Light-operated gas release type antibacterial microneedle patch and preparation method thereof

Abstract

The invention relates to a light-operated gas release type antibacterial microneedle patch and a preparation method thereof, and the light-operated gas release type antibacterial microneedle patch comprises the following components in percentage by mass: 5%-10% of sodium alginate and 90%-95% of ferroferric dodecacarbonyl. The compactness of a bacterial biofilm of an infected wound surface can be destroyed through the puncture effect of the microneedle patch so as to overcome the hindering effect of the bacterial biofilm on medicine permeation, and effective penetration of an infected layer of the wound surface is achieved. Then, by regulating and controlling the irradiation power and the irradiation duration of near-infrared laser and combining with the change of the loading capacity of the dodecacarbonyl ferroferric nano material in the microneedle patch, the transition of electrons of the material from a valence band to a conduction band of the dodecacarbonyl ferroferric nano material rich in carbonyl in the microneedle under the action of near-infrared light irradiation can be realized; therefore, a large number of carbonyl groups in the dodecacarbonyl ferroferric nano material form carbon monoxide to kill bacteria.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a light-controlled gas-releasing antibacterial microneedle patch and a preparation method thereof. Background Art

[0002] Wound infection is one of the important factors affecting wound healing. As a common wound complication, infection seriously affects the healing speed and quality of wounds. Generally, wound infection can be divided into acute wound infection and chronic wound infection. Acute wound infection is clinically manifested as redness, swelling, pain, increased skin temperature, exudation of limb fluid or bloody fluid, and even the formation of sinus tracts or swelling. Chronic wound infection has a longer course, usually more than one month, with multiple recurrences and lack of specific manifestations. Common chronic wound infection symptoms include diabetic foot, pressure sores, etc. Wound infection can cause many hazards, such as difficulty in wound healing, formation of cellulitis, and even sepsis, mental disorder, etc., which seriously threaten the life and health of patients.

[0003] In the related art, the methods for preventing and treating wound infection mainly include the use of antibiotics, antibacterial preparations, medical hydrogen peroxide, phage therapy, etc., but they all still have different problems. For example, bacteria are prone to develop resistance to antibiotics, and the use of antibacterial preparations is accompanied by certain toxic side effects. In addition, the use of medical hydrogen peroxide is limited to external use, and hydrogen peroxide is easy to decompose to produce high concentrations of reactive oxygen species (ROS), which will damage normal tissues while killing pathogenic microorganisms. Phage therapy has problems such as insufficient broad-spectrum antibacterial properties and incomplete sterilization.

[0004] Therefore, methods for treating wound infection still need to be improved. Summary of the invention

[0005] The embodiments of the present application provide a light-controlled gas-releasing antibacterial microneedle patch and a preparation method thereof, and provide a new dressing product for treating wound infection, which has a good promoting effect on the recovery of the wound and can accelerate the healing of infected wounds.

[0006] The first aspect of the present application provides a light-controlled gas-releasing antibacterial microneedle patch, which comprises, by mass percentage: sodium alginate (5%-10%) and triiron dodecacarbonyl (90%-95%).

[0007] In some optional embodiments, the light-controlled gas-releasing antibacterial microneedle patch comprises:

[0008] a base substrate having a microneedle side and an opposing back side;

[0009] A solid microneedle extends from the microneedle side of the base substrate, and the solid microneedle comprises, by mass percentage: sodium alginate (5%-10%) and triiron dodecacarbonyl (90%-95%).

[0010] In some alternative embodiments, the solid microneedles have a height of 200 μm to 900 μm.

[0011] In some optional embodiments, the average spacing between the solid microneedle tips is 280 μm to 320 μm.

[0012] In a second aspect, the present application provides a method for preparing a light-controlled gas-releasing antibacterial microneedle patch, comprising:

[0013] Mixing a sodium alginate solution with a solution containing triiron dodecacarbonyl nanomaterial to prepare a mixed solution;

[0014] Filling the mixture at least once into a negative mold containing solid microneedle cavities,

[0015] The mixture placed in the negative mold is dried to prepare the light-controlled gas-releasing antibacterial microneedle patch containing solid microneedles.

[0016] In some optional embodiments, the light-controlled gas-releasing antibacterial microneedle patch comprises:

[0017] a base substrate having a microneedle side and an opposing back side;

[0018] A solid microneedle extends from the microneedle side of the base substrate.

[0019] In some optional embodiments, the mass concentration of sodium alginate is 0.5%-3%.

[0020] In some optional embodiments, the molar concentration of triiron dodecacarbonyl is 10 μM-100 μM.

[0021] In some optional embodiments, the mixture is filled into a negative mold containing solid microneedle cavities at least once in an environment of negative pressure of 0.01-0.2 MPa; optionally, the mixture is left to stand for 30-60 minutes to remove bubbles in the mixture in the negative mold.

