A bacteria-targeted microneedle patch, preparation and application

Abstract

The present invention relates to a bacteria-targeted microneedle patch, preparation and application, and belongs to the field of biomedicine. In the present invention, the microneedle patch is composed of gelatinase-responsive antibacterial nanoparticles Ce6@GNP-Van and methacrylated hyaluronic acid (HAMA) hydrogel matrix. The enzyme-responsive antibacterial nanoparticles Ce6@GNP-Van use type A gelatin as raw material, synthesize enzyme-responsive gelatin nanoparticles (GNPs) by a two-step desolvation method, and load the photosensitizer dihydrochlorin e6 (Chlorin e6, Ce6) therein, and then modify vancomycin on its surface to obtain, apply the antibacterial nanoparticles to the microneedle patch, and prepare enzyme-responsive microneedles. The present invention combines antibacterial treatment with a microneedle drug delivery system to meet the requirements of precise bacterial targeting and stimulus-responsive drug release, thereby providing a new strategy for the treatment of abscess wounds.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to a bacteria-targeting microneedle patch, its preparation and application. Background Art

[0002] Staphylococcus aureus (S. aureus) is a common pathogen that can easily cause severe abscesses and chronic wounds, posing severe challenges to human health and a heavy economic burden. Common treatments for abscesses include puncture therapy and antibiotic therapy. However, puncture therapy not only causes large wounds, but also makes it difficult to completely remove the bacteria in the abscess and prone to recurrence. More importantly, there is a stubborn biofilm in the abscess wound, which is an organized colony combination surrounded by a self-generated matrix of extracellular polymers. It has stronger antibiotic resistance than planktonic bacteria. On the other hand, antibiotic treatment is an important cause of bacterial resistance, which will directly lead to treatment failure and cause more serious recurrence of the disease. Therefore, the development of new abscess treatment methods is urgent.

[0003] Microneedle patches, composed of tiny needles, have emerged as promising therapeutic tools, capable of penetrating the stratum corneum and delivering drugs to the dermis in a minimally invasive manner. To this end, microneedles have been further developed into various application modalities. Currently, template molding and cross-linking processes are commonly used to prepare hydrogel microneedles. Hyaluronic acid (HA) is the most commonly used polymer for hydrogel microneedle preparation due to its biocompatibility and gel-forming properties. HA can be modified with methacrylate and photocrosslinked to produce hydrogel microneedles. Upon entry into the skin, the microneedles swell and dissolve upon contact with the interstitial fluid, distributing the pre-loaded substance into the surrounding tissue. The photocrosslinked hydrogel network also provides mechanical strength to the microneedles, maintaining their integrity and facilitating their removal from the skin. Furthermore, microneedles are more convenient for medical staff and even patients to operate, thus combining microneedles with therapeutic systems has the potential for wider biomedical applications.

[0004] With advances in optical technology and the development of novel photosensitizers, photodynamic antibacterial therapy (PDAT) has become one of the most promising treatments for bacterial infections due to its low resistance to drug resistance. Irradiating photosensitizers with appropriate excitation light effectively kills bacteria by generating reactive oxygen species (ROS), which can cause oxidative damage to bacteria. However, common photosensitizers have poor water solubility and tend to aggregate in physiological environments, resulting in a weakened PDAT effect. Therefore, how to deliver photosensitizers to the epidermis / dermis via nanoparticles (MNs) is a key issue in achieving their full potential. As nanoparticle-based drug delivery systems have attracted significant attention in disease diagnosis and treatment, various biodegradable nanoparticles, both natural and synthetic, have been applied in the biomedical field. Among various natural polymers, gelatin has been used as a plasma expander for decades. Gelatin nanoparticles (GNPs) exhibit excellent biocompatibility, are easily metabolized, and can be conjugated to a variety of drugs, targeting ligands, and shielding substances. Functionalized GNP surfaces can be used for a variety of applications, including enhancing targeting capabilities or responding to surrounding stimuli (pH, enzymes, or temperature). Therefore, GNPs are expected to be loaded with photosensitizers with poor water solubility and then loaded into MNs as drug carriers for the treatment of bacterial infections. Summary of the Invention

[0005] The present invention aims to provide a gelatinase-responsive microneedle patch and its application. By combining enzyme-responsive drug-loaded nanoparticles with microneedles, a biodegradable microneedle patch composed of methacrylated hyaluronic acid (HAMA) has been developed. This patch can deliver enzyme-responsive drug-loaded nanoparticles to the lesions of abscess wounds. The microneedle patch prepared by the present invention exhibits excellent antibacterial and abscess-repairing effects, and has broad application prospects in antibacterial and photodynamic therapy of abscess wounds.

