A core-shell microneedle for removing bacterial biofilms and its preparation method

Abstract

The present invention discloses a core-shell microneedle for clearing bacterial biofilms and a preparation method thereof, belonging to the technical field of biomedicine. The core-shell microneedle comprises a shell layer and a core layer. The shell layer comprises a shell layer material and cuprous oxide nanoparticles loaded on the shell layer material. The core layer comprises a core layer material and mesoporous polydopamine nanoparticles loaded on the core layer material, which are wrapped with citraconate and can target neutrophils. The present invention can penetrate the biofilm and preferentially release cuprous oxide nanoparticles, disrupt the redox homeostasis inside the biofilm, kill bacteria and disperse the biofilm; it can target neutrophils, relieve the immunosuppressive state of innate immune cells, and restore their bacterial killing ability, so as to effectively phagocytose the escaped bacteria; the synergistic effect of the two enables the present invention to completely clear the biofilm, has clinical application prospects, and reduces the bacterial drug resistance caused by long-term use of antibiotics.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a core-shell microneedle for removing bacterial biofilms and a preparation method thereof. Background Art

[0002] Clinical epidemiological statistics show that approximately 80% of chronic infection cases are closely related to biofilm formation, in which pathogenic microbial communities colonize at the host tissue interface in the form of single-species or multi-species biofilms. Typical pathogens include Gram-positive bacteria (such as Staphylococcus epidermidis, Staphylococcus aureus) and Gram-negative bacteria (such as Pseudomonas aeruginosa, Enterobacteriaceae bacteria). Such biofilms preferentially form on the surfaces of indwelling medical devices (catheters, artificial joints, etc.) and mucosal tissues, and their pathogenic characteristics are reflected in two key dimensions: on the one hand, the biofilm-state strains have significantly enhanced tolerance to antibacterial drugs compared to planktonic strains (up to 1,000 times), and on the other hand, they evade immune clearance through mechanisms such as interfering with the chemotactic migration of immune cells and inhibiting phagocytic function. Research shows that the unique three-dimensional physical barrier of biofilms and their dual defense mechanisms pose a dual dilemma in clinical diagnosis and treatment, which is also an important inducement for the high recurrence rate of infections. The physical barrier prolongs the residence time of pathogens, providing a time window for microenvironment regulation; the disorder of redox homeostasis further weakens the immune clearance ability and promotes the expansion of biofilms. Therefore, wound biofilm infections form a vicious cycle and are difficult to cure, and the global death toll continues to rise.

[0003] The multi-dimensional defense characteristics of biofilms make it difficult for a single treatment strategy to efficiently clear infections. Currently, treatment methods mainly rely on physical, chemical, biological, and combination therapies. However, these technologies still face many challenges in clinical translation, mainly including problems such as penetrability, stability, and adaptability to the biofilm microenvironment. Therefore, it is urgent to develop therapeutic drugs that can break the vicious cycle of "biofilm barrier - oxidative stress - immunosuppression". Summary of the Invention

[0004] In view of the above problems, the present invention aims to provide a core-shell microneedle for removing bacterial biofilms and a preparation method thereof.

[0005] The technical solution of the present invention is as follows:

[0006] On the one hand, a core-shell microneedle for removing bacterial biofilms is provided, including a shell layer and a core layer. The shell layer includes a shell layer material and cuprous oxide nanoparticles loaded on the shell layer material, and the core layer includes a core layer material and mesoporous polydopamine nanoparticles loaded on the core layer material and coated with citraconate and capable of targeting neutrophils.

[0007] Preferably, the shell layer material is any one of hyaluronic acid, hyaluronic acid methacrylate, and polyacrylic acid.

[0008] Preferably, the core layer material is any one of polyvinyl alcohol, chitosan, and polyvinylpyrrolidone.

[0009] Preferably, the mesoporous polydopamine nanoparticles encapsulated with citrates and capable of targeting neutrophils are prepared by the following steps:

[0010] S1: Prepare mesoporous polydopamine nanoparticles and citrate solution respectively;

[0011] S2: Modify the mesoporous polydopamine nanoparticles with a positively charged substance containing more amino groups than the exposed amino groups of the mesoporous polydopamine to obtain amino-functionalized mesoporous polydopamine nanoparticles;

[0012] S3: Modify the amino-functionalized mesoporous polydopamine nanoparticles with a substance capable of targeting neutrophils to obtain mesoporous polydopamine nanoparticles capable of targeting neutrophils;

[0013] S4: Mix the citrate solution with the mesoporous polydopamine nanoparticles capable of targeting neutrophils to obtain the mesoporous polydopamine nanoparticles encapsulated with citrates and capable of targeting neutrophils.

[0014] Preferably, in step S1, the mesoporous polydopamine nanoparticles are prepared by any one of the soft template method, hard template method, self-assembly method, and electrochemical polymerization method.

