Degradable microneedle patch CeO2-CIP MN and preparation method and application thereof

Abstract

The invention discloses a degradable microneedle patch CeO2-CIP MN as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Comprising the following steps: dissolving ciprofloxacin, mixing the dissolved ciprofloxacin with CeO2 and gelatin to prepare a mixed solution, pouring the mixed solution into a microneedle mold, and vacuumizing to fill the tip of the microneedle with the mixed solution; and removing the redundant solution, adding the mixed solution to fill the backing, drying overnight at room temperature, and finally stripping the microneedle from the mold to obtain the degradable microneedle patch. According to the invention, the degradable microneedle patch loaded with the cerium dioxide nanoparticles and the ciprofloxacin is constructed, and the microneedle patch has antibacterial and antioxidant properties at the same time. In a vibrio vulnificus infected wound model of a mouse soaked in seawater, the CeO2-CIP MN can effectively kill vibrio vulnificus, remove active oxygen of the wound, promote cell proliferation and accelerate repair of the infected wound. Therefore, CeO2-CIP MN has great potential in the aspect of treatment of wounds infected by vibrio vulnificus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a degradable microneedle patch CeO2-CIP MN and a preparation method and application thereof. Background Art

[0002] When living in coastal areas or sailing at sea, broken skin directly contacts seafood or seawater, which may cause Vibrio vulnificus infection. Vibrio vulnificus (V. vulnificus) is a Gram-negative bacterium with a fast spread and strong toxicity. The average incubation period is 24 hours. The patient's condition will worsen with each hour of delay. Within one or two days after infection, it can cause skin redness and swelling, severe pain, and tissue necrosis or sepsis in severe cases, with a mortality rate of up to 50%. In addition, soaking in high-salt, hypertonic seawater will aggravate tissue damage and cause slow wound healing. Therefore, it is particularly important to develop an antibacterial material that is convenient to carry and use at sea when Vibrio vulnificus infection is promptly treated and treated in the early stage.

[0003] In infected wounds, bacteria usually form biofilms to provide a barrier for bacteria to resist external damage. Recently, microneedles (MNs) have been applied to the regeneration and repair of skin wounds as a minimally invasive, painless and efficient new drug delivery system. Microneedle patches have many tiny needles that can break through the barriers of the stratum corneum and biofilm, forming micron-scale channels on the skin surface, thereby guiding antibacterial drugs to deeper parts and improving drug delivery efficiency. Studies have shown that Vibrio vulnificus is sensitive to multiple types of antibacterial drugs, such as third-generation cephalosporins, quinolones, and tetracyclines. Currently, antibiotic treatment is an effective method to kill Vibrio vulnificus. Since Vibrio vulnificus infection is prone to occur in people with low immunity, it is necessary to use broad-spectrum antibiotics to treat Vibrio vulnificus infection and eliminate other potential bacteria. Ciprofloxacin, as a third-generation quinolone antibacterial drug, has a broad-spectrum antibacterial property and has high antibacterial activity against Gram-negative bacteria. It is also used clinically to treat skin infections.

[0004] Bacterial infection triggers the body's immune response. After being activated, immune cells release a large amount of reactive oxygen species (ROS) that kill bacteria, such as superoxide anions (O2 - ), hydrogen peroxide (H2O2), hydroxyl radical (·OH), etc. However, excessive ROS can disrupt the redox balance in cells and lead to oxidative stress. In recent years, nano-cerium dioxide has attracted extensive attention in the biomedical field due to its good redox properties. 3+ and Ce 4+ The rapid transition between valence states enables it to exhibit a variety of enzyme mimicking activities, such as catalase (CAT) and superoxide dismutase (SOD) activities, thus having good ROS scavenging function.

[0005] However, there are no reports on combining ciprofloxacin, CeO2 and microneedle drug delivery system. Summary of the invention

[0006] The purpose of the present invention is to provide a degradable microneedle patch CeO2-CIP MN and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a method for preparing a degradable microneedle patch CeO2-CIP MN, comprising the following steps:

[0009] After ciprofloxacin is dissolved, it is mixed with CeO2 and gelatin to prepare a mixed solution, which is poured into a microneedle mold and vacuumed to allow the mixed solution to fill the microneedle tip; after removing excess solution, the mixed solution is added to fill the backing, dried overnight at room temperature, and finally the microneedle is peeled off from the mold to obtain the degradable microneedle patch.

