Riboflavin soluble microneedle and application thereof in photodynamic antibacterial treatment

Abstract

The invention discloses a riboflavin soluble microneedle and application of the riboflavin soluble microneedle in photodynamic antibacterial treatment. The invention provides a soluble microneedle patch with an antibacterial function. The soluble 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 riboflavin and nano-silver; the needle bodies are arranged on the backing layer in an array mode. The microneedle prepared by the invention is good in mechanical strength, can quickly release the medicine, can penetrate through the skin to deliver the medicine to a certain depth, has a photodynamic antibacterial effect, and is suitable for being applied to photodynamic antibacterial treatment.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a riboflavin soluble microneedle and application thereof in photodynamic antibacterial treatment. Background Art

[0002] Riboflavin, also known as vitamin B2, is a water-soluble yellow-green fluorescent substance. Riboflavin has the effects of diuresis, lowering blood lipids and improving heart function. It is an essential vitamin in the life activities of organisms. Its physiological functions mainly include: as a participant in mitochondrial energy metabolism, riboflavin can not only improve its energy metabolism, but also act as a free radical scavenger to remove the damage to mitochondrial function caused by free radicals produced by lipid peroxidation, thereby protecting the mitochondria; it has the function of hydrogen transfer in the body and has extremely strong oxidizing ability. In addition, riboflavin is also a photosensitizer that can play a photodynamic role under certain light effects. Photodynamic antibacterial therapy refers to the fact that under certain light effects, photosensitizers can produce reactive oxygen, thereby having a certain killing effect on bacteria.

[0003] As an emerging drug delivery system, microneedles (MNs) can effectively deliver drugs through the skin, increase the distribution of drugs in deeper tumor sites, and reduce the leakage of therapeutic drugs to adjacent tissues, thereby improving the therapeutic effect. In addition, compared with traditional drug release methods, microneedle drug release systems have the advantages of being simple, safe, and less painful.

[0004] Microneedles can be classified into five types: solid, coated, dissolving, hollow and hydrogel, based on their size, shape and range of production materials. Metals such as silicon, stainless steel and titanium alloys are the most commonly used materials for microneedles, but these materials may cause safety issues after being inserted into the skin. Polymers are the preferred material for making soluble microneedles because they do not trigger an immune response in the body, degrade in the body, and can be adjusted to different strengths and functions. Summary of the invention

[0005] The purpose of the present invention is to improve a riboflavin soluble microneedle and its application in photodynamic antibacterial treatment.

[0006] In a first aspect, the present invention claims a soluble microneedle patch with antibacterial function.

[0007] The soluble microneedle patch with antibacterial function claimed in the present invention comprises a needle body and a backing layer; the needle body comprises a needle body matrix material and a drug; the drug is riboflavin (RB) and nanosilver; the needle body is arranged in an array on the backing layer.

[0008] Furthermore, in the needle body, the mass ratio of riboflavin to nanosilver can be 1000:1.

[0009] Furthermore, the needle body matrix material may be composed of polyvinyl pyrrolidone K29-32 (PVP K29-32) and polyvinyl alcohol (PVA).

[0010] Exemplarily, in the needle body matrix material, the mass ratio of polyvinyl pyrrolidone K29-32 and polyvinyl alcohol may be 3:1.

[0011] Furthermore, the material of the backing layer may consist of polyvinyl alcohol and polylysine (PL).

[0012] For example, in the backing layer, the mass ratio of polyvinyl alcohol to polylysine may be 12:(1-7), such as 12:7.

[0013] Furthermore, in the needle body, the mass ratio of riboflavin, nanosilver, polyvinyl pyrrolidone K29-32 and polyvinyl alcohol can be 2×10 -2 : 2×10 -5 :3:1.

[0014] In an example case of the present invention, when preparing the needle body of the soluble microneedle patch, the composition of the prepared unit dose of the needle body material solution is as follows: 20 mg riboflavin, 0.2 mL of a nanosilver solution with a nanosilver content of 100 μg / mL, 3 g of polyvinyl pyrrolidone K29-32, 1 g of polyvinyl alcohol and 20 mL of water. The needle body material solution is fully stirred and mixed at room temperature, centrifuged at 3000 rpm for 5 minutes to remove bubbles, and set aside.