[0015] Preferably, in step S2, the positively charged substance containing more amino groups than the exposed amino groups of the mesoporous polydopamine is any one or more of polyethyleneimine, polyvinylamine, polylysine, and chitosan.

[0016] Preferably, in step S3, the substance capable of targeting neutrophils is any one or more of sialic acid, α-1 antitrypsin, and neutrophil cell membrane.

[0017] Preferably, in step S1, the concentration of the citrate solution is 10 - 20 mM; in step S4, the mass ratio of the mesoporous polydopamine nanoparticles capable of targeting neutrophils to the mass of citrate in the citrate solution is 1:1 - 2.

[0018] On the other hand, there is also provided a preparation method of the core-shell microneedles for removing bacterial biofilms described in any one of the above, including the following steps:

[0019] S1': Prepare cuprous oxide nanoparticles and mesoporous polydopamine nanoparticles encapsulated with citrates and capable of targeting neutrophils respectively;

[0020] S2': Disperse the copper peroxide nanoparticles in the shell material to obtain Mixture 1; disperse the mesoporous polydopamine nanoparticles encapsulated with citraconate and capable of targeting neutrophils in the core material to obtain Mixture 2;

[0021] S3': Add Mixture 1 into a microneedle mold and perform vacuum drying to obtain the shell layer of the core-shell microneedles;

[0022] S4': Add Mixture 2 into the microneedle mold of the shell layer of the core-shell microneedles obtained in step S3 and perform vacuum drying to obtain the core layer of the core-shell microneedles;

[0023] S5': Load the backing with the core material, dry and demold to obtain the core-shell microneedles.

[0024] Preferably, in step S2', in Mixture 1, the concentration of the copper peroxide nanoparticles is 32 - 64 μg / mL; in Mixture 2, the concentration of the mesoporous polydopamine nanoparticles encapsulated with citraconate and capable of targeting neutrophils is 100 - 200 μg / mL.

[0025] The beneficial effects of the present invention are as follows:

[0026] The present invention can penetrate the biofilm and preferentially release copper peroxide nanoparticles, disrupt the redox homeostasis inside the biofilm, kill bacteria and disperse the biofilm; it can target neutrophils, relieve the immunosuppressive state of innate immune cells, and restore their bacterial killing ability, thereby effectively phagocytosing the escaped bacteria; the synergistic effect of the two enables the present invention to completely remove the biofilm. The present invention has clinical application prospects and is beneficial to reducing the bacterial drug resistance caused by the long-term use of antibiotics. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a schematic diagram of the preparation process of CMPS nanoparticles in Example 1;

[0029] Figure 2 It is a schematic diagram of the preparation process of the core-shell microneedles in Example 1;

[0030] Figure 3Morphology pictures of CuO2 nanoparticles in Example 1; among them, a is a field emission electron scanning microscope photograph, and b is a transmission electron microscope photograph;

[0031] Figure 4 Schematic diagram of the test results of the •OH generation ability of CuO2 nanoparticles in Example 1; among them, a is the absorption spectrum of 3,3',5,5'-tetramethylbenzidine, and b is the ESR spectrum of 5,5-dimethyl-1-pyrroline-N-oxide capturing •OH;

[0032] Figure 5 Morphology pictures of CMPS nanoparticles in Example 1; among them, a is a field emission electron scanning microscope photograph, and b is a transmission electron microscope photograph;

[0033] Figure 6 Schematic diagram of the test results of the targeting ability of CMPS nanoparticles in Example 1; among them, a is the flow cytometry of neutrophils incubated for 2 h after different treatments, and b is the corresponding mean fluorescence intensity;

[0034] Figure 7 Schematic diagram of the test results of citric acid salt restoring the IRG1 level of neutrophils in Example 1; among them, a is the mortality rate of neutrophils, and b is the relative expression rate of IRG1 mRNA;

[0035] Figure 8 Schematic SEM morphology diagram of the core-shell microneedles in Example 1;

[0036] Figure 9 Morphology diagram of the core-shell microneedles in Example 1; among them, a is the morphology diagram of the microneedles loaded with dye, and b is the representative fluorescence image of the core-shell microneedles;

[0037] Figure 10 Schematic diagram of the comparison results of the biofilm removal performance of microneedles in different groups; among them, a is the MRSA biofilm image under different treatments stained by the crystal violet method, and b is the relative biofilm content;

[0038] Figure 11 Photos of the wound healing process in mice; among them, a is the picture of the wound site of different treatment groups during 14 days of treatment, and b is the healing trajectory diagram;

[0039] Figure 12 Schematic diagram of the comparison results of different isomers in restoring the phagocytic function of neutrophils. Detailed implementation manners

[0040] The present invention is further described below with reference to the accompanying drawings and examples. It should be noted that, in the absence of conflict, the embodiments in this application and the technical features in the embodiments can be combined with each other. It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The use of similar words such as "include" or "comprising" in the present invention means that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0041] On the one hand, the present invention provides a core-shell microneedle for removing bacterial biofilms, comprising a shell layer and a core layer, wherein the shell layer comprises a shell layer material and copper peroxide nanoparticles loaded on the shell layer material, and the core layer comprises a core layer material and mesoporous polydopamine nanoparticles loaded on the core layer material and coated with citraconate and capable of targeting neutrophils.