[0010] The second technical solution of the present invention is the degradable microneedle patch CeO2-CIP MN prepared by the preparation method.

[0011] The third technical solution of the present invention is the use of the degradable microneedle patch CeO2-CIP MN in the preparation of a drug for treating wounds infected with Vibrio vulnificus.

[0012] A fourth technical solution of the present invention is a medicine for treating wounds infected with Vibrio vulnificus, comprising the degradable microneedle patch CeO2-CIP MN.

[0013] A fifth technical solution of the present invention is the use of the degradable microneedle patch CeO2-CIP MN in the preparation of a drug for promoting wound healing.

[0014] The sixth technical solution of the present invention is a medicine for promoting wound healing, comprising the degradable microneedle patch CeO2-CIP MN.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] The present invention constructs a degradable microneedle patch (CeO2-CIP MN) loaded with cerium dioxide nanoparticles (CeO2NPs) and ciprofloxacin (CIP). The good broad-spectrum antibacterial effect of CIP and the multiple enzyme-mimicking activities of CeO2 make CeO2-CIP MN have both antibacterial and antioxidant properties. In a Vibrio vulnificus infection wound model of mice immersed in seawater, CeO2-CIP MN can effectively kill Vibrio vulnificus, remove wound reactive oxygen species (ROS), promote cell proliferation, and accelerate the repair of infected wounds. Therefore, CeO2-CIP MN has great potential in the treatment of Vibrio vulnificus infection wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Preparation and characterization of CeO2-CIP MNs. (a) Preparation process of CeO2-CIP MNs; (b) Schematic diagram of microneedle dimensions; (c) Overall photo of CeO2-CIP MNs; (d) Representative optical images of CeO2-CIP MNs; (e, f) SEM images of CeO2-CIP MN arrays and tips; (g) Image of CeO2-CIP MNs inserted into the back skin of living mice; (h) H&E staining image of CeO2-CIP MNs inserted into the back skin of mice; (i) Image of CeO2-CIP MNs inserted into agarose gel; (j) Schematic diagram of microneedle mechanical strength test; (k) Stress-displacement curve during microneedle compression; (l) In vitro degradation curve of CeO2-CIP MNs; (m) In vitro release curve of CIP in CeO2-CIP MNs.

[0019] Figure 2The in vitro antibacterial and antioxidant properties of the microneedle patch. Among them, (a) Representative colony images of V. vulnificus, E. coli and S. aureus cultured on agar plates after treatment under different conditions; (b) Representative inhibition zone images of V. vulnificus, E. coli and S. aureus under different treatment conditions; (c) Inhibition rates of V. vulnificus, E. coli and S. aureus under different treatment conditions; (d) Inhibition zone diameters of V. vulnificus, E. coli and S. aureus under different treatment conditions; (e) Representative live / dead images of V. vulnificus under different treatment conditions; (f) Schematic diagram of ABTS+· free radical scavenging process; (g) The ability of CeO2-CIP MN to scavenge ABTS+· free radicals; (h) Image of CeO2-CIP MN scavenging OH·; (i) The ability of CeO2-CIP MN to scavenge OH·.

[0020] Figure 3 The in vitro biosafety of CeO2-CIP MNs. (a) Biocompatibility of different groups of microneedles on L929 cells; (b) Biocompatibility of different groups of microneedles on HUVEC cells; (c) Biocompatibility of different groups of microneedles on RAW264.7 cells; (d) Migration rate of L929 cells under different treatment conditions; (e) Representative migration images of L929 cells under different treatment conditions; (f) Recovery of micropores in the back skin of mice after CeO2-CIP MNs were applied.

[0021] Figure 4 CeO2-CIP MN promotes wound healing in Vibrio vulnificus infection. (a) Schematic diagram of in vivo experiment of microneedle application in mouse Vibrio vulnificus infection wound model; (b) Representative images of wounds in mice in different treatment groups (uninfected control group, Vibrio vulnificus infection control group and CeO2-CIP MN group) within 13 days; (c) Simulation diagram of wound healing process in different treatment groups; (d) Wound healing rate in different groups; (e) Changes in body temperature of mouse wounds within 13 days; (f) Changes in weight of mice within 13 days; (g) Histological analysis results of wounds in different treatment groups on the 13th day.

[0022] Figure 5 The in vivo biosafety of CeO2-CIP MNs. (a) Representative images of H&E staining of mouse organs (heart, liver, spleen, lung, and kidney); (b) Blood routine test data of mice on day 13.