[0015] In an example case of the present invention, when preparing the backing layer of the soluble microneedle patch, the solvent of the prepared backing layer material solution is water, and the solute is polyvinyl alcohol and polylysine, wherein the mass percentage of polyvinyl alcohol is 12%, and the mass percentage of polylysine is 1-7%, such as 7%. After the backing layer material solution is prepared, it is centrifuged at 3000rpm for 5 minutes to remove bubbles and set aside.

[0016] In an example case of the present invention, the needle body of the soluble microneedle patch is a quadrangular pyramid, the length of the microneedle is 900 μm, the bottom side length is 400×400 μm, the needle tip distance is 700 μm, and it forms a 20×20 array.

[0017] In one embodiment of the present invention, the molecular weight of the polyvinyl alcohol is 44.05 (MW).

[0018] The soluble microneedle patch is an integrated microneedle patch prepared by a polydimethylsiloxane mold method.

[0019] More specifically, the soluble microneedle patch can be prepared according to the method described in the second aspect below.

[0020] In a second aspect, the present invention claims a method for preparing the soluble microneedle patch described in the first aspect above.

[0021] The method for preparing the soluble microneedle patch described in the first aspect above as claimed in the present invention may include the following steps:

[0022] (A1) adding the needle material solution required for preparing the soluble microneedle patch (as described in the first aspect above) into a polydimethylsiloxane (PDMS) mold, and removing bubbles by vacuuming and centrifuging; the process also includes the step of carefully scraping off the residual solution on the surface of the mold;

[0023] (A2) After the liquid in the mold becomes gel, add the backing layer material solution required for preparing the soluble microneedle patch (as described in the first aspect above) into the mold, spread it over the entire surface of the mold, dry it, and demold it to obtain the soluble microneedle patch.

[0024] For example, in step (A1), the vacuuming may be performed at 27° C. for 2 times, each time for 3 minutes. The centrifugation may be performed at 4500 rpm for 2 times, each time for 5-10 minutes (eg, 10 minutes).

[0025] Exemplarily, in step (A2), the drying may be performed at 27° C. for more than 12 hours, such as 12-20 hours (specifically, 12 hours).

[0026] The prepared soluble microneedle patch is stored in a dry and dark environment for future use.

[0027] In a third aspect, the present invention claims protection for the use of the soluble microneedle patch described in the first aspect above in any of the following:

[0028] (B1) preparing products for inhibiting bacteria;

[0029] (B2) Preparation of a product for treating skin wound abscesses.

[0030] Furthermore, the product is a photodynamic therapy product.

[0031] Furthermore, the skin wound abscess is a skin wound abscess caused by bacterial infection or a skin wound abscess caused by other reasons combined with bacterial infection.

[0032] Furthermore, the bacteria are Gram-positive bacteria and / or Gram-negative bacteria. In the implementation case of the present invention, the bacteria are Staphylococcus aureus and / or Escherichia coli.

[0033] The needle body of the soluble microneedle patch prepared by the present invention contains riboflavin, a photosensitive antibacterial agent, and nanosilver. Riboflavin and nanosilver work together to exert antibacterial effects. The backing layer also contains polylysine, which also has antibacterial effects. The three antibacterial components added to the microneedles of the present invention maximize the antibacterial effect of the obtained microneedles.

[0034] Experiments have shown that the microneedles prepared by the present invention have good mechanical strength, can release drugs quickly, can penetrate the skin to deliver drugs to a certain depth, and have photodynamic antibacterial effects, and are suitable for the application of photodynamic antibacterial therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The morphological characterizations of the soluble microneedle array of the present invention were performed using a digital camera (A, B), a stereo microscope (C, D), and a scanning electron microscope (E, F, G, H).

[0036] Figure 2 This is an image of the microneedle of the present invention under a laser confocal microscope.

[0037] Figure 3 The results of the penetration of the soluble microneedles of the present invention on the isolated mouse skin. A is a layer-by-layer scan of the mouse skin treated with the microneedles of the present invention for 5 minutes using a confocal laser scanning microscope, and the distribution of the drug at different depths in the mouse skin; B is a 3D reconstructed image (scale: 100 μm).

[0038] Figure 4 The XRD images of the microneedle of the present invention and its components are shown in Figure 1. A is the XRD image of RB, PVP, PVA, MN, and PL; B is the XRD image of nanosilver.

[0039] Figure 5 The infrared spectra of the microneedle and its components are shown in Figure 1. A is the infrared spectra of RB, PVP, PVA, and MNPL; B is the infrared spectra of nanosilver.