[0022] In a third aspect, the present application provides a method for using a light-controlled gas-releasing antibacterial microneedle patch, comprising:

[0023] A light-controlled gas-releasing antibacterial microneedle patch having a solid microneedle side is applied to the wound surface, wherein the light-controlled gas-releasing antibacterial microneedle patch comprises, by mass percentage, sodium alginate (5%-10%) and triiron dodecacarbonyl (90%-95%); the patch is irradiated with a near-infrared laser of 808nm-1064nm to make the carbonyl of triiron dodecacarbonyl form carbon monoxide for killing bacteria, thereby achieving antibacterial effect on the wound surface.

[0024] This application has at least the following beneficial effects:

[0025] The light-controlled gas-release antibacterial microneedle patch of the present application mainly includes sodium alginate and dodecacarbonyl triiron nanomaterials, and its composition ratio is clear. Sodium alginate itself has certain antibacterial properties and the effect of promoting wound recovery. When using the light-controlled gas-release antibacterial microneedle, the compactness of the bacterial biofilm of the infected wound can be destroyed by the puncture of the microneedle patch to overcome its hindering effect on drug penetration, and achieve effective penetration of the wound infection layer. Subsequently, by regulating the irradiation power and irradiation duration of the near-infrared laser (808nm-1064nm) and combining the loading amount of the dodecacarbonyl triiron nanomaterial in the microneedle patch, the dodecacarbonyl triiron nanomaterial rich in carbonyl (C=O) in the microneedle can be controlled to realize the electron transition of the material from the valence band to the conduction band under the action of light energy, so that the carbonyl in the dodecacarbonyl triiron nanomaterial forms carbon monoxide to kill bacteria. It is matched with sodium alginate, which has a good promoting effect on the recovery of the wound and can accelerate the healing of the infected wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The features, advantages and technical effects of exemplary embodiments of the present application will be described below with reference to the accompanying drawings.

[0027] Figure 1 A schematic diagram of the structure of a light-controlled gas-releasing antibacterial microneedle patch in an embodiment of the present application is shown;

[0028] Figure 2 A schematic diagram of the process of preparing a light-controlled gas-releasing antibacterial microneedle patch in an embodiment of the present application is shown;

[0029] Figure 3 A microscopic observation image of a light-controlled gas-releasing antibacterial microneedle patch in an embodiment of the present application is shown;

[0030] Figure 4 A diagram showing the heating effect of the light-controlled gas-releasing antibacterial microneedle patch using near-infrared light irradiation in an embodiment of the present application is shown;

[0031] Figure 5 The graph shows the carbon monoxide release test result after using near-infrared light to illuminate the light-controlled gas-releasing antibacterial microneedle patch in the embodiment of the present application;

[0032] Figure 6 The biotoxicity of the light-controlled gas-releasing antibacterial microneedle patch against wound infection to human immortalized Hacat cells using the Cell Counting Kit-8 cell counting method in the embodiment of the present application is shown;

[0033] Figure 7 The antibacterial performance test of the light-controlled gas-releasing antibacterial microneedle patch on the wound surface according to the embodiment of the present application is shown.

[0034] Figure 8 The live-dead fluorescent staining observation effect of Escherichia coli after being treated with different experimental groups in the examples of the present application is shown.

[0035] Fig. 9 The photographic observation pictures of the wounds of mice infected with different treatment groups at different time points in the examples of the present application are shown.

[0036] Fig.10 A diagram showing the healing of wound tissue after wound treatment in mice observed using the hematoxylin-eosin staining method in an embodiment of the present application is shown. DETAILED DESCRIPTION

[0037] The following is a further detailed description of the implementation of the present application in conjunction with the drawings and examples. The following description of the examples and the drawings are used to exemplarily illustrate the principles of the present application, but cannot be used to limit the scope of the present application, that is, the present application is not limited to the described embodiments.

[0038] For ease of description, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unambiguous range; and any lower limit can be combined with other lower limits to form an unambiguous range, and any upper limit can be combined with any other upper limit to form an unambiguous range. In addition, although not explicitly stated, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unambiguous range.

[0039] In the description of the present application, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number, and the “multiple” in “one or more” means two or more than two.

[0040] In the description of the present application, it should be noted that, unless otherwise specified, “first”, “second”, “third”, etc. are only used for distinction and do not indicate their order.

[0041] The above application content of the present application is not intended to describe each disclosed embodiment or each implementation in the present application. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided by a series of examples, which can be used in various combinations. In each example, enumeration is only used as a representative group and should not be interpreted as exhaustive.

[0042] In the process of wound infection, pathogenic microorganisms gradually form biofilms through adsorption, reproduction, secretion of glycoproteins, mucopolysaccharides, etc., which are "passivated" and escape the killing of drugs. The light-controlled gas-release antibacterial microneedle patch proposed in the patent of this invention can effectively penetrate the bacterial biofilm and kill pathogenic microorganisms in infected wounds of a certain depth, which is of great significance for the prevention and treatment of wound infection.

