Double-layer microneedle patch and application thereof in preparation of medicine for promoting wound healing of old people

Abstract

The invention discloses a double-layer microneedle patch and application thereof in preparation of a medicine for promoting wound healing of old people. The double-layer microneedle patch comprises an outer layer, an inner layer and an outer layer, wherein the outer layer is hyaluronic acid (HA) loaded lidocaine hydrochloride (LiH); and the inner layer is prepared by loading hydroxytyrosol (HT) on methacrylated gelatin (GelMA). The wound healing process of the old mice is remarkably accelerated, the wound area is small, the natural crust falling speed is higher, the wound reepithelization degree is higher, collagen arrangement is more compact and ordered, and hair papilla cell regeneration in the wound area is increased. The double-layer microneedle patch has a great application prospect in preparation of medicines for promoting wound healing of old people.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of materials, in particular to a double-layer microneedle patch and application thereof in preparation of a drug for promoting wound healing in the elderly. BACKGROUND

[0002] With the continuous growth of the aging population, the incidence of trauma in the elderly population is also increasing, and the prognosis of the elderly is worse than that of young people, with increased morbidity and mortality. The number and diameter of collagen fiber bundles in the skin of the elderly decrease, the junction between the dermis and the epidermis becomes flat, the nutrient transport between the dermis and the epidermis decreases, and the fragility of the skin increases. This results in reduced resistance to injury in aged skin and poor self-repairing ability. There are a series of coordinated stages in skin wound repair, mainly including thrombosis, inflammation, new tissue formation (including re-epithelialization and granulation tissue formation and contraction), and finally tissue remodeling. Any disorder at any stage will lead to delayed healing or non-healing of the wound.

[0003] Local pain caused by wounds not only causes psychological trauma to patients, but also causes many physical inconveniences, affecting hundreds of millions of people worldwide. Among the many analgesic drugs, lidocaine shows significant effects in relieving acute pain through subcutaneous injection due to its ion channel blocking characteristics, and thus has been widely used in the field of local analgesia. However, the operation process of subcutaneous injection is not only cumbersome, but also easily intensifies the fear of patients. With the advent of lidocaine hydrochloride (LiH) transdermal anesthetic products, a convenient and painless alternative solution is provided for daily analgesic treatment. However, both sprays and topical ointments face the challenge of low drug absorption efficiency.

[0004] Therefore, it is necessary to develop a microneedle patch for wound repair in diabetic patients. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a double-layer microneedle patch, which can release LiH through the outer layer HA to instantly relieve pain, and slowly release HT through the inner layer GelMA to target senescent cells and treat wounds in the elderly, so as to balance pain relief and wound repair, significantly accelerate wound healing in the elderly, and have great application prospect in preparation of a drug for promoting wound healing in the elderly.

[0006] To achieve the purpose, the present application adopts the following technical solutions: In the first aspect of the present application, a double-layer microneedle patch is provided, comprising: The outer layer is hyaluronic acid (HA) loaded with lidocaine hydrochloride (LiH); The inner layer is methacrylated gelatin (GelMA) loaded with hydroxytyrosol (HT).

[0007] Further, the microneedle has a needle height of 576.19 ± 50 μm; and a needle spacing of 552.52 ± 50 μm.

[0008] The needle height can ensure accurate delivery of the dermis layer, targeting the senescent cell-rich area; In a second aspect of the present application, a preparation method of a double-layer microneedle patch is provided, the method comprising: Inner layer forming: injecting a GelMA solution containing HT into the outer layer of the mold, vacuum degassing, drying, and then ultraviolet curing; Outer layer coating: coating a HA solution containing LiH on the inner layer of the mold and drying; After demolding, a double-layer microneedle patch is obtained.

[0009] Further, the concentration of lidocaine hydrochloride (LiH) is 5-20 mg / mL.

[0010] The reason for the concentration of lidocaine hydrochloride (LiH) being 5-20 mg / mL: a concentration < 15 mg / mL can avoid crystallization affecting the sharpness of the needle tip; a concentration > 5 mg / mL can guarantee the effective analgesic threshold. Preferably, the concentration of LiH is 10 mg / mL.

[0011] Further, the concentration of HT is 50-100 μM.