[0023] Figure 6 Representative live / dead images of E. coli under different treatment conditions.

[0024] Figure 7 Representative live / dead images of S. aureus under different treatment conditions. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0031] The embodiment of the present invention provides a method for preparing a degradable microneedle patch CeO2-CIP MN, comprising the following steps:

[0032] After ciprofloxacin is dissolved, it is mixed with CeO2 and gelatin to prepare a mixed solution, which is poured into a microneedle mold and vacuumed to allow the mixed solution to fill the microneedle tip; after removing excess solution, the mixed solution is added to fill the backing, dried overnight at room temperature, and finally the microneedle is peeled off from the mold to obtain the degradable microneedle patch.

[0033] In some specific embodiments, the concentration of ciprofloxacin in the mixed solution is 400 μg / mL.

[0034] In some specific embodiments, the concentration of CeO2 in the mixed solution is 100 μg / mL.

[0035] In some specific embodiments, the mass volume ratio of the gelatin to the mixed solution is 1:4 (g / mL).

[0036] The embodiment of the present invention also provides a degradable microneedle patch CeO2-CIP MN prepared by the preparation method.

[0037] The embodiment of the present invention also provides the use of the degradable microneedle patch CeO2-CIP MN in preparing a medicine for treating bacterially infected wounds.

[0038] In some specific embodiments, the bacteria include Vibrio vulnificus, Escherichia coli, and Staphylococcus aureus.

[0039] The embodiment of the present invention further provides a medicine for treating bacterially infected wounds, comprising the degradable microneedle patch CeO2-CIP MN.

[0040] The embodiment of the present invention also provides the use of the degradable microneedle patch CeO2-CIP MN in preparing a medicine for promoting wound healing.

[0041] The embodiment of the present invention further provides a medicine for promoting wound healing, comprising the degradable microneedle patch CeO2-CIP MN.

[0042] The present invention prepares an antibacterial and antioxidant degradable microneedle patch (CeO2-CIP MN) for accelerating the healing of wounds infected with Vibrio vulnificus. Gelatin (Gel) has good biocompatibility and degradability, can promote cell adhesion, proliferation and differentiation, and is selected as the substrate of the microneedle patch. When applied to the wound, the microneedle patch can effectively deliver the loaded ciprofloxacin and CeO2 to the infected site, thereby playing a rapid bactericidal and antioxidant role and reducing the inflammatory response of the wound. In the wound model infected with Vibrio vulnificus, CeO2-CIP MN showed good therapeutic effect. At the same time, the prepared CeO2-CIP MN also showed high antibacterial activity against Escherichia coli (Escherichia coli, E.coli) and Staphylococcus aureus (Staphylococcus aureus, S.aureus).

[0043] Example 1

[0044] 1 Materials

[0045] All chemical reagents were purchased commercially and used directly in this work without further purification. Gelatin (Gel, biotechnology grade) and nano-cerium oxide (CeO2, 20nm-50nm spherical, 99.5%) were purchased from Macklin, Shanghai, China. Ciprofloxacin was purchased from Heowns, Tianjin, China. Luria-Bertani (LB) broth, LB agar, tryptone soy broth (TSB), and 3% sodium chloride tryptone soy agar (TSA) were purchased from HKM, Guangdong, China. Total antioxidant capacity test kit (ABTS method) and CCK-8 kit were purchased from Beyotime, Shanghai, China. Hydroxyl free radical test kit was purchased from Nanjing Jiancheng Bioengineering Institute. Bacterial live and dead dyes were purchased from ThermoFisher Scientific, USA.

[0046] Preparation and morphology characterization of 2CeO2-CIP MN

[0047] The microneedle molds were purchased from Henan Benteng Biological Company, China.

[0048] Ciprofloxacin (CIP) was dissolved in 2% acetic acid solution (400 μg / mL) and mixed with CeO2 (100 μg / mL) and 25% (w / v, g / mL) gelatin (Gel) in deionized water to prepare a mixed solution. 300 μL of the mixed solution was poured into the microneedle mold, and the mixed solution was filled to the tip of the microneedle by vacuum. After removing the excess solution, 800 μL of the mixed solution was added to fill the backing, dried at room temperature overnight, and finally the microneedle was peeled off from the mold. The surface morphology of the microneedle was observed by stereomicroscope and field emission scanning electron microscope (SEM).