[0043] The first aspect of the present application provides a light-controlled gas-releasing antibacterial microneedle patch, which comprises, by mass percentage: sodium alginate (5%-10%) and triiron dodecacarbonyl (90%-95%).

[0044] Sodium alginate is a natural polysaccharide extracted from the cell wall of brown algae (such as kelp and laver). It is an anionic bio-glue with many carboxyl and hydroxyl groups in its molecular structure, which gives it good adhesion properties. These functional groups can interact with some groups on the surface of microorganisms, causing sodium alginate to form a protective covering layer on the surface of microorganisms, preventing microorganisms from attaching and growing. Sodium alginate has certain antioxidant properties, which may help inhibit the growth of some microorganisms that require an oxidative environment. Sodium alginate has good water absorption, water retention, and moisturizing properties, and can absorb and retain moisture. Forming a moist environment on the wound surface helps prevent the wound from drying out. At the same time, sodium alginate can promote cell proliferation and migration, which helps accelerate the wound healing process. In addition, sodium alginate has a certain anti-inflammatory effect, which can reduce the inflammatory response around the wound, reduce pain, and help provide better wound comfort.

[0045] On the other hand, triiron dodecarbonyl is an organic iron compound with the molecular formula Fe 3 (CO) 12 It is an organic metal compound with an iron bond structure, which contains a large number of carbonyl groups (CO). In this compound, the carbonyl group forms a coordination bond with metallic iron through the electron pairs on its carbon and oxygen. Under high-energy infrared radiation, CO molecules may be desorbed from the carbonyl group to produce free CO gas.

[0046] In the examples of the present application, sodium alginate, which is highly biocompatible and friendly to wound healing, is used as the main material for preparing the microneedle patch. The microneedle patch is prepared by mixing sodium alginate with iron trioxide dodecacarbonyl nanomaterials, and the prepared microneedle patch is irradiated with near-infrared light to accurately release carbon monoxide on demand to kill bacteria and promote wound repair.

[0047] In some optional embodiments, the light-controlled gas-releasing antibacterial microneedle patch comprises:

[0048] a base substrate having a microneedle side and an opposing back side;

[0049] A solid microneedle extends from the microneedle side of the base substrate, and the solid microneedle comprises, by mass percentage: sodium alginate (5%-10%) and triiron dodecacarbonyl (90%-95%).

[0050] According to the embodiments of the present application, on the one hand, sodium alginate, which is highly biocompatible and friendly to wound healing, is used as the main material for preparing microneedle patches. Microneedle patches are prepared by mixing sodium alginate with iron trioxide dodecacarbonyl nanomaterials, and the prepared microneedle patches are irradiated with near-infrared light to achieve on-demand precise release of carbon monoxide to kill bacteria and promote wound repair. With the help of the unique needle tip array structure of the microneedle patch, it can easily penetrate the dense biofilm formed by bacteria on the infected wound surface, and achieve carbon monoxide gas release and sterilization with precisely controllable penetration depth, which can effectively avoid the problems of insufficient penetration performance of conventional therapeutic drugs on the bacterial biofilm formed on the wound surface and poor treatment effect.

[0051] See also Figure 1 The light-controlled gas-releasing antibacterial microneedle patch 1 is composed of a patch substrate and a microneedle tip 2. Figure 1 The middle left picture shows a top view of the light-controlled gas-releasing antibacterial microneedle patch 1. Figure 1 The middle right picture shows a front view of the light-controlled gas-releasing antibacterial microneedle patch 1. Figure 1 The lower middle figure shows a side view of the light-controlled gas-releasing antibacterial microneedle patch 1 .

[0052] In the following examples, the microneedle patch group indicated that the microneedle patch used was prepared from sodium alginate solution only and did not contain triiron dodecacarbonyl. The microneedle patch containing triiron dodecacarbonyl indicated that the microneedle patch used contained sodium alginate and triiron dodecacarbonyl.

[0053] In some optional embodiments, the tip length of the patch microneedle is 800 μm to 900 μm in height.

[0054] According to the embodiments of the present application, the needle tip length of the patch microneedle is within the above-mentioned value, which is conducive to the microneedle penetrating the dense biofilm formed by bacteria on the infected wound surface, and realizing the carbon monoxide gas release and sterilization with precisely controllable penetration depth. At the same time, when the microneedle patch is used on the wound surface, the needle tip of the above-mentioned length will not produce obvious tingling sensation.

[0055] In some optional embodiments, the average spacing between the solid microneedle tips is 280 μm to 320 μm.