[0042] In this invention, copper peroxide nanoparticles are placed within the shell layer, enabling the core-shell microneedles to penetrate biofilms and preferentially release the copper peroxide nanoparticles. Under acidic conditions, the copper peroxide nanoparticles continuously catalyze the conversion of endogenous hydrogen peroxide (H2O2) into long-lasting hydroxyl radicals (•OH), disrupting the redox homeostasis within the biofilm, killing bacteria and dispersing the biofilm. Mesoporous polydopamine nanoparticles encapsulated with citraconic acid and capable of targeting neutrophils are placed within the core layer. This allows them to target neutrophils, release citraconic acid, and relieve the immunosuppressive state of innate immune cells, restoring their bacterial killing capacity and effectively engulfing escaping bacteria. The synergistic effect of these two factors enables the present invention to completely eliminate biofilms.

[0043] It should be noted that bacterial biofilm infection forms a physical barrier, and treatment faces two major problems: the difficulty for bactericides and immune cells to enter the inner layer of the biofilm, and the immunosuppressive state of innate immune cells. If the immune function of innate immune cells is restored first, they will not be able to penetrate the extracellular matrix network of the biofilm due to the volume effect. Therefore, the first step is to disperse the biofilm and kill bacteria. The core-shell microneedle will first release the shell material. Therefore, the copper peroxide nanoparticles that can disperse the biofilm and kill bacteria are placed in the shell layer rather than the core layer.

[0044] In a specific embodiment, the shell layer material is any one of hyaluronic acid, hyaluronic acid methacrylate, and polyacrylic acid, and the core layer material is any one of polyvinyl alcohol, chitosan, and polyvinyl pyrrolidone.

[0045] It should be noted that the shell materials and core materials in the above embodiments are only the preferred materials of the present invention, and other water-soluble polymer materials suitable for microneedles in the prior art (such as dextran, carboxymethyl cellulose, gelatin, sodium alginate, etc.) can also be applied to the present invention. In addition, it should be noted that since hyaluronidase exists in the biological membrane and can degrade hyaluronic acid, using hyaluronic acid as the shell material can promote the release of cupric peroxide nanoparticles in the shell. The core material can also be selected from the same material as the shell material, but selecting different materials can enable the drug to be further released in a graded manner.

[0046] In a specific embodiment, the mesoporous polydopamine nanoparticles encapsulated with citrates and capable of targeting neutrophils are prepared by the following steps:

[0047] S1: Prepare mesoporous polydopamine nanoparticles and a citrate solution respectively.

[0048] In a specific embodiment, the mesoporous polydopamine nanoparticles are prepared by any one of the soft template method, hard template method, self-assembly method, and electrochemical polymerization method. It should be noted that the above-mentioned preparation methods of mesoporous polydopamine nanoparticles in the embodiments are all prior art, and the specific steps will not be elaborated here.

[0049] S2: Modify the mesoporous polydopamine nanoparticles with a positively charged substance containing more amino groups than the exposed amino groups of mesoporous polydopamine to obtain amino-functionalized mesoporous polydopamine nanoparticles.

[0050] In a specific embodiment, the positively charged substance containing more amino groups than the exposed amino groups of mesoporous polydopamine is any one or more of polyethyleneimine, polyvinylamine, polylysine, and chitosan.

[0051] S3: Modify the amino-functionalized mesoporous polydopamine nanoparticles with a substance capable of targeting neutrophils to obtain mesoporous polydopamine nanoparticles capable of targeting neutrophils.

[0052] In a specific embodiment, the substance capable of targeting neutrophils is any one or more of sialic acid, α-1 antitrypsin, and neutrophil cell membrane.

[0053] In the above embodiments, the sialic acid can target L-selectin of neutrophils, the α-1 antitrypsin can target neutrophil elastase-binding peptide, and the neutrophil membrane can target neutrophils homologously. All of them can selectively recognize on the surface of neutrophils to achieve targeting neutrophils. It should be noted that the substances in the above embodiments are only the preferred substances of the present invention that can target neutrophils, and other substances that can target neutrophils in the prior art can also be applicable to the present invention.

[0054] S4: Mix the citraconate solution with the mesoporous polydopamine nanoparticles capable of targeting neutrophils to obtain the mesoporous polydopamine nanoparticles wrapped with citraconate and capable of targeting neutrophils.

[0055] In the present invention, the citraconate captures escaped bacteria by inhibiting IRG1 expression and restoring the phagocytic function of neutrophils. It should be noted that although itaconate and mesaconate are isomers of citraconate, they cannot achieve the technical effects that citraconate of the present invention can achieve. The present invention can only use citraconate to restore the phagocytic function of neutrophils.

