Photosensitive antibacterial and hemostatic patch with activated immune response, preparation method and application

Abstract

This invention belongs to the field of biomedical engineering and discloses a photo-activated immune response antibacterial hemostatic patch, its preparation method, and its application. The invention involves modifying silk fibroin sequentially with methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate and methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate, followed by electrospinning to prepare a fiber membrane. This membrane is then treated with anhydrous ethanol to obtain the antibacterial hemostatic patch. This antibacterial silk fibroin hemostatic patch adheres tightly to damaged tissue under ultraviolet light, achieving rapid hemostasis. After application, under near-infrared light irradiation, it precisely activates the immune response at the site of infection and, together with the reactive oxygen species generated by dihydroporphyrin E6 in the fiber membrane, synergistically kills drug-resistant bacteria on the wound surface, significantly improving the photodynamic therapy effect and promoting wound repair, achieving good wound healing results in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and relates to an antibacterial and hemostatic patch that activates immune response by light, its preparation method, and its application. Background Technology

[0002] Commercial wound dressings, such as gauze, gelatin sponges, and bandages, can effectively stop bleeding from superficial wounds by sealing the wound surface. However, deep tissue wounds, including those involving the liver, fragile tissues, or vascular tissues, are difficult to treat with manual pressure. Therefore, using these commercial hemostatic agents often fails to achieve ideal hemostasis. Furthermore, injured sites are usually associated with a high risk of bacterial infection. For example, after burns, antibiotic-resistant bacterial infection is a major complication in burn patients, with a high incidence rate and a leading cause of death. Therefore, combating antibiotic-resistant bacterial infection is a crucial aspect of burn patient treatment.

[0003] Silk fibroin, a natural protein extracted from silkworm cocoons, possesses adjustable mechanical strength, low immunogenicity, and good biocompatibility, and has been widely used in regenerative medicine. In hemostasis, silk fibroin can bind to fibrinogen and platelets, promoting the body's coagulation response. However, its lack of effective antibacterial activity limits its further application in hemostasis and wound healing.

[0004] Photodynamic therapy (PDT) is considered an effective treatment for killing drug-resistant bacteria. It utilizes the reactive oxygen species generated by photosensitizers under light irradiation to accelerate bacterial membrane rupture and inactivate bacterial DNA and proteins, thereby killing bacteria (such as Staphylococcus aureus) and preventing abscesses. It boasts advantages such as high efficiency, precise timing and location, and low invasiveness to normal tissues. However, photosensitizers are rapidly metabolized by the body, resulting in short-lasting and poor antibacterial effects. Furthermore, PDT alone has relatively poor bacterial killing efficacy in clinical settings.

[0005] Therefore, there is an urgent need to develop multifunctional wound hemostatic materials that combine rapid hemostasis and excellent antibacterial activity to improve the prognosis of clinical patients. Summary of the Invention

[0006] In view of this, the present invention aims to provide a photo-activated immune response antibacterial hemostatic patch, its preparation method, and its application. This antibacterial hemostatic patch can adhere tightly to damaged tissue under ultraviolet light irradiation, achieving rapid hemostasis. After application, utilizing the high positioning accuracy of light, under near-infrared light irradiation, it precisely activates the immune response at the patient's infection site. This, along with the reactive oxygen species generated by dihydroporphyrin E6 in the antibacterial hemostatic patch, synergistically kills drug-resistant bacteria on the wound surface, significantly improving the photodynamic therapy effect and promoting wound repair, achieving good wound healing results in clinical practice.

[0007] The technical solution adopted in this invention is as follows:

[0008] A method for preparing a photosensitive, immune-activated antibacterial and hemostatic patch includes the following steps:

[0009] S1. Methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate was added to an aqueous solution of silk fibroin and stirred at room temperature in the dark until completely dissolved. After the reaction was complete, the solution was dialyzed and lyophilized to obtain silk fibroin modified with 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate.

[0010] S2,4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyric acid-modified silk fibroin was dissolved in water, and dihydroporphyrin E6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark until completely dissolved. After the reaction was complete, the solvent was removed by rotary evaporation, dialyzed, and lyophilized to obtain dihydroporphyrin E6-modified o-nitrobenzyl silk fibroin.

[0011] S3. The dihydroporphyrin e6-modified o-nitrobenzyl fibroin is dissolved in hexafluoroisopropanol to obtain a spinning solution, and electrospinned to obtain a fiber membrane. The fiber membrane is treated with anhydrous ethanol and then air-dried to obtain an antibacterial hemostatic patch.