[0012] The reason for the concentration of HT being 50-100 μM: > 150 μM, the cell viability is significantly decreased (inhibition rate > 15%); < 50 μM, the anti-aging effect is insufficient. Preferably, the concentration of HT is 100 μM, and HT restores the migration ability of senescent cells to 89% (an increase of 117% compared to the H2O2 group).

[0013] Further, the hyaluronic acid (HA) has a molecular weight of 50-200 kDa, a pH value of 5.5-6.0, and a concentration of 25-35% (preferably 30%).

[0014] This concentration range is beneficial for the balance between fast release of LiH and structural stability, and the porous structure accelerates dissolution (LiH 30min release > 80%, Figure 8 B).

[0015] Further, the concentration of GelMA is 8-12% (preferably 10%). This concentration range is beneficial for slow-release control (avoiding burst release), and the cross-linked network of the present application blocks diffusion, so that HT is released slowly over 24h Figure 9 B).

[0016] Further, the LAP photoinitiator concentration is 0.25±0.05%, and the ultraviolet light curing wavelength is 405±5 nm. The concentration of the initiator can accurately control the GelMA crosslinking density, avoiding HT burst release (sustained release rate of 76.3%). The ultraviolet light excitation LAP generates free radicals with maximum efficiency, ensuring the mechanical strength of the needle tip.

[0017] Further, the pressure in the vacuum debubbling is-0.08~-0.1 MPa.

[0018] In the embodiments of the present application, by using GelMA 10±2% + LAP 0.25±0.05%, the crosslinking density is ensured to be 15-20%, the HT release kinetics is optimized, and the HT sustained release retention rate is >95% at 24h (single-layer mixed microneedle inactivation rate >40%).

[0019] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: The LiH / HT@HA-GelMA MN developed by the double-layer microneedle patch has good biocompatibility due to the combination of water-soluble material HA and non-water-soluble material GelMA. On the one hand, it can dissolve in the skin interstitial fluid and exhibit biodegradability, and on the other hand, it can continuously release drugs through swelling properties. This drug delivery method can effectively solve the local multi-drug delivery of skin damage repair sites. By simulating drugs with double-layer fluorescent dyes, we successfully constructed a double-layer microneedle delivery system loaded with drugs under a live light sectioning microscope. Moreover, the LiH / HT@HA-GelMA MN can successfully penetrate the skin and reach the dermis. The drug release experiment proves that the microneedle can effectively deliver the loaded drugs. With the dissolution of the microneedle, sequential drug delivery is achieved. This avoids multiple injections at the same site, meeting the principles of comfort and effectiveness. Based on a series of in vitro evaluation results, it is confirmed that the prepared LiH / HT@HA-GelMA MN can control the gradual release of drugs in the local area, enhance the therapeutic effect, and prolong the duration of the effect. This drug delivery method is simple to operate and relatively quick in the treatment process, without the need for frequent drug replacement, and can achieve rapid and painless local treatment. LiH / HT@HA-GelMA MN has good application prospects in wound treatment targeting senescent cells. Specifically, it has the following advantages: (1) LiH / HT@HA-GelMA MN has a good double-layer structure, neat needle body arrangement, uniform size, and good mechanical strength that can penetrate the skin.

[0020] (2) LiH / HT@HA-GelMA MN can achieve layered drug release, with LiH being rapidly released within 30 min and HT being slowly released within 24 h.

[0021] (3) LiH / HT@HA-GelMA MN significantly accelerated the wound healing process of aged mice, with smaller wound area, faster natural exfoliation of scab, and higher degree of re-epithelialization, more dense and ordered collagen arrangement, and increased regeneration of hair papilla cells in the wound area. In vivo experiments in mice also confirmed that LiH / HT@HA-GelMA MN could up-regulate the expression of SIRT4 in KCs and inhibit ferroptosis in KCs.

[0022] (4) LiH / HT@HA-GelMA MN could also accelerate the wound healing of photoaged mouse skin, increase the number of new blood vessels, and improve the healing quality. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Preparation process of LiH / HT@HA-GelMA MN.

[0024] Figure 2 Morphology of HA-GelMA MN and LiH / HT@HA-GelMA MN under different magnifications of a digital microscope.

[0025] Figure 3 Morphology of HA-GelMA MN and LiH / HT@HA-GelMA MN under different magnifications of a scanning electron microscope (scale bar: 300 µm).

[0026] Figure 4 Confocal scanning electron microscopy of LiH / HT@HA-GelMA MN.