[0049] 3 Mechanical properties of CeO2-CIP MN

[0050] The mechanical properties of CeO2-CIP MN were tested by an electronic universal testing machine. The microneedle substrate was cut flat and placed on the test platform with the needle tip facing up. The base plate movement speed was set to 0.5 mm / min. The force measurement started from the first contact of the sensor with the tip of the needle tip and ended when the sensor moved 0.8 mm. The loading force was standardized to the loading force of each needle, that is, the total loading force divided by the number of needles, and the force-displacement curve was plotted.

[0051] 4CeO2-CIP MN skin puncture experiment

[0052] CeO2-CIP MN loaded with rhodamine 123 (rhodamine 123 was added to the CeO2-CIP MN precursor mixed solution and mixed to a final concentration of 2 mg / mL) was inserted into 3% (w / v) agarose gel. The microneedle was pulled out after 1 min, and the micropores formed after the microneedle was inserted into the skin were observed and photographed.

[0053] CeO2-CIP MN was inserted into the skin tissue of the back of mice, and the microneedle was pulled out after 1 minute. The micropores formed after the microneedle was inserted into the skin were observed and photographed. The skin tissue at the insertion site was collected, immersed in 4% paraformaldehyde, and stained with H&E. The micropores formed after the microneedle was inserted were observed under an optical microscope.

[0054] In addition, to evaluate the recovery of skin microporous channels after CeO2-CIP MN insertion, CeO2-CIP MN was inserted into the dorsal skin tissue of mice, the microneedles were removed after 1 min, and photos were taken at 0, 1, 3, 5, 10, 15, 20, 30, and 45 min to record the skin recovery until the mark completely disappeared.

[0055] 5CeO2-CIP MN in vitro degradation experiment

[0056] In order to observe the in vitro degradation behavior of microneedles, CeO2-CIP MN was dried to constant weight and placed in 10 mL of 37°C preheated PBS (pH = 7.4) solution. The microneedles were taken out at 10, 20, 30, 45, 60, and 90 min, and the microneedle mass was recorded after drying to constant weight. The in vitro degradation rate of microneedles was calculated by the following formula:

[0057] In vitro degradation rate = (w0-w t ) / w0×100%;

[0058] Where w0 represents the initial mass of the microneedle, w t Indicates the mass of the microneedles after being taken out and dried at a predetermined time.

[0059] 6CeO2-CIP MN in vitro release experiment

[0060] CeO2-CIP MN was placed in 10 mL PBS (pH 7.4) solution and continuously shaken at 37°C and 100 rpm on a shaker. 2 mL of solution was taken for analysis at a predetermined time, and 2 mL of fresh PBS solution was added to keep the volume constant. The UV-visible absorbance of the solution was measured at 269 nm using a UV spectrophotometer to calculate the cumulative release rate of CIP.

[0061] 7 In vitro antibacterial test

[0062] The experiment was divided into five groups: (1) Control (without microneedles); (2) Blank MN; (3) CeO2 MN; (4) CIP MN; (5) CeO2-CIP MN.

[0063] 7.1 Dilution plate test

[0064] The in vitro antibacterial activity of different groups of microneedles was studied.

[0065] First, Vibrio vulnificus was cultured in TSB medium containing 2% NaCl until the logarithmic growth phase, and then the bacterial solution was diluted to 1×10 8 CFU / mL. 1 mL of Vibrio vulnificus solution (1×10 8 CFU / mL), the above different groups of ultraviolet sterilized microneedles (each piece) were added to the bacterial solution, incubated on a shaker at 37°C for 2 h, the bacterial solution was gradiently diluted and 30 μL was evenly spread on the TSA agar plate, and the colony growth on the plate was observed after incubation at 37°C for 24 h, and the colony number was calculated.

[0066] Escherichia coli and Staphylococcus aureus were cultured in LB broth until the logarithmic growth phase, and then the bacterial solution was diluted to 1 × 10 6 After incubation with the above-mentioned different groups of microneedles (each piece) for 2 hours, 30 μL of the bacterial solution was evenly spread on the LB agar plate after gradient dilution. After incubation at 37°C for 24 hours, the growth of the colonies on the plate was observed and the number of colonies was calculated.

