silk in the mixed solution at 80°C, place the dissolved solution in a dialysis bag after cooling, dialyze with distilled water for three days, and obtain silk fibroin after freeze-drying;
[0033] Step 2: Weigh 0.9 g of silk fibroin, 0.1 g of gelatin and 0.2 g of polylactic acid, and dissolve all of them in a mixed solution of 3.80 mL of hexafluoroisopropanol and 4.97 mL of formic acid, then add TPIS with a mass concentration of 1.2% (14.4 mg) to the mixed solution, stir thoroughly at room temperature until completely dissolved, and prepare a spinning precursor solution;
[0034] Step 3: Add the spinning precursor solution prepared in step 2 into a 5 mL syringe (needle diameter 0.7 mm), and install it on a portable electrospinning device. Hold the device with your hand, turn on the power switch, set the high-voltage electrostatic field to 15 KV, the flow rate to 16 μL / min, adjust the distance between the spinneret and the receiving device to 15 cm, and perform spinning. Deposit the nanofiber dressing TPIS@ND directly on the drum receiving device with a receiving speed of 80 r / min, and the deposition time is 240 min.
[0035] Embodiment 3:
[0036] A preparation method of a photodynamic nanofiber dressing:
[0037] Step 1: Weigh 2.96 g of calcium chloride and dissolve it in a mixed solution of 4.61 mL of anhydrous ethanol and 3.36 mL of deionized water, then dissolve 1 g of degummed silk in the mixed solution at 80°C, place the dissolved solution in a dialysis bag after cooling, dialyze with distilled water for three days, and obtain silk fibroin after freeze-drying;
[0038] Step 2: Weigh 0.9 g of silk fibroin, 0.2 g of gelatin and 0.1 g of polylactic acid, and dissolve all of them in a mixed solution of 5.32 mL of hexafluoroisopropanol and 2.98 mL of formic acid, then add TPIS with a mass concentration of 1.8% (21.6 mg) to the mixed solution, stir thoroughly at room temperature until completely dissolved, and prepare a spinning precursor solution;
[0039] Step 3: Add the spinning precursor solution prepared in step 2 into a 5 mL syringe (needle diameter 0.7 mm), and install it on a portable electrospinning device. Hold the device with your hand, turn on the power switch, set the high-voltage electrostatic field to 20 KV, the flow rate to 16 μL / min, adjust the distance between the spinneret and the receiving device to 18 cm, and perform spinning. Deposit the nanofiber dressing TPIS@ND directly on the drum receiving device with a receiving speed of 100 r / min, and the deposition time is 360 min.
[0040] Embodiment 4:
[0041] In vitro reactive oxygen species testing, hemolysis experiments, cytotoxicity studies, antibacterial performance testing, and in vivo wound healing experiments:
[0042] (1) 2′,7′-dichlorofluorescein diacetate test for reactive oxygen species
[0043] 2′,7′-dichlorofluorescein diacetate was used as an ROS indicator to evaluate the ROS generation capacity of nanofiber dressings with different TPIS contents. 2 ) was attached to the bottom of a 6-well plate, and 50 μL of 40 μmol 2′,7′-dichlorofluorescein diacetate and 1950 μL of phosphate buffered saline solution were added to each well. Recording of white light irradiation (6 mW / cm 2 ) as a function of time.
[0044] Figure 2 2′,7′-dichlorofluorescein diacetate was used as an indicator to evaluate the generation of reactive oxygen species in nanofiber dressings with different TPIS contents. 2,7-Dichlorofluorescein diacetate showed a generally enhanced photoluminescence intensity when interacting with reactive oxygen species. 2 ), with the increase of TPIS content, the photoluminescence intensity of 2′,7′-dichlorofluorescein diacetate gradually increased, indicating that TPIS has excellent reactive oxygen species generating ability.