[0056] In a specific embodiment, in step S1, the concentration of the citraconate solution is 10 - 20 mM; in step S4, the mass ratio of the mesoporous polydopamine nanoparticles capable of targeting neutrophils to the mass of citraconate in the citraconate solution is 1:1 - 2.

[0057] On the other hand, the present invention also provides a preparation method of the core-shell microneedles for removing bacterial biofilms described in any one of the above, including the following steps:

[0058] S1': Prepare cupric peroxide nanoparticles and mesoporous polydopamine nanoparticles wrapped with citraconate and capable of targeting neutrophils respectively;

[0059] S2': Disperse the cupric peroxide nanoparticles in the shell material to obtain mixture one; disperse the mesoporous polydopamine nanoparticles wrapped with citraconate and capable of targeting neutrophils in the core material to obtain mixture two;

[0060] S3': Add the mixture one into a microneedle mold and vacuum dry to obtain the shell layer of the core-shell microneedles;

[0061] [[ID=2�]]S4': Add the mixture two into the microneedle mold of the shell layer of the core-shell microneedles obtained in step S3 and vacuum dry to obtain the core layer of the core-shell microneedles;

[0062] S5': Load the backing with the core material, dry and demold to obtain the core-shell microneedles.

[0063] It should be noted that in the present invention, the microneedle mold is a prior art, and a common PDMS mold can be used. The mold cavity of the microneedle mold includes a groove portion and inverted conical blind holes connected below the groove portion and distributed in an array.

[0064] In a specific embodiment, in step S2', in the first mixture, the concentration of the cupric peroxide nanoparticles is 32 - 64 μg / mL; in the second mixture, the concentration of the mesoporous polydopamine nanoparticles coated with citraconate and capable of targeting neutrophils is 100 - 200 μg / mL.

[0065] Example 1

[0066] A core - shell microneedle for removing bacterial biofilms, as Figure 1 and Figure 2 shown, is prepared by the following steps:

[0067] (1) Prepare cupric peroxide (CuO2) nanoparticles by a liquid - phase synthesis method. First, dissolve cupric chloride dihydrate (CuCl2·2H2O) and sodium hydroxide (NaOH) in ultrapure water to prepare solutions with concentrations of 0.01 mol / L and 0.03 mol / L respectively. Then, place polyvinyl alcohol (PVP, 2 g), CuCl2·2H2O (20 mL, 0.01 mol / L), NaOH (20 mL, 0.03 mol / L), and hydrogen peroxide (H2O2, 400 μL, 30%) in a glass bottle and stir at room temperature for 30 min. Finally, collect the brownish - black CuO2 nanoparticle precipitate by centrifugation (8000 rpm, 5 min), wash it three times with ultrapure water to obtain CuO2 nanoparticles, and freeze - dry them for standby.

[0068] (2) Synthesize mesoporous polydopamine (MPDA) nanoparticles by the "soft - template" method. First, mix 60 mL of ethanol with 65 mL of deionized water (containing 0.36 g of poly(ethylene glycol) - block - poly(propylene glycol) - block - poly(ethylene glycol) (F127) and 417 μL of 1,3,5 - trimethylbenzene solution). After stirring for 30 min, add 60 mg of dopamine hydrochloride and 90 mg of tris(hydroxymethyl)aminomethane (Tris) to the mixture. After reacting at room temperature for 24 h, centrifuge to collect the crude product. Then wash the crude product 3 times in an ethanol / acetone mixed solvent (volume ratio 2:1) to remove the template, and finally obtain MPDA nanoparticles with good monodispersity.

[0069] (3) Polyethyleneimine (PEI) was used to modify MPDA nanoparticles. Through Michael addition and Schiff base reaction, amino-functionalized modification of PEI was achieved on the surface of MPDA. Specifically: MPDA nanoparticles were dispersed in phosphate buffered saline (PBS buffer, pH 7.4, 1 mg / mL), and then 1 mL of PEI solution (1 mg / mL) was added. The mixture was stirred for 24 h at room temperature in the dark. After the reaction, the reaction mixture was centrifuged and washed three times with water to obtain MPDA-PEI nanoparticles (abbreviated as MP nanoparticles).

[0070] (4) Sialic acid (SA) was used to modify MP nanoparticles. Grafting of sialic acid was achieved on the surface of MP particles through amide reaction. Specifically: MP nanoparticles were dispersed in PBS buffer (pH 5.5, 1 mg / mL), and then 2 mL of SA solution (1 mg / mL) was added. The mixture was stirred for 12 h at room temperature. Subsequently, the reaction mixture was centrifuged and washed three times with water to obtain MPDA-PEI-SA nanoparticles (abbreviated as MPS nanoparticles).

