Lysozyme hydrogel microneedle and application thereof in burn wound healing

Abstract

The invention discloses a lysozyme hydrogel microneedle and application of the lysozyme hydrogel microneedle in burn wound healing. The invention provides a hydrogel microneedle patch with an antibacterial function. The hydrogel microneedle patch comprises a needle body and a backing layer, the needle body comprises a needle body matrix material and a medicine; the medicine is lysozyme; the needle bodies are arranged on the backing layer in an array mode. The lysozyme hydrogel microneedle prepared by the invention shows good mechanical properties and good mechanical strength, can slowly release drugs, has an antibacterial effect, and shows an obvious curative effect in burn wound healing.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a lysozyme hydrogel microneedle and its application in burn wound healing. Background Art

[0002] Lysozyme (LY) is a protein that exists in almost all human biological fluids, such as serum, saliva, tears, sweat, and synovial fluid. In addition, it has been isolated from avian eggs and mammalian milk. Lysozyme is a class of enzymes widely present in the biological world. As an antibacterial enzyme that hydrolyzes the bacterial cell wall, lysozyme mainly achieves its dissolution by catalyzing the hydrolysis of the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetyl-d-glucosamine residues in the peptidoglycan of the bacterial cell wall. Since the peptidoglycan network is lacking in animal cell membranes, peptidoglycan has become a unique antibacterial target. Importantly, lysozyme has a broad-spectrum antibacterial effect, has bactericidal or bacteriostatic effects on a variety of bacteria, and has a high bactericidal effect on drug-resistant bacteria. At present, some lysozyme preparations have been used in clinical treatment, such as peptic ulcer, respiratory tract infection, skin infection, etc. The food additive standard of the European Union has included it in the category of functional factors with antibacterial, bacteriolytic, and bactericidal activities. At the same time, the US Food and Drug Administration has also listed it as a conventional safe substance. In China, lysozyme has also been approved to be added to infant milk powder as an additive, which helps to enhance the ability of infants' intestines to resist infection.

[0003] Hydrogel microneedles are constructed using polymer materials. When applied, their needle tips penetrate the skin by a specific force to form microporous channels in the skin tissue. Based on the unique physical and chemical properties of the polymer material, drugs can uptake interstitial fluid through the formed microporous channels by means of this material, thereby causing its own swelling phenomenon. In this dynamic process, the drugs are gradually released continuously from the carrier material and accurately delivered to the predetermined action site to achieve the expected therapeutic or physiological regulation function. Hyaluronic acid itself is a natural polysaccharide polymer with good biocompatibility and plays a certain role in various biological processes such as cell proliferation, differentiation, morphogenesis, inflammation, and wound healing. Methacryloylated hyaluronic acid (HAMA) has been given the ability of photocuring by introducing methacryloyl groups on the hyaluronic acid molecular chain. Moreover, it has good biocompatibility and can be used as a sustained-release carrier for drugs to achieve the sustained-release effect of drugs and improve the therapeutic effect of drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a lysozyme hydrogel microneedle and its application in burn wound healing.

[0005] In a first aspect, the present invention claims to protect a hydrogel microneedle patch with antibacterial function.

[0006] The hydrogel microneedle patch with antibacterial function claimed by the present invention may include a needle body and a backing layer; the needle body may include a needle body matrix material and a drug; the drug may be lysozyme; the needle bodies may be arranged in an array on the backing layer.

[0007] Further, the needle body matrix material may be methacrylated hyaluronic acid (HAMA).

[0008] Further, in the needle body, the mass ratio of lysozyme to methacrylated hyaluronic acid (HAMA) may be 1:5.

[0009] Further, the material of the backing layer may be polyvinyl alcohol (PVA).

[0010] Further, when preparing the needle bodies of the hydrogel microneedle patch, the composition of the needle body material solution per unit dose may be as follows: 1 g of methacrylated hyaluronic acid (HAMA), 200 mg of lysozyme, 10 mL of a 2.5 g / L LAP initiator solution (the solvent of the LAP initiator solution may be PBS). Stir at room temperature until completely dissolved, sterilize with a 0.22 μm sterile needle filter, and store at 4°C for later use.

[0011] Further, when preparing the backing layer of the hydrogel microneedle patch, the solvent of the backing layer material solution may be water, and the solute may be polyvinyl alcohol (PVA), with a concentration of polyvinyl alcohol (PVA) of 120 g / L. During the preparation of this solution, it needs to be heated and stirred at 60°C until completely dissolved, left to stand overnight, and stored at 4°C for later use.

[0012] Further, the needle bodies of the hydrogel microneedle patch may be quadrangular pyramids, the length of the microneedles may be 900 μm, the side length of the bottom surface may be 400×400 μm, the distance between the tips may be 700 μm, and they form a 20×20 array.

[0013] In one embodiment of the present invention, the molecular weight of the polyvinyl alcohol is 44.05 (MW).

[0014] In one embodiment of the present invention, the hydrogel microneedle patch is an integrated microneedle patch prepared by the polydimethylsiloxane mold method.

[0015] More specifically, the hydrogel microneedle patch may be prepared according to the method described in the second aspect hereinafter.

[0016] In a second aspect, the present invention claims a method for preparing the hydrogel microneedle patch described in the first aspect above.