[0012] Furthermore, in step S1, the concentration of the silk fibroin aqueous solution is 0.15 g / mL.

[0013] Furthermore, in step S1, the ratio of methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate to the aqueous solution of silk fibroin is (0.15~1.5g):100mL.

[0014] Furthermore, in step S1, the reaction time is 48 hours.

[0015] Furthermore, in step S2, the mass ratio of dihydroporphyrin e6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1:1, and the ratio of the silk fibroin modified with 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyric acid, dihydroporphyrin e6, and water is 15g:(0.01~0.1)g:100mL.

[0016] Furthermore, in step S2, the reaction time is 12 hours.

[0017] Furthermore, the dialysis is performed using ultrapure water for 3 hours.

[0018] Furthermore, the ratio of the dihydroporphyrin e6-modified o-nitrobenzyl fibroin to hexafluoroisopropanol is (5-25) g: 100 mL.

[0019] Furthermore, the electrospinning specifically involves spraying the material at a feed rate of 1 mL / h onto a flat collector 15 cm away from the spray nozzle under a high voltage electric field of 10 kV.

[0020] The present invention also provides an antibacterial hemostatic patch prepared by the above-described preparation method.

[0021] The present invention also provides the application of the above-mentioned antibacterial hemostatic patch in functional hemostatic materials. The specific method of application is as follows: irradiation with 350-400nm ultraviolet light for 2-5 minutes, followed by irradiation with 600-700nm near-infrared light for 5-10 minutes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The product of this invention exhibits enhanced mechanical properties through increased internal chemical cross-linking after ultraviolet light irradiation. Within two minutes of ultraviolet light irradiation, it undergoes a photochemical reaction with amino groups in tissues, adhering to the tissue and creating a wound-sealing effect for effective hemostasis. Simultaneously, utilizing the ability of silk fibroin to recruit macrophages, this product generates inflammatory-activating molecules (reactive oxygen species) under near-infrared light irradiation, activating the immune response at the affected area. This, combined with the generated reactive oxygen species, further treats bacterial infections of the wound, significantly improving the efficiency of photodynamic antibacterial therapy. Attached Figure Description

[0024] Figure 1 The graph shows the in vivo hemostatic performance test results of the antibacterial hemostatic patch prepared in Example 1.

[0025] Figure 2 The image shows the results of the photoactivated immune response test of the antibacterial hemostatic patch prepared in Example 1.

[0026] Figure 3 This refers to the in vivo anti-drug-resistant bacterial properties of the antibacterial hemostatic patch prepared in Example 1. Detailed Implementation

[0027] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0028] Example 1

[0029] Silk fibroin was dissolved in ultrapure water to obtain a solution of 0.15 g / mL. Then, methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate (1.5 g) was added to 100 mL of the silk fibroin solution. The mixture was stirred at room temperature in the dark until completely dissolved and reacted for 48 hours. After dialyzing with ultrapure water for 3 days, the silk fibroin modified with 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate was obtained by lyophilization.

[0030] The 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyric acid-modified silk fibroin was dissolved in ultrapure water to obtain a solution of 0.15 g / mL. Then, dihydroporphyrin E6 (0.1 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.1 g), and N-hydroxysuccinimide (0.1 g) were added to 100 mL of the solution. The mixture was stirred at room temperature in the dark until completely dissolved and reacted for 12 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was dialyzed against ultrapure water for 3 days. The resulting product was then lyophilized to obtain dihydroporphyrin E6-modified o-nitrobenzyl silk fibroin.

[0031] Using electrospinning technology, the above-mentioned dihydroporphyrin E6-modified o-nitrobenzyl fibroin was dissolved in hexafluoroisopropanol to obtain a solution with a concentration of 0.25 g / mL. Under high voltage (10 kV) electric field conditions, it was sprayed onto a flat collector 15 cm away from the spray nozzle at a feed rate of 1 mL / h to obtain a fiber membrane. The membrane was then soaked in anhydrous ethanol solution for 4 hours to remove impurities and then air-dried.

[0032] Example 2

[0033] Silk fibroin was dissolved in ultrapure water to obtain a solution of 0.15 g / mL. Then, methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate (0.15 g) was added to 100 mL of the silk fibroin solution. The mixture was stirred at room temperature in the dark until completely dissolved and reacted for 48 hours. After dialyzing with ultrapure water for 3 days, the silk fibroin modified with 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate was obtained by lyophilization.

