Double-layer detachable microneedle as well as preparation method and application thereof

Abstract

The invention relates to a double-layer detachable microneedle which comprises a soluble microneedle tip and a detachable hydrogel substrate layer, and the microneedle tip layer comprises at least one of drugs, extracellular vesicles, macromolecular substances and combinations of the drugs, the extracellular vesicles and the macromolecular substances. The extracellular vesicles are one or a combination of a plurality of exosomes, microvesicles, apoptotic bodies, autophagy-related extracellular vesicles, matrix vesicles and stress-related extracellular vesicles, and the macromolecular substances are one or a combination of a plurality of polypeptides, proteins, antigens, enzymes and antibodies. According to the double-layer detachable microneedle, early-stage antibiosis and pH adjustment can be achieved, infection is reduced, and inflammation is inhibited; macrophages are induced to be polarized from M1 to M2, and inflammation is promoted to be transited to a proliferation stage; needle tip loading is continuously released, and angiogenesis, cell proliferation and tissue regeneration are enhanced; the double-layer structure can sequentially respond to different stage requirements of the wound, and the healing efficiency of the diabetic wound is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a double-layer detachable microneedle, its preparation method, and its application in promoting the healing of diabetic wounds. Background Technology

[0002] Diabetic wounds are a common and serious complication of diabetes, typically characterized by an alkaline environment and persistent high-grade inflammation, leading to delayed healing. Traditional wound treatments are ill-suited to the complex and dynamic environment of diabetic wounds, resulting in poor treatment outcomes. In recent years, microneedling technology, as a painless and non-invasive drug delivery method, has been increasingly applied in wound healing. However, most existing microneedling techniques promote wound healing solely through encapsulation and controlled drug release, which is insufficient to address the complex and dynamic environment of diabetic wounds, thus limiting their therapeutic effectiveness. Summary of the Invention

[0003] To address the technical problems existing in the above-mentioned technologies, this invention provides a double-layer detachable microneedle, its preparation method, and its application. The specific technical solution is as follows:

[0004] A dual-layer removable microneedle includes a soluble microneedle tip and a removable hydrogel base layer.

[0005] Furthermore, the microneedle tip layer includes at least one of a drug, extracellular vesicles, macromolecules, and combinations thereof.

[0006] Furthermore, the extracellular vesicles are one or a combination of several of the following: exosomes, microvesicles, apoptotic bodies, autophagy-associated extracellular vesicles, matrix vesicles, and stress-associated extracellular vesicles.

[0007] Furthermore, the macromolecule is one or a combination of several of the following: polypeptides, proteins, antigens, enzymes, and antibodies.

[0008] Furthermore, the drug comprises nanoparticles or a composition of different nanoparticles.

[0009] Furthermore, the hydrogel backing layer is made of a combination of pH-regulating and antibacterial components such as hyaluronic acid, polyvinyl alcohol, graphene oxide, and silver nanowires.

[0010] Furthermore, the microneedle tip is conical, with a tip height of 200-1000μm, a bottom diameter of 200-500μm, and a tip spacing of 500-1200μm, and is distributed in an array within the substrate region.

[0011] The present invention also relates to the use of the above-mentioned double-layer detachable microneedles in wound healing drugs.

[0012] When used in wound healing, the dual-layer detachable microneedles are applied to the wound surface, allowing the microneedle tips to dissolve within the skin and release the loaded components. The hydrogel base provides an acidic microenvironment and modulates the state of immune cells in the early stages of wound healing; the hydrogel base layer is removed at a preset time to promote subsequent tissue repair and collagen remodeling.

[0013] This invention also relates to the application of the above-mentioned double-layer detachable microneedles in the repair of skin wounds in chronic diabetes.

[0014] Beneficial effects

[0015] Compared with existing microneedles, the beneficial effects of this invention are:

[0016] 1. Achieve early antibacterial and pH regulation, reduce infection and suppress inflammation;

[0017] 2. Induces macrophage polarization from M1 to M2, promoting the transition from inflammation to the proliferative phase;

[0018] 3. Continuous release of needle tip-loaded material enhances angiogenesis, cell proliferation, and tissue regeneration;