[0017] The method for preparing the hydrogel microneedle patch described in the first aspect above, for which the present invention claims protection, may include the following steps:

[0018] (A1) Take the needle body material solution required for preparing the hydrogel microneedle patch (as described in the first aspect above) and add it to the microneedle mold, and remove air bubbles by vacuum pumping; then perform "heating and concentration, adding the needle body material solution" and repeat this step (this step refers to "heating and concentration, adding the needle body material solution") until the needle body part of the microneedle mold is filled; then perform photo-curing;

[0019] (A2) Add the backing layer material solution required for preparing the hydrogel microneedle patch (as described in the first aspect above) to the microneedle mold, dry it, and demold to obtain the hydrogel microneedle patch.

[0020] In one embodiment of the present invention, in step (A1), the amount of the needle body material solution added to the microneedle mold each time is 200 μL; the step of "heating and concentration, adding the needle body material solution" is repeated 2 times.

[0021] Further, in step (A1), the vacuum pumping can be performed at 27 °C for 2 - 3 times, 3 min each time. The heating can be performed at 30 °C for 2 h. The photo-curing can be performed by irradiating with a 405 nm light source for 5 seconds.

[0022] Further, in step (A2), the drying condition can be drying at 27 °C for more than 12 hours, specifically 16 hours.

[0023] In one embodiment of the present invention, the microneedle mold is polydimethylsiloxane (PDMS).

[0024] In the third aspect, the present invention claims protection for the application of the hydrogel microneedle patch described in the first aspect above in any of the following:

[0025] (B1) Preparing a product for inhibiting bacteria;

[0026] (B2) Preparing a product for promoting the healing of burn wounds.

[0027] Further, the burn wound can be a second-degree burn wound or a second-degree burn wound complicated with bacterial infection.

[0028] Further, the bacteria can be Gram-positive bacteria and / or Gram-negative bacteria.

[0029] Furthermore, the Gram-positive bacteria can be Staphylococcus aureus, and the Gram-negative bacteria can be Escherichia coli.

[0030] Experimental results show that the lysozyme hydrogel microneedles prepared by the present invention exhibit good mechanical properties, have good mechanical strength, can slowly release drugs, have antibacterial effects, and show obvious curative effects in the healing of burn wounds. Brief Description of the Drawings

[0031] Figure 1 These are the preparation steps of the lysozyme hydrogel microneedles of the present invention.

[0032] Figure 2 These are the morphological characterizations of the lysozyme hydrogel microneedles of the present invention by a stereomicroscope (A, B) and a scanning electron microscope (C, D).

[0033] Figure 3 These are the surface scanning elemental analyses of the lysozyme hydrogel microneedles of the present invention by an energy spectrometer.

[0034] Figure 4 These are the images of the lysozyme hydrogel microneedles of the present invention under a laser confocal microscope and their penetration on ex vivo mouse skin. Among them, A is the distribution diagram of the drug at different depths in the mouse skin, and B is the 3D reconstruction image.

[0035] Figure 5 These are the XRD images of the lysozyme hydrogel microneedles of the present invention and their individual components. Among them, A is the XRD image of LY, LY-MNs, and PVA, and B is the XRD image of HAMA.

[0036] Figure 6 These are the infrared spectrum images of the lysozyme hydrogel microneedles of the present invention and their individual components.

[0037] Figure 7 These are the DSC images of the lysozyme hydrogel microneedles of the present invention and their individual components.

[0038] Figure 8 These are the force-displacement curves of the lysozyme hydrogel microneedles of the present invention and the lysozyme soluble microneedles as a control. Among them, A is the force-displacement curve of the lysozyme hydrogel microneedles of the present invention; B is the force-displacement curve of the lysozyme soluble microneedles as a control.

[0039] Figure 9 These are the in vitro swelling morphology diagrams of the lysozyme hydrogel microneedles of the present invention. Among them, A is before the microneedles swell; B is after the microneedles swell.

[0040] Figure 10 These are the drug release curves of the lysozyme hydrogel microneedles of the present invention.

[0041] Figure 11 These are the bacterial growth curves. Among them, A is the OD 600 growth curve of Staphylococcus aureus; B is the OD 600Growth curve. In the figure, HAMA-MNs is the lysozyme hydrogel microneedle group, and Control is the control group.

[0042] Figure 12 Are the colonies of Staphylococcus aureus and Escherichia coli on the agar plate after different treatments.

[0043] Figure 13 Are the CLSM images of live and dead stained Staphylococcus aureus and Escherichia coli after different treatments.

[0044] Figure 14 Are the morphologies of Staphylococcus aureus and Escherichia coli in different treatment groups photographed by SEM.

[0045] Figure 15 Are the wound change diagrams of each group of mice within 7 days after different treatments.

[0046] Figure 16 Are the H&E staining images of skin tissues on the 7th day after different treatments.

[0047] Figure 17 Are the Masson staining images of skin tissues on the 7th day after different treatments. Detailed implementation manners

[0048] The present invention will be further described in detail below in conjunction with the specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0049] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0050] The room temperature mentioned in the following examples all refers to 25°C.

[0051] The polyvinyl alcohol (PVA) used in the following examples has a degree of alcoholysis of 87.0 - 89.0 mol%, a viscosity of 3.2 - 3.6 mPa·s, and a molecular weight of 44.05 (MW). Aladdin Biotechnology (Shanghai, China), CAS: 9002 - 89 - 5.

[0052] The average molecular weight of the methacrylated hyaluronic acid (HAMA) used in the following examples is 150 KD, Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

[0053] The lysozyme used in the following examples is from Beijing Honghu United Chemical Products Co., Ltd., CAS: 12650 - 88 - 3.