[0027] Figure 5 Double-layer morphology of LiH / HT@HA-GelMA MN under a live light-sectioning microscope (scale bar: 200 µm).

[0028] Figure 6 Stress-displacement curves of HA-GelMA MN and LiH / HT@HA-GelMA MN.

[0029] Figure 7 Mouse skin puncture experiment: (A) Changes in skin after insertion of LiH / HT@HA-GelMA MN; (B) Punctured skin stained with Ponceau red; (C) HE staining of mouse punctured skin (scale bar: 100 µm).

[0030] Figure 8 (A) LiH drug concentration standard curve; (B) LiH release curve in LiH / HT@HA-GelMA MN.

[0031] Figure 9(A) HT drug concentration standard curve; (B) HT release curve of LiH / HT@HA-GelMA MN.

[0032] Figure 10 The dissolution of LiH / HT@HA-GelMA MN within 3 min.

[0033] Figure 11 The wound area graph of different treatment mice at 0, 3, 7, 14 days (scale: 7.5 mm).

[0034] Figure 12 (A) Comparison of wound area of different treatment mice at 0, 3, 7, 14 days; (B) Quantitative statistics of wound area of different treatment mice at 0, 3, 7, 14 days.

[0035] Figure 13 HE staining graph of 14-day wound of different treatment mice (scale: 500 µm).

[0036] Figure 14 Masson staining graph of 14-day wound of different treatment mice (scale: 500 µm).

[0037] Figure 15 Detection of 14-day hair follicle cell proliferation of different treatment mice (scale: 200 µm).

[0038] Figure 16 (A) Expression of p53 in the skin of photoaging model mice (scale: 100 µm); (B) Expression of p21 in the skin of photoaging model mice (scale: 100 µm).

[0039] Figure 17 Wound area graph of photoaging mice at 0, 3, 7, 14 days (scale: 7.5 mm) Figure 18 (A) Comparison of wound area of photoaging mice at 0, 3, 7, 14 days; (B) Quantitative statistics of wound area of photoaging mice at 0, 3, 7, 14 days.

[0040] Figure 19 HE staining graph of 14-day wound of photoaging mice (scale: 200 µm).

[0041] Figure 20 Masson staining graph of 14-day wound of photoaging mice (scale: 200 µm).

[0042] Figure 21 CD31 staining graph of 14-day wound of photoaging mice (scale: 200 µm).

[0043] Figure 22 SIRT4 staining in 14-day wounds of aged mice (scale bar: 200 µm).

[0044] Figure 23 ACSL4 and GPX4 staining in KCs in 14-day wounds of aged mice (scale bar: 200 µm).

[0045] Figure 24 HE staining of major organs in 14-day post-modeling aged mice (scale bar: 200 µm). DETAILED DESCRIPTION

[0046] The advantages and various effects of the embodiments of the present application will be more clearly presented hereinafter with specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the embodiments of the present application, rather than limit the embodiments of the present application.

[0047] Throughout the specification, unless otherwise specifically indicated, the terms used herein are understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the embodiments of the present application belong. If there is a contradiction, the present specification takes priority.

[0048] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment, etc. used in the embodiments of the present application can be obtained by market purchase or can be obtained by existing methods.

[0049] To solve the above technical problems, the general idea of the present application is as follows: The present application prepared LiH / HT@HA-GelMA MN patches with a size of 1x1 cm, with HA as the outer layer and GelMA as the inner layer. The morphology was characterized in detail by digital microscope and SEM, and the results showed that the synthesized microneedles were double-layered conical shape and arranged in order. The results of compression test and skin penetration experiment related to mechanical properties showed that LiH / HT@HA-GelMA MN could effectively penetrate the epidermis layer of the skin, reach the dermis area, and leave micropores on the skin, which facilitated the release of drugs. In addition, the LiH loaded by LiH / HT@HA-GelMA MN could be rapidly released within 30 min to relieve pain, while HT was slowly released within 24 h to target senescent cells for treatment. In summary, the present application successfully prepared LiH / HT@HA-GelMA microneedle patches, which may play an important role in the treatment of wounds targeting senescent cells.