[0006] According to a first aspect of the present invention, a bacteria-targeted microneedle patch is provided, comprising a microneedle tip and a substrate; the microneedle tip contains drug-loaded gelatin nanoparticles, the drug-loaded gelatin nanoparticles are gelatinase-responsive, and the substrate is methacrylated hyaluronic acid; the drug-loaded gelatin nanoparticles are loaded with a photosensitizer, and the surface of the drug-loaded gelatin nanoparticles is modified with vancomycin.

[0007] Preferably, the photosensitizer can generate singlet oxygen under the action of light.

[0008] Preferably, the photosensitizer is dihydrochlorin e6.

[0009] According to another aspect of the present invention, there is provided a method for preparing any one of the bacteria-targeting microneedle patches, comprising the following steps:

[0010] (1) preparing gelatin nanoparticles loaded with a photosensitizer by a two-step desolvation method, and then modifying the surface of the gelatin nanoparticles with vancomycin to obtain drug-loaded gelatin nanoparticles;

[0011] (2) pouring the mixture of the drug-loaded gelatin nanoparticles obtained in step (1) and methacrylated hyaluronic acid onto the microneedle mold and drying to obtain a microneedle layer;

[0012] (3) Filling the microneedle layer obtained in step (2) with an aqueous solution of methacrylated hyaluronic acid, removing bubbles and drying the solution, then photocuring the solution, and peeling the solution off from the microneedle mold to obtain the microneedle patch.

[0013] Preferably, the photosensitizer can generate singlet oxygen under the action of light.

[0014] Preferably, the photosensitizer is dihydrochlorin e6.

[0015] According to another aspect of the present invention, there is provided a use of any one of the bacteria-targeting microneedle patches for preparing an antibacterial dressing.

[0016] Preferably, the antibacterial dressing is a dressing for treating abscess wounds.

[0017] Preferably, the application is specifically as follows: the vancomycin on the microneedle patch targets the bacterial community, the gelatinase secreted by the bacterial community causes the drug-loaded gelatin nanoparticles on the microneedle patch to disintegrate and release photosensitizers, and singlet oxygen is generated under laser irradiation, thereby achieving specific killing of bacteria by the drug-loaded gelatin nanoparticles.

[0018] Preferably, the bacterium is Staphylococcus aureus.

[0019] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0020] (1) The microneedle patch of the present invention can target bacterial communities and disintegrate and release drugs only in response to gelatinase secreted by bacterial communities. It also produces a large amount of singlet oxygen under laser irradiation, which enhances the specific killing of bacteria by nanoparticles, thereby achieving the purpose of promoting wound repair.

[0021] (2) GNP has good biocompatibility and contains many amino groups on its surface. After loading Ce6, it can not only improve its solubility, but also be grafted with carboxyl Van that can selectively identify and inactivate Staphylococcus aureus to obtain Ce6@GNP-Van. During the delivery of this composite nanoparticle, the singlet oxygen generation ability of Ce6 is significantly inhibited, avoiding the phototoxicity problem during delivery. Since common photosensitizers have poor water solubility and are prone to aggregation in physiological environments, resulting in a weakened PDAT effect, how to deliver photosensitizers to the epidermis / dermis through MN is the key to exerting their effect. Ce6 has a high efficiency in generating singlet oxygen and is widely developed for photodynamic therapy. However, like most photosensitizers to date, Ce6 is hydrophobic and easily aggregates in solution, so it has certain difficulties in practical application. Gelatin nanoparticles (GNPs) have good biocompatibility, are easily metabolized, and can be coupled with a variety of drugs, targeting ligands, and shielding substances.

[0022] (3) Staphylococcus aureus (S. aureus) is the main cause of abscess wounds, and S. aureus is a Gram-positive bacterium that can secrete gelatinase to cleave gelatin nanoparticles. Staphylococcus aureus (S. aureus) is a common pathogen that can easily cause severe abscesses and chronic wounds. Common treatments for abscesses include puncture therapy and antibiotic therapy. However, puncture therapy not only causes large wounds, but also makes it difficult to completely remove the bacteria in the abscess and prone to recurrence. More importantly, there is a stubborn biofilm in the abscess wound, which is an organized colony combination surrounded by an extracellular polymer matrix (EPS). It has stronger antibiotic resistance than planktonic bacteria. On the other hand, antibiotic treatment is an important reason for bacterial resistance, which will directly lead to treatment failure and cause more serious recurrence of the disease. Therefore, the development of new abscess treatment methods is imminent.