[0054] Example 1. Preparation of the lysozyme hydrogel microneedle patch of the present invention

[0055] (1) Prepare a 0.25% (W / V) initiator standard solution: Accurately measure 10 mL of PBS and add the initiator LAP into a brown reagent bottle. There is 0.025 g of LAP pre-loaded in the bottle. Then place the reagent bottle in a constant temperature water bath at 40 - 50 °C and heat for 15 minutes. During the heating process, gently shake the reagent bottle at regular intervals to ensure that the initiator is fully dissolved.

[0056] (2) Prepare a lysozyme mixed solution (microneedle body material solution) containing 10% (W / V) methacrylated hyaluronic acid (HAMA): Weigh 1 g of HAMA and 200 mg of lysozyme precisely and put them into a beaker. Add 10 mL of the 0.25% (W / V) initiator standard solution prepared in step (1) into the beaker, and place the beaker on a magnetic stirrer to stir at room temperature until completely dissolved. The prepared solution is sterilized using a 0.22 μm sterile needle filter and stored in a 4 °C refrigerator for later use.

[0057] (3) Prepare a 12% PVA backing layer solution: Weigh 2.4 g of polyvinyl alcohol (PVA) precisely, add 20 mL of pure water, heat and stir at 60 °C until completely dissolved, let it stand overnight, and store in a 4 °C refrigerator for later use.

[0058] As Figure 1 shown. In a polydimethylsiloxane microneedle mold, add 200 μL of the lysozyme mixed solution (microneedle body material solution) containing 10% (W / V) HAMA prepared in the above step (2), evacuate the air bubbles at 27 °C, and then scrape off the surface bubbles. This operation is repeated 2 - 3 times, 3 minutes each time. Then heat and concentrate at 30 °C, add 200 μL of the microneedle body material solution each time, and this operation is repeated twice (the microneedle part of the microneedle mold is filled), and the heating concentration time is 2 h each time. After heating is completed, irradiate with a 405 nm light source for 5 seconds for curing, and add 1 mL of the 12% PVA backing layer solution prepared in step (3). After drying at 27 °C for 16 h, demold to obtain the lysozyme hydrogel microneedle patch.

[0059] The microneedle body of the hydrogel microneedle patch prepared by the present invention can be quadrangular pyramid-shaped, the length of the microneedles can be 900 μm, the bottom side length can be 400×400 μm, the distance between the needle tips can be 700 μm, and it forms a 20×20 array.

[0060] Example 2. Observation of the geometric structure, morphology and elemental analysis of the microneedles on the lysozyme hydrogel microneedle patch of the present invention

[0061] The characteristics of the lysozyme hydrogel microneedles prepared in Example 1 were observed using a digital camera, a stereomicroscope (Motic, Beijing Khanmeng Zixing Instrument Co., Ltd.), a Nikon AXR laser confocal microscope (China), and a field emission scanning electron microscope (ZEISS, Gemini SEM 300, Germany). Subsequently, the surface of the microneedles was scanned using Energy Dispersive X-ray Spectroscopy (EDS) for elemental analysis. Figure 2 Figure 2 shows the morphological characteristics of the lysozyme hydrogel microneedles under a stereomicroscope and a scanning electron microscope. In the present invention, the needle body part of the prepared microneedles contains lysozyme (LY) and methacrylated hyaluronic acid (HAMA). The stereomicroscope and the scanning electron microscope were used to observe the formed microneedles. The results showed that the microneedles presented a good regular square pyramid shape. Each microneedle had a sharp tip. Under microscopic observation, no tip breakage was found, and the distribution of the microneedles in the overall structure was uniform.

[0062] To further explore the chemical composition of the microneedles, energy dispersive spectroscopy (EDS) was used to perform area scan elemental analysis on the microneedles. The analysis results showed that the elements detected by area scanning of the microneedle tip were C, O, N ( Figure 3 ), which was consistent with the elements contained in the structures of lysozyme (LY) and HAMA, strongly proving that these two substances were uniformly distributed on the surface of the microneedle tip.

[0063] Example 3: Observation of the fluorescence on the lysozyme hydrogel microneedles of the present invention and its penetration in ex vivo mouse skin under a laser confocal microscope

[0064] Fluorescein Isothiocyanate (FITC) was encapsulated in the lysozyme hydrogel. It is a commonly used fluorescent labeling reagent with a high fluorescence quantum yield, strong and stable green fluorescence signal emission, and is easy to detect and analyze. The preparation method of the hydrogel microneedles carrying FITC is referred to Example 1, with the only difference being that 0.5 mg of FITC (Fluorescein Isothiocyanate, Shanghai Yuanye Bio-Technology Co., Ltd.) was additionally added when preparing the lysozyme mixed solution (the needle body material solution) containing 10% (W / V) methacrylated hyaluronic acid (HAMA) in step (2).

