Phage-antibiotic co-loaded gradient-degrading microneedle device and methods thereof

Abstract

The application provides a phage-antibiotic co-loaded gradient degradation microneedle device and a method thereof, and relates to the technical field of medical microneedles.The microneedle device comprises a microneedle array and a backing.The microneedles in the microneedle array have a double-layer structure: an outer layer structure comprising phages for target bacteria and a matrix loaded with the phages; and an inner layer structure comprising antibiotics for target bacteria and a matrix loaded with the antibiotics.Through the double-layer microneedle structure, differential degradation of the phages and the antibiotics is achieved, the purpose of first resisting biofilms and then resisting bacteria is achieved, and the treatment efficiency and effect of PJI are improved.

Description

Technical Field

[0001] This invention relates to the field of medical microneedle technology, and in particular to a phage-antibiotic co-loaded gradient degradation microneedle device and method thereof. Background Technology

[0002] Periprosthetic joint infection (PJI) is a complication following joint replacement surgery. PJI significantly increases the revision rate and mortality rate after joint replacement surgery.

[0003] The main challenges in treating PJI include the following aspects: Joint prostheses, as implants, lack a blood supply and are located deep within the tissue, making it difficult for antibacterial drugs to reach the site of infection via blood circulation, thus hindering drug accumulation to achieve effective antibacterial concentrations. Without early intervention, acute PJI will turn into a persistent chronic infection. Long-term use of antibiotics can easily lead to drug-resistant bacterial infections, further increasing the difficulty of later treatment. During long-term chronic infection, due to the nature of the prosthesis and the biological characteristics of the bacteria, the bacteria will form a dense biofilm around the prosthesis (the main chemical components include water, extracellular polysaccharides, extracellular DNA and proteins). Because the biofilm has high antibiotic resistance and immune escape ability, it will further increase the difficulty of fighting infection.

[0004] Currently, various polymer materials are widely used in the design and manufacture of medical microneedles due to their excellent designability. Their biodegradability and biocompatibility can compensate for the shortcomings of traditional materials (such as residue problems). Through targeted design of materials, the mechanical and solubility properties of the needle can be controlled, thereby achieving purposes such as controlled drug release. Common polymer materials include hyaluronic acid and polylactic acid. Among them, polylactic acid microneedles, while possessing biodegradability and biocompatibility, also exhibit stronger toughness (i.e., fracture resistance) compared to traditional microneedles. Soluble microneedles, represented by materials such as hyaluronic acid, polyethylene glycol, and polyvinyl alcohol, can absorb interstitial fluid and dissolve after being inserted subcutaneously, thereby releasing the loaded drug.

[0005] In particular, hydrogels, as functionalized polymer materials, have also been applied to the preparation of microneedles. By changing the cross-linking method and degree, the internal structure of the hydrogel (including pore size, shape, and distribution) can be adjusted, thereby achieving the purpose of regulating mechanical and degradation properties. Under the action of shock waves, it can continuously maintain physical pathways, disrupt the multilayer structure of biomembranes, and thus achieve anti-infective treatment. However, simply loading antibiotics and other drugs onto such microneedles can cause the drugs to be released prematurely and attenuate before they have a chance to pierce the biomembrane, thus seriously affecting the therapeutic effect of the drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a gradient degradation microneedle device and method for phage-antibiotic co-carrying, so as to solve at least one technical problem existing in the prior art.

[0007] In a first aspect, to solve the above-mentioned technical problems, the present invention provides a phage-antibiotic co-loaded gradient degradation microneedle device, comprising a microneedle array and its backing; The microneedles in the microneedle array have a double-layer structure: The outer structure includes a phage targeting the target bacteria and a matrix on which the phage is loaded; The inner structure includes an antibiotic targeting the target bacteria and a matrix loaded with the antibiotic.

[0008] In this way, the phage in the outer layer structure is used to quickly destroy the biofilm matrix, and then the antibiotics in the inner layer structure are used to slowly release antibiotics to lyse the exposed target bacteria, thereby ensuring the antibiotic concentration during antibacterial treatment and thus improving the antibacterial effect.

[0009] In one feasible implementation, the microneedles are conical in shape.

[0010] In one feasible implementation, the microneedles are 1 mm in height to fit subcutaneous and periarticular tissues.