[0019] 4. The dual-layer structure can sequentially respond to the needs of different stages of wound healing, significantly improving the healing efficiency of diabetic wounds. The microneedles of this invention can precisely cover the key time window of the "inflammatory phase – proliferative phase" in the wound healing process, promoting the healing of diabetic wounds in a time sequence. During the inflammatory phase, it adjusts the pH to acidic, promotes macrophage transformation, and reduces inflammation. During the proliferative phase, it promotes angiogenesis. By day 10 post-surgery, the wound closure rate of the DSH MN group was significantly better than that of the control group, the simple pH adjustment group, and the simple drug delivery group. The microneedles can significantly improve the repair efficiency of diabetic wounds. Attached Figure Description

[0020] Figure 1 These are images showing the preparation and characterization of the bilayer microneedles of this invention, where: A) microneedle morphology; B) drug distribution at the microneedle tip; C) microneedle backing layer morphology; D) swelling capacity of the microneedle backing layer; E) pH adjustment capacity of the microneedle backing layer; F) microneedle mechanical strength; G) microneedle skin penetration capability; H) microneedle subcutaneous drug delivery capability.

[0021] Figure 2 This is a schematic diagram of the in vitro biological effects of the DSH-MN needle tip of the present invention, wherein A) cell migration; B) angiogenesis; CD) quantitative analysis of cell migration and angiogenesis; EG) flow cytometry results of macrophage polarization and their quantitative analysis; HL) gene expression of iNOS, Arg-1, IL-6, IL-10, and TNF-α.

[0022] Figure 3This is a schematic diagram illustrating the antibacterial properties of DSH-MN of the present invention, wherein: AB) inhibition zones formed by the hydrogel backing layer against Escherichia coli and Staphylococcus aureus and their average diameter; C) images of colony formation of Escherichia coli and Staphylococcus aureus on agar plates after treatment with the hydrogel backing layer; DE) quantitative analysis of the survival rate of Escherichia coli and Staphylococcus aureus after treatment with the hydrogel backing layer; FG) live / dead fluorescence images of Escherichia coli and Staphylococcus aureus after treatment with the hydrogel backing layer; H) scanning electron microscope images of Escherichia coli and Staphylococcus aureus after treatment with the hydrogel backing layer; I) GSEA analysis related to apoptosis clearance; J) schematic diagram of the antibacterial mechanism of the microneedles.

[0023] Figure 4 This is a schematic diagram illustrating the results of a study on the wound healing performance of the micro-target in diabetic mice according to the present invention. A) Schematic diagram of the wound healing process; B) Photographs of wounds in different treatment groups; C) Simulated wound area in different treatment groups; D) Statistical analysis of wound area in different treatment groups; E) Statistical analysis of wound healing rate in different treatment groups; F) HE staining results of wounds in different treatment groups on day 10; G) Quantitative analysis of granulation tissue width in different treatment groups on day 10; H) Quantitative analysis of collagen deposition area; I) Masson staining results in different treatment groups on day 10.

[0024] Figure 5 This is a schematic diagram of the results of the study on the wound healing mechanism of the micro-target in diabetic mice according to the present invention, wherein AB) pH changes at the wound site; CJ) changes in macrophages, inflammatory factors (IL-6, TNF-α) and CD31; Detailed Implementation

[0025] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0026] Reference Figure 1-5 The preparation method of the double-layer detachable microneedle of the present invention includes the following steps:

[0027] Step 1: Synthesize microneedles

[0028] A novel bilayer detachable microneedle (DSH-MN) was prepared by preparing a tip solution and a backing layer solution using a stepwise microforming method. The tip of the microneedle includes at least one of a drug, extracellular vesicles, macromolecules, or combinations thereof, along with hyaluronic acid. The backing layer includes hyaluronic acid, polyvinyl alcohol, graphene oxide, and silver nanowires.

[0029] Step 2: Characterizing the microneedles

[0030] The morphology of the microneedles was characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Each microneedle patch contained 100 tips distributed in a 10×10 array within a 1.48 cm × 1.48 cm area. SEM showed that the tips were conical, with a height of 540 μm, a base diameter of 470 μm, and a tip-to-tip spacing of 1100 μm. The microneedles exhibited good morphology. The hydrogel backing layer displayed a three-dimensional porous network structure, with a surface covered by GO-AgNWs. Fluorescence imaging indicated that the tip-encapsulated components were uniformly distributed on the tips. Hyaluronic acid endowed the microneedles with excellent hydrophilicity, achieving a swelling rate of up to 500%, effectively absorbing wound exudate and maintaining a moist environment. Experiments showed that the hydrogel rapidly reduced the pH to 6.5 in a solution with a pH of 8 and remained stable, confirming its pH regulation capability. The mechanical strength of the microneedles was tested using a universal testing machine. When a pressure of 1.6 N / needle was applied, the microneedle tips only bent without breaking, indicating their ability to penetrate the stratum corneum. To investigate the effects of microneedles on skin tissue, treated skin sections were prepared and stained with H&E. The results showed that the microneedles induced significant tissue damage in the skin. Using female BALB / c mice as a model, microneedles were fixed to the skin on the mouse's back for 2 minutes and then removed. Skin samples from the puncture sites were then scanned using laser confocal microscopy. The microneedles could penetrate subcutaneously, and the fluorescence intensity decreased with increasing depth. The 3D reconstructed image showed a conical shape, consistent with the morphology of the microneedles. In conclusion, the synthesized microneedles can effectively penetrate the skin, enabling transdermal drug delivery.