[0071] (5) Citraconate was encapsulated in MPS nanoparticles. First, in an ice bath environment, 20 mM citraconic acid (Citra) was mixed with NaOH solution and adjusted to pH 6.5 to obtain a stable citraconate solution. The obtained Citra solution (2 mL) was mixed with MPS nanoparticles (1 mg / mL), and the mixture was stirred for 12 h at room temperature to enable Citra to be encapsulated inside MPS nanoparticles through π-π stacking interaction. The product was collected by centrifugation to obtain Citra@MPDA-PEI-SA nanoparticles (abbreviated as CMPS nanoparticles).

[0072] (6) Core-shell microneedles were prepared. CuO2 nanoparticles were dispersed in hyaluronic acid (HA, 2%) gel to obtain mixture one. 2 mL of mixture one was cast into a PDMS mold, and under vacuum negative pressure, mixture one was filled down to the tip of the microneedle (MN). Then, it was air-dried overnight at room temperature to obtain the outer shell of Cu / CMPS MN. Secondly, CMPS nanoparticles were mixed with polyvinyl alcohol (PVA, 15%) gel to obtain mixture two. Mixture two was added above the hollow MN, and the cavity was filled under vacuum. After drying overnight at room temperature in the dark, the core of Cu / CMPS MN was obtained. Excessive PVA (15%) gel was used as the substrate of the MN patch, and after drying, Cu / CMPS MN was obtained.

[0073] Example 2

[0074] Different from Example 1, this example does not include step (1), and the CuO2 nanoparticles in step (6) were directly purchased commercially available CuO2 nanoparticles.

[0075] Example 3

[0076] Different from Example 1, this example does not include step (2), and the MPDA nanoparticles in step (3) are directly purchased from the market.

[0077] Example 4

[0078] Different from Example 1, in this example, the material for modifying the MPDA nanoparticles in step (3) is polyvinylamine.

[0079] Example 5

[0080] Different from Example 1, in this example, the material for modifying the MPDA nanoparticles in step (3) is polylysine.

[0081] Example 6

[0082] Different from Example 1, in this example, the material for modifying the MPDA nanoparticles in step (3) is chitosan.

[0083] Example 7

[0084] Different from Example 1, in this example, the material for modifying the MP nanoparticles in step (4) is α-1 antitrypsin.

[0085] Example 8

[0086] Different from Example 1, in this example, the material for modifying the MP nanoparticles in step (4) is neutrophil cell membrane.

[0087] Example 9

[0088] Different from Example 1, in this example, the core layer material in step (6) is chitosan.

[0089] Example 10

[0090] Different from Example 1, in this example, the core layer material in step (6) is polyvinylpyrrolidone.

[0091] Comparative Example 1

[0092] Different from Example 1, this comparative example does not include steps (1)-(5), and CuO2 nanoparticles and CMPS nanoparticles are not added in step (6), that is, only a blank microneedle patch is obtained by using hyaluronic acid (HA, 2%) gel to prepare the outer shell and polyvinyl alcohol (PVA, 15%) gel to prepare the inner core, denoted as MN.

[0093] Comparative Example 2

[0094] Different from Example 1, this comparative example only includes step (1) and step (6), and no CMPS nanoparticles are added in step (6). The obtained core-shell microneedles are denoted as Cu MN.

[0095] Comparative Example 3

[0096] Different from Example 1, this comparative example does not include step (1), and no CuO2 nanoparticles are added in step (6). The obtained core-shell microneedles are denoted as CMPS MN.

[0097] Test Example 1 Observation of the Morphology of CuO2 Nanoparticles

[0098] Drop the prepared CuO2 nanoparticle solution (1 mg / mL) in Example 1 onto a silicon wafer and let it air-dry naturally. After sputter coating with gold (the thickness of the gold film is about 10 nm), observe the surface morphology of the CuO2 nanoparticles under a scanning electron microscope (SEM) and take pictures; Drop 5 μL of the CuO2 nanoparticle solution (1 mg / mL) onto a copper grid. After deposition for 10 min, suck away the excess solution from the edge of the copper grid with a clean filter paper. After natural air-drying, perform transmission electron microscope (TEM) imaging and local high-resolution imaging to observe its overall morphology and lattice structure. The results are as Figure 3 shown. As Figure 3 can be seen, the obtained CuO2 nanoparticles are spindle-shaped, with good uniformity and dispersibility.

[0099] Test Example 2 Measurement of the •OH Generation Ability of CuO2 Nanoparticles

[0100] The 3,3',5,5'-tetramethylbenzidine (TMB) probe molecule can undergo a single-electron transfer reaction with strongly oxidizing ROS to generate a characteristic blue oxidation product (ox-TMB). Given that an acidic microenvironment can trigger the decomposition of CuO2 nanoparticles to produce •OH, this test example selects TMB as a specific detection probe for ROS. The ability of the material to generate ROS at different pH values is detected using TMB reagent and ESR spectroscopy. After co-incubating the CuO2 solution (100 μg / mL) with TMB solutions having different pH values (7.4 or 5.6) for 20 min, measure their absorption spectra in the range of 400 - 800 nm. The generation of ROS is determined by the increase in the absorbance of the solution at 650 nm. In addition, the CuO2 solution (100 μg / mL) was added to PBS solutions with different pH values (7.4 or 5.6), and the above supernatant was subjected to ESR spectroscopy with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a •OH scavenger. The results are as Figure 4 shown.