[0067] 7.2 Inhibition zone experiment

[0068] V. vulnificus was cultured in LB broth containing 2% NaCl and incubated overnight at 37°C in a shaking incubator. The culture was then diluted to 3 × 10 8 CFU / mL, take 40 μL of bacterial solution and evenly spread it on LB agar plate containing 2% NaCl. Put each group of microneedles after UV sterilization into the agar plate, incubate at 37°C for 24 hours, and measure the size of the inhibition zone.

[0069] E. coli (1×10 7CFU / mL) and Staphylococcus aureus (1×10 8 CFU / mL), 40 μL of bacterial solution was evenly spread on LB agar plate. Each group of microneedles (each piece) after UV sterilization was placed on the agar plate, incubated at 37°C for 24 hours, and the size of the inhibition zone was measured.

[0070] 7.3 Bacterial live / dead staining experiment

[0071] The above different groups of microneedles (each piece) were mixed with 1 mL 3×10 8 CFU / mL Vibrio vulnificus / Escherichia coli / Staphylococcus aureus solution was incubated for 3 hours and washed twice with PBS. Then SYTO9 / PI dye was added and incubated on a shaker at 37°C in the dark for 30 minutes, and excess dye was washed off with PBS. Finally, 10 μL of bacterial suspension was placed on a slide and observed by super-resolution spinning disk confocal microscopy.

[0072] 8CeO2-CIP MN Antioxidant Experiment

[0073] The total antioxidant capacity test kit (ABTS method) and hydroxyl radical (OH - ) assay kit was used to detect the antioxidant properties of CeO2-CIP MN.

[0074] 9CeO2-CIP MN cytotoxicity assessment

[0075] The cytotoxicity of microtargets to different cell lines was evaluated by CCK-8 assay.

[0076] Blank MN / CeO2MN / CIP MN / CeO2-CIP MN after UV sterilization were diluted to 0.25 / 0.5 / 1 / 2 / 4 mg / mL with DMEM medium containing 10% FBS and 1% double antibody. 100 μL L929, HUVEC and RAW264.7 cells (5000 cells / well) were inoculated in 96-well plates and cultured for 24 hours. After the cells were completely attached to the wall, the medium in the original 96-well plate was discarded, and 100 μL of fresh medium containing different concentrations of drugs was added. After 24 hours of culture, the medium containing drugs was discarded, and 100 μL of fresh medium (containing 10 μL CCK-8) was added to each well. After incubation at 37°C, the absorbance at a wavelength of 450 nm was measured.

[0077] 10 Scratch test

[0078] Dissolve UV-sterilized Blank MN / CeO2MN / CIP MN / CeO2-CIP MN in DMEM medium containing 1% FBS to a final concentration of 4 mg / mL. Inoculate 1.5 mL of L929 cells in a 6-well plate and incubate in a 37°C incubator for 24 h. After the cells adhere to the wall, draw a straight line in the 6-well plate with a sterile 10 μL pipette tip and wash with PBS to remove cell debris. Observe the same position under an inverted microscope and take pictures at 0, 12, and 24 h. Use Image J to analyze cell migration rate.

[0079] 11 In vivo wound healing assessment

[0080] The feeding and experiments of mice were carried out in strict accordance with the standards of the "Guangdong Provincial Laboratory Animal Management Regulations" and the approval requirements of the Experimental Animal Ethics Committee of Guangdong Medical University. The experimental device use license number is SYXK (Guangdong) 2020-0147. Female Balb / c mice (8 weeks old, 20-22 g) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and the mice were housed in a room under a light / dark cycle for three weeks.

[0081] A seawater immersion (SWI) Vibrio vulnificus infection model was constructed to evaluate the healing effect of CeO2-CIP MN.

[0082] Balb / c mice were randomly divided into 3 groups (8 mice in each group): ① uninfected control group; ② Vibrio vulnificus infected control group; ③ CeO2-CIP MN treated group.

[0083] After the mice were anesthetized, their backs were depilated and disinfected. A circular full-thickness skin wound with a diameter of 10 mm was made on the back of each mouse using a skin puncher, and the wound was covered with sterile gauze soaked in seawater for 1 h to simulate SWI.

[0084] The uninfected control group was not given any bacteria or drugs.

[0085] In the control group infected with Vibrio vulnificus, 30 μL of Vibrio vulnificus solution (1×10 8 CFU / mL).

[0086] In the CeO2-CIP MN treatment group, 30 μL of Vibrio vulnificus solution (1×10 8 CFU / mL). CeO2-CIP MNs were treated 6 h after surgery.