[0011] In one feasible implementation, the matrix of the outer structure includes poloxamer 407 (P407); poloxamer 407 is a thermosensitive hydrogel that, after depolymerization and dissolution in the human body, can be excreted with urine, etc., and as a conventional biocompatible material, it can be maintained for a minimum of several minutes in vivo and in vitro.

[0012] In one feasible implementation, the bacteriophage includes an anti-Staphylococcus epidermidis bacteriophage; the specific concentration of the anti-Staphylococcus epidermidis bacteriophage can be 9 × 10⁻⁶. 9 PFU / mL.

[0013] In one feasible implementation, the matrix of the inner layer structure comprises sodium alginate-polyvinyl alcohol hydrogel, the degradation rate of which is affected by cross-linking mode, strength, size and other conditions, with a minimum degradation time of one day and usually not exceeding one month.

[0014] In one feasible implementation, the antibiotic includes vancomycin; the specific concentration of the vancomycin may be 1.2 mg / ml.

[0015] In one feasible implementation, the backing material includes sodium alginate-polyvinyl alcohol hydrogel, which not only allows for slower degradation but also enables flexible adhesion to the curved surface of joint prostheses (such as knee joints, hip joints, etc.), making it easier to prepare simultaneously with microneedle arrays.

[0016] In one feasible implementation, the edge of the backing is evenly distributed with several through holes; the diameter of the through holes is larger than the diameter of the arthroscopic probe, so that the doctor can use the arthroscopic probe to hook the through holes of the backing, assist in moving or opening the microneedle device, and perform treatment on the target site.

[0017] In one feasible implementation, the backing is provided with several air bladders on the side without the microneedle array. In body fluid, the side with the air bladders will float up, causing the tips of the microneedle array to point downwards so that they can quickly reach the target area below. After the doctor confirms that the orientation is correct, the air bladders can be punctured by a key endoscope hook (or probe) so that the microneedle device sinks and fits the target area.

[0018] In one feasible implementation, the backing material also includes biocompatible paramagnetic particles, so that doctors can use an arthroscopic hook (or probe) with controllable magnetism (e.g., an electromagnet) to magnetically pull the microneedle device and perform treatment on the target site.

[0019] In one feasible implementation, the paramagnetic particles are made of Fe3O4 nanoparticles. These nanoparticles are gradually acidified by the phagocytic action of lysosomes or macrophages in the body, releasing iron ions, which then bind to transferrin and are transported to the bone marrow to synthesize heme.

[0020] In one feasible implementation, the microneedle device further includes a (degradable) sleeve for protecting the microneedle array and its backing when rolled into a cylinder for passage through surgical instruments such as arthroscopy.

[0021] In one feasible implementation, the microneedle device further includes a substrate; the substrate is fixed to the side of the backing that is not provided with the microneedle array; the area of ​​the substrate is larger than the area of ​​the backing so that the microneedle device can be applied to the skin surface for the treatment of superficial infections.

[0022] In one feasible implementation, the substrate is a medical silicone membrane.

[0023] Secondly, based on the same inventive concept, this application also provides a method for manufacturing the above-mentioned microneedle device, including a method for preparing an outer layer structure and an inner layer structure.

[0024] In one feasible implementation, the method for preparing the outer layer structure includes the following steps: Step b1: At 4°C, prepare a 30% (w / v, weight / volume) poloxamer 407 solution by mixing poloxamer 407 powder with ultrapure water; then add the sensitive phage stock solution to the poloxamer 407 solution, with the sensitive phage stock solution titer being 10. 9 PFU / mL was used to obtain a phage-loaded poloxamer 407 solution; Step b2: Pour the phage-loaded poloxamer 407 solution into the injection hole of a (conventional) microneedle mold; incubate at 37°C for 10 minutes to form an outer layer structure with a conical cavity.