[0101] As Figure 4It can be seen that under acidic conditions (pH 5.6), TMB exhibits a significant characteristic absorption peak at 652 nm, confirming that the decomposition process of CuO2 is accompanied by the continuous generation of ROS. Moreover, typical •OH characteristic signals (quadruplet, and the peak intensity ratio is 1:2:2:1) were detected by ESR spectroscopy, and the signal intensity was significantly stronger than that under neutral conditions (pH 7.4).

[0102] Test Example 3 Observation of the morphology of CMPS nanoparticles

[0103] Using the CMPS nanoparticles in Example 1, 10 μL of CMPS (100 μg / mL) was dropped on a silicon wafer, and after gold spraying treatment, its morphology was observed and analyzed by SEM; in addition, 5 μL of CMPS (100 μg / mL) was dropped on a copper mesh, and the morphological structure was observed and photographed by TEM. The results are as Figure 5 shown. From Figure 5 It can be seen that the CMPS nanoparticles described in the present invention have regular spherical morphology, and TEM confirms that their particle size distribution is 250 nm.

[0104] Test Example 4 Testing the targeting ability of CMPS nanoparticles

[0105] The targeting behavior of CMPS nanoparticles to neutrophils was investigated using a flow cytometer (FCM). First, the extracted neutrophils (1 × 10 7 cell / mL) were inoculated in a 24-well plate for culture. Then, DMEM medium containing MPDA and CMPS (100 µg / mL) encapsulated with Rhm B was added for 2 h, centrifuged and collected, and after filtering through a neutral filter for neutrophils, it was tested by FCM. The data was analyzed using FlowJo software and the corresponding mean fluorescence intensity was statistically analyzed. The results are as Figure 6 shown. From Figure 6 It can be seen that the phagocytosis amount of neutrophils for CMPS is significantly higher than that of non-targeted MPDA. At the same time, when the L-selectin on the surface of neutrophils is inhibited, the phagocytosis amount of neutrophils for CMPS also decreases significantly. Quantitative analysis of the Rhm B signal in cells also shows the same results. These results further indicate that SA plays an important role in mediating the uptake of CMPS nanodrugs by neutrophils.

[0106] Test Example 5 Testing the restoration of the IRG1 level in neutrophils by citrates

[0107] Methicillin-resistant Staphylococcus aureus (MRSA, 1 × 10 6 cell / mL) was added to a medium containing neutrophils (1× 10 7In 24-well plates (cell / mL), after co-incubating with 0 or 10 mM citraconate for 1 h, the death of neutrophils in vitro was monitored by measuring the release of lactate dehydrogenase (LDH). Take 50 μL of the treated neutrophil supernatant sample, centrifuge at 500 g for 5 min to remove fragmented cells, and then store at -20 °C. The next day, take 10 μL of each sample and mix it with 100 μL of LDH Reaction Mix (prepared according to the kit instructions), place it in a 96-well plate with a transparent bottom, incubate at room temperature for 30 min, and then measure the absorbance at 450 nm using a microplate reader. At the same time, perform RT-qPCR experiments. Collect neutrophils from each group and extract total RNA using a total RNA extraction kit. Immediately perform the removal reaction of genomic DNA and the reverse transcription reaction through primer design and real-time fluorescence quantitative PCR reaction. Use QuantStudioTM Design&Analysis SE Software (Thermo) to analyze the CT (Threshold cycle) values of each test sample during the PCR process. This test example is through 2 -△△CT (where △△CT is the difference between the △CT value of each test sample and the △CT value of the control group, and the △CT value is the difference between the CT value of the target gene and the CT value of the internal reference gene) to calculate the relative mRNA expression level of X, quantify each sample, and the results are as Figure 7 shown.

[0108] It can be seen from Figure 7 that MRSA infection induced neutrophil death, and the addition of citraconate inhibited this phenomenon. At the same time, the qRT-PCR results showed that the expression of IRG1 mRNA in neutrophils increased after infection with MRSA compared with the control group, and the addition of citraconate restored the IRG1 mRNA level in neutrophils to the normal level, showing no significant difference from the control group. The above results indicate that citraconate inhibits the expression of the IRG1 gene and restores the phagocytic function of neutrophils against MRSA.

[0109] Test Example 6 Observation of the morphology of core-shell microneedles

[0110] The overall morphology of the core-shell microneedles in Example 1 was observed using a scanning electron microscope, and the results are as Figure 8 shown. It can be seen from Figure 8 that the core-shell microneedle patch prepared in Example 1 consists of a 15 × 15 array of conical needles with a height of 600 μm and a base diameter of 300 μm.