[0023] (4) Methacrylated hyaluronic acid (HAMA) has good biocompatibility, biodegradability and non-immunogenicity. The hydrogel formed after photocrosslinking has a certain mechanical strength. The prepared MN is sufficient for insertion into the skin. The preparation method is gentle and can well retain the pharmacological activity of the loaded drug. After penetrating the skin, HAMA swells when it contacts the interstitial fluid of the skin tissue. The incorporated drug will diffuse into the skin tissue. HAMA can also increase the hydration of the surface skin, causing the drug to tend to be retained in the more hydrophilic epidermis, reducing the possibility of the drug penetrating further through the dermis into the blood and thus being lost. Therefore, the use of HAMA as a matrix material to prepare microneedle patches loaded with antibacterial drugs has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Schematic diagram of the preparation of degradable MN patch based on enzyme-responsive nanoparticles Ce6@GNP-Van and its application in the treatment of bacterially infected abscess wounds.

[0025] Figure 2 Transmission electron microscopy images, particle size distribution and actual images of the enzyme-responsive nanoparticles GNP, Ce6@GNP, GNP-Van and Ce6@GNP-Van in Example 1.

[0026] Figure 3 This is a diagram showing the phenomenon of Staphylococcus aureus degrading gelatin into liquid in Example 2.

[0027] Figure 4 The production of reactive oxygen species under laser irradiation after the enzyme-responsive nanoparticles Ce6@GNP-Van in Example 3 were co-incubated with 1,3-diphenylisobenzofuran (DPBF).

[0028] Figure 5 The in vitro antibacterial properties of Ce6@GNP-Van in Example 4. Figure 5 Figure a is a photo of colonies on LB agar plates with or without laser irradiation at different concentrations. Figure 5 Middle b is the plate counting method for quantitative analysis of the effect of Ce6@GNP-Van on the activity of S. aureus.

[0029] Figure 6 This is the in vitro anti-biofilm activity of the enzyme-responsive nanoparticle Ce6@GNP-Van in Example 5.

[0030] Figure 7 Schematic diagram of the preparation and drug loading of the microneedle patch of Example 6.

[0031] Figure 8 This is a morphology characterization diagram of the microneedle patch of Example 6: Figure 8 a in the figure is a photo of a microneedle patch. Figure 8 b is the scanning electron microscope image and magnified image of the microneedle. Figure 8 In figure c is a schematic diagram of the structure of the needle tip.

[0032] Figure 9 This is the degradation diagram of the microneedle patch in mice in Example 7: Figure 9 a in the figure shows the microneedle patch on the mouse skin and the traces after the patch was removed. Figure 9 Figure b shows the morphology of the microneedle at different time points after insertion into the mouse skin.

[0033] Figure 10 This is a diagram showing the therapeutic effect of the drug-loaded microneedle patch in Example 8 on promoting the repair of abscess wounds in vivo.

[0034] Figure 11These are bacterial cultures of skin tissue infected with S. aureus in eight different treatment groups after 13 days of treatment in Example 8. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0036] The microneedle patch provided by the present invention comprises enzyme-responsive drug-loaded nanoparticles Ce6@GNP-Van and a methacrylated hyaluronic acid (HAMA) hydrogel matrix. The gelatinase-responsive microneedle patch of the present invention is prepared by photocuring the active ingredient (gelatinase)-responsive drug-loaded nanoparticles Ce6@GNP-Van and the methacrylated hyaluronic acid (HAMA) hydrogel matrix.

[0037] In some embodiments, the enzyme-responsive drug-loaded nanoparticles Ce6@GNP-Van in the microneedle patch of the present invention are made from type A gelatin. Bacteria-responsive gelatin nanoparticles GNP are synthesized by a two-step desolvation method, and the photosensitizer dihydrochlorin e6 (Chlorin e6, Ce6) is loaded into it. Vancomycin (Van) is then modified on its surface to obtain composite nanoparticles (Ce6@GNP-Van). GNP has good biocompatibility and contains many amino groups on its surface. After loading Ce6, it can not only improve its solubility, but also be grafted with carboxyl-containing Van that can selectively identify and inactivate Staphylococcus aureus to obtain Ce6@GNP-Van. During the transportation of the composite nanoparticles, the singlet oxygen production ability of Ce6 is significantly suppressed, avoiding the problem of phototoxicity during transportation. Ce6@GNP-Van is applied to the microneedle patch to make enzyme-responsive microneedles. The microneedle patch of the present invention, the enzyme-responsive drug-loaded nanoparticles can target bacterial communities and disintegrate and release drugs only in response to gelatinase secreted by the bacterial communities, and generate a large amount of singlet oxygen under laser irradiation, thereby enhancing the specific killing of bacteria by the nanoparticles, thereby achieving the purpose of promoting wound repair. Figure 1 Schematic diagram of the preparation of degradable MN patch based on enzyme-responsive nanoparticles Ce6@GNP-Van and its application in the treatment of bacterially infected abscess wounds.