[0056] According to the embodiments of the present application, the average spacing between the needle tips of the solid microneedles is within the above range, which is conducive to penetrating the dense biofilm formed by bacteria on the infected wound surface and achieving carbon monoxide gas release sterilization with precisely controllable penetration depth.

[0057] In a second aspect, the present application provides a method for preparing a light-controlled gas-releasing antibacterial microneedle patch, comprising:

[0058] Mixing a sodium alginate solution with a solution containing triiron dodecacarbonyl nanoparticles to prepare a mixed solution;

[0059] Filling the mixture at least once into a negative mold containing solid microneedle cavities,

[0060] The mixture placed in the negative mold is dried to prepare the light-controlled gas-releasing antibacterial microneedle patch containing solid microneedles.

[0061] In some embodiments, Figure 2 As shown, the preparation method of the light-controlled gas-releasing antibacterial microneedle patch comprises:

[0062] Step 1: Prepare a sodium alginate solution, the concentration of which can be controlled within a range of 0.5% to 3% (mass to volume ratio), and the solvent is deionized water. Preferably, a 1.5% sodium alginate solution is prepared.

[0063] Step 2: The concentration of triiron dodecacarbonyl is 10 μM-100 μM, and the solvent is deionized water. A 60 μM concentration of triiron dodecacarbonyl solution is optional.

[0064] Step 3: Mix the liquids prepared with the optimal concentrations selected in step 1 and step 2 at a volume ratio of 1:1 for 10 minutes, and stir on a magnetic stirrer at a speed of 700-1000 rpm.

[0065] Step 4: Inject the mixed liquid from step 3 into a specially made microneedle mold (such as Figure 2 As shown), the mold is poured for the first time and placed in a negative pressure environment of 0.01-0.2 MPa, preferably 0.07 MPa, and allowed to stand for 1 hour. The excess bubbles in the liquid in the mold are then removed and the mold is placed in an oven at 65 degrees Celsius for 0-3 hours, preferably 1 hour.

[0066] Step 5: Perform a second infusion, inject a certain amount of the liquid prepared in the third step into the microneedle mold again, and put it into a 65 degree Celsius oven to dry for 0-3 hours, preferably 1 hour.

[0067] Step 6: Perform a third injection, inject the liquid prepared in the third step into the microneedle mold again until the microneedle mold is just filled up and placed at room temperature for 3-24 hours, preferably 12 hours.

[0068] Step 7: Use a hair dryer to demould the microneedle mold containing the material obtained in step 6, thereby obtaining a light-controlled gas-releasing antibacterial microneedle patch.

[0069] In some optional embodiments, a light-controlled gas-releasing antibacterial microneedle patch base substrate, the base substrate having a microneedle side and an opposite back side;

[0070] A solid microneedle extends from the microneedle side of the base substrate.

[0071] In some optional embodiments, the mass percentage of the sodium alginate solution is 0.5%-3%.

[0072] In some optional embodiments, the molar concentration of triiron dodecacarbonyl is 10 μM-100 μM.

[0073] In some optional embodiments, the mixture is filled into a negative mold containing solid microneedle cavities at least once in an environment of negative pressure of 0.01-0.2 MPa, and allowed to stand for 30-60 minutes to remove bubbles in the mixture in the negative mold.

[0074] In a third aspect, the present application provides a method for using a light-controlled gas-releasing antibacterial microneedle patch, comprising:

[0075] The light-controlled gas-releasing antibacterial microneedle patch is pressed against the wound surface on the side away from the solid microneedle, wherein the light-controlled gas-releasing antibacterial microneedle patch includes, by mass percentage, sodium alginate (5%-10%) and triiron dodecacarbonyl (90%-95%). Then, irradiation is performed with a near-infrared laser of 808nm-1064nm to make the carbonyl of triiron dodecacarbonyl form carbon monoxide for killing bacteria, thereby achieving antibacterial effect on the wound surface.

[0076] Example

[0077] The following examples more specifically describe the disclosure of the present application, which are intended for illustrative purposes only, as it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the disclosure of the present application. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0078] Example 1

[0079] This embodiment provides a method for preparing a light-controlled gas-releasing antibacterial microneedle patch, comprising:

[0080] The first step: preparing a sodium alginate solution, the concentration of which can be controlled within a concentration range of 1.5% (mass to volume ratio), and the solvent is deionized water.

[0081] Step 2: The concentration of triiron dodecacarbonyl is 60 μM, and the solvent is deionized water. The average particle size of triiron dodecacarbonyl is 200-500 nm.

[0082] Step 3: Mix the liquids prepared with the optimal concentrations selected in step 1 and step 2 at a volume ratio of 1:1 for 10 minutes, and stir on a magnetic stirrer at a speed of 700-1000 rpm.

[0083] Step 4: Inject the mixed liquid from the third step into a specially prepared microneedle mold for the first injection, place it in a negative pressure environment of 0.01-0.2 MPa, preferably 0.07 MPa, and let it stand for 1 hour. Then remove excess bubbles from the liquid in the mold and place it in a 65 degree Celsius oven to dry for 1 hour.