[0045] (2) Hemolysis experimental study
[0046] In order to evaluate the hemolysis rate of TPIS@ND, 1 mL of fresh rat blood was first washed three times with saline by centrifugation (3000 rpm, 15 min) and resuspended in phosphate buffered saline to prepare a 2% red blood cell suspension. Then 1 mL of red blood cell suspension was mixed with TPIS@ND with different TPIS contents (mass concentration 0%, 0.6%, 1.2% and 1.8%, Φ = 10 mm) in a centrifuge tube. 9.8 mL of phosphate buffered saline was added to 200 μL of red blood cell suspension as a negative control; 9.8 mL of deionized water was added to 200 μL of red blood cell suspension as a positive control. After incubating each mixed solution at 37 ° C for 2 h, it was centrifuged at 2000 rpm for 5 min, and the absorbance of the supernatant at 540 nm was measured using an enzyme reader. The hemolysis rate was calculated.
[0047] Figure 3 AHemolysis evaluation showed that the nanofiber dressing had no hemolysis and no damage to rat erythrocytes.
[0048] (3) Cytotoxicity test
[0049] The cytotoxicity of nanofiber dressings on L929 and NIH-3T3 cells was investigated by CCK-8 staining and live / dead kit staining. Briefly, nanofiber dressing samples with a diameter of 6 mm were placed in a 96-well culture plate and sterilized using UV light for 24 h. Subsequently, the cell suspension was incubated at 1 × 10 4 The density of the bacteria was inoculated on the sample surface and incubated at 37°C and 5% CO2 for 24, 48, and 72 h to study the viability.
[0050] Figure 3 B-3C are the cell viability counts of NIH-3T3 cells and L929 cells cultured on nanofiber dressings with different TPIS contents on days 1, 2, and 3. It can be seen that there is no statistical difference in cell proliferation at three time points on nanofiber dressings with different TPIS contents, proving that they have good biocompatibility.
[0051] (4) Antibacterial performance test
[0052] The plate colony counting method was used to evaluate the in vitro antibacterial activity of TPIS@ND against Escherichia coli and methicillin-resistant Staphylococcus aureus. Escherichia coli and methicillin-resistant Staphylococcus aureus were cultured in liquid culture medium at 37°C overnight and collected after centrifugation. Then, the obtained bacterial suspension (10 8 CFU / mL) was diluted tenfold twice, and 1 mL of the diluted bacterial suspension was mixed with TPIS@ND (2×2 cm 2 ) were mixed in a 6-well plate and incubated at 37°C for 1 h. A fluorescent lamp was used to illuminate the plate with 6 mW / cm 2 Irradiation for 15 min. After incubation at 37 °C for 2 h, the suspension was diluted tenfold twice, and then 100 μL of the diluted bacterial suspension was smeared on the solid culture medium. After culturing in a constant temperature incubator at 37 °C for 24 h, the colonies were counted using the conventional plate counting method. The antibacterial activity was evaluated at a fixed irradiation time of 15 min and TPIS@ND with different TPIS contents (mass concentrations of 0%, 0.6%, 1.2% and 1.8%). The morphology of bacteria was observed by scanning electron microscopy imaging. Double staining of cell apoptosis was performed with AO / PI and observed using a confocal microscope. In order to quantify the amount of protein released by bacteria treated with TPIS@ND, the bacterial protein concentration after TPIS@ND treatment was measured using a BCA protein concentration assay kit. The changes in the level of intracellular ATP synthesis after TPIS@ND treatment were measured using an enhanced ATP detection kit.
[0053] Figure 4 A~ Figure 4E are photos of Escherichia coli and methicillin-resistant Staphylococcus aureus grown on plates treated with nanofiber dressings with different TPIS contents, scanning electron microscope images, bacterial survival rate, protein content released by bacteria after BCA detection, and changes in intracellular ATP synthesis levels. The results show the excellent antibacterial activity of nanofiber dressings, indicating that they can be used as a powerful antibacterial material to prevent wound infection.