[0040] Figure 6 The DSC images of the microneedle of the present invention and its components are shown in Figure 4. AF are the DSC images of nanosilver, PL, RB, MN, PVP, and PVA, respectively.

[0041] Figure 7 Force and compression displacement curves of microneedles with different contents of polylysine (PL).

[0042] Figure 8 Stereoscopic microscope images of the soluble microneedles of the present invention after being inserted into mouse skin at different time periods and the statistical results of the dissolution of the microneedles.

[0043] Fig. 9 The graph is a graph showing the average cumulative penetration amount of riboflavin per unit area versus time in the two groups of microneedles (RB-MN) of the present invention and riboflavin solution (RB-Solution). The data are expressed as mean±SD (n=3).

[0044] Fig.10 The OD600 growth curves of Staphylococcus aureus (A) and Escherichia coli (B) in different treatment groups in Example 11.

[0045] Fig.11 These are the colonies of Staphylococcus aureus and Escherichia coli on the agar plate after different treatments in Example 11.

[0046] Fig.12 The following are graphs showing the wound changes of mice in each group at different times after treatment in Example 12.

[0047] Fig.13 These are H&E (first row) and Masson (second row) staining images of infected skin tissues on the 7th day after different treatments in Example 12.

[0048] Fig.14 Fluorescence spectra of 10 μg / mL riboflavin (A) and 10 μg / mL riboflavin mixed with different concentrations of nanosilver (nanosilver concentration: 0.001 μg / mL in B; 0.01 μg / mL in C; 0.1 μg / mL in D; excitation wavelength 440 nm, emission wavelength 531 nm). DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] The room temperature described in the following examples refers to 25°C.

[0052] 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).

[0053] Example 1. Preparation of the soluble microneedle patch of the present invention

[0054] The soluble microneedle patch prepared in this embodiment includes a needle body and a backing layer; the needle body includes a needle body matrix material and a drug; the drug is riboflavin and nanosilver; the needle body is arranged in an array on the backing layer.

[0055] 1. Prepare needle material solution

[0056] The main drug of the needle body of the soluble microneedle patch of the present invention is riboflavin, and there is also nano silver that plays a synergistic antibacterial role. The matrix material is polyvinyl pyrrolidone (PVP) K29-32 (average molecular weight 58000) and polyvinyl alcohol (PVA).

[0057] Weigh 3 g of PVP K29-32, 1 g of PVA, 20 mg of riboflavin, and 0.2 mL of 100 μg / mL nanosilver solution (Xianfeng Nano, product number: 100695), add them into 20 mL of water, and stir and mix them thoroughly at room temperature to obtain a needle material solution.

[0058] Before preparing the soluble microneedle patch, the needle material solution needs to be centrifuged at 3000 rpm for 5 minutes to fully remove bubbles.

[0059] 2. Prepare the backing layer material solution

[0060] The material of the backing layer of the soluble microneedle patch of the present invention consists of polyvinyl alcohol (PVA) and polylysine (PL).

[0061] A mixed solution containing PVA and PL is prepared, wherein the solvent is water and the solutes are PVA and PL. In the mixed solution, the mass percentage of PVA is 12% and the mass percentage of PL is 7%. Thus, a backing layer material solution is obtained.

[0062] Before preparing the soluble microneedle patch, the backing layer material solution needs to be centrifuged at 3000 rpm for 5 minutes to fully remove bubbles.

[0063] 3. Preparation of soluble microneedle patches

[0064] The polydimethylsiloxane (PDMS) mold was cleaned with ultrapure water and set aside.

[0065] In the mold, first add 200μL of the needle material solution prepared in step 1, fill the PDMS mold cavity, and evacuate at 27℃ for 3min, twice (purpose: to remove bubbles); suck away the excess bubbles on the upper layer, and carefully scrape off the residual solution on the surface of the mold; then centrifuge at 4500rpm for 10min at room temperature, twice (purpose: to remove bubbles). After the liquid becomes gel, add 1mL of the backing layer solution prepared in step 2 to the mold, then dry it at 27℃ for 12h. After demolding, cut off the thick part of the edge with scissors. The prepared soluble microneedle patch is stored in a dry and dark environment for use.

[0066] The microneedle tips of the soluble microneedle patch prepared by the present invention are in the shape of a quadrangular pyramid, the length of the microneedle is 900 μm, the side length of the bottom surface is 400×400 μm, the distance between the tips is 700 μm, and the array is 20×20.