[0025] In one feasible implementation, the method for preparing the inner layer structure includes the following steps: Step c1: Mix 2% (w / v) sodium alginate and 10% (w / v) polyvinyl alcohol with the sensitive antibiotics screened by the target bacteria drug sensitivity test to obtain an antibiotic mixture. Step c2: At room temperature, the antibiotic mixture is stirred until completely clear, then centrifuged to remove bubbles, and then poured into the injection hole of the microneedle mold with the outer layer structure already prepared, so that it enters and fills the conical cavity (composed of the outer layer structure) and the backing cavity completely; then, a preset number of cross-linking and curing cycles (e.g., 5 times) are performed to achieve hydrogel cross-linking and curing, and the inner layer structure and backing are obtained; the cross-linking and curing cycle refers to freezing at -20°C for 8 hours and then thawing at room temperature for 4 hours; Of course, the concentration and material of the mixture can be adjusted according to actual needs and mold conditions, and the backing can be cast separately to adjust the density and toughness of the backing, thereby adjusting the center of gravity and extensibility of the microneedle device, and thus improving the rate at which the microneedle device unfolds from a rolled-up cylindrical shape to a sheet shape in practical applications.

[0026] In one feasible implementation, the method for screening and purifying the sensitive phage reservoir specifically includes the following steps: Step a1: After obtaining the joint cavity puncture fluid from the patient's infection focus, perform microbial culture, isolation and identification to obtain the target bacteria (i.e., pathogenic bacteria); for the target bacteria, after isolating single bacteria through drug sensitivity test, culture them in culture medium (e.g., 12-18 hours) to obtain the target bacteria culture; Step a2: Add the target bacterial culture to (molten) soft agar, mix thoroughly, and then spread it on a culture dish containing solid agar to obtain a bacterial plate, thereby ensuring the uniform distribution and growth of the target bacteria; the weight / volume concentration of agar powder in the soft agar ranges from 0.3% to 0.8%; the weight / volume concentration of agar powder in the solid agar is 1.5%; Step a3: Amplify and culture multiple candidate phages and filter them to obtain a concentration of 10. 7 -10 9 Multiple candidate phage stock solutions with PFU / mL are available to remove bacterial residues and are suitable for sensitivity screening. Step a4: Multiple candidate phage stock solutions are dropped onto the surface of several bacterial plates on solid agar. Each solid agar plate contains both a negative control area and a positive control area for bacterial growth culture, allowing for clearer comparison of effects. The negative control area refers to the area where only buffer solution is added; the positive control area refers to the area where both buffer solution and lysozyme are added. After culture, the spots on the solid agar surface are visually observed and classified according to their transparency. When the spotted area is completely transparent, it means that the bacteria have been completely removed and are considered to have completely lysed; When the spotted area is translucent, it means that the bacteria have been partially removed, and this is considered partial lysis. When the spotted area is opaque, it means that the bacteria have not been removed, and it is judged as no lysis. Candidate phages in the completely transparent areas of the spotted region were identified as sensitive phages, and the sensitive phage reservoir was determined.

[0027] In one feasible embodiment, the method for preparing the microneedle device further includes a method for preparing an airbag, specifically comprising: Step d1: Construct a (conventional) three-layer microneedle mold: Place the microneedle female mold (the mold material can be PDMS, i.e., polydimethylsiloxane) at the bottom as the bottom mold; place the molding frame containing the backing cavity on the microneedle female mold as the middle mold; place the airbag molding cap on the backing molding frame as the top mold. Specifically, the top of the airbag forming cover is provided with several upward-facing hemispherical recesses, which serve as airbag cavities; Step d2: First, through the first injection hole of the mold, the outer layer structure is obtained according to the method in step b2; then, the antibiotic mixture is poured into the first injection hole, and through one cross-linking curing cycle, the preliminary cross-linked inner layer structure is obtained. Step d3: Inject a backing mixture containing sodium alginate, polyvinyl alcohol, and sodium bicarbonate into the middle and top molds through the second injection hole of the mold, thereby filling the backing cavity and the air bladder cavity; the concentration of sodium bicarbonate can be 0.5-1% (w / v). Step d4: Inject citric acid solution through the second injection hole and vibrate the three-layer microneedle mold so that the citric acid reacts with sodium bicarbonate to generate carbon dioxide bubbles, which then gradually gather upward into the air bladder cavity to form an air bladder; the concentration of the citric acid solution can be 0.1% (w / v). Step d5: Perform a preset number of cross-linking and curing cycles (e.g., 4 times) on the three-layer microneedle mold to fully cross-link the inner structure, backing and airbag arm. Step d6: Demold the three-layer microneedle mold to obtain a microneedle array with an airbag backing; In this way, a microneedle device with an airbag can be prepared by using a three-layer microneedle mold and citric acid and sodium bicarbonate, which are safe for the human body.