[0065] The percutaneous penetration behavior of hydrogel microneedles carrying FITC was visually studied using confocal laser scanning microscopy (CLSM) technology. The hydrogel microneedles were vertically inserted into the dorsal skin of ex vivo BALB / c mice at a constant pressure of 12 N / needle, and the microneedles were removed after 5 minutes. Fluorescence imaging analysis was performed using a Nikon C2+ confocal laser scanning microscope (Japan), and the instrument parameters were set as follows: laser power 49.3 W / 4.161 W, detection mode GaAsP, and exposure time 7 s. To obtain three-dimensional distribution information, continuous tomographic scanning was performed at a step size of 2 μm along the Z-axis direction starting from the skin surface until the fluorescence signal disappeared. High-resolution fluorescence images of 512×512 pixels were collected for each X-Y plane. The NIS-Elements AR software (Version 4.60) was used to perform three-dimensional reconstruction on the collected two-dimensional image sequences, and the visualization effect of the fluorescence signal was enhanced through pseudocolor processing. By analyzing the fluorescence intensity distribution and penetration depth, the distribution characteristics of the drug in each skin layer could be evaluated.

[0066] Figure 4 Figure A shows the penetration and distribution of lysozyme at different depths in the skin under FITC fluorescence labeling. It can be clearly observed from the figure that the fluorescence signal gradually diffuses from the epidermis to the dermis, indicating that lysozyme can be effectively released from the microneedles and penetrate into the skin tissue. This result intuitively reflects the delivery efficiency and distribution range of lysozyme in the skin. Figure 4 Figure B is a three-dimensional reconstructed image based on the fluorescence signal, further showing the spatial distribution characteristics of the drug in the skin. It can be seen from the three-dimensional image that the fluorescence signal is mainly distributed in the microneedle insertion area and its surrounding tissues, and the signal intensity gradually weakens with the increase in depth. The maximum penetration depth of the drug reaches 167.11 μm. This result fully demonstrates that the lysozyme hydrogel microneedles can effectively penetrate the skin barrier and deliver the drug to the subcutaneous tissue.

[0067] Example 4. XRD study of the components of the lysozyme hydrogel microneedles of the present invention

[0068] To deeply explore the interaction mechanism between lysozyme (LY) and other components in the lysozyme hydrogel microneedles, XRD detection and analysis were carried out using an X-ray diffractometer (XRD-6100) from Shimadzu Corporation. In this experiment, a Cu target was selected as the ray source, and the incident X-ray wavelength was accurately set to The working voltage of the instrument was maintained at 40 kV, and the working current was 40 mA. During the scanning process, two scanning angle 2θ ranges were set, namely 5°-40° and 5°-80°, respectively, to comprehensively obtain diffraction information at different angles. The scanning rate was uniformly set to 4° / min to ensure the accuracy and stability of the experimental data collection.

[0069] The XRD test results of the lysozyme hydrogel microneedles prepared in Example 1 of the present invention and the materials they contain are as follows Figure 5 shown. Polyvinyl alcohol (PVA) and lysozyme (LY) have a diffraction peak at 2θ = 21°. In the lysozyme hydrogel microneedles (LY-MNs), the diffraction peak intensity increases and the position slightly changes to 2θ = 19.52° ( Figure 5 A in). The reason may be that when the addition amount of LY is small, its covalent cross-linking effect may trigger the structural reconstruction of the polymer system. Experimental data show that this intermolecular interaction will lead to the formation of a new hydrogen bond network between LY and PVA chain segments. It should be noted that this newly formed interfacial hydrogen bond may gradually replace the original intramolecular and intermolecular hydrogen bond interactions in PVA molecules. The van der Waals force and electrostatic force between LY and PVA will also hinder the formation of crystals, resulting in an amorphous structure. Figure 5 B in shows that HAMA is in an amorphous state, and its XRD pattern clearly exhibits the diffraction characteristics of typical amorphous substances. When the 2θ value is close to 20°, a broad and diffuse diffraction peak appears. The morphology of this diffraction peak is a significant sign of an amorphous polymer, which is completely different from the sharp and clear diffraction peaks presented by crystalline substances. In crystalline substances, atoms or molecules are arranged in a periodic and orderly manner, enabling X-rays to strongly diffract at specific angles, thus generating sharp diffraction peaks; while in amorphous substances such as HAMA hydrogels, the internal molecular arrangement lacks long-range order, and X-ray scattering is more dispersed, so a broad and diffuse diffraction peak is formed, which fully indicates that the molecular arrangement inside the HAMA hydrogel is in a relatively disordered state.

[0070] Example 5. Infrared spectroscopy study of the components of the microneedles of the present invention

[0071] To deeply explore the molecular interaction mechanism between lysozyme (LY) and the other components in the lysozyme hydrogel microneedles (LY-MNs) prepared in Example 1 of the present invention, the present invention selects Fourier transform infrared spectroscopy (FTIR) technology. The specific experimental operation steps are as follows: First, place the lysozyme microneedle sample in a dry environment and grind it into a uniform powder state using a mortar to ensure uniform particle size of the sample. Then, weigh the sample and spectroscopic grade potassium bromide (KBr) crystal according to a mass ratio of 1:100. Subsequently, place the weighed sample and KBr crystal in an agate mortar and mix them thoroughly by grinding to ensure uniform dispersion at the microscopic level. After mixing, carefully transfer the uniformly mixed sample powder into a tablet press mold and apply a stable pressure of 10 MPa, and continue pressing until a transparent and uniform texture thin film is formed. After the thin film is formed, place it in the sample chamber of the infrared spectrometer and collect spectral data under standard conditions at room temperature (25 ± 1°C). The spectral scanning range of this experiment is set to 4000 - 500 cm-1 , so as to comprehensively cover the vibration absorption range of relevant chemical bonds. During the whole experiment, the background spectrum of blank KBr was collected synchronously. After the experiment, the background subtraction and baseline correction operations were carried out using the professional software built in the instrument, so as to obtain accurate and reliable experimental data.