[0087] In addition, all groups were bandaged with sterile gauze, and the wound conditions of the mice were observed and photographed on days 0, 3, 5, 7, 9, 11, and 13. The weight and wound temperature of the mice were also recorded. The wound area was analyzed using ImageJ software. The wound healing rate was calculated using the following formula:

[0088] Wound healing rate = (A0-A t ) / A0×100%;

[0089] Among them, A0 represents the wound area on day 0, A t Indicates the wound area at the measurement time point.

[0090] After the wound healed, the blood of the mice was taken for routine blood analysis. After the mice were euthanized, the skin tissues and the heart, liver, spleen, lungs, and kidneys of the wounds were cut and fixed in 4% paraformaldehyde solution. The wound tissues of the mice were stained with H&E, Masson, IL-6, TNF-α, and the heart, liver, spleen, lungs, and kidneys of the mice were stained with H&E.

[0091] 12 Statistical analysis

[0092] All quantitative data were obtained from the analysis results of at least three samples and are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) or T test, and P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns no difference).

[0093] 13 Results and Discussion

[0094] 13.1 Preparation and Characterization of CeO2-CIP MN

[0095] The present invention successfully prepared CeO2-CIP MN ( Figure 1 (a) Figure 1 Figures c and d are the actual images of CeO2-CIP MN and the photos taken under a stereo microscope. The microneedles are arranged in an orderly manner on the substrate in a 20×20 array. The SEM image shows that the tip of CeO2-CIP MN is conical, with a tip height of 800μm, a bottom diameter of 300μm, and a tip distance of 700μm ( Figure 1 The above results demonstrate the successful preparation of CeO2-CIP MNs.

[0096] As a transdermal drug delivery tool, microneedles must penetrate the stratum corneum or bacterial biofilm barrier to effectively deliver drugs. In order to evaluate the skin penetration ability of microneedles, CeO2-CIP MNs were applied to mouse skin for 1 minute and then removed. Figure 1In the middle g, neatly arranged micro-channels can be observed on the back skin of the mouse. The H&E staining results show that the CeO2-CIP MN needle tip successfully penetrated the skin ( Figure 1 h); In addition, CeO2-CIP MN loaded with rhodamine 123 was applied to agarose gel simulating skin, and a good puncture effect was also obtained. The mechanical strength of the microneedle tip was tested by an electronic universal testing machine ( Figure 1 j), the mechanical properties of CeO2-CIP MN were further verified. Figure 1 As shown in Figure k, the loading of CeO2 and CIP improves the mechanical properties of the microneedles compared to Blank MNs. CeO2-CIP MNs have sufficient mechanical strength to successfully penetrate the skin. The above results indicate that CeO2-CIP MNs have sufficient mechanical properties for effective transdermal drug delivery.

[0097] Next, the in vitro degradation and cumulative release behavior of CeO2-CIP MNs were studied. CeO2-CIP MNs were immersed in PBS buffer (pH 7.2-7.4), and the weight change of the microneedles and the cumulative release of CIP were recorded within a predetermined time. Figure 1 As shown in Figures l and m, CeO2-CIP MN can dissolve within 90 minutes and release CIP to kill bacteria in the wound.

[0098] 13.2 In vitro antimicrobial and antioxidant properties

[0099] 13.2.1 In vitro antibacterial properties

[0100] Vibrio vulnificus is highly toxic, with high disability and mortality rates, and is known as a hidden killer in the ocean. The in vitro antibacterial activity of CeO2-CIP MN was evaluated by incubating microneedles with Vibrio vulnificus solution and then diluting and coating the plate. Figure 2 As can be seen in Figure a, there are a large number of Vibrio vulnificus colonies on the TSA agar plate of the Control group, while almost no colonies are generated on the agar plates of the CIP MN and CeO2-CIP MN groups. The quantitative results show that the inhibition rate of CIP MN and CeO2-CIP MN against Vibrio vulnificus is close to 100% ( Figure 2 The results of the inhibition zone experiment showed that the diameter of the inhibition zone of CIP MN and CeO2-CIP MN against Vibrio vulnificus was close to 40 mm ( Figure 2 b, d). At the same time, the in vitro antibacterial effect of CeO2-CIP MN was further evaluated by SYTO9 / PI bacterial live / dead staining experiment. Figure 2As shown in Figure e, the Vibrio vulnificus in the Control group showed a large amount of green fluorescence, while the Vibrio vulnificus treated with CIP MN and CeO2-CIPMN showed strong red fluorescence. The above results prove that the prepared CeO2-CIP MN can effectively kill Vibrio vulnificus.