[0054] (5) In vivo wound healing experimental study
[0055] 6-8 week old SD male rats weighing 200-220g were selected. After three days of feeding with ordinary feed, a diabetes model was established by intraperitoneally injecting streptozotocin (60mg / kg) into rats that had fasted overnight for three consecutive days. The blood glucose level was measured through the tail vein using a blood glucose meter on the second day after the injection. Rats with blood glucose higher than 16.7mmol / L were identified as diabetic models for further experiments. Next, a diabetic chronic wound model infected with methicillin-resistant Staphylococcus aureus was established using diabetic rats. The SD rats were randomly divided into 4 groups: blank control group, ND group, TPIS@ND(-L) group and TPIS@ND group (treated with 6mW / cm 2 White light treatment). The rats were fed with a normal diet and had free access to water. After anesthesia, the SD rats were fixed on the operating table, and the diabetic rats were shaved and depilated with a razor and depilatory cream to fully expose the back. The surgical area was disinfected with 75% alcohol. Then a wound with a diameter of 1.2 cm was cut on the back of each rat with a scalpel, and the MRSA solution (100 μL per wound, 1×10 8 CFU / mL), the wound was covered with gauze for 8 hours to prevent wound infection and establish a diabetic chronic wound model infected with methicillin-resistant Staphylococcus aureus. The wounds in the experimental group were covered with in situ electrospun nanofiber dressings, while the control group was not treated and covered with gauze. The light group was irradiated with 6mW / cm 2 The wounds of each group were treated with white light for 30 min, and the other groups were kept in the dark. The macroscopic images of the wounds of each group were observed and recorded using a mobile phone camera on days 0, 3, 6, 9, and 12, and the wound area was measured by wound boundary using Image J to evaluate the wound healing process.
[0056] Figure 5 A shows the appearance photos of the blank group, ND group, TPIS@ND group under dark conditions, and TPIS@ND group on days 0, 3, 6, 9, and 12 after surgery. Figure 5 B is the relative wound area of wounds at different time points (the wound area of all groups on day 0 is set as 100%). -2 Under light, the TPIS@ND group had a significant effect in promoting wound healing compared with other groups.
[0057] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. Preparation of an in situ electrospun photodynamic nanofiber dressing for promoting diabetic wound healing, characterized in that: The photodynamic nanofiber dressing is composed of silk fibroin, gelatin, and polylactic acid, and is prepared by in-situ electrostatic spinning with the photosensitizer TPIS.
2. The method for preparing the photodynamic nanofiber dressing according to claim 1, characterized in that: The photodynamic nanofiber dressing is made by the following method: (1) dissolving the degummed silk in a ternary dissolution system of calcium chloride, anhydrous ethanol and deionized water, removing excess impurities by dialysis, and freeze-drying the resulting solution to obtain silk fibroin; (2) dissolving silk fibroin, gelatin, and polylactic acid in hexafluoroisopropanol and formic acid, and stirring to obtain a mixed solution; (3) performing in-situ electrospinning on the spinning precursor solution of step (2) to collect and obtain a nanofiber dressing, which is named ND; (4) adding photosensitizer TPIS to the mixed solution and stirring to obtain a spinning precursor solution; (5) The spinning precursor solution is subjected to in-situ electrospinning, and the photodynamic nanofiber dressing is collected and named TPIS@ND.
3. The method according to claim 2, characterized in that In step (1), the dissolution temperature is 78-82° C., and the molar ratio of calcium chloride, anhydrous ethanol and deionized water is 1:(1-3):(7-9).
4. The method according to claim 2, characterized in that In step (2), the mass ratio of the silk fibroin, gelatin and polylactic acid is 9:(2-0):(1-3).
5. The method according to claim 2, characterized in that In step (2), the mass ratio of hexafluoroisopropanol to formic acid is (3-7): (7-3).
6. The method according to claim 2, characterized in that In step (4), the mass concentration of the added photosensitizer TPIS is 0.6%-1.8%.
7. The method according to claim 2, characterized in that In step (3) and step (5), the electrospinning conditions are: a high voltage electrostatic field of 10-20 KV, a flow rate of 8-24 μL / min, a distance between the spinneret and the receiving device of 12-18 cm, a deposition time of 120-360 min, and a receiving device of a roller with a receiving speed of 60-100 r / min.
8. The method according to claim 1, characterized in that The wound is a wound caused by diabetes, and the diabetes is type I diabetes.
9. The method according to claim 1, characterized in that The fiber diameter of the prepared photodynamic nanofiber dressing is 300-800nm.
10. The preparation of an in situ electrospun photodynamic nanofiber dressing for promoting diabetic wound healing as claimed in claim 1, wherein the photodynamic nanofiber dressing is directly electrospun and deposited on the wound without directly contacting the wound through other routes.