[0072] Lysozyme is a macromolecule and a compact protein (14 kDa) composed of 129 amino acids and folded into a globular structure. Lysozyme has a typical α-helix and β-sheet structure. Its core structure is composed of about 60 amino acids, forming a stable three-dimensional structure of two main α-helices and several β-sheet fragments. In the infrared spectrum, the stretching vibration peak of C=O (amide I band) is at 1656 cm -1 ; 1531 cm -1 (amide II band) is related to C-N stretching and N-H bending vibrations. HAMA is obtained by esterifying the hydroxyl part of hyaluronic acid molecules with methacrylic acid. According to the test results of the infrared spectrum, it is analyzed that there is a certain stretching vibration peak near 3349 cm -1 for HAMA, which is the absorption band of O-H in the HAMA structure; 2900 cm -1 is the stretching vibration of -CH2-; 1417 cm -1 is the vibration of acetamido; 1058 cm -1 and 949 cm -1 are the stretching vibration bands of C-O-C. Polyvinyl alcohol (PVA) is composed of a large number of vinyl alcohol chain segments, and has a large number of hydroxyl groups, intermolecular hydrogen bonds and terminal unsaturated olefins in its molecular structure. Since the characteristic functional group in PVA is hydroxyl group, 3379 cm -1 is the stretching vibration absorption peak of -OH, and 2940 cm -1 is the stretching vibration of -CH. There are certain stretching vibrations in LY-MNs at 1262 cm -1 and 1031 cm -1 . There are certain offsets in the characteristic peaks of HAMA and LY, and it is analyzed that electrostatic interaction and hydrophobic interaction may occur. The infrared spectrum images of the hydrogel microneedles of the present invention and their individual components of each sample are as Figure 6 shown.

[0073] Example 6. DSC study on each component of the lysozyme hydrogel microneedles of the present invention

[0074] The present invention uses a differential scanning calorimetry system to explore the thermodynamic interaction characteristics among the components of the lysozyme (LY) hydrogel microneedle system prepared in Example 1 of the present invention. The experiment was carried out on a TA Instruments DSC 250 differential scanning calorimeter (USA), and the thermodynamic characterizations were performed on pure lysozyme (LY), lysozyme hydrogel microneedles (LY-MNs), methacrylated hyaluronic acid (HAMA), and polyvinyl alcohol (PVA), respectively. The specific experimental method is as follows: Accurately weigh 3-5 mg of the sample to be tested and place it in a standard aluminum sealed crucible (diameter 6.8 mm). An empty aluminum crucible of equal mass is placed in the reference cell to eliminate systematic errors. The linear heating mode is adopted, with the starting temperature of 25 °C, the final temperature of 350 °C, and the constant heating rate of 10 °C / min. To ensure the reliability of the thermodynamic data, each sample is tested in parallel 3 times. During the test, high-purity nitrogen (purity ≥ 99.999%) is continuously introduced, and the flow rate is strictly controlled at 60.0 mL / min. This measure can effectively avoid the oxidation reaction during the thermal decomposition of the sample and maintain the stability of the instrument baseline at the same time.

[0075] The endothermic peaks of the DSC curve of lysozyme at about 96.4 °C and 202.7 °C are attributed to the thermal degradation of lysozyme. The DSC curve of HAMA shows an endothermic phenomenon at 99.3 °C. This phenomenon may be related to the desorption of bound water in the HAMA molecule or the local movement of the molecular chain. As a polysaccharide derivative, HAMA contains a large number of hydrophilic groups (such as hydroxyl and carboxyl groups) on its molecular chain, and these groups may release bound water during heating, resulting in an endothermic phenomenon.

[0076] In the DSC thermogram, distinct endothermic peaks appeared at 163.9 °C and 203 °C for the PVA sample, indicating that PVA might have undergone certain structural changes or physical transformations at these temperatures, such as water evaporation or rearrangement of intermolecular structures. This endothermic phenomenon might be related to the hydrates within the PVA molecules or the movement of molecular chains, and these changes would affect the thermal behavior of the material. The LY-MNs sample showed a prominent endothermic peak at 205.9 °C. This endothermic peak might be associated with certain physical processes in the LY-MNs sample, such as phase transition, molecular rearrangement, or evaporation of the solvent. At this temperature point, LY-MNs might have undergone a transition from the solid state to the liquid state or other states, resulting in the appearance of the endothermic phenomenon. Additionally, protein molecules have abundant hydrogen bond interactions and hydrophobic interactions in their structures. These non-covalent forces form strong binding forces between protein molecules. These interactions can effectively enhance the stability between molecules, enabling proteins to exhibit strong thermal stability at high temperatures. The synergistic effect of these non-covalent interactions not only strengthens the intermolecular attraction but also may provide important support for the thermal stability of the complex. Therefore, protein complexes tend to maintain the stability of their structures at relatively high temperatures and are not easily subject to drastic thermal degradation or decomposition. The DSC images of the hydrogel microneedles of the present invention and the individual samples of their respective components are as Figure 7 shown.