[0111] The shell layer and core layer of the Cu / CMPS MN core-shell microneedles were respectively labeled with the fluorescent dyes rhodamine B (Rhm B) and fluorescein isothiocyanate (FITC). The distributions of the Rhm B (red) and FITC (green) fluorescence signals in the Cu / CMPS MN core-shell microneedles were observed under a fluorescence microscope, and the results are as Figure 9 shown. It can be seen from Figure 9 that the outer shell is loaded with Rhm B and the inner core is loaded with FITC, which confirms that the Cu / CMPS MN of the present invention has a core-shell structure.

[0112] Test Example 7 Testing the performance of core-shell microneedles in removing biofilms

[0113] To construct a MRSA biofilm, first, MRSA was cultured in tryptic soy broth (TSB) containing 0.2% glucose to obtain a bacterial suspension with an OD value (600 nm) of 0.8. Then, the bacterial suspension was dispersed in a 24-well plate, 1 mL per well, and cultured at 37 °C for two days, with the TSB medium replaced once a day. Subsequently, the wells were washed with PBS to remove planktonic bacteria, and the formed biofilms were respectively added with fresh TSB containing sterile PBS, MN (Comparative Example 1), CMPS MN (Comparative Example 3), Cu MN (Comparative Example 2), and Cu / CMPS MN (Example 1) to the culture plate (1 mL per well), and incubated for 6 h under the conditions simulating the microenvironment of infected tissues (pH 5.6 and 100 μM H2O2). Then, the medium in each well was removed, the well plate was washed with PBS, and neutrophils (1 × 10 7 Cells / mL) were added to or not added to each well, and then incubated at 37 °C for 1 h, and then washed 3 times with frozen PBS to stop bactericidal action. Crystal violet (0.1%, 200 μL) was added to each well, and incubated at 37 °C for 15 min for photographing and observation. After washing 4 times with PBS, 200 μL of 30% glacial acetic acid solution was added, and the absorbance was measured at a wavelength of 595 nm. The results are as Figure 10 shown. It can be seen from Figure 10 that the combined treatment with neutrophils and Cu / CMPS MN significantly reduced the bacterial count in the Staphylococcus aureus biofilm, and the biofilm biomass decreased to 0.99%, which was better than the simple material treatment group.

[0114] Test Example 8 Using the core-shell microneedle patch for in vivo biofilm removal

[0115] Establishment of an animal model: After shaving the back of a Kunming mouse, the full-thickness skin (including the epidermis and dermis) was excised using a biopsy punch (8 mm in diameter). Immediately after the wound was created, a MRSA suspension (containing 1 × 10 8CFU / mL, 100 μL) was applied to the wound surface. A sterile 3M film was covered to maintain a moist environment, and a stable biofilm was formed after 72 h. The mice with wound biofilms were randomly divided into 5 groups (6 mice in each group), and on the first day, they were treated with PBS, MN, CMPS MN, Cu MN, and Cu / CMPS MN respectively, and histological analysis was performed on the second and fourteenth days.

[0116] Within 14 days of treatment, the wound healing process and the body weight changes of the mice were recorded by taking pictures of the mice on days 0, 4, 7, 11, and 14 and weighing their body weights at the above time points. The wound area was statistically analyzed using ImageJ software to evaluate the wound healing rate, and the wound healing rate was calculated according to formula (1). On the fourteenth day, the wound tissues of each treatment group were fixed, embedded, and sectioned, and hematoxylin-eosin staining and Masson staining were performed on them. Then, image acquisition was carried out using a pathological section scanner to observe the wound healing situation.

[0117] (1)

[0118] In the formula: S0 and S t are the initial wound area and the wound area at time point t, respectively.

[0119] On the fourteenth day of treatment, the wound tissue was homogenized and then diluted and plated to quantify the viable bacteria count. In addition, the wound tissue was taken out, fixed, dehydrated, and sputter-coated with gold, and then the biofilm status on the wound surface, the degree of bacterial aggregation, and the EPS structure after treatment were observed by SEM. The results are as Figure 11 shown.

[0120] From Figure 11 it can be seen that during the 14-day treatment process, compared with other groups, the administration of Cu / CMPS MN significantly accelerated the wound closure. The healing trajectory diagrams outlined can visually observe the sizes of the wound healing areas of each treatment group. On the fourteenth day of treatment, the wound closure rate of the Cu / CMPS MN group was as high as 100%, significantly higher than that of other groups, and a complete, connected, and thickened regenerated epidermis appeared at the wound tissue edge.