[0101] In addition, the present invention also studies the antibacterial effect of CeO2-CIP MN on Escherichia coli and Staphylococcus aureus. The results show that CeO2-CIP MN also has good antibacterial effect on Escherichia coli and Staphylococcus aureus ( Figure 2 a, b, c, d, Figure 6 , 7 ). In contrast, the antibacterial activity of CeO2-CIP MN against Vibrio vulnificus and Escherichia coli was better than that against Staphylococcus aureus, because ciprofloxacin belongs to the third-generation quinolone antibiotics and has better antibacterial effect against Gram-negative bacteria than against Gram-positive bacteria.

[0102] 13.2.2 In vitro antioxidant properties

[0103] During wound healing, excessive ROS will lead to slow wound healing. In the present invention, the commonly used 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) probe and hydroxyl radical kit were used to detect the antioxidant properties of CeO2-CIP MN. ABTS+ is blue-green and is reduced to colorless ABTS( Figure 2 f). Figure 2 As shown in Figure g, CeO2-CIP MN can effectively remove ROS, and with the increase of CeO2-CIP MN concentration, the ability to remove ABTS free radicals gradually increases. The Fenton reaction produces hydroxyl free radicals. After the electron acceptor is given, the red substance is formed by color development with Griess reagent. Under the action of antioxidants, the hydroxyl free radicals are removed, and the red color of the reaction solution gradually becomes lighter ( Figure 2 h). Figure 2 As shown in Figure i, CeO2-CIP MN can significantly remove OH·, and the OH· removal rate gradually increases with the increase of CeO2-CIP MN concentration. The above results show that CeO2-CIP MN has good antioxidant activity and can effectively remove ROS in wounds.

[0104] 13.3 In vitro biosafety

[0105] Microneedles come into direct contact with the skin during use, so it is necessary to test their biosafety. The cell compatibility of different groups of microneedles with mouse fibroblasts (L929), human umbilical vein endothelial cells (HUVEC), and mouse mononuclear macrophage leukemia cells (RAW264.7) was tested by CCK-8 experiments. Different groups of microneedles were prepared into extracts (0.25-4 mg / mL) and incubated with L929, HUVEC, and RAW264.7, respectively. Figure 3 As shown in a, b, and c, the microneedle extracts in each group with a concentration of 0.25 to 4 mg / mL have good cell compatibility with L929, HUVEC, and RAW264.7, and the cell viability is above 80%, which can be used for subsequent animal experimental studies. During wound healing, fibroblasts migrate to the site of injury, proliferate, differentiate to synthesize extracellular matrix, and form granulation tissue with capillaries and inflammatory cells to fill the defective tissue. The effect of CeO2-CIPMN on the migration of L929 cells was evaluated by cell scratch experiment. Figure 3 As shown in Figures d and e, compared with the control group, CeO2-CIP MN significantly promoted the migration of L929 cells, with the migration rate within 24 hours approaching 40%.

[0106] In addition, in order to evaluate the application safety of CeO2-CIP MN, CeO2-CIP MN was inserted into the back skin of mice for 1 min and then removed to observe the skin redness and swelling and the recovery of micropores. Figure 3 As shown in middle f, the skin did not show redness, swelling or bleeding, and the microchannels left by the microneedles on the skin were completely restored within 45 minutes. The results show that CeO2-CIP MN has good application safety and will not harm skin tissue during use, which is conducive to improving patient compliance.

[0107] 13.4 Effect of Promoting Infected Wound Healing in Vivo

[0108] In order to simulate the situation of Vibrio vulnificus infection at sea and evaluate the in vivo wound healing effect of CeO2-CIP MN, a Vibrio vulnificus infection wound model was established in mice immersed in seawater. Figure 4 As shown in Figure a, a circular wound with a diameter of about 10 mm was first created on the back of the mouse, and the wound was covered with sterile gauze soaked in seawater to simulate seawater intrusion, and then the wound was infected with Vibrio vulnificus. The results showed that on the 7th day, the wound area of ​​the CeO2-CIP MN group was significantly smaller than that of the other two groups ( Figure 4 b, c). On the 13th day, the wounds in the CeO2-CIP MN group were almost completely healed, with a wound healing rate of 93.9% ± 0.9%, while the wound healing rates in the uninfected control group and the Vibrio vulnificus infected control group were only 87.3% ± 3.2% and 79% ± 2.2%, respectively ( Figure 4During the treatment, the wound temperature and body weight of the mice remained basically stable ( Figure 4 The above results show that CeO2-CIP MN has a good healing effect on wounds infected with Vibrio vulnificus.