[0067] Example 2: Observation of the geometric structure and morphology of the microneedles on the soluble microneedle patch of the present invention

[0068] The specific operation is as follows: use a digital camera equipped with a macro lens, a scanning electron microscope (SEM), and a stereo microscope to take an optical image of the soluble microneedle patch of the present invention prepared in Example 1, and observe and characterize the length, bottom width, and needle tip spacing of the microneedles.

[0069] The results are as follows Figure 1 As shown, the soluble microneedle array of the present invention, when examined under a stereo microscope, shows well-structured morphological features, sharp tips, and complete needle arrangement. SEM characterization shows that all microneedles are quadrangular pyramidal and can be evenly distributed on the backing layer. The SEM image confirms that the microneedle tips are sharp without any visible breaks, which is necessary for sufficient skin penetration.

[0070] Example 3: Observation of fluorescence on the microneedles of the present invention under a laser confocal microscope

[0071] Utilizing the yellow-green fluorescence property of riboflavin under 440-500 nm wavelength light irradiation, the soluble microneedle patch of the present invention prepared in Example 1 was observed under a laser confocal microscope (Nikon, Japan, laser power: 49.3 W / 4.161 W, mode: GaAsP, exposure time: 7 s).

[0072] The results are as follows Figure 2 As shown in the figure, the microneedles of the soluble microneedle patch prepared by the present invention exhibit yellow-green fluorescence, have good needle shape, and have uniform drug distribution.

[0073] Example 4: Penetration of the soluble microneedles of the present invention on isolated mouse skin

[0074] Riboflavin is a photosensitizer with yellow-green fluorescence. In order to visualize the penetration and distribution of riboflavin under the skin, the soluble microneedles prepared in Example 1 of the present invention were inserted into the skin of rats and removed after 5 minutes. A confocal laser scanning microscope was used to obtain fluorescence images of each x-plane in the vertical direction (z-axis) from the skin surface at increasing heights. Subsequently, the x-plane images were superimposed along the z-axis to obtain a three-dimensional reconstructed image.

[0075] Figure 3 Center A shows the penetration and distribution of riboflavin at different depths of the skin. Figure 3 Figure B is a three-dimensional reconstruction of riboflavin after it penetrated the mouse skin. The results show that the yellow-green fluorescence signal of riboflavin can be detected within 389.93μm, indicating that the drug penetration depth can reach 389.93μm. The penetration depth of the drug is less than the height of the microneedle. The reason for this analysis may be the elasticity of the skin. At the same time, it can be observed that the intensity and coverage of the fluorescence signal change with the scanning depth, and the reconstructed three-dimensional distribution of riboflavin is similar to the geometric shape of the microneedle. These results prove that the microneedle can penetrate the stratum corneum (thickness of 10-20μm) and deliver drugs to the dermis.

[0076] Example 5: XRD study of the components of the microneedle of the present invention

[0077] The soluble microneedle (MN) of the present invention prepared in Example 1 was detected by an X-ray diffraction analyzer (XRD). X-ray diffraction (XRD) is a technique used to study the physical phase and crystal structure of a substance. It can also be used to analyze the composition, intramolecular bonds, molecular configuration, conformation and particle size of a substance, which is based on the diffraction pattern produced when the substance is irradiated with x-rays. At the same time, individual samples of each component of the microneedle of the present invention - riboflavin (RB), nanosilver, polyvinyl pyrrolidone (PVP) K29-32, polyvinyl alcohol (PVA) and polylysine (PL) were used as controls.

[0078] In the soluble microneedles prepared by the present invention, riboflavin and nanosilver belong to a crystalline structure with certain characteristic peaks. The other materials belong to an amorphous structure. The XRD test results of the soluble microneedles of the present invention show that riboflavin and nanosilver no longer have the peak characteristics after encapsulation and become amorphous, which also proves that the drug encapsulation is successful. The specific XRD test results are as follows Figure 4 shown.

[0079] Example 6: Infrared spectroscopy study of the components of the microneedles of the present invention

[0080] In this example, infrared spectroscopy was performed on the soluble microneedles (MN) of the present invention prepared in Example 1. At the same time, individual samples of the components of the microneedles of the present invention, riboflavin (RB), nanosilver, polyvinyl pyrrolidone (PVP) K29-32, polyvinyl alcohol (PVA) and polylysine (PL), were used as controls.