[0077] Example 7. Mechanical properties of the lysozyme hydrogel microneedles of the present invention

[0078] In terms of mechanical property characterization, a universal material testing machine (INSTRON 5969, USA) was used to conduct compression property tests on the lysozyme hydrogel microneedles prepared in Example 1 of the present invention. The test parameters were set as follows: loading rate 0.5 mm / min, maximum load 140 N, and displacement resolution 0.1 μm. The experimental data were processed using Origin 9.0 software to plot the compression displacement-force curve. At the same time, the lysozyme hydrogel microneedles prepared in Example 1 of the present invention and soluble microneedles were simultaneously subjected to mechanical tests to compare the mechanical properties of the two types of microneedles.

[0079] Among them, the preparation method of the soluble microneedles is as follows: Lysozyme and polyvinylpyrrolidone (PVP) K29-32 (average molecular weight 58,000) are dissolved in ultrapure water, stirred and mixed evenly at room temperature, and fully dissolved. After standing overnight, it is placed in a 4°C refrigerator for later use to obtain the tip solution (in the tip solution, the content of lysozyme is the same as that in the needle body material solution when preparing the hydrogel microneedles, and the content of PVP K29-32 is 0.3 g / mL). The preparation method of the backing layer solution is the same as above, using a 12% PVA solution. During the preparation of the microneedles, first, the pre-prepared tip solution is accurately injected into the cavity of the PDMS mold, and then the residual bubbles in the solution are completely removed through two cycles of vacuum degassing operations (5 minutes each time). To improve the filling efficiency, a high-speed centrifugation technique (4500 rpm, 10 minutes) is used to drive the solution to fully penetrate into the gaps of the mold microstructure. Subsequently, the PVA solution is injected as the base support layer, and the mold is transferred to a constant-temperature drying environment (27°C) for 12 hours of drying treatment. After the material is completely dried and cured, the microneedle array is taken out from the PDMS mold, and finally, a microneedle array without structural defects is obtained.

[0080] Figure 8 In A, the lysozyme hydrogel microneedles of the present invention can withstand a force of 132 N at 1.4 mm, and each needle is 0.587 N. Figure 8 In B, the lysozyme soluble microneedles can withstand a force of about 1.5 N at a displacement of 2 mm, and each needle is 0.0067 N. This shows that the mechanical strength of the lysozyme hydrogel microneedles of the present invention is significantly greater than that of the soluble microneedles, and the mechanical properties of the material are improved after photocuring.

[0081] Example 8. In vitro dissolution of the hydrogel microneedle patch of the present invention

[0082] To systematically evaluate the in vitro dissolution properties of the lysozyme hydrogel microneedles (LY-MNs) prepared in Example 1 of the present invention, an in vitro evaluation method based on ex vivo skin was established in the present invention. The specific experimental steps are as follows: The LY-MNs array is vertically pressed on the pre-treated dorsal skin of BALB / c mice ex vivo, and a constant pressure is applied to maintain contact. The microneedle samples are taken out at the time points of 1, 2, and 8 minutes respectively, and the morphological changes of the microneedles are immediately observed and recorded with a stereomicroscope. To obtain quantitative analysis data, the microscopic images are processed using Image J software.

[0083] The changes in the swelling of the lysozyme microneedles before and after insertion into the skin were photographed with a stereomicroscope. Figure 9Show the swelling morphology of the microneedles. After the hydrogel microneedles are inserted into the skin, they will rapidly absorb interstitial fluid or body fluid. Due to their high hydrophilicity and porous structure, they can swell significantly within a short time. This rapid swelling property helps the microneedles to form a tight contact within the skin, thus improving the drug delivery or sampling efficiency. After the hydrogel microneedles absorb water, their volume will increase significantly. This swelling behavior can enhance the mechanical interaction between the microneedles and the surrounding tissues, prevent the microneedles from falling off, and also contribute to the sustained release of drugs in the tissues. After swelling, the mechanical properties of the hydrogel microneedles will change, and their modulus usually decreases and becomes softer. This softening property can reduce the mechanical damage to the surrounding tissues and improve the comfort of the patients.

[0084] Example 9. In vitro drug release experiment of the lysozyme hydrogel microneedles of the present invention

[0085] In the study of in vitro drug release, the lysozyme hydrogel microneedles (LY-MNs) prepared in Example 1 of the present invention were placed in a pH 7.4 phosphate buffer solution (PBS) and stirred magnetically at a constant temperature of 37 °C (100 rpm). Samples of 1 mL were taken at predetermined time points (15 min, 0.5, 1, 2, 4, 6, 10, 12, 24, 48 h) (while supplementing an equal amount of fresh release medium at the same temperature), filtered through a 0.22 μm filter membrane and then injected for analysis. The sampling time points for the lysozyme hydrogel microneedles were: 0.5, 1, 3, 5, 8, 15, 30 min, and the rest of the operations were the same. All experiments were repeated three times, and the data were expressed as mean ± standard deviation (Mean ± SD). Four mathematical models were used to fit and analyze the release curves: (1) Zero-order kinetic equation: Q = kt; (2) First-order kinetic equation: ln(1 - Q) = -kt; (3) Higuchi equation: Q = kt 1 / 2 ; (4) Ritger-Peppas equation: Q = kt n . By comparing the correlation coefficient (R 2 ), and the release exponent (n), the best release mechanism of lysozyme (LY) was determined.