[0031] Example 1

[0032] The double-layer detachable microneedles in this embodiment have conical tips with a tip height of 540 μm, a bottom diameter of 470 μm, a tip spacing of 1100 μm, and are distributed in an array within the substrate area.

[0033] It includes soluble microneedle tips and a removable hydrogel base layer:

[0034] The microneedle tip layer comprises the drug and extracellular vesicles. Extracellular vesicles are exosomes and microvesicles. The drug comprises nanoparticles or a composition of different nanoparticles.

[0035] The hydrogel backing layer is made of a combination of hyaluronic acid, polyvinyl alcohol, graphene oxide, and antibacterial ingredients.

[0036] Example 2

[0037] The double-layer detachable microneedles in this embodiment have conical tips with a tip height of 1000μm, a bottom diameter of 500μm, and a tip spacing of 1200μm, and are distributed in an array within the substrate area.

[0038] It includes soluble microneedle tips and a removable hydrogel base layer:

[0039] The microneedle tip layer consists of a combination of extracellular vesicles and macromolecules. Extracellular vesicles are a combination of exosomes, microvesicles, apoptotic bodies, and autophagy-related extracellular vesicles. Macromolecules are a combination of enzymes and antibodies.

[0040] The hydrogel backing layer is made of a combination of pH-regulating and antibacterial components such as graphene oxide and silver nanowires.

[0041] Example 3

[0042] In this embodiment, the double-layer detachable microneedles have conical tips with a tip height of 800 μm, a bottom diameter of 300 μm, and a tip spacing of 1000 μm, and are distributed in an array within the substrate area.

[0043] It includes soluble microneedle tips and a removable hydrogel base layer:

[0044] The tip layer of a microneedle comprises drugs and macromolecules. The macromolecules are combinations of peptides, proteins, antigens, and antibodies. The drugs consist of nanoparticles or compositions of different nanoparticles.

[0045] The hydrogel backing layer is made of a combination of pH-regulating and antibacterial components such as graphene oxide and silver nanowires.

[0046] Example 4

[0047] In this embodiment, the method for preparing the double-layer detachable structure is as follows:

[0048] First, PEG-PLGA-secreting nanoparticles were prepared. PEG-PLGA was dissolved in ethyl acetate to prepare the oil phase. The aqueous phase was formed by mixing the secretion group aqueous solution (containing exosomes, cytokines, and other active ingredients) with the oil phase. The resulting colostrum was treated with a probe-type sonicator for 594 s (3 s sonication, 7 s interval) to obtain a homogeneous emulsion. Subsequently, the emulsion was poured into a beaker containing 1% PVA solution and F68 and sonicated in a water bath for 5 min to form a two-emulsion mixture. After evaporating ethyl acetate at room temperature, the system was centrifuged at 14,800 rpm for 10 min, the precipitate was collected, and washed three times with ultrapure water. Then, it was centrifuged at 500 rpm for 5 min to remove large particles, and the supernatant was lyophilized for later use. When preparing the microneedle tip solution, PEG-PLGA-secreting nanoparticles were dissolved in deionized water, hyaluronic acid (HA) was added, and the mixture was stirred until completely dissolved to obtain a homogeneous solution. The backing layer solution was prepared by dissolving PVA and HA in distilled water and stirring vigorously at 90 °C; after complete dissolution, AgNWs-GO solution was added and mixed well for later use.

[0049] The microneedles were fabricated using a two-step casting process with a PDMS mold. First, the needle tip solution was added to the PDMS mold, and a vacuum was applied for 15 minutes to ensure complete entry of the solution into the needle cavity. After drying at room temperature, an AgNWs-GO-PVA-HA backing layer solution was added to the mold. The mold was then frozen at -20 °C for 10 h, followed by thawing at 25 °C for 1 h, and this freeze-thaw cycle was repeated three times to form a hydrogel backing layer with good mechanical properties. Finally, the mold was dried at room temperature for 12 h until fully formed, and then demolded to obtain the microneedle patch.