[0081] Riboflavin is -OH at 3317cm -1 The characteristic absorption peak at 2939cm -1 Symmetrical and asymmetric stretching vibrations of methyl, methylene and methylene; 1732cm -1 It is the characteristic peak of carbonyl stretching vibration; 1543cm -1 The stretching vibration of the aromatic ring skeleton; 1245cm -1 and 1079cm -1 It is the stretching vibration of aromatic amine. In polylysine, 2950cm -1 and 1994cm -1 It is the stretching vibration of -NH2, 1679cm -1 and 1540cm -1 It is the vibration of amide bond region I and II. 2346cm in the infrared spectrum of nanosilver -1 、1702cm -1 and 1394cm -1 It may be the characteristic absorption peak of -CH3, 3040cm -1 It may be the -OH peak that absorbs air moisture during the test. In PVP, due to the presence of keto carbonyl in the structure, the characteristic peak is at 1663cm -1 PVA is composed of a large number of vinyl alcohol segments, and its molecular structure contains a large number of hydroxyl groups, intermolecular hydrogen bonds, and terminal unsaturated olefins. Since the characteristic functional group of PVA is hydroxyl, 3340cm -1 is the absorption peak of -OH stretching vibration, 2937cm -1 is the absorption peak of -CH stretching vibration. In the microneedle of the present invention, the change of the mode may be due to the formation of intermolecular hydrogen bonds. The specific infrared spectrum detection results are as follows Figure 5 shown.

[0082] Example 7: DSC study of the components of the microneedle of the present invention

[0083] Differential scanning calorimetry (DSC) was used to detect the soluble microneedles (MN) of the present invention prepared in Example 1. At the same time, individual samples of the components of the microneedles of the present invention, riboflavin (RB), nanosilver, polyvinyl pyrrolidone (PVP) K29-32, polyvinyl alcohol (PVA) and polylysine (PL), were used as controls.

[0084] In the DSC test results, the endothermic peaks of riboflavin (RB) are at 303°C, and the endothermic peaks of polylysine (PL) are at 304°C. These characteristic melting peaks disappear from the MN curve, indicating that they no longer exist in the form of crystals in the PVP and PVA mixture, indicating that the microneedle preparation process inhibits the crystallization of riboflavin and the drug is distributed in the microneedle in an amorphous molecular state. The specific DSC image results are as follows: Figure 6 shown.

[0085] Example 8 Mechanical properties of the microneedles of the present invention at different polylysine (PL) contents

[0086] The compressive strength of the soluble microneedle of the present invention under compression mode was determined using a universal testing machine. The sensor was connected to the plane of the stainless steel substrate by a mobile sensor. A constant speed of 10 mm / min was selected, and the initial distance from the needle tip to the bracket was set to 10 mm. Then, the force of the mobile bracket was recorded as a function of the compression displacement. The mechanical strength of the soluble microneedle is a key indicator, and the size of the mechanical properties of the microneedle determines whether the microneedle can successfully break through the skin stratum corneum barrier. This embodiment uses a universal testing machine to test the compressive performance of the microneedle of the present invention.

[0087] Test microneedles: In addition to the soluble microneedles with a PL content of 7% prepared in Example 1 of the present invention, microneedles with PL contents of 1%, 3%, and 5% were also prepared with reference to Example 1 (except for the different PL contents, all other operations were exactly the same as in Example 1).

[0088] The results are as follows Figure 7 As shown, the microneedles prepared by the present invention with PL contents of 1%, 3%, 5%, and 7% have an average compressive stress of 13.51 N, and each needle can withstand a force of 0.060 N, which is greater than the minimum required force of 0.058 N per needle, and can ensure that the microneedle patch has sufficient strength to penetrate the skin without breaking.

[0089] Example 9: In vitro dissolution of the soluble microneedle patch of the present invention

[0090] In order to study the in vitro dissolution of the microneedles of the present invention, the soluble microneedles of the present invention prepared in Example 1 were inserted into the ex vivo mouse skin and peeled off at 1, 2, and 8 minutes respectively. A stereo microscope was used to observe the morphological changes of the needle body before and after the microneedles were inserted into the mouse skin, and then the changes in the needle height were analyzed using Image J software, and the percentage change of the needle head at each time point was calculated. The purpose of the present invention is to prepare soluble microneedles that can be dissolved in the interstitial fluid of the skin. Therefore, it is very important to observe the dissolution rate of the microneedles after insertion.