[0086] Figure 10 This is the release curve of LY-MNs. The release degree reached 99.44% at 48 h. Fitting the drug release degree data in Table 1, LY-MNs conformed to the Higuchi equation. The Higuchi equation mainly describes the process of drug release from the matrix by diffusion. The drug release conforming to the Higuchi equation means that the drug release is mainly dominated by the diffusion mechanism. Drug molecules are gradually released into the external environment by diffusion in the matrix, and its release rate is proportional to the square root of time. It usually indicates that there will be no obvious disintegration or erosion and other phenomena during the release process of the drug preparation, and the drug is slowly released by diffusion in the medium. This also conforms to the phenomenon that lysozyme is slowly released from the pores of the HAMA material.

[0087] Table 1. Fitting of the drug release data of the lysozyme hydrogel microneedles of the present invention

[0088] Fitting model Release equation <![CDATA[Fitness (R 2 )]]> Zero order y = 38.77 + 1.41x 0.8136 First order <![CDATA[y = 58.96*(1 - e -5.06 )]]> 0.2847 Higuchi <![CDATA[y = 2.81x 1 / 2 + 38.77]]> 0.8137 Ritger - peppas <![CDATA[y = 41.93x 0.19 > 0.7565

[0089] Example 10. In vitro antibacterial effect of the hydrogel microneedles of the present invention

[0090] The present invention systematically evaluated the antibacterial effect of the lysozyme hydrogel microneedles (LY-MNs) prepared in Example 1. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were selected as model strains for the experiment. The specific experimental steps are as follows:

[0091] (1) Bacterial culture and grouping: The two strains were inoculated into LB medium and cultured with shaking at 37 °C and 180 rpm for 12 h until the logarithmic growth phase. The bacterial suspension was divided into two groups: control group (only adding PBS); LY-MNs group (adding the LY-MNs dilution). After mixing 3 mL of the bacterial suspension with 3 mL of PBS (or 3 mL of the LY-MNs dilution, that is, dissolving LY-MNs in 10 mL of water, and the content of lysozyme in the dilution was 79.72 μg / mL), corresponding treatments were carried out according to the groups.

[0092] (2) Bacterial growth kinetics analysis: The treated bacterial suspension was continuously cultured at 37 °C, and the OD 600 value was measured every hour using a microplate reader to plot the bacterial growth curve and evaluate the inhibitory effect of the control group and the group added with the LY-MNs dilution on bacterial proliferation.

[0093] (3) Colony-forming unit (CFU) determination: The treated bacterial suspension was diluted 10 4 times, 100 μL was taken and evenly spread on an LB agar plate. After culturing at 37 °C for 24 h, the growth of colonies was recorded using a digital camera and the CFU was counted.

[0094] (4) Bacterial viability detection: Using the SYTO9 / PI double staining method, after mixing the sample with the dye, the fluorescence images of live / dead bacteria were observed using a confocal laser scanning microscope (CLSM). Live bacteria showed green fluorescence (SYTO9 staining), and dead bacteria showed red fluorescence (PI staining).

[0095] (5) Bacterial morphological observation: The bacterial suspension was centrifuged at 3000 rpm for 5 min to collect the bacteria, and fixed with 2.5% glutaraldehyde at 4 °C overnight. Subsequently, gradient dehydration was carried out successively with 30%, 50%, 70%, 80%, 90% and 100% ethanol, and the dehydration time for each time was 15 minutes. After dehydration was completed, the sample was subjected to critical point drying, and then sputter coating was carried out. Finally, a field emission scanning electron microscope was used to observe the changes in the ultrastructure of the bacteria.

[0096] The results showed that:

[0097] This example studied the antibacterial effects of LY-MNs against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli. Through the analysis of the bacterial growth curve, the results showed that under the action of LY-MNs, lysozyme exhibited significant antibacterial activity. After incubation at 37 °C for 12 hours, LY-MNs could effectively and completely inhibit the growth of Staphylococcus aureus and Escherichia coli (see Figure 11 ). In the control group, the samples without microneedle treatment had a significantly faster rising rate of the bacterial growth curve, indicating that in the absence of LY-MNs treatment, the proliferation ability of bacteria was stronger. Further through Figure 12 the standard agar plate experiment shown, the results indicated that LY-MNs had significant inhibitory effects on both Staphylococcus aureus and Escherichia coli and could kill more than 99% of the bacteria.

[0098] To further evaluate the antibacterial effect of LY-MNs, the present invention adopted the live / dead bacterial staining technique to observe the survival state of bacteria. In this experimental method, live bacterial cells were stained with the green fluorescent dye SYTO 9, while dead bacterial cells were stained with the red fluorescent dye PI35. Figure 13 The confocal laser scanning microscope (CLSM) images shown demonstrated that after treating bacteria with LY-MNs, we observed strong red fluorescence signals, indicating obvious bacterial death, which reflected the good effect of LY-MNs in inhibiting bacterial growth. In addition, Figure 14 the scanning electron microscope (SEM) images further confirmed the antibacterial activity of LY-MNs. The cell walls of the treated Staphylococcus aureus and Escherichia coli showed obvious deformation, wrinkling, breakage, and incomplete structure, while the bacteria in the control group maintained normal bacterial morphology with intact surfaces. These observation results indicated that LY-MNs showed significant activity in antibacterial treatment.

[0099] Example 11. In vivo burn wound treatment effect of the hydrogel microneedles of the present invention

[0100] To further study the antibacterial ability of the lysozyme hydrogel microneedles (LY-MNs) prepared in Example 1 of the present invention in vivo, a burn and scald model was established on the skin of mice. The specific experimental steps are as follows:

[0101] 1. Establishment of the animal model: C57 mice (6 - 8 weeks old) were depilated on the back one day before modeling. They were anesthetized before modeling, and then the back was scalded with medium-grade steam in a wall-mounted machine for 5 seconds to cause second-degree burns.