[0050] Example 5

[0051] In this embodiment, the method for preparing the double-layer detachable structure is as follows:

[0052] To prepare the microneedle tip solution, angiogenic peptides and exosomes were dissolved in deionized water, and hyaluronic acid (HA) was added. The solution was stirred until completely dissolved to obtain a homogeneous solution. The backing layer solution was prepared by dissolving PVA and HA in distilled water and stirring vigorously at 90 °C. After complete dissolution, AgNWs-GO solution was added and mixed thoroughly for later use.

[0053] The microneedles were fabricated using a two-step casting process with a PDMS mold. First, the needle tip solution was added to the PDMS mold, and a vacuum was applied for 15 minutes to ensure complete entry of the solution into the needle cavity. After drying at room temperature, an AgNWs-GO-PVA-HA backing layer solution was added to the mold. The mold was then frozen at -20 °C for 10 h, followed by thawing at 25 °C for 1 h, and this freeze-thaw cycle was repeated three times to form a hydrogel backing layer with good mechanical properties. Finally, the mold was dried at room temperature for 12 h until fully formed, and then demolded to obtain the microneedle patch.

[0054] Example 6

[0055] In this embodiment, the method for preparing the double-layer detachable structure is as follows:

[0056] To prepare the microneedle tip solution, microvesicles and recombinant human epidermal growth factor were dissolved in deionized water, and hyaluronic acid (HA) was added. The solution was stirred until completely dissolved to obtain a homogeneous solution. The backing layer solution was prepared by dissolving PVA and HA in distilled water and stirring vigorously at 90 °C. After complete dissolution, AgNWs-GO solution was added and mixed thoroughly for later use.

[0057] The microneedles were fabricated using a two-step casting process with a PDMS mold. First, the needle tip solution was added to the PDMS mold, and a vacuum was applied for 15 minutes to ensure complete entry of the solution into the needle cavity. After drying at room temperature, an AgNWs-GO-PVA-HA backing layer solution was added to the mold. The mold was then frozen at -20 °C for 10 h, followed by thawing at 25 °C for 1 h, and this freeze-thaw cycle was repeated three times to form a hydrogel backing layer with good mechanical properties. Finally, the mold was dried at room temperature for 12 h until fully formed, and then demolded to obtain the microneedle patch.

[0058] Example 7

[0059] In vitro biological effects of DSH-MN needle tips

[0060] To evaluate the in vitro function of the DSH-MN needle tip, microneedles containing only the needle tip component were first prepared. Scratch assays showed that the microneedle tip group achieved almost complete closure within 12 hours, with a significantly faster migration rate than the control group. Subsequently, its pro-angiogenic capacity was evaluated using a HUVEC lumen formation assay. The results showed that the number of intact tubular structures formed in the microneedle tip-treated group was approximately four times that of the control group, suggesting a significant pro-angiogenic effect. Further investigation was conducted on the effect of the microneedle tip on macrophage polarization. Flow cytometry analysis showed that LPS / IFN-γ stimulation increased the proportion of M1 macrophages in RAW264.7 to 15.1%; while after the addition of the microneedle tip, the proportion of M2 cells significantly increased to 43.1%, and the proportion of M1 decreased to 6.01%. Under non-inflammatory conditions, the microneedle tip had little effect on the distribution of M1 and M2 macrophages, indicating that its immunomodulatory function mainly operates in an inflammatory environment. Gene expression results further showed that SMN significantly upregulated M2 markers (Arg1, IL-10) and inhibited LPS / IFN-γ-induced M1-related genes (iNOS, IL-6, TNF-α), consistent with flow cytometry results, confirming its immunomodulatory effect.