[0038] The microneedle patch of the present invention uses methacrylated hyaluronic acid (HAMA) because of its good biocompatibility, biodegradability and non-immunogenicity. The hydrogel formed after photocrosslinking has a certain mechanical strength. The prepared microneedles are sufficient for insertion into the skin. The preparation method is gentle and can well retain the pharmacological activity of the loaded drug.

[0039] The microneedle patch of the present invention can physically penetrate and destroy the bacterial biofilm structure when applied to the infected area, swell and degrade after contacting body fluids, and promote the release of antibacterial agents.

[0040] In some embodiments, the preparation method of the microneedle patch of the present invention is prepared by a two-step casting process, using HAMA hydrogel loaded with enzyme-responsive nanoparticles Ce6@GNP-Van to make the MN tip, and the HAMA hydrogel is made into the second layer of the support structure.

[0041] Specifically:

[0042] First, HAMA hydrogel loaded with Ce6@GNP-Van was used to make the microneedle tip, and HAMA hydrogel was made into the second layer of the support structure.

[0043] First, 1 g of HAMA was dissolved in 20 mL of PBS and stirred at 37°C for 1 hour to prepare a 5% (w / v) HAMA solution; a photoinitiator LAP solution (2.5 mg / mL) was added to the HAMA solution and mixed thoroughly to obtain a uniform solution; 1 mL of the Ce6@GNP-Van and HAMA mixture was poured onto the microneedle PDMS mold. After covering, the mold was centrifuged at 2000 g and dried under air flow three times. The mold was placed in a 30°C oven and dried for 6 hours to obtain the microneedle layer.

[0044] Next, to form a dissolvable support, a 5% (w / v) aqueous solution of HAMA was refilled onto the dried microneedle layer in the PDMS mold and treated at 1 bar pressure for 5 minutes to remove air bubbles. The mold was then dried in a 30°C oven for 6 hours, then cured with 405nm light for 2 seconds and dried at 30°C for another 12 hours.

[0045] Finally, the MN patch was gently peeled off from the PDMS mold to obtain a microneedle patch loaded with Ce6@GNP-Van (MN / Ce6@GNP-Van). The resulting MN patch was stored in a sealed 12-well plate for further study. This composite microneedle patch provides a new strategy for the treatment of abscess wounds.

[0046] The application of the microneedle patch in the present invention can meet the requirements of precise bacterial targeting and stimulus-responsive drug release, and can be used as a wound dressing to eradicate bacterial infection on the wound surface and promote the stagnant healing of abscess wounds.

[0047] The following are specific embodiments

[0048] Example 1: Preparation of enzyme-responsive nanoparticles

[0049] First, bacteria-responsive gelatin nanoparticles (GNPs) were synthesized using type A gelatin via a two-step desolvation method. The photosensitizer chlorin e6 (Ce6) was then loaded onto these nanoparticles, and vancomycin was then modified onto their surfaces to create composite nanoparticles (Ce6@GNP-Van). GNPs have excellent biocompatibility and contain numerous amino groups on their surfaces. Loading Ce6 not only improves their solubility but also allows for grafting with carboxyl groups on Van, which can selectively recognize and inactivate Staphylococcus aureus, to create Ce6@GNP-Van. Preparation was performed according to the following steps:

[0050] (1) Preparation and characterization of Ce6@GNPs

[0051] Ce6@GNPs were prepared using a two-step desolvation method. First, 1.25 g of type A gelatin was dissolved in 25 mL of deionized water at 40°C. Then, 25 mL of acetone was added with stirring at 600 rpm (5 mL / min), which produced a partially high-molecular-weight gelatin precipitate. The supernatant was discarded, and the precipitate was dissolved in 15 mL of water. 4 mg of Ce6 was added, and the pH was adjusted to 2.5 with 1 M HCl. With continued stirring, approximately 40 mL of acetone was added dropwise to the solution at 1 mL / min. After the mixture turned milky white again, 50% glutaraldehyde (125 μL, 1 μL / s) was added dropwise and stirred for 16 h to crosslink the gelatin and form stable particles. The reaction mixture was rotary evaporated to remove the acetone and excess glutaraldehyde, and the solution was concentrated to approximately 10 mL. The Ce6@GNPs were collected by centrifugation and stored in a refrigerator at 4°C until further use.

[0052] (2) Preparation and characterization of Ce6@GNP-Van

[0053] 2 mL of vancomycin hydrochloride (0.084 mg / mL) was dissolved in 6 mL of MES buffer (pH 4-6). 0.2045 g of EDC and 0.2316 g of NHS were then added for activation for 30 minutes. Next, 2 mL of Ce6@GNP dispersion (20 mg / mL) was added, the pH was adjusted to 7.4, and the mixture was stirred for 3 hours. After the reaction, the Ce6@GNP-Van was purified by ultrafiltration and centrifugation, and its particle size and morphology were characterized.