[0034] The 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyric acid-modified silk fibroin was dissolved in ultrapure water to obtain a solution of 0.15 g / mL. Then, dihydroporphyrin E6 (0.1 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.1 g), and N-hydroxysuccinimide (0.1 g) were added to 100 mL of the solution. The mixture was stirred at room temperature in the dark until completely dissolved and reacted for 12 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was dialyzed against ultrapure water for 3 days. The resulting product was then lyophilized to obtain dihydroporphyrin E6-modified o-nitrobenzyl silk fibroin.

[0035] Using electrospinning technology, the above-mentioned dihydroporphyrin E6-modified o-nitrobenzyl fibroin was dissolved in hexafluoroisopropanol to obtain a solution with a concentration of 0.25 g / mL. Under high voltage (10 kV) electric field conditions, it was sprayed onto a flat collector 15 cm away from the spray nozzle at a feed rate of 1 mL / h to obtain a fiber membrane. The membrane was then soaked in anhydrous ethanol solution for 4 hours to remove impurities and then air-dried.

[0036] Example 3

[0037] Silk fibroin was dissolved in ultrapure water to obtain a solution of 0.15 g / mL. Then, methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate (1.5 g) was added to 100 mL of the silk fibroin solution. The mixture was stirred at room temperature in the dark until completely dissolved and reacted for 48 hours. After dialyzing with ultrapure water for 3 days, the silk fibroin modified with 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyrate was obtained by lyophilization.

[0038] The 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyric acid-modified silk fibroin was dissolved in ultrapure water to obtain a solution of 0.15 g / mL. Then, dihydroporphyrin E6 (0.01 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.01 g), and N-hydroxysuccinimide (0.01 g) were added to 100 mL of the solution. The mixture was stirred at room temperature in the dark until completely dissolved and reacted for 12 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was dialyzed against ultrapure water for 3 days. The resulting product was then lyophilized to obtain dihydroporphyrin E6-modified o-nitrobenzyl silk fibroin.

[0039] Using electrospinning technology, the above-mentioned dihydroporphyrin E6-modified o-nitrobenzyl fibroin was dissolved in hexafluoroisopropanol to obtain a solution with a concentration of 0.25 g / mL. Under high voltage (10 kV) electric field conditions, it was sprayed onto a flat collector 15 cm away from the spray nozzle at a feed rate of 1 mL / h to obtain a fiber membrane. The membrane was then soaked in anhydrous ethanol solution for 4 hours to remove impurities and then air-dried.

[0040] Test Example 1: Tensile Strength Test of Fiber Membrane After Ultraviolet Irradiation

[0041] The samples (antibacterial hemostatic patches) prepared in Examples 1-3 were cut into rectangles with a length of 3 cm and a width of 1 cm, and then subjected to 365 nm ultraviolet light (50 mW cm⁻¹). -2 After irradiation for 2 minutes, a universal electronic tensile tester was used to measure the tensile strength at a displacement rate of 1 mm / min until the material broke. The results are shown in Table 1.

[0042] Test Example 2: Test of tissue adhesion strength of fibrous membrane after ultraviolet light irradiation

[0043] The sample (antibacterial hemostatic patch) prepared in Example-3 was cut into rectangles with a length of 1 cm and a width of 3 cm. One end of each rectangle was placed on pig skin tissue and exposed to 365 nm ultraviolet light (50 mW cm⁻¹). -2 After irradiation for 2 minutes, the tissue was stretched at one end using a universal electronic stretching machine, and the other end was stretched at a displacement rate of 1 mm / min to determine its tensile strength. The results are shown in Table 1.

[0044] Table 1. Tensile strength and tissue adhesion of the samples prepared in Examples 1-3

[0045] sample Example 1 Example 2 Example 3 Tensile strength (MPa) 0.27 0.1 0.3 Tissue adhesion strength (kPa) 68 5 74

[0046] Test Example 3: Using a mouse liver model to test the in vivo hemostatic performance of the product.

[0047] ICR mice were anesthetized, and their livers were exposed. The liver was punctured using a syringe, and the wound was covered with the antibacterial hemostatic dressing prepared in Example 1. The wound was then irradiated with ultraviolet light (50mW cm⁻²) for 2 minutes. Finally, pre-weighed filter paper was used to absorb the blood seeping from the treated wound, and the amount of bleeding was recorded and compared with the amount of bleeding from an untreated wound. Figure 1 As shown, untreated mice ( Figure 1 The left figure shows a blood loss of 26.5 mg. Mice treated with the antibacterial hemostatic patch prepared in Example 1 ( Figure 1 (See right figure) Blood loss was only 3.7 mg, proving that it has good hemostatic properties.