[0028] In one feasible implementation, the vibration mode in step d4 is ultrasonic vibration, with a frequency range of 1-3MHz, so that citric acid and sodium bicarbonate can react better, promote the generation of carbon dioxide bubbles, and gather upward more quickly.

[0029] Thirdly, based on the same inventive concept, this application also provides a method for using the above-mentioned microneedle device in arthroscopic surgery, comprising the following steps: Step e1: After demolding, the microneedle array is rolled into a cylindrical shape and inserted into a sleeve; then, the sleeve is frozen and fixed by liquid nitrogen, and then freeze-dried for 45 hours to remove excess moisture. Step e2: At the start of the arthroscopic surgery, the sleeve is inserted into the joint cavity through the arthroscope; Step e3: After the sleeve dissolves in the joint cavity, the microneedle array unfolds into a sheet shape, and then adheres to the target area by its own gravity or manual traction, so that the tip of the microneedle can pierce the biomembrane of the target area. Step e4: Press the microneedle array from outside the body to promote further penetration of the microneedles into the biomembrane; In this way, the outer structure rapidly dissolves and releases bacteriophages to quickly destroy the biofilm matrix. After the biofilm is destroyed (about 24 hours), the inner structure slowly releases antibiotics to treat the infection foci at the target site.

[0030] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention provides a phage-antibiotic co-carrying gradient degradation microneedle device and method, which achieves differential degradation of phage and antibiotic through a double-layer microneedle structure, thereby achieving the purpose of first resisting biofilm and then antibacterial, thus improving the treatment efficiency and effect of PJI. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 This is a schematic diagram of a gradient degradation microneedle device co-loaded with bacteriophage and antibiotic provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of an airbag provided in an embodiment of the present invention; Figure 3 A schematic diagram of a sleeve provided in an embodiment of the present invention; Figure 4 A schematic diagram of the substrate provided in an embodiment of the present invention; Figure 5 A flowchart illustrating the application of arthroscopic surgery in an embodiment of the present invention; Figure 6 This is a comparison chart of release rates provided in an embodiment of the present invention.

[0033] Figure label: 1-Outer layer structure; 2-Inner layer structure; 3-Backing; 31-Airbag; 4-Sleeve; 5-Base. Detailed Implementation

[0034] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0036] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0037] The present invention will be further explained below with reference to specific embodiments.

[0038] Example 1: like Figure 1 As shown, this embodiment provides a phage-antibiotic co-loaded gradient degradation microneedle device, including a microneedle array and its backing 3; The microneedles in the microneedle array have a double-layer structure: The outer structure 1 includes a bacteriophage targeting the target bacteria and a matrix loaded with the bacteriophage; The inner structure 2 includes an antibiotic targeting the target bacteria and a matrix loaded with the antibiotic.

[0039] In this way, the phage in the outer structure 1 is used to quickly destroy the biofilm matrix, and then the antibiotics in the inner structure 2 are used to slowly release antibiotics to lyse the exposed target bacteria, thereby ensuring the antibiotic concentration during antibacterial treatment and thus improving the antibacterial effect.

[0040] Furthermore, the microneedles are conical in shape.

[0041] Furthermore, the microneedles are 1 mm in height to fit subcutaneous and periarticular tissues.

[0042] Furthermore, the thickness of the backing 3 is 0.5 mm; the wall thickness of the outer layer structure 1 is 200 μm; and the maximum diameter of the inner layer structure 2 is 300 μm.

[0043] Furthermore, the matrix of the outer structure 1 includes poloxamer 407 (P407); poloxamer 407 is a thermosensitive hydrogel that can be excreted in urine after depolymerization and dissolution in the human body. As a conventional biocompatible material, it can be maintained for a minimum of several minutes in vivo and in vitro.

[0044] Furthermore, the bacteriophage includes an anti-Staphylococcus epidermidis bacteriophage; the specific concentration of the anti-Staphylococcus epidermidis bacteriophage can be 9 × 10⁻⁶. 9 PFU / mL.

[0045] Furthermore, the matrix of the inner structure 2 includes sodium alginate-polyvinyl alcohol hydrogel, whose degradation rate is affected by cross-linking mode, strength, size and other conditions, with a minimum degradation time of one day and usually no more than one month.

[0046] Furthermore, the antibiotic includes vancomycin; the specific concentration of the vancomycin may be 1.2 mg / ml.