[0054] The results are as follows Figure 2 As shown, transmission electron microscopy observed that the prepared GNPs and drug-loaded Ce6@GNP, GNP-Van, and Ce6@GNP-Van were all discrete and uniform spherical, with average particle sizes of approximately 159.1, 176.9, 169.5, and 182.6 nm, respectively.

[0055] Example 2: Experiment on liquefaction of gelatin by Staphylococcus aureus

[0056] Staphylococcus aureus (ATCC 6538) was cultured in LB medium and the bacteria in the exponential growth phase were collected by centrifugation for subsequent experiments. The bacterial concentration was monitored by measuring the optical density (OD) at 600 nm using a microplate reader. 600 The value was adjusted to 0.1, and the S. aureus bacterial concentration corresponding to this OD value was determined to be 2×10 8 CFU / mL. S. aureus was inoculated into LB medium containing 3% (w / w) gelatin. The inoculated tubes were cultured together with uninoculated tubes at 37°C. After 24 hours, the tubes were placed in a 4°C refrigerator for 30 minutes. The gelatin was tilted and observed for liquefaction. A positive result was observed when the medium no longer solidified.

[0057] The results are as follows Figure 3 As shown, S. aureus can produce an extracellular gelatinase that breaks down gelatin into amino acids, thereby losing its coagulation power. Therefore, a gelatin liquefaction test can be performed using a semi-solid gelatin culture medium to examine S. aureus's degradation of gelatin. After S. aureus was inoculated into the gelatin culture medium and incubated at 37°C for 24 hours, the test tube was cooled to 4°C. The results showed that the culture medium without bacteria had solidified, while the culture medium with bacteria was in a liquefied state, confirming that S. aureus can indeed degrade gelatin. This lays the foundation for the subsequent degradation of drug-loaded GNPs in response to microbial gelatinase, which then triggers the release of the loaded drug into the biofilm.

[0058] Example 3: Singlet oxygen production from enzyme-responsive nanoparticles Ce6@GNP-Van after light treatment

[0059] Quantitative analysis of singlet oxygen production by Ce6@GNP-Van using DPBF 1To determine the O2 capacity, a microplate reader was used to measure the absorbance at 415 nm of the DPBF and Ce6@GNP-Van mixture after various treatments. The samples were divided into six groups: PBS, PBS + L, Ce6@GNP-Van, Ce6@GNP-Van + L, Ce6@GNP-Van + S. aureus, and Ce6@GNP-Van + S. aureus + L, where L represents laser irradiation. The remaining amount of DPBF is calculated as 1 - (initial mixture absorbance - final absorbance) / initial mixture absorbance × 100%.

[0060] The experimental results are as follows Figure 4 As shown in the figure, with the extension of laser irradiation time, the absorption of Ce6@GNP-Van+L group decreased significantly, indicating that Ce6 was photodynamically generated. 1 O2 gradually increased and oxidized DPBF; while the absorbance of Ce6@GNP-Van group without laser irradiation remained basically unchanged, indicating that Ce6@GNP-Van can produce a large amount of 1 O2, laying the foundation for subsequent photodynamic antibacterial therapy. More importantly, the co-incubation of Ce6@GNP-Van with bacteria significantly enhanced the 1 The generation of O2 (p < 0.01) may be due to the fact that after the photosensitizer is loaded into GNPs, the 1 The ability to produce O2 is inhibited to a certain extent. When Ce6@GNP-Van is targeted and enriched in bacteria, the gelatinase secreted by the bacteria will degrade the nanoparticles and release Ce6. Under laser irradiation, the ability to produce ROS is significantly enhanced, which will be able to kill bacteria more effectively.

[0061] Example 4: In vitro antibacterial and photodynamic antibacterial activities of enzyme-responsive nanoparticles Ce6@GNP-Van

[0062] In order to explore the antibacterial effect of Ce6@GNP-Van on S. aureus, different concentrations of Ce6@GNP-Van were mixed with S. aureus culture (10 7 After incubation for 0.5 h, 100 μL of each bacterial suspension was applied to LB agar plates and cultured at 37 °C for 18 h. The number of colonies on the LB agar plates was recorded and the inhibition rate was calculated. 7 CFU / mL of S. aureus was added to LB medium and incubated for 0.5 h. The mixture was then irradiated with a laser (660 nm, 0.8 W / cm 2) for 5 minutes (all subsequent laser irradiations were performed under these conditions unless otherwise specified). Subsequently, 100 μL of each bacterial suspension was plated onto LB agar plates and incubated at 37°C for 18 hours. The number of colonies on the LB agar plates was recorded, and the inhibition rate was calculated. The bacterial survival rate (S%) was calculated according to the equation S% = Nb / Na, where Na and Nb represent the number of bacteria before and after incubation, as determined by the gold standard colony count method.