[0091] Figure 8The height reduction changes of the microneedle of the present invention at different time points after being inserted into the mouse skin are shown in the stereo microscope. From the initial stage to 8 minutes, the height of the needle decreases significantly over time, indicating that the needle body can dissolve in the skin. The height reduction in 8 minutes reaches 72%. These results show that the microneedle dissolves rapidly after contacting the skin interstitial fluid.

[0092] Example 10: Evaluation of the permeability of the soluble microneedle patch of the present invention for transdermal administration

[0093] The in vitro permeability of the microneedles of the present invention through mouse skin was studied using a Franz diffusion cell. The microneedles prepared in Example 1 were inserted into the ex vivo mouse skin by applying a certain force for 30 seconds. The mouse skin was mounted in a Franz diffusion cell. The mouse skin was fixed in the donor chamber of the diffusion cell using a sealing film. The diffusion cell was continuously stirred and maintained in a 37°C circulating water bath. Sample solutions (1 mL) were extracted from it at predetermined time intervals (0.5, 1, 2, 3, 4, 6, 8, 12, 24 hours), and an equal amount of PBS was supplemented at each time point.

[0094] LC-MS / MS was used to determine the drug permeation concentration at each time point, and Microsoft office Excel and GraphPad Prism5 software were used for data statistics and processing.

[0095] The cumulative drug penetration per unit area is calculated according to the formula:

[0096]

[0097] Note: In the formula, Qn is the cumulative permeation amount of the drug per unit area (μg / cm 2 ); Cn is the drug concentration (μg / mL) measured at the nth time point; A is the permeation area; Ci is the drug concentration (μg / mL) measured at the ith (i≤n-1)th sampling point.

[0098] First, we established riboflavin liquid phase and mass spectrometry methods for quantitative detection of drugs in transdermal penetration experiments. The mass spectrometry method suitable for riboflavin is as follows: the ion source is an electrospray ion source (ESI), the detection mode is a positive ion multiple reaction monitoring mode (MRM), and the ion pairs used for quantitative analysis are m / z 377 / 243. Liquid phase method: methanol-1.0% glacial acetic acid aqueous solution (45:55, volume ratio), flow rate: 1mL / min, column temperature: 25℃, detection wavelength: 444nm. Transdermal penetration is a key detection indicator for transdermal drug preparations. The present invention uses a Franz diffusion cell to evaluate transdermal drug penetration and detects the average cumulative permeation per unit area of ​​riboflavin through the skin after 24 hours. At the same time, a riboflavin solution group was set as a control (the sampling time of the riboflavin solution group was consistent with that of the riboflavin microneedle group, except that a 1mg / mL riboflavin solution was added to the mouse skin).

[0099] The results showed that the average cumulative permeability of the riboflavin solution group after 24 hours was 159.527±3.425ug / cm 2 The average cumulative permeability of the soluble microneedle group after 24 hours was as high as 286.414±7.742ug / cm 2 , which is significantly higher than that of the solution group. Fig. 9 .

[0100] Example 11: In vitro antibacterial effect of the soluble microneedle patch of the present invention

[0101] Escherichia coli (E.coil) and Staphylococcus aureus (S.aureus) were inoculated into LB medium and cultured at 37°C with shaking at 180 rpm for 12 h. 5 cfu / mL) were divided into 4 groups. The four groups are:

[0102] Control group: 3 mL of bacterial suspension was mixed with 3 mL of PBS and no light treatment was performed.

[0103] Light group (Light(+)): 3 mL of bacterial suspension was mixed with 3 mL of PBS and then immediately subjected to light treatment (the wavelength of the excitation light during photodynamic therapy was 470 nm and the light intensity was 0.45 W / cm 2 , 10min).

[0104] Microneedle group of the present invention (MN): 3 mL of bacterial suspension was mixed with the soluble microneedles prepared in Example 1, and no light treatment was performed.

[0105] Microneedle + light irradiation group (MN + L): 3 mL of bacterial suspension was mixed with the soluble microneedles prepared in Example 1, and then immediately subjected to light irradiation (the wavelength of the excitation light during photodynamic therapy was 470 nm and the light intensity was 0.45 W / cm 2 , 10min).