[0047] Furthermore, the material of the backing 3 includes sodium alginate-polyvinyl alcohol hydrogel, which not only allows for slower degradation but also enables it to flexibly conform to the curved surface of the joint prosthesis (such as the knee joint, hip joint, etc.).

[0048] Example 2: Based on Embodiment 1, this embodiment also includes several through holes evenly distributed along the edge of the backing 3; the diameter of the through holes is larger than the diameter of the arthroscopic probe, so that the doctor can use the arthroscopic probe to hook the through holes of the backing 3 to assist in moving or opening the microneedle device and perform treatment on the target area.

[0049] Example 3: like Figure 2 As shown (microneedle array not shown), in this embodiment, based on embodiment one, several air bladders 31 are provided on the side of the backing 3 that does not have a microneedle array. In this way, when the microneedle device is in body fluid, the side with air bladders 31 will float up, causing the tip of the microneedle array to point downwards so that it can quickly move towards the target area below. After the doctor confirms that the orientation is correct, the air bladders 31 can be punctured by a key endoscope hook (or probe) so that the microneedle device sinks down and fits against the target area.

[0050] Example 4: Based on Example 1, this embodiment adds biocompatible paramagnetic particles to the material of the backing 3. In this way, doctors can use an arthroscopic hook (or probe) with controllable magnetism (e.g., an electromagnet) to magnetically pull the microneedle device and perform treatment on the target area.

[0051] Furthermore, the paramagnetic particles are made of Fe3O4 nanoparticles. These nanoparticles are gradually acidified by the phagocytic action of lysosomes or macrophages in the body, releasing iron ions, which then bind to transferrin and are transported to the bone marrow to synthesize heme.

[0052] Example 5: like Figure 3 As shown (microneedle array not shown), in this embodiment, based on embodiment one, the microneedle device further includes a biodegradable sleeve 4, which is used to protect the backing 3 when it is rolled into a cylinder and passes through the arthroscope.

[0053] Example 6: like Figure 4 As shown (microneedle array not shown), in this embodiment, based on embodiment one, the microneedle device further includes a base 5; the base 5 is fixed to the side of the backing 3 where the microneedle array is not provided; the area of ​​the base 5 is larger than the area of ​​the backing 3 so that the microneedle device can be applied to the skin surface, which is suitable for treating superficial infections.

[0054] Furthermore, the substrate 5 is a medical silicone membrane.

[0055] Example 7: This embodiment provides a method for manufacturing any of the above-mentioned microneedle devices, including methods for preparing the outer layer structure and the inner layer structure.

[0056] Furthermore, the method for preparing the outer layer structure includes the following steps: Step b1: At 4°C, prepare a 30% (w / v) poloxamer 407 solution by mixing poloxamer 407 powder with ultrapure water; then add the sensitive phage stock solution to the poloxamer 407 solution, with the sensitive phage stock solution titer being 10. 9 PFU / mL was used to obtain a phage-loaded poloxamer 407 solution; Step b2: Pour the phage-loaded poloxamer 407 solution into the injection hole of the microneedle mold; incubate at 37°C for 10 minutes to form an outer layer structure with a conical cavity.

[0057] Furthermore, the method for preparing the inner layer structure includes the following steps: Step c1: Mix 2% (w / v) sodium alginate and 10% (w / v) polyvinyl alcohol with the sensitive antibiotics screened by the target bacteria drug sensitivity test to obtain an antibiotic mixture. Step c2: At room temperature, the antibiotic mixture is stirred until completely clear, then centrifuged to remove bubbles, and then poured into the injection hole of the microneedle mold with the outer layer structure already prepared, so that it enters and fills the conical cavity (composed of the outer layer structure) and the backing cavity completely; then, a preset number of cross-linking and curing cycles (e.g., 5 times) are performed to achieve hydrogel cross-linking and curing, and the inner layer structure and backing are obtained; the cross-linking and curing cycle refers to freezing at -20°C for 8 hours and then thawing at room temperature for 4 hours; Of course, the concentration and material of the mixture can be adjusted according to actual needs and mold conditions, and the backing can be cast separately to adjust the density and toughness of the backing, thereby adjusting the center of gravity and extensibility of the microneedle device, and thus improving the rate at which the microneedle device unfolds from a rolled-up cylindrical shape to a sheet shape in practical applications.