[0063] The results showed that there was no significant difference in the number of colonies on LB agar plates with or without light exposure under different concentrations of GNPs ( Figure 5 a) in the figure, basically no antibacterial effect; there was no obvious antibacterial difference between different concentrations of Ce6@GNP under no light conditions, but after light treatment, with the increase of Ce6@GNP concentration, the number of colonies on the LB agar plate gradually decreased, showing a concentration-dependent antibacterial inhibition effect; the antibacterial effect of different concentrations of GNP-Van in the same concentration group was not much different regardless of whether it was exposed to light or not, but with the increase of GNP-Van concentration, the number of colonies on the LB agar plate gradually decreased, which showed that Van played a targeted bactericidal role to a certain extent; compared with the first three control groups, Ce6@GNP-Van showed good antibacterial effect under both light and no light conditions, and the antibacterial effect was concentration-dependent, and the minimum inhibitory concentration MIC of Ce6@GNP-Van was 1.3847 W / v. 90 The value is about 15μM ( Figure 5 b), while the minimum inhibitory concentration MIC of Ce6@GNP-Van+L 90 The value was as low as 5 μM, indicating that Ce6@GNP-Van had the best antibacterial results under light conditions.

[0064] Example 5: Determination of anti-biofilm activity of enzyme-responsive nanoparticles Ce6@GNP-Van

[0065] Live / dead bacterial staining was used to study the changes in bacterial viability before and after incubation with Ce6@GNP-Van. The biofilm was treated with Ce6@GNP-Van for different times (0, 0.5 and 1h), and half of the samples in each group were exposed to light for 5 minutes after treatment as the Ce6@GNP-Van+L group. After that, they were carefully washed with PBS, and then a certain proportion of SYTO 9 and PI mixed dyes were added to the surface of the biofilm and incubated in the dark at room temperature for 0.5h. After staining, the dye was discarded and washed with sterile PBS solution. The biofilm was imaged using an FV3000 confocal laser scanning microscope to evaluate the damage of Ce6@GNP-Van to the S. aureus biofilm. At the same time, the morphological changes of bacteria before and after incubation with Ce6@GNP-Van were studied using an SU8010 field emission scanning electron microscope (FESEM). Biofilms were treated with Ce6@GNP-Van for different time periods (0, 0.5, and 1 hour). Half of each sample was then exposed to light for an additional 5 minutes after treatment to form the Ce6@GNP-Van+L group. The bacterial suspensions, before and after treatment, were dripped onto silicon wafers and fixed with 2% glutaraldehyde. The cells were then dehydrated using a series of graded ethanol solutions (30%-100%). Finally, the silicon wafers with attached bacteria were dried with nitrogen and imaged using FESEM after platinum sputter coating.

[0066] SYTO 9 can enter all bacteria and show green fluorescence. PI cannot pass through the membrane of living cells, but can embed into DNA through the membrane of dead cells to produce red fluorescence. However, this will cause the fluorescence of SYTO 9 to weaken. Therefore, by mixing SYTO 9 and PI in an appropriate ratio, it is possible to achieve green fluorescence in living bacteria and red fluorescence in dead bacteria. The experimental results are as follows Figure 6The biofilm without antimicrobial agents showed thick, dense live bacteria, exhibiting bright green fluorescence. After 0.5 hours of Ce6@GNP-Van treatment of the S. aureus biofilm, in addition to green fluorescence, a significant amount of red fluorescence also appeared. This is likely due to the gradual penetration of Ce6@GNP-Van into the biofilm. Van's targeting ability for S. aureus increased its specificity for bacteria within the biofilm, contributing to a certain degree of bacterial killing. The presence of green fluorescence also suggests that the antimicrobial alone cannot completely kill the bacteria. Laser irradiation of the sample at this point further reduced the green fluorescence and increased the red fluorescence, indicating that the antimicrobial combined with PDAT enhances the bactericidal effect. After 1 hour of Ce6@GNP-Van treatment, a small amount of green fluorescence was still observed in the biofilm, while the S. aureus biofilm, after light treatment, almost completely turned red, demonstrating that the antimicrobial combined with PDAT is highly effective in killing bacteria. SEM observations revealed that bacteria without Ce6@GNP-Van treatment exhibited a normal spherical morphology. In stark contrast, the bacterial capsules of some treated with Ce6@GNP-Van exhibited deformation and contraction. This phenomenon became more pronounced after further laser irradiation, and with increasing treatment time, the bacteria were observed to undergo complete shrinkage and lysis. These results suggest that the combination of the antimicrobial agent and PDAT exhibits enhanced synergistic antimicrobial and anti-biofilm activity.