[0106] The mixed suspension of each group was cultured at 37°C, and the OD was measured every hour using an enzyme labeling instrument. 600 In order to observe the growth of different bacterial colonies on agar plates, we diluted the bacterial suspensions after different treatments by 10 4 100 μL of the suspension was added to the agar plate and evenly spread. After incubation at 37°C for 24 hours, we took images of colony growth on all agar plates. The photodynamic antibacterial effect of the microneedle of the present invention on Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains was evaluated.

[0107] The bacterial growth curve shows that the microneedles of the present invention exhibit a strong antibacterial effect under 440nm light irradiation, and can completely inhibit the growth of Staphylococcus aureus or Escherichia coli after incubation at 37°C for 12 hours ( Fig.10 ). The other groups without microneedles showed a faster growth curve due to the lack of antibacterial effect. In addition, Fig.11 The standard agar plate experiment showed that the microneedle of the present invention had an inhibitory effect on Staphylococcus aureus and Escherichia coli, killing more than 99% of the bacteria.

[0108] Example 12: In vivo antibacterial therapeutic effect of the soluble microneedle patch of the present invention

[0109] In order to study the antibacterial properties of the soluble microneedle patch of the present invention in vivo, a subcutaneous abscess model was established under the skin of mice in this example. We injected 50 μL (10 8 cfu / mL) were injected subcutaneously into mice (Babl / c female mice, 6-8 weeks old). One day later, subcutaneous abscesses appeared in the mice. These mice were randomly divided into five groups (3 mice in each group). The five groups were:

[0110] Control group (Control): mice with subcutaneous abscesses were not treated in any way;

[0111] Light group: mice with subcutaneous abscesses were treated with light only (the wavelength of the excitation light during photodynamic therapy was 470 nm and the light intensity was 0.45 W / cm 2 , 15min);

[0112] Microneedle group of the present invention (MN): The microneedle patch prepared in Example 1 of the present invention was applied to the abscess of mouse skin (the microneedle patch was inserted into the wound and administered twice a day), without light treatment;

[0113] Riboflavin solution + light treatment group (Solution+L): 1 mg / mL riboflavin aqueous solution was prepared and applied to the skin wound of mice with a cotton swab (twice a day). Light treatment was performed immediately after each medication (the wavelength of the excitation light during photodynamic therapy was 470 nm and the light intensity was 0.45 W / cm 2 , 15min);

[0114] Microneedle + light treatment group (MN + L): The microneedle patch prepared in Example 1 of the present invention was first applied to the abscess of the mouse skin (the microneedle patch was inserted into the wound and the drug was administered twice a day), and light treatment was performed immediately after each medication (the wavelength of the excitation light during photodynamic therapy was 470 nm and the light intensity was 0.45 W / cm 2 , 15min).

[0115] Each group was treated for 7 days, and abscesses were imaged on days 1, 3, 5, and 7. After 7 days, mice were euthanized, and subcutaneous abscesses were excised and histologically analyzed using hematoxylin and eosin (H&E) and Masson's trichrome staining.

[0116] The changes of wounds in each group of mice at different time after treatment are as follows Fig.12 H&E staining of infected skin tissue of mice on day 7 after different treatments ( Fig.13 The first row in the figure shows that the skin structure of the MN+L group is similar to that of healthy skin, with an intact epidermal surface and fewer inflammatory cells. Masson staining results ( Fig.13 The second row in the figure also shows that the epidermis and subcutaneous tissue of the mice in the MN+L treatment group were completely formed. In addition, the collagen fibers in the MN+L group increased, indicating that the infected tissue had recovered. It can be seen that the microneedle of the present invention has an excellent photodynamic biofilm ablation effect.

[0117] Comparative Example 1

[0118] Before preparing the soluble microneedle patch of the present invention, we conducted fluorescence spectrum detection on the selected riboflavin and nanosilver at different mass ratios. A total of 4 samples were obtained, which are as follows:

[0119] Sample A: Riboflavin aqueous solution with a final concentration of 10 μg / mL.

[0120] Sample B: a mixed aqueous solution of riboflavin and nanosilver, wherein the final concentration of riboflavin is 10 μg / mL and the final concentration of nanosilver is 0.001 μg / mL.

[0121] Sample C: a mixed aqueous solution of riboflavin and nanosilver, wherein the final concentration of riboflavin is 10 μg / mL and the final concentration of nanosilver is 0.01 μg / mL.

[0122] Sample D: a mixed aqueous solution of riboflavin and nanosilver, wherein the final concentration of riboflavin is 10 μg / mL and the final concentration of nanosilver is 0.1 μg / mL.