[0058] Furthermore, the method for screening and purifying the sensitive phage reservoir specifically includes the following steps: Step a1: After obtaining the joint cavity puncture fluid from the patient's infection focus, perform microbial culture, isolation and identification to obtain the target bacteria (i.e., pathogenic bacteria); for the target bacteria, after isolating single bacteria through drug sensitivity test, culture them in culture medium (e.g., 12-18 hours) to obtain the target bacteria culture; Step a2: Add the target bacterial culture to (molten) soft agar, mix thoroughly, and then spread it on a culture dish containing solid agar to obtain a bacterial plate, thereby ensuring the uniform distribution and growth of the target bacteria; the weight / volume concentration of agar powder in the soft agar ranges from 0.3% to 0.8%; the weight / volume concentration of agar powder in the solid agar is 1.5%; Step a3: Amplify and culture multiple candidate phages and filter them to obtain a concentration of 10. 7 -10 9 Multiple candidate phage stock solutions with PFU / mL are available to remove bacterial residues and are suitable for sensitivity screening. Step a4: Multiple candidate phage stock solutions are dropped onto the surface of several bacterial plates on solid agar. Each solid agar plate contains both a negative control area and a positive control area for bacterial growth culture to compare the effects. The negative control area is the area where only buffer solution is added; the positive control area is the area where both buffer solution and lysozyme are added. After culture, the spots on the solid agar surface are visually observed and classified according to their transparency. When the spotted area is completely transparent, it means that the bacteria have been completely removed and are considered to have completely lysed; When the spotted area is translucent, it means that the bacteria have been partially removed, and this is considered partial lysis. When the spotted area is opaque, it means that the bacteria have not been removed, and it is judged as no lysis. Candidate phages in the completely transparent areas of the spotted region were identified as sensitive phages, and the sensitive phage reservoir was determined.

[0059] Example 8: The method for preparing the microneedle device in Example 3 also includes a method for preparing an airbag, specifically including: Step d1: Construct a three-layer microneedle mold: Place the microneedle female mold (the mold material can be PDMS, i.e., polydimethylsiloxane) at the bottom as the bottom mold; place the molding frame containing the backing cavity on the microneedle female mold as the middle mold; place the airbag molding cap on the backing molding frame as the top mold. Specifically, the top of the airbag forming cover is provided with several upward-facing hemispherical recesses, which serve as airbag cavities; Step d2: First, through the first injection hole of the mold, the outer layer structure is obtained according to the method in step b2; then, the antibiotic mixture is poured into the first injection hole, and through one cross-linking curing cycle, the preliminary cross-linked inner layer structure is obtained. Step d3: Inject a backing mixture containing sodium alginate, polyvinyl alcohol, and sodium bicarbonate into the middle and top molds through the second injection hole of the mold, thereby filling the backing cavity and the air bladder cavity; the concentration of sodium bicarbonate can be 0.5-1% (w / v). Step d4: Inject citric acid solution through the second injection hole and vibrate the three-layer microneedle mold so that the citric acid reacts with sodium bicarbonate to generate carbon dioxide bubbles, which then gradually gather upward into the air bladder cavity to form an air bladder; the concentration of the citric acid solution can be 0.1% (w / v). Step d5: Perform a preset number of cross-linking and curing cycles (e.g., 4 times) on the three-layer microneedle mold to fully cross-link the inner structure, backing and airbag arm. Step d6: Demold the three-layer microneedle mold to obtain a microneedle array with an airbag backing; In this way, a microneedle device with an airbag can be prepared by using a three-layer microneedle mold and citric acid and sodium bicarbonate, which are safe for the human body.