[0067] Example 6: Preparation of microneedle patch

[0068] The microneedle patch was fabricated using a two-step solvent casting process with a dissolvable support substrate. The microneedle tip was made of HAMA hydrogel loaded with Ce6@GNP-Van, and the HAMA hydrogel was used as the second layer of the support structure.

[0069] First, 1 g of HAMA was dissolved in 20 mL of PBS and stirred at 37°C for 1 h to prepare a 5% (w / v) HAMA solution; a photoinitiator LAP solution (2.5 mg / mL) was added to the HAMA solution and mixed thoroughly to obtain a uniform solution; 1 mL of the Ce6@GNP-Van and HAMA mixture was poured onto the microneedle PDMS mold. After covering, the mold was centrifuged at 2000 g and dried under air flow three times. The mold was placed in a 30°C oven and dried for 6 h to obtain the microneedle layer.

[0070] To form a soluble support substrate, a 5% (w / v) HAMA aqueous solution was refilled onto the dried microneedle layer in the PDMS mold and treated at 1 bar pressure for 5 minutes to remove bubbles. The mold was placed in a 30°C oven for 6 hours, then cured with 405nm light for 2 seconds and continued to dry at 30°C for 12 hours. Finally, the microneedle patch was gently peeled from the PDMS mold to obtain a microneedle patch loaded with Ce6@GNP-Van (MN / Ce6@GNP-Van). Similarly, microneedle patches loaded with only GNPs, Ce6-GNPs, and GNP-Van (MN / GNP, MN / Ce6-GNP, MN / GNP-Van) were prepared as controls. The resulting microneedle patches were stored in sealed 12-well plates for further study.

[0071] Figure 7 Schematic diagram of the preparation and drug loading of the MN patch of Example 6; Figure 8 The morphology characterization diagram of the microneedle of Example 5: The digital picture of the microneedle is as follows: Figure 8 As shown in a, the SEM image clearly shows that the MN patch consists of 400 (20×20) microneedles on the bottom plate ( Figure 8 b), the needle height is about 600 μm ( Figure 8 In (c), the base diameter is 250 μm, the center-to-center spacing between adjacent needles is 550 μm, and the microneedles are conical in shape, uniform in size, and have sharp tips, which are necessary for insertion into the skin.

[0072] Example 7: Skin Insertion and Degradation Performance of Microneedle Patch

[0073] To evaluate the skin insertion ability of microneedles, the microneedles were inserted into the mouse skin for 10 minutes. After the supporting substrate was completely melted, photos were taken and observed. The morphology of the microneedle patch at different time points was observed using an ultra-deep three-dimensional microscope to evaluate the biodegradability of the patch.

[0074] The results are as follows Figure 9 As shown, in order to test whether the prepared microneedles can be fully inserted into the skin, mice were used as a model. After 10 minutes of microneedle application, the skin surface was wiped clean to remove the degraded HAMA support substrate. It can be observed that the mouse skin surface showed a complete array of spots (20×20) corresponding to the MN microneedle sites, indicating that the MNs have been inserted into the skin ( Figure 9 To evaluate the degradation ability of Ce6@GNP-Van-loaded MNs in the interstitial fluid of the skin, MN patches were removed from the back skin of BALB / c mice at different time points during the application process for microscopic imaging. The results showed that the needle tips of the MNs were blunted after being inserted into the skin ( Figure 9In b), due to the high biodegradability of HAMA, the microneedle can basically melt within 10 minutes, proving that MN can penetrate the skin and quickly degrade after contact with interstitial fluid. It also has good hygroscopic properties. This property enables MN to absorb excess tissue penetrating fluid produced by the wound, providing favorable conditions for wound healing.

[0075] Example 8: Abscess wound repair experiment

[0076] Female mice (Balb / c, 6 weeks, ~20 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. First, a mouse model of S. aureus infection was established. The mice were anesthetized by intraperitoneal injection, and the backs were depilated and disinfected with iodine. 50 μL of 10 7 A bacterial solution containing 100 CFU / mL of S. aureus was injected at a depth of 5 mm. A mouse abscess wound model was established 16 hours after infection. All animal experiments were approved by the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology (IACUC Number: S904).