[0123] Fluorescence spectra of the above four samples were measured. The results are as follows Fig.14 As shown. Among them, curve A is the fluorescence spectrum of riboflavin (corresponding to sample A), with an excitation wavelength of 440nm and an emission wavelength of 531nm. Curves B, C and D are all fluorescence spectrum curves after riboflavin and nanosilver are mixed, corresponding to samples B, C and D respectively. The results show that when the amount of riboflavin is determined, the intensity of the fluorescence spectrum is weakened as the amount of nanosilver increases, which will affect the subsequent photodynamic properties of riboflavin. In order to ensure the fluorescence properties of riboflavin itself and enhance the antibacterial effect of riboflavin and nanosilver, we finally selected sample C, that is, the mass ratio of riboflavin to nanosilver is 1000:1 as the best mass ratio. The best mass ratio of riboflavin and nanosilver (mass ratio of 1000:1) can be mixed to ensure that the synergistic antibacterial effect of the two can be exerted, and the fluorescence properties of riboflavin will not be destroyed due to excessive use of nanosilver, thereby affecting the photodynamic therapy effect.

[0124] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A soluble 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 riboflavin and nanosilver; the needle body is arranged in an array on the backing layer.

2. The soluble microneedle patch according to claim 1, characterized in that: In the needle body, the mass ratio of riboflavin to nanosilver is 1000:1; and / or The needle body matrix material is composed of polyvinyl pyrrolidone K29-32 and polyvinyl alcohol; Furthermore, in the needle body matrix material, the mass ratio of polyvinyl pyrrolidone K29-32 and polyvinyl alcohol is 3:1; and / or The material of the backing layer is composed of polyvinyl alcohol and polylysine; Furthermore, in the backing layer, the mass ratio of polyvinyl alcohol to polylysine is 12:(1-7).

3. The soluble microneedle patch according to claim 1 or 2, characterized in that: In the needle body, the mass ratio of riboflavin, nanosilver, polyvinyl pyrrolidone K29-32 and polyvinyl alcohol is 2×10 -2 : 2×10 -5 :3:

1.

4. The soluble microneedle patch according to any one of claims 1 to 3, characterized in that: When preparing the needle body of the soluble microneedle patch, the prepared unit dose of needle body material solution has the following composition: 20 mg riboflavin, 0.2 mL of nanosilver solution with a nanosilver content of 100 μg / mL, 3 g of polyvinyl pyrrolidone K29-32, 1 g of polyvinyl alcohol and 20 mL of water.

5. The soluble microneedle patch according to any one of claims 1 to 4, characterized in that: When preparing the backing layer of the soluble microneedle patch, the solvent of the prepared backing layer material solution is water, and the solutes are polyvinyl alcohol and polylysine, wherein the mass percentage of polyvinyl alcohol is 12% and the mass percentage of polylysine is 1-7%.

6. The soluble microneedle patch according to any one of claims 1 to 5, characterized in that: The needle body of the soluble microneedle patch is in the shape of a quadrangular pyramid, the length of the microneedle 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 soluble microneedle patch according to any one of claims 1 to 6, comprising the following steps: (A1) adding a needle material solution required for preparing the soluble microneedle patch into a polydimethylsiloxane mold, and removing bubbles by vacuuming and centrifuging; (A2) After the liquid in the mold becomes gel, a backing layer material solution required for preparing the soluble microneedle patch is added to the mold, dried, and demolded to obtain the soluble microneedle patch.

8. The method according to claim 7, characterized in that: In step (A1), the vacuuming is performed at 27° C. for 2 times, each time for 3 minutes; and / or the centrifugation is performed at 4500 rpm for 2 times, each time for 5 minutes; and / or In step (A2), the drying condition is drying at 27° C. for more than 12 hours.

9. Use of the soluble microneedle patch according to any one of claims 1 to 6 in any of the following: (B1) preparing products for inhibiting bacteria; (B2) Preparation of a product for treating skin wound abscesses.

10. The use according to claim 9, characterized in that: The product is a photodynamic therapy product; and / or The skin wound abscess is a skin wound abscess caused by bacterial infection or a skin wound abscess caused by other reasons combined with bacterial infection; and / or The bacteria are Gram-positive bacteria and / or Gram-negative bacteria; Furthermore, the Gram-positive bacteria is Staphylococcus aureus, and the Gram-negative bacteria is Escherichia coli.

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