[0060] Example 9: like Figure 5 As shown, this embodiment provides a method for using any of the above-mentioned microneedle devices in arthroscopic surgery, including the following steps: Step e1: After demolding, the microneedle array is rolled into a cylindrical shape and inserted into a sleeve; then, the sleeve is frozen and fixed by liquid nitrogen, and then freeze-dried for 45 hours to remove excess moisture. Step e2: At the start of the arthroscopic surgery, the sleeve is inserted into the joint cavity through the arthroscope; Step e3: After the sleeve dissolves in the joint cavity, the microneedle array unfolds into a sheet shape, and then adheres to the target area by its own gravity or manual traction, so that the tip of the microneedle can pierce the biomembrane of the target area. Step e4: Press the microneedle array from outside the body to promote further penetration of the microneedles into the biomembrane; Thus, as Figure 6 As shown, a differential release effect was formed between antibiotics and bacteriophages: the outer structure rapidly dissolves and releases bacteriophages to quickly destroy the biofilm matrix, and after the biofilm is destroyed (about 24 hours), the inner structure slowly releases antibiotics to treat the infection foci at the target site.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A phage-antibiotic co-loaded gradient degradation microneedle device, comprising a microneedle array and a backing, characterized in that: The microneedles in the microneedle array have a double-layer structure: The outer structure includes a phage targeting the target bacteria and a matrix on which the phage is loaded; The inner structure includes an antibiotic targeting the target bacteria and a matrix loaded with the antibiotic.

2. The microneedle device according to claim 1, characterized in that, The matrix of the outer structure includes poloxamer 407.

3. The microneedle device according to claim 2, characterized in that, The matrix of the inner layer structure comprises sodium alginate-polyvinyl alcohol hydrogel.

4. The microneedle device according to claim 1, characterized in that, It also includes a sleeve to protect the microneedle array and its backing when rolled into a cylinder.

5. The microneedle device according to claim 1, characterized in that, The microneedle device also includes a substrate; the substrate is fixed to the side of the backing that is not provided with the microneedle array; the area of ​​the substrate is larger than the area of ​​the backing.

6. The microneedle device according to claim 1, characterized in that, The edge of the backing is evenly distributed with several through holes.

7. The microneedle device according to claim 1, characterized in that, The backing has several airbags on the side without the microneedle array.

8. A method for manufacturing the microneedle device as described in claim 3, characterized in that, Including methods for preparing the outer and inner layer structures; The method for preparing the outer layer structure specifically includes the following steps: Step b1: At 4°C, prepare a 30% (w / v) poloxamer 407 solution by mixing poloxamer 407 powder with ultrapure water; then add the screened and purified sensitive phage stock solution to the poloxamer 407 solution, with a sensitive phage stock solution titer of 10. 9 PFU / mL was used to obtain a phage-loaded poloxamer 407 solution; Step b2: Pour the phage-loaded poloxamer 407 solution into the injection hole of the microneedle mold; incubate at 37°C for 10 minutes to form an outer layer structure with a conical cavity.

9. The preparation method according to claim 8, characterized in that, The method for preparing the inner layer structure specifically includes the following steps: Step c1: Mix sodium alginate (2% by weight / volume) and polyvinyl alcohol (10% by weight / volume) with the sensitive antibiotics screened by the target bacteria susceptibility test to obtain an antibiotic mixture. Step c2: At room temperature, stir the antibiotic mixture until it is completely clear, then centrifuge to remove bubbles, and then pour it into the injection hole of the microneedle mold with the prepared outer layer structure. Then, a preset number of cross-linking and curing cycles are performed to obtain the inner layer structure and backing; the cross-linking and curing cycle refers to freezing at -20℃ for 8 hours and then thawing at room temperature for 4 hours.

10. The preparation method according to claim 8, characterized in that, The method for screening and purifying the sensitive phage stock solution includes the following steps: Step a1: After obtaining the joint cavity puncture fluid from the patient's infection focus, perform microbial culture, isolation and identification to obtain the target bacteria; for the target bacteria, after isolating single colonies through drug sensitivity test, culture them in culture medium to obtain the target bacteria culture; Step a2: Add the target bacterial culture to soft agar, mix thoroughly, and then spread it on a culture dish containing solid agar to obtain a bacterial plate. Step a3: Amplify and culture multiple candidate phages and filter them to obtain a concentration of 10. 7 -10 9 Multiple candidate phage stock solutions with PFU / mL; Step a4: Multiple candidate phage stock solutions are dropped onto the surface of several bacterial plates on solid agar. Each solid agar plate contains both negative and positive control areas for bacterial growth culture. After culture, the spots on the solid agar surface are visually observed and classified according to their transparency. When the spot area is completely transparent, it is considered to be completely lysed; When the spotted area is translucent, it is considered partial lysis; When the spot area is opaque, it is determined that there is no lysis; Candidate phages in the completely transparent areas of the spotted region were identified as sensitive phages, and the sensitive phage reservoir was determined.

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