[0077] To investigate the antibacterial efficacy of the MN system against S. aureus-infected mice, infected mice were divided into eight treatment groups, each containing five mice: PBS, PBS+L, MN, MN / GNP, MN / GNP-Van, MN / Ce6@GNP, MN / Ce6@GNP-Van, and MN / Ce6@GNP-Van+L, where L represents laser irradiation. The microneedle system was applied to infected abscess wounds, and the wounds were photographed every two days to document the extent of ulceration. After 13 days of treatment, the mice were sacrificed, and skin tissue from the infected wounds was obtained and abraded and plated to count bacterial colonies to evaluate the in vivo photodynamic antibacterial efficacy of the microneedle system.

[0078] The results are as follows Figure 10 As shown in the figure, by observing the changes in the wounds during the treatment, it can be seen that the wounds in the PBS group and PBS+L group have not healed. Due to severe bacterial infection, the wound surface was in an inflammatory and suppurative state throughout the experiment, with visible scabs and bacterial pus to the naked eye, indicating that relying solely on autoimmunity is far from enough to repair the infected skin; the abscess sites of the mice in the MN group, MN / GNP group, MN / Ce6@GNP group and MN / Ce6@GNP+L group showed obvious scabs, but the recovery was incomplete; while the MN / Ce6@GNP-Van group and MN / Ce6@GNP-Van+L group had re-epithelialization, and the abscess area of ​​the mice was effectively controlled. The treatment effects of these two groups were significantly better than those of the first 6 groups, which was due to the effect of the loaded antibacterial agent; compared with the MN / Ce6@GNP-Van group, the MN / Ce6@GNP-Van+L group had a relatively better complete re-epithelialization effect and the smallest scar area.

[0079] To further confirm the in vivo antibacterial properties of MN / Ce6@GNP-Van, we extracted skin tissue from the abscess wound 13 days after treatment, crushed the skin area evenly, and diluted it with PBS to perform a smear test to determine the residual bacteria in the abscess wound and check whether the infection in the wound was cleared. Figure 11 As shown, consistent with the wound healing results, MN / Ce6@GNP-Van+L showed almost complete bacterial eradication, in stark contrast to the other groups of mice. This result suggests that wound healing and antibacterial therapy are inseparable.

[0080] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bacteria-targeting microneedle patch, characterized in that: The microneedle patch includes a microneedle tip and a substrate; the microneedle tip contains drug-loaded gelatin nanoparticles, the drug-loaded gelatin nanoparticles are gelatinase responsive, and the substrate is methacrylated hyaluronic acid; the drug-loaded gelatin nanoparticles are loaded with a photosensitizer, and the surface of the drug-loaded gelatin nanoparticles is modified with vancomycin; the drug-loaded gelatin nanoparticles and the methacrylated hyaluronic acid are photocured to obtain the microneedle patch.

2. The bacteria-targeting microneedle patch according to claim 1, wherein: The photosensitizer can generate singlet oxygen under the action of light.

3. The bacteria-targeting microneedle patch according to claim 2, wherein: The photosensitizer is dihydrochlorin e6.

4. The method for preparing the bacteria-targeting microneedle patch according to any one of claims 1 to 3, wherein: The following steps are involved: (1) preparing gelatin nanoparticles loaded with a photosensitizer by a two-step desolvation method, and then modifying the surface of the gelatin nanoparticles with vancomycin to obtain drug-loaded gelatin nanoparticles; (2) pouring the mixture of the drug-loaded gelatin nanoparticles obtained in step (1) and methacrylated hyaluronic acid onto the microneedle mold and drying to obtain a microneedle layer; (3) Filling the microneedle layer obtained in step (2) with an aqueous solution of methacrylated hyaluronic acid, removing bubbles and drying the solution, then photocuring the solution, and peeling the solution off from the microneedle mold to obtain the microneedle patch.

5. The method for preparing the bacteria-targeting microneedle patch according to claim 4, wherein: The photosensitizer can generate singlet oxygen under the action of light.

6. The method for preparing the bacteria-targeting microneedle patch according to claim 5, wherein: The photosensitizer is dihydrochlorin e6.

7. Use of the bacteria-targeting microneedle patch according to any one of claims 1 to 3 for preparing an antibacterial dressing.

8. The use according to claim 7, characterized in that The antibacterial dressing is a dressing for treating abscess wounds.

9. The use according to claim 7 or 8, characterized in that The application is specifically as follows: the vancomycin on the microneedle patch targets the bacterial community, and the gelatinase secreted by the bacterial community causes the drug-loaded gelatin nanoparticles on the microneedle patch to disintegrate and release photosensitizers, and singlet oxygen is generated under laser irradiation, thereby achieving the specific killing of bacteria by the drug-loaded gelatin nanoparticles.

10. The use according to claim 9, characterized in that The bacteria is Staphylococcus aureus.

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