[0109] On day 13, the wound tissues of mice were histologically analyzed ( Figure 4 (g). H&E staining results showed that the wound surface of CeO2-CIP MN group had a higher degree of re-epithelialization and less inflammatory cell infiltration compared with the uninfected control group and the Vibrio vulnificus infected control group on day 13. Masoon staining results showed that a large amount of collagen deposition appeared in the wound surface of CeO2-CIP MN group, showing a darker blue staining.

[0110] 13.5 Biosafety of CeO2-CIP MN in vivo

[0111] In order to study whether CeO2-CIP MN has toxic side effects on mice, H&E staining was performed on the heart, liver, spleen, lung and kidney of the mice on the 13th day, and blood was taken for routine blood analysis. The results of H&E staining showed that there were no abnormalities in the organs of the mice, and no obvious damage was observed ( Figure 5 The blood routine test data showed that the blood routine indexes of mice treated with CeO2-CIP MN were normal, with no significant difference compared with the control group ( Figure 5 The above results show that CeO2-CIP MN has good biosafety in vivo.

[0112] The results of bacterial live / dead staining showed that the CeO2-CIP MN treatment group had obvious red fluorescence, indicating that CeO2-CIPMN had an effective effect on Escherichia coli ( Figure 6 ) and Staphylococcus aureus ( Figure 7 ) has strong antibacterial activity.

[0113] In summary, the present invention successfully prepared a degradable microneedle patch (CeO2-CIP MN) with antibacterial and antioxidant functions. CeO2-CIP MN has sufficient mechanical strength and can penetrate the skin stratum corneum barrier to achieve effective drug delivery of CeO2 and CIP. The results of antibacterial experiments show that CeO2-CIP MN has good antibacterial activity against Vibrio vulnificus, Escherichia coli and Staphylococcus aureus. In addition, the loaded CeO2 has antioxidant function and can effectively remove ROS, thereby reducing oxidative stress damage and inflammatory response at the wound. At the same time, CeO2-CIP MN has good biological and application safety. Finally, the healing effect of CeO2-CIP MN was verified by the Vibrio vulnificus infection wound model of mice immersed in seawater. The results show that CeO2-CIP MN can significantly promote the healing of Vibrio vulnificus infected wounds. In summary, the present invention provides ideas for the future treatment of Vibrio vulnificus infected wounds.

[0114] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a degradable microneedle patch CeO2-CIP MN, characterized in that: The following steps are involved: After ciprofloxacin is dissolved, it is mixed with CeO2 and gelatin to prepare a mixed solution, which is poured into the microneedle mold and vacuumed to allow the mixed solution to fill the microneedle tip; After removing the excess solution, the mixed solution is added to fill the backing, and the mixture is dried overnight at room temperature. Finally, the microneedles are peeled off from the mold to obtain the degradable microneedle patch.

2. The preparation method according to claim 1, characterized in that: The concentration of ciprofloxacin in the mixed solution is 400 μg / mL.

3. The preparation method according to claim 1, characterized in that: The concentration of CeO2 in the mixed solution is 100 μg / mL.

4. The preparation method according to claim 1, characterized in that: The mass volume ratio of the gelatin to the mixed solution is 1:4 (g / mL).

5. The degradable microneedle patch CeO2-CIP MN prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the degradable microneedle patch CeO2-CIP MN as claimed in claim 5 in the preparation of a drug for treating bacterially infected wounds.

7. The use according to claim 6, characterized in that: The bacteria include Vibrio vulnificus, Escherichia coli and Staphylococcus aureus.

8. A drug for treating bacterially infected wounds, characterized in that: Including the degradable microneedle patch CeO2-CIP MN as described in claim 5.

9. Use of the degradable microneedle patch CeO2-CIP MN as claimed in claim 5 in preparing a medicine for promoting wound healing.

10. A drug for promoting wound healing, characterized in that: Including the degradable microneedle patch CeO2-CIP MN as described in claim 5.

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