[0084] Step 5: Perform a second infusion, inject a certain amount of the liquid prepared in the third step into the microneedle mold again, and put it into a 65 degree Celsius oven to dry for 1 hour.

[0085] Step 6: Perform the third injection, inject the liquid prepared in the third step into the microneedle mold again until the microneedle mold is just filled up and place it at room temperature for 12 hours.

[0086] Step 7: Use a hair dryer to blow the microneedle mold containing the material obtained in step 6 to demould, thereby obtaining a light-controlled gas-releasing antibacterial microneedle patch. The solid microneedle tip portion corresponding to the negative mold in the microneedle mold is a quadrangular pyramid, and the corresponding tip specifications are 300 microns in length, 300 microns in width, 800 microns in height, and the distance between the two tips is 300 microns.

[0087] Example 2

[0088] The difference between this embodiment and embodiment 1 is that the concentration of the sodium alginate solution is different, which is 3%. The concentration of triiron dodecacarbonyl is different, which is 15 μM.

[0089] Example 3

[0090] The difference between this embodiment and embodiment 1 is that the concentration of the sodium alginate solution is different, which is 0.5%, and the concentration of the triiron dodecacarbonyl solution is different, which is 80 μM.

[0091] Example 4

[0092] The difference between this embodiment and embodiment 1 is that only two pourings are performed, and after the second pouring, the mixture is placed in a 65 degree Celsius oven for drying for 2 hours.

[0093] Comparative Example 1

[0094] The difference between this comparative example and Example 1 is that only the sodium alginate solution of Example 1 is used, and triiron dodecacarbonyl is not added.

[0095] Performance Testing

[0096] 1) Microscopic observation: The light-controlled gas-releasing antibacterial microneedle patch prepared in Example 1 was subjected to microscopic observation, and images were taken using a stereo microscope and a scanning electron microscope.

[0097] like Figure 3 As shown, it is a light-controlled gas-releasing antibacterial microneedle patch prepared in Example 1. Figure a is a stereoscopic image of the microneedle patch and Figure b is a scanning electron microscopic image. As shown in the figure, the microneedle patch is composed of a base substrate and a plurality of microneedle tips. The number and size of the needle tips in the patch can be obtained by preparing different microneedle molds as needed. In an embodiment of the present invention, the needle tip portion of the prepared microneedle patch is a quadrangular pyramid, and the needle tip specifications are 300 microns in length, 300 microns in width, and 800 microns in height, and the distance between the two needle tips is 300 microns.

[0098] 2) Use of light-controlled gas-releasing antibacterial microneedle patch

[0099] The photo-controlled gas-releasing antibacterial microneedle patch prepared in Example 1 was irradiated with 808 nm near-infrared lasers of different powers (0.2, 0.4, 0.6, 0.8, and 1.0 W / cm2), and it was found that the patch had good photothermal temperature rise response characteristics. Figure 4As shown in Figure a, Figure a is the temperature rise curve of near-infrared laser irradiation with different powers; when the prepared microneedle patch is irradiated with 808nm near-infrared lasers of different powers (0.2, 0.4, 0.6, 0.8, and 1.0 W / cm2, respectively), the temperature rise of the microneedle patch becomes more obvious with the increase of the irradiation laser power. When the laser irradiation time reaches 180 seconds, the heat exchange of the microneedle patch tends to balance, and the temperature rise of the microneedle patch tends to be gentle. This shows that the temperature rise response of the microneedle patch is positively correlated with the irradiation power of the near-infrared laser. Using high-power near-infrared irradiation can rapidly increase the temperature of the microneedle patch. On the other hand, Figure 4 As shown in Figure b, Figure b shows the temperature rise curve of microneedle patches containing different concentrations of nano-dodecyl iron carbonyl under near-infrared light irradiation. The content of dodecyl iron carbonyl nanomaterials in the microneedle patch is also very important for the temperature rise of the microneedle patch. Under the same power of near-infrared laser irradiation, when the content of dodecyl iron carbonyl nanomaterials in the microneedle patch is (30-60 micromoles), after 180 seconds of near-infrared laser irradiation, the temperature changes of the microneedle patch are △T 30 =7.7℃, △T 45 =9.1℃, △T 60 =10.9℃. As the content of dodecacarbonyl iron nanomaterials gradually increased, the temperature of a 2 square centimeter microneedle patch containing 75 micromoles of dodecacarbonyl iron nanomaterials increased by 14.4 degrees Celsius. Under the same conditions, the temperature of a microneedle patch containing 90 micromoles of dodecacarbonyl iron nanomaterials increased by 16.8 degrees Celsius. This shows that under near-infrared light irradiation, the content of dodecacarbonyl iron nanomaterials in the microneedle patch is positively correlated with the temperature increase.