[0061] Example 8

[0062] Microneedle antibacterial effect

[0063] Inhibition zone experiments on *Escherichia coli* and *Staphylococcus aureus* revealed significant inhibition zones in both experimental groups, while the PBS control showed no antibacterial effect. The diameter of the inhibition zones increased with prolonged pre-soaking time (6-48 h), indicating that silver ion release was time-dependent, enhancing the antibacterial effect. Further analysis using a Transwell co-culture system assessed the bactericidal effect. The survival rates of both bacteria decreased over time, with significant antibacterial effects observed after 12 h. Live / dead staining showed abundant red fluorescence in *E. coli* at 6 h, which increased over time; the bactericidal process of *Staphylococcus aureus* was slower, but the number of dead cells increased significantly after 12 h. Scanning electron microscopy images showed that *E. coli* changed from rod-shaped to aggregated and collapsed; *Staphylococcus aureus* exhibited surface wrinkling, membrane depression, and eventual rupture. These results indicate that the hydrogel exerts its antibacterial effect by disrupting bacterial structure. Furthermore, macrophages treated with microneedles also exhibited significant antibacterial activity, reducing bacterial release and spread by phagocytosing apoptotic cells containing bacteria.

[0064] Example 9

[0065] Microneedling for Diabetic Wounds

[0066] Diabetic mice were randomly divided into four groups, with medication changed every two days. Wound conditions were observed and photographed on days 0, 2, 4, 6, 8, and 10 of treatment. The remaining wound area was calculated using ImageJ software. Simultaneously, the pH value at the wound site was measured using a skin pH meter. Mice were sacrificed on days 2, 4, 6, 8, and 10 after treatment, and skin tissue around the wounds was collected. The skin tissue was fixed in 4% paraformaldehyde solution for subsequent hematoxylin and eosin (HE) staining, Masson staining, immunohistochemical staining for IL-6 and TNFα, and immunofluorescence staining for CD31, CD86, and CD206.

[0067] During the healing process of diabetic wounds, DSH MN achieved temporal regulation of the local microenvironment and cellular processes. For the first four days, the group with the hydrogel backing plate was acidic, while the group without the backing plate remained alkaline, demonstrating its early pH-regulating effect. From days 4 to 6 after backing plate removal, the pH of DSHMN changed from acidic to neutral, consistent with the transition of the wound from the inflammatory phase to the proliferative phase and the shift of macrophage metabolism from glycolysis to oxidative phosphorylation. Western blot analysis showed a significant decrease in lactate dehydrogenase on day 6. Immunofluorescence indicated that the DSH MN group had a large number of M2 macrophages by day 6, while other groups were still in the inflammatory phase; the CD206 / CD86 ratio was significantly higher in the DSH MN group on days 6 and 8. IL-6 and TNF-α staining showed that inflammation was almost completely resolved by day 8 with DSH MN. The angiogenesis marker CD31 showed neovascularization on day 6, peaked on day 8, and decreased on day 10, indicating a significant acceleration in the remodeling phase. In summary, DSH MN can precisely regulate pH, inflammation, polarization, and angiogenesis at different stages, thereby systematically promoting the healing of diabetic wounds.

[0068] This specification uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A double-layered detachable microneedle, characterized in that, It includes soluble microneedle tips and a removable hydrogel base layer.

2. The double-layer detachable microneedle according to claim 1, characterized in that, The microneedle tip layer includes at least one of a drug, extracellular vesicles, macromolecules, and combinations thereof.

3. The double-layer detachable microneedle according to claim 2, characterized in that, The extracellular vesicles are one or a combination of several of the following: exosomes, microvesicles, apoptotic bodies, autophagy-associated extracellular vesicles, matrix vesicles, and stress-associated extracellular vesicles.

4. The double-layer detachable microneedle according to claim 2, characterized in that, The macromolecules are one or a combination of several of the following: polypeptides, proteins, antigens, enzymes, and antibodies.

5. The double-layer detachable microneedle according to claim 2, characterized in that, The drug comprises nanoparticles or a composition of different nanoparticles.

6. The double-layer detachable microneedle according to claim 1, characterized in that, The hydrogel backing layer is made of a combination of pH-regulating and antibacterial components such as hyaluronic acid, polyvinyl alcohol, graphene oxide, and silver nanowires.

7. The double-layer detachable microneedle according to claim 1, characterized in that, The microneedles have conical tips with a height of 200-1000 μm, a bottom diameter of 200-500 μm, and a tip spacing of 500-1200 μm, and are distributed in an array within the substrate area.

8. The double-layer detachable microneedle according to claim 7, characterized in that, The microneedles have a tip height of 540 μm, a bottom diameter of 470 μm, a tip spacing of 1100 μm, and are distributed in a 10 × 10 array within a base area of ​​1.48 cm × 1.48 cm.

9. The use of the double-layered detachable microneedles according to any one of claims 1 to 8 in a wound healing medicament.

10. The application of the double-layer detachable microneedles according to any one of claims 1 to 8 in the repair of skin wounds in chronic diabetes.

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