[0102] 2. Experimental grouping and treatment: The successfully modeled mice were randomly divided into 3 groups (n = 5): (1) Control group: No treatment was given; (2) Solution group: Treated by applying an aqueous solution of lysozyme (the concentration of lysozyme was 20 mg / mL) once a day on the wound; (3) Microneedle group: Treated by administering the lysozyme hydrogel microneedles of the present invention to the wound surface once a day with microneedles.

[0103] 3. Observation indicators: (1) Recording of wound healing: The morphological changes of the wound surface were recorded using a digital camera; (2) Body weight monitoring: The body weight of the mice was weighed every two days to evaluate the treatment safety; (3) Histopathological analysis: The mice were sacrificed after 7 days, and the skin tissues at the modeling site were taken, fixed with 4% paraformaldehyde, and embedded in paraffin for sectioning. Hematoxylin-eosin (H&E) staining was performed to observe the infiltration of inflammatory cells, and Masson trichrome staining was used to evaluate the deposition of collagen fibers and tissue repair.

[0104] The results showed that:

[0105] In Figure 15 , it can be seen that after modeling, the skin showed redness and swelling. Subsequently, there were ulceration and scabbing. The wound healing in the microneedle treatment group was relatively better. The body weight statistics of the mice within 7 days showed that there was no difference in the body weight changes among the groups, indicating that the treatment with lysozyme microneedles was safe for the mice and had no side effects. From Figure 16 The H&E staining results shown, in the control group, the red arrow pointed out that the surface of the dermis was incomplete, with ulceration and necrosis. The black arrow pointed out that there was a large amount of inflammatory cell infiltration. There were also a small number of inflammatory cells in the solution group. In contrast, the skin pathological section structure of the lysozyme microneedle group was relatively complete. In the Masson staining, collagen fibers were stained blue, and cytoplasm and muscle fibers were stained red. From Figure 17 The Masson staining results of each group shown, in the control group, there was a large amount of red inflammatory cell infiltration around, and the collagen fibers might appear loose due to edema and were arranged slightly disorderly. In the solution group, there were some healed collagen fibers in the dermis layer. The collagen fiber area in the microneedle group was relatively larger, and the degree of healing was better compared.

[0106] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A hydrogel microneedle patch with antibacterial function, comprising a needle body and a backing layer; the needle body comprises a needle body matrix material and a drug; the drug is lysozyme; the needle bodies are arranged in an array on the backing layer.

2. The hydrogel microneedle patch according to claim 1, wherein: The needle body matrix material is methacrylated hyaluronic acid.

3. The hydrogel microneedle patch according to claim 1 or 2, characterized in that: In the needle body, the mass ratio of lysozyme to methacrylated hyaluronic acid is 1:

5.

4. The hydrogel microneedle patch according to any one of claims 1-3, characterized in that: The material of the backing layer is polyvinyl alcohol.

5. The hydrogel microneedle patch according to any one of claims 1-4, characterized in that: When preparing the needle bodies of the hydrogel microneedle patch, the composition of the unit-dose needle body material solution prepared is as follows: 1 g of methacrylated hyaluronic acid, 200 mg of lysozyme, and 10 mL of a 2.5 g / L LAP initiator solution; and / or When preparing the backing layer of the hydrogel microneedle patch, the solvent of the backing layer material solution prepared is water, and the solute is polyvinyl alcohol, wherein the concentration of polyvinyl alcohol is 120 g / L.

6. The hydrogel microneedle patch according to any one of claims 1-5, characterized in that: The needle bodies of the hydrogel microneedle patch are quadrangular pyramids, the length of the microneedles is 900 μm, the side length of the bottom surface is 400×400 μm, the distance between the needle tips is 700 μm, and they form a 20×20 array.

7. A method for preparing the hydrogel microneedle patch according to any one of claims 1-6, comprising the following steps: (A1) Take the needle body material solution required for preparing the hydrogel microneedle patch and add it to a microneedle mold, and remove air bubbles by vacuum pumping; then perform "heating and concentration, and adding the needle body material solution" and repeat this step until the needle body part of the microneedle mold is filled; then perform light curing; (A2) Add the backing layer material solution required for preparing the hydrogel microneedle patch to the microneedle mold, dry it, and demold to obtain the hydrogel microneedle patch.

8. The method according to claim 7, wherein: In step (A1), the vacuum pumping is performed at 27°C for 2-3 times, 3 minutes each time; and / or, the heating is performed at 30°C for 2 h; and / or, the light curing is performed by irradiating with a 405 nm light source for 5 seconds; and / or In step (A2), the drying condition is drying at 27°C for more than 12 hours.

9. The application of the hydrogel microneedle patch according to any one of claims 1-6 in any one of the following: (B1) Preparing a product for inhibiting bacteria; (B2) Preparing a product for promoting the healing of burn wounds.

10. The application according to claim 9, characterized in that: The burn wound is a second-degree burn wound or a second-degree burn wound complicated with bacterial infection; and / or The bacteria are Gram-positive bacteria and / or Gram-negative bacteria; Furthermore, the Gram-positive bacteria are Staphylococcus aureus, and the Gram-negative bacteria are Escherichia coli.

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