[0100] 3) Detection of carbon monoxide release during use of light-controlled gas-releasing antibacterial microneedle patch

[0101] In order to detect whether 808nm near-infrared laser irradiation can induce the microneedle patch to release carbon monoxide, in this embodiment, 11mg of hemoglobin was added to 3mL of phosphate buffer (pH=7.4) to obtain a hemoglobin mixed solution, 1.6mg of sodium dithionite was added under nitrogen protection, and 1mL of a microneedle patch sample containing 15 micromoles of triiron dodecacarbonyl nanomaterial was further added to obtain 4mL of a mixed solution. The mixed solution was divided into two groups, one group was illuminated (sealed with plastic wrap, the power was 0.6 watts per square centimeter, and the near-infrared light was irradiated for 50 minutes), and the other group was not illuminated. Afterwards, the absorbance of the solution at 420nm and 432nm was detected. The test results are as follows: Figure 5As shown in the figure, after 10 seconds of near-infrared light irradiation, the microneedle patch quickly released carbon monoxide gas, but after 30 seconds of irradiation, the amount of carbon monoxide released tended to be flat. This shows that near-infrared light irradiation can promote the release of carbon monoxide gas from the microneedle patch in a shorter period of time, and the amount of carbon monoxide released can be controlled within a reasonable and safe range by regulating the amount of iron trioxide added. In wound treatment, many research reports have shown that a certain degree of carbon monoxide gas can play a cytoprotective, immunomodulatory and anti-inflammatory role. At the same time, it can also accelerate the healing of infected wounds by regulating cytokine activation.

[0102] 4) Cytotoxicity test: In order to detect the biocompatibility of the light-controlled gas-releasing antibacterial microneedle patch, the commercial Cell Counting Kit-8 cell counting detection kit (purchased from Beyotime Biotechnology Co., Ltd.) was used in the examples of this patent for detection. Example 1 Preparation of light-controlled gas-releasing antibacterial microneedle patch (referred to as microneedle patch containing triiron dodecacarbonyl in the figure) and comparison of microneedle patch made without adding triiron dodecacarbonyl nanomaterial (referred to as microneedle patch in the figure) to test the toxicity of Hacat (human immortalized epidermal cells) cells from the American Type Culture Collection (ATCC). Hacat cells in the logarithmic growth phase were cultured at 1.2×10 6 The cells were added to a 96-well cell culture plate (200 microliters / well) at a density of 100 microliters / well, and DMEM high-glucose cell culture medium (penicillin and streptomycin 10,000 U / mL) containing 10% fetal bovine blood was used as the cell culture medium. The cells were placed in a carbon dioxide incubator containing 5% volume fraction and cultured at 37 degrees Celsius for 24 hours. When the cell monolayer covered the bottom of the 96-well plate, microneedle solutions of different concentrations (0, 1, 3, 5, 10, 20, 40, 60, 80, 100 micromoles) were added respectively, and PBS was used as a blank group and the cell culture medium was used as a control group. After the cells were cultured in the incubator for 12 hours, 10 microliters of CCK-8 reagent was added to each well and continued to be cultured in the cell culture incubator at 37 degrees Celsius for 1.5 hours. Subsequently, the optical density (OD) of the solution in the 96-well cell culture plate at 450 nanometers was measured using an enzyme marker. Cell survival rate (%) = (experimental well-blank well) / (negative control well-blank well) × 100%. The cell toxicity results were statistically shown in Figure 6 As shown, both the microneedle patches containing and not containing triiron dodecacarbonyl exhibited good cell compatibility, and the addition of triiron dodecacarbonyl was more conducive to cell growth and proliferation.