[0121] Test Example 9 Testing the performance of citraconate and its isomers in restoring the phagocytic function of neutrophils

[0122] The MRSA transfected with green fluorescent protein (GFP) was co-incubated with neutrophils, and then neutrophils were labeled with lymphocyte antigen 6 complex locus G (LY6G) antibody and 4',6-diamidino-2-phenylindole (DAPI). The phagocytosis of bacteria by neutrophils under 10 mM itaconate (Ita), mesaconate (Mesa), and citraconate (Citra) was detected by fluorescence co-localization. The results are as Figure 12as shown, where Merge is the merged image of GFP, LY6G, and DAPI. From Figure 12 it can be seen that only citraconate can restore the phagocytic function of neutrophils.

[0123] It should be noted that the above test examples are only the test results of some embodiments of the present invention. Other embodiments have similar performances to Embodiment 1 and can completely remove the biofilm. In addition, the above embodiments are only some embodiments of the present invention. The core-shell microneedles of the present invention prepared by changing the temperature, time, etc. of the core layer material, shell layer material, and preparation method also have similar performances.

[0124] In summary, the present invention can achieve the complete removal of the biofilm. Compared with the prior art, the present invention has significant progress.

[0125] The above are only representative embodiments of the present invention and do not limit the present invention in any form. Any person skilled in the art, within the scope of the technical solution of the present invention, makes some modifications or variations using the disclosed technical content, which are equivalent embodiments of the present invention. However, any simple modification, equivalent change, and variation made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A core-shell microneedle for removing bacterial biofilms, characterized in that, It includes a shell layer and a core layer. The shell layer includes a shell layer material and cuprous oxide nanoparticles loaded on the shell layer material. The core layer includes a core layer material and mesoporous polydopamine nanoparticles wrapped with citrates and capable of targeting neutrophils, wherein the substance capable of targeting neutrophils is sialic acid.

2. The core-shell microneedles for removing bacterial biofilms according to claim 1, wherein The shell layer material is any one of hyaluronic acid, hyaluronic acid methacrylate, and polyacrylic acid.

3. The core-shell microneedle for removing bacterial biofilms according to claim 1, wherein The core layer material is any one of polyvinyl alcohol, chitosan, and polyvinylpyrrolidone.

4. The core-shell microneedle for removing bacterial biofilms according to any one of claims 1-3, characterized in that, The mesoporous polydopamine nanoparticles wrapped with citrates and capable of targeting neutrophils are prepared by the following steps: S1: Prepare mesoporous polydopamine nanoparticles and a citrate solution respectively; S2: Modify the mesoporous polydopamine nanoparticles with a positively charged substance containing more amino groups than the exposed amino groups of mesoporous polydopamine to obtain amino-functionalized mesoporous polydopamine nanoparticles; S3: Modify the amino-functionalized mesoporous polydopamine nanoparticles with a substance capable of targeting neutrophils to obtain mesoporous polydopamine nanoparticles capable of targeting neutrophils; S4: Mix the citrate solution with the mesoporous polydopamine nanoparticles capable of targeting neutrophils to obtain the mesoporous polydopamine nanoparticles wrapped with citrates and capable of targeting neutrophils.

5. The core-shell microneedle for clearing bacterial biofilms according to claim 4, characterized in that, In step S1, the mesoporous polydopamine nanoparticles are prepared by any one of the soft template method, hard template method, self-assembly method, and electrochemical polymerization method.

6. The core-shell microneedle for removing bacterial biofilms according to claim 4, wherein In step S2, the positively charged substance containing more amino groups than the exposed amino groups of mesoporous polydopamine is any one or more of polyethyleneimine, polyvinylamine, polylysine, and chitosan.

7. The core-shell microneedle for removing bacterial biofilms according to claim 4, characterized in that, In step S1, the concentration of the citrate solution is 10 - 20 mM; in step S4, the mass ratio of the mesoporous polydopamine nanoparticles capable of targeting neutrophils to the mass of citrate in the citrate solution is 1:1 - 2.

8. The preparation method of the core-shell microneedles for removing bacterial biofilms according to any one of claims 1-7, characterized in that, It includes the following steps: S1': Prepare cuprous oxide nanoparticles and mesoporous polydopamine nanoparticles wrapped with citrates and capable of targeting neutrophils respectively; S2': Disperse the cuprous oxide nanoparticles in the shell layer material to obtain mixture one; disperse the mesoporous polydopamine nanoparticles wrapped with citrates and capable of targeting neutrophils in the core layer material to obtain mixture two; S3': Add mixture one into a microneedle mold and vacuum dry to obtain the shell layer of the core-shell microneedle; S4': Add mixture two into the microneedle mold of the shell layer of the core-shell microneedle obtained in step S3 and vacuum dry to obtain the core layer of the core-shell microneedle; S5': Load a backing with the core layer material, dry and demold to obtain the core-shell microneedle.

9. The preparation method of the core-shell microneedles for removing bacterial biofilms according to claim 8, characterized in that, In step S2', in mixture one, the concentration of the cuprous oxide nanoparticles is 32 - 64 μg / mL; in mixture two, the concentration of the mesoporous polydopamine nanoparticles wrapped with citrates and capable of targeting neutrophils is 100 - 200 μg / mL.

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