[0048] Test Example 4: Using a mouse back skin injury model, we tested the product's ability to activate the immune response at the affected area under near-infrared light.

[0049] The antibacterial hemostatic patch prepared in Example 1 was used to cover the wound on the back of mice, and ultraviolet light (50mW cm⁻¹) was applied. -2 After irradiating for 2 minutes to allow the fibrous membrane to adhere to the wound, 660nm near-infrared light (20mW cm⁻¹) was used. -2 The affected area was irradiated with fibrous membrane for 10 minutes, and 12 hours later, tissue from the affected area was taken for immunofluorescence staining to observe the activation of immune cells, and compared with mouse tissue with fibrous membrane attached but not treated with 660nm near-infrared light. Figure 2 As shown, the red fluorescent markers represent activated macrophages. The left image shows mouse tissue that has not been treated with 660nm near-infrared light, while the right image shows mouse tissue that has been treated with 660nm near-infrared light. It can be seen that after near-infrared light treatment, macrophages at the wound site are significantly activated, proving that the product has good photoactivated immune response performance.

[0050] Test Example 5: The product's anti-drug resistance performance under near-infrared light was tested using a mouse dorsal skin lesion infection model.

[0051] The antibacterial hemostatic patch prepared in Example 1 was used to cover the infected wound on the back of mice, and ultraviolet light (50mW cm⁻¹) was applied. -2 After irradiating for 2 minutes to allow the fibrous membrane to adhere to the wound, 660nm near-infrared light (20mW cm⁻¹) was used. -2 The affected area was irradiated with fibrous membrane for 10 minutes, and 3 days later, tissue samples were taken from the affected area for bacterial culture and compared with mouse tissue that had fibrous membrane attached but had not been treated with 660nm near-infrared light. Figure 3 As shown, the left image is the result of mouse tissue without 660nm near-infrared light treatment, and the right image is the result of mouse tissue treated with 660nm near-infrared light. It can be seen that after near-infrared light treatment, the number of drug-resistant bacteria at the wound site decreased significantly, proving that the antibacterial and hemostatic patch provided by this invention has good photo-antibacterial properties.

Claims

1. A method of preparing an antibacterial hemostatic patch film for photoactivation of immune response, characterized in that, Comprising the following steps: S1. Methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoate is added to a silk fibroin aqueous solution, stirred at room temperature in the dark until completely dissolved, after the reaction is complete, dialysis, lyophilization, to obtain 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid modified silk fibroin; S2. The 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid modified silk fibroin is dissolved in water, and chlorin e6, 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added, stirred at room temperature in the dark until completely dissolved, after the reaction is complete, dialysis after removing the solvent by rotary evaporation, lyophilization, to obtain chlorin e6 modified o-nitrobenzyl silk fibroin; S3. The chlorin e6 modified o-nitrobenzyl silk fibroin is dissolved in hexafluoroisopropanol to obtain a spinning solution, electrospinning to obtain a fiber membrane, the fiber membrane is treated with anhydrous ethanol solution, then air dried, to obtain an antibacterial hemostatic patch membrane.

2. The production method according to claim 1, characterized by, In step S1, the concentration of the silk fibroin aqueous solution is 0.15 g / mL.

3. The production method according to claim 1, characterized by, In step S1, the amount ratio of methyl 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoate to the silk fibroin aqueous solution is (0.15-1.5) g:100 mL.

4. The method of claim 1, wherein, In step S1, the reaction time is 48 h.

5. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of chlorin e6, 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:1:1, and the amount ratio of the 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid modified silk fibroin, chlorin e6 and water is 15 g:(0.01-0.1) g:100 mL.

6. The method of claim 1, wherein, In step S2, the reaction time is 12 h.

7. The preparation method according to claim 1, characterized in that, The amount ratio of the chlorin e6 modified o-nitrobenzyl silk fibroin to hexafluoroisopropanol is (5-25) g:100 mL.

8. The method of claim 1, wherein, The electrospinning is specifically: under the condition of an electric field high voltage of 10 kV, the feeding speed is 1 mL / h, and the planar collector is sprayed to a distance of 15 cm from the spraying port.

9. The antibacterial hemostatic patch membrane prepared by the preparation method of any one of claims 1-8.

10. The application of the antibacterial hemostatic patch membrane of claim 9 in functional hemostatic materials, and the application method is specifically: irradiating with 350-400 nm ultraviolet light for 2-5 minutes, and then irradiating with 600-700 nm near-infrared light for 5-10 minutes.

Images