[0103] 5) Antibacterial performance test

[0104] In order to promote the healing of infected wounds, it is first necessary to kill the pathogenic microorganisms in the wounds. Therefore, it is very important to detect the antibacterial efficacy of the prepared microneedle patch. In this example, Escherichia coli is selected as the model bacteria to test the antibacterial efficacy of the microneedle patch prepared in Example 1 and the microneedle patch prepared in Comparative Example 1 without the addition of triiron dodecacarbonyl nanomaterials. First, pick a monoclonal Escherichia coli colony and add 5 ml of bacterial culture solution to incubate on a constant temperature shaker (6 hours), draw 100 microliters of bacterial solution in a 96-well plate and place it in an ELISA instrument to detect the absorbance of the bacterial solution. At the same time, draw 10 microliters of bacterial solution and dilute it 10 times step by step, diluting it to about 1×10 -6 After the dilution factor is reached, 100 μl of bacterial solution is taken for plating. The plate is placed in an incubator for 24 hours and the number of colonies is observed. The bacterial concentration of the original bacterial suspension is inferred based on the number of colonies. Colony forming unit = number of colonies × dilution factor. Repeat the above conditions to re-cultivate the bacteria and detect the OD value. Dilute the bacterial suspension and keep the absorbance value the same as the initial test. The bacterial concentration at this time is the same as the bacterial concentration calculated in the previous step. The bacterial solution is centrifuged to remove the culture medium, resuspended in sterile phosphate buffer solution, and 1×10 5 / mL concentration of bacteria was aspirated with 1mL and 100 microliters of the extract of the microneedle patch containing triiron dodecacarbonyl (referred to as the microneedle patch containing triiron dodecacarbonyl) prepared in Example 1 and the microneedle patch prepared in Comparative Example 1 without adding triiron dodecacarbonyl nanomaterial (referred to as the microneedle patch) were added respectively. Another group of microneedle patches containing triiron dodecacarbonyl (referred to as the microneedle patch containing triiron dodecacarbonyl) prepared in Example 1 were irradiated with near-infrared for 10 minutes and then left to stand for 1 hour to allow carbon monoxide to fully react with bacteria. The bacterial solution after the action and other control groups were diluted and plated by the same multiples, placed in a bacterial incubator for 24 hours, and then the monoclonal colonies were observed and the antibacterial rate was calculated. The following results are obtained: Figure 7 The results are shown in Figure a, which shows the number of bacterial colonies in different experimental groups observed by the plate colony counting method; Figure b shows the statistical results of the antibacterial rate of different experimental groups. Through the final experimental results, it can be concluded that the microneedle patch containing triiron dodecacarbonyl for anti-wound infection has significantly better antibacterial performance than other experimental groups under the irradiation of near-infrared light, and its antibacterial efficiency can reach 99.57% compared with the blank control group.

[0105] 6) Bacterial fluorescent live-dead staining observation

[0106] A commercial bacterial live / dead fluorescent staining kit (LIVE / DEAD BacLightTM, Thermo Fisher Scientific, USA) was used to observe the live / dead fluorescent staining of Escherichia coli after treatment in different experimental groups. Figure 8As shown, Escherichia coli in the logarithmic growth phase were collected by centrifugation after different treatments, rinsed with sterile phosphate buffer, and then the bacterial suspension was thoroughly mixed with the detection kit dye in a 1:1 ratio according to the instructions of the manual. 3 micro bacterial live and dead detection reagent mixture was added per milliliter of bacteria and incubated in a 37-degree bacterial incubator for 15 minutes. Each time, 5 micro bacterial suspensions were aspirated onto a glass slide and observed under a fluorescence microscope. Among them, the dye solution of the kit used contained SYTO-9 and propidium iodide. After the bacteria are stained, SYTO-9 can penetrate bacteria with intact and damaged membranes, while propidium iodide can only penetrate cells with damaged membranes. At the same time, the penetration of propidium iodide will cause a decrease in SYTO-9 fluorescence staining. Therefore, bacteria with intact membrane structures exhibit green fluorescence after staining, while bacteria with damaged membrane structures exhibit red fluorescence. As shown Figure 8 As shown, the antibacterial properties of the blank control group, the microneedle patch group (the microneedle patch prepared in Comparative Example 1 without adding triiron dodecacarbonyl nanomaterials), and the microneedle patch group containing triiron dodecacarbonyl (the light-controlled gas-release antibacterial microneedle patch prepared in Example 1, referred to as the microneedle patch containing triiron dodecacarbonyl) are relatively weak, and most bacteria show green fluorescence. On the contrary, after near-infrared irradiation, a large number of red fluorescent bacteria can be clearly observed in the illumination group containing triiron dodecacarbonyl (the light-controlled gas-release antibacterial microneedle patch prepared in Example 1, referred to as the microneedle patch containing triiron dodecacarbonyl). This indicates that the membrane structure of most bacteria is damaged and the growth activity of bacteria is inhibited. This result more intuitively verifies the good antibacterial property of the microneedle patch.

[0107] 7) Repair effect on infected wounds in mice

[0108] The repair effect of different treatment groups on the infected wound of mice was observed. The microneedle patch group (sodium alginate) prepared in comparative example 1 was treated with medication one day after the wound was formed, and the patch was placed on the wound and fixed with a 3M film for one day. The microneedle patch group containing triiron dodecacarbonyl prepared in Example 1 adopted the same treatment method as above. The illumination group of the microneedle patch containing triiron dodecacarbonyl adopted the microneedle patch containing triiron dodecacarbonyl prepared in Example 1, which was irradiated with near-infrared light.

[0109] After processing, the results are as follows Fig. 9 As shown, the blank control group was treated with saline and no bacteria were added to the wound. Since the wound was not infected by bacteria, the blank control group showed a faster wound healing speed and was completely healed 18 days after the wound was formed. In contrast, 10 μl of 1×10 6 / mL Staphylococcus aureus concentration of microneedle patch group and containing iron dodecacarbonyl triiron group due to the failure to effectively inhibit the occurrence of wound infection caused by wound healing quality and speed significantly deteriorated, 18 days after treatment of the wound of mice still failed to completely heal. On the contrary, the light group containing iron dodecacarbonyl triiron prepared in Example 1 can significantly inhibit the proliferation of Staphylococcus aureus in the wound of mice after near-infrared irradiation, and the infection of the wound is significantly inhibited. 18 days after treatment, the healing of the wound infected by mice is very close to the degree of wound healing of the blank control group of mice, showing that the light-controlled gas release type antibacterial microneedle patch can significantly kill bacteria in the wound under near-infrared light irradiation and can significantly promote the healing of the wound.

[0110] 8) Observation of tissue section staining of mice with infected wounds

[0111] The healing of the wounds of mice in different groups after treatment was observed by staining the tissue sections. The wounds of mice in different treatment groups were sampled and sliced, and the healing of the wounds of mice was observed by hematoxylin-eosin staining. Fig.10 As shown, 21 days after treatment, the tissue granulation and epithelialization of the blank control group and the illumination group of the microneedle patch containing triiron dodecacarbonyl prepared in Example 1 were significantly faster than those of the microneedle patch group and the microneedle patch group containing triiron dodecacarbonyl. At the same time, as shown by the black arrows in the figure, the wound closure of the blank control group and the illumination group of the microneedle patch containing triiron dodecacarbonyl was also faster, the wound was smaller, and the infiltration of inflammatory cells in the tissue was also significantly lower than that of the microneedle patch group prepared in Comparative Example 1 and the microneedle patch group containing triiron dodecacarbonyl not exposed to light prepared in Example 1, indicating that the infection of the wound was suppressed and the healing of the wound was good.

[0112] Although the present application has been described with reference to preferred embodiments, various modifications may be made thereto and parts thereof may be replaced with equivalents without departing from the scope of the present application. In particular, the various technical features mentioned in the various embodiments may be combined in any manner as long as there is no structural conflict. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A light-controlled gas-releasing antibacterial microneedle patch, characterized in that: The light-controlled gas-releasing antibacterial microneedle patch comprises, by mass percentage, 5%-10% of sodium alginate and 90%-95% of triiron dodecacarbonyl.

2. The light-controlled gas-releasing antibacterial microneedle patch according to claim 1, characterized in that: The light-controlled gas-releasing antibacterial microneedle patch comprises: a base substrate having a microneedle side and an opposing back side; A solid microneedle extends from the microneedle side of the base substrate, and the solid microneedle comprises, by mass percentage, 5%-10% sodium alginate and 90%-95% triiron dodecacarbonyl.

3. The light-controlled gas-releasing antibacterial microneedle patch according to claim 2, characterized in that: The solid microneedles have a height of 200 μm to 900 μm.

4. The light-controlled gas-releasing antibacterial microneedle patch according to claim 2, characterized in that: The average spacing between the solid microneedle tips is 280 μm to 320 μm.

5. A method for preparing a light-controlled gas-releasing antibacterial microneedle patch, characterized in that: include: Mixing a sodium alginate solution with a solution containing triiron dodecacarbonyl nanomaterial to prepare a mixed solution; Filling the mixture at least once into a negative mold containing solid microneedle cavities, The mixture placed in the negative mold is dried to prepare the light-controlled gas-releasing antibacterial microneedle patch containing solid microneedles.

6. The preparation method according to claim 5, characterized in that: The light-controlled gas-releasing antibacterial microneedle patch comprises: a base substrate having a microneedle side and an opposing back side; A solid microneedle extends from the microneedle side of the base substrate.

7. The preparation method according to claim 5, characterized in that: The mass concentration of the sodium alginate solution is 0.5%-3%.

8. The preparation method according to claim 5, characterized in that: The molar concentration of the triiron dodecacarbonyl is 10 μM-100 μM.

9. The preparation method according to claim 5, characterized in that: The mixture is filled into a negative mold containing solid microneedle cavities at least once in an environment of negative pressure of 0.01-0.2 MPa, and allowed to stand for 30-60 minutes to remove bubbles in the mixture in the negative mold.

10. A method for using a light-controlled gas-releasing antibacterial microneedle patch, characterized in that: include: The light-controlled gas-releasing antibacterial microneedle patch having a solid microneedle side is closely attached to the wound surface, wherein the light-controlled gas-releasing antibacterial microneedle patch comprises, by mass percentage, 5%-10% of sodium alginate and 90%-95% of triiron dodecacarbonyl; Irradiation is performed with a near-infrared laser of 808 nm to 1064 nm, so that the carbonyl group of dodecacarbonyltriiron in the light-controlled gas-releasing antibacterial microneedle patch forms carbon monoxide for killing bacteria, thereby achieving antibacterial effect on the wound surface.

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