[0050] The effects of LiH / HT@HA-GelMA MN on wound repair in naturally aging and photoaged mice were evaluated through in vivo animal experiments. The results showed that this MN significantly promoted high-quality wound healing, manifested as reduced wound area, enhanced angiogenesis, increased epithelialization, and the formation of accessory organs such as hair follicles. Furthermore, in mouse wound tissue, LiH / HT@HA-GelMA MN also upregulated SIRT4 expression in KCs and inhibited ferroptosis in KCs, consistent with the results of cell experiments. This study provides a theoretical basis for the development of novel microneedle drug delivery systems and offers new therapeutic approaches for improving the treatment of wounds in the elderly and for skin aging-related diseases.

[0051] Addressing the long-standing challenges in treating wounds in the elderly, namely the drug incompatibility (the weakly alkaline environment of LiH leads to the oxidative inactivation of HT) and the uncontrollable timing release (analgesia requires rapid release within 0-30 minutes vs. anti-aging requires sustained release within 6-24 hours), this invention achieves a breakthrough innovation through a dual-material layered timing-controlled release structure: (1) Innovative design: Utilizing the rapid dissolution properties of HA (releasing >80% LiH within 30 minutes), Figure 8 (B)

[0052] Combining the GelMA sustained-release network (HT 24h sustained release conforms to the Higuchi model, Figure 9 (B)

[0053] This invention features an innovative structure (layered controlled release) that solves the problem of drug incompatibility (HT inactivation rate <5% vs. injection group >40%).

[0054] (2) Resolving the essence of the contradiction: The outer HA layer rapidly releases LiH, providing immediate analgesia and eliminating the alkaline environment, thus avoiding the risk of HT inactivation. The inner GelMA layer provides sustained HT release, creating a neutral environment for long-lasting anti-aging effects, thereby activating the SIRT4 / inhibiting the ferroptosis pathway. This invention features a time-series innovation (LiH rapid release / HT sustained release) to meet the clinical needs of "analgesia first - repair later". (3) Synergistic gain effect: Wound healing rate increased by 41% (vs. mixed injection group).

[0055] The number of hair follicles regenerated increased by 64% (breaking through the bottleneck of functional regeneration of wounds in the elderly).

[0056] Conclusion: By adopting the innovative approach of "HA-GelMA property complementarity, hierarchical time-sequential controlled release, and drug-mechanism dual synergy", the contradiction between drug compatibility and release kinetics that cannot be overcome by existing technologies is resolved, resulting in technical effects that exceed the expectations of those skilled in the art.

[0057] A double-layer microneedle patch according to the present application will be described in detail below in combination with examples and experimental data.

[0058] Example 1, synthesis of a double-layer microneedle patch 1. Synthesis of LiH / HT@HA-GelMA MN 1) Preparation of required solutions (1) 0.25% LAP: Take out the lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) in the GelMA kit, add 20 mL of PBS to 0.05 g of LAP powder, and place it in a 50 °C water bath for 20 min. Shake the bottle several times during heating to accelerate dissolution. After dispensing, store it at 4 °C in the dark.

[0059] (2) 10% GelMA solution: Add 10 mL of LAP solution to 1 g of GelMA, shake it evenly, and then place it in a 70 °C water bath for 20 min. Repeat the shaking to accelerate dissolution. After complete dissolution, immediately filter the GelMA using a 0.22 μm filter, and store it at -20 °C in the dark after dispensing.

[0060] (3) 30% HA solution: Add 10 mL of deionized water to 3 g of HA powder, and vortex repeatedly to form a transparent liquid. Filter it using a 0.22 μm filter, and store it at -20 °C in the dark after dispensing.

[0061] (4) GelMA solution containing 100 μmol / L HT: Add 1 μL of sterile 100 mmol / L HT to 10 mL of 10% GelMA, and mix it to prepare a GelMA solution with a final concentration of 100 μmol / L HT.

[0062] (5) HA solution containing 10 mg / mL LiH: Add 20 mg / mL LiH to HA to make the final concentration 10 mg / mL.

[0063] 2) Synthesis of LiH / HT@HA-GelMA MN (1) Place the microneedle mold in a biological safety cabinet, and sterilize it using ultraviolet and ozone for 1 h.

[0064] (2) Use a pipette to take 500 μL of GelMA solution and slowly add it along the side wall of the mold until the entire liquid evenly covers the surface of the mold.

[0065] (3) The mold is placed in a vacuum defoaming machine, and the liquid fills the needle tip through vacuum negative pressure, while the gas floats on the surface to form bubbles.

[0066] (4) Use a 200 μL pipette to suck the surface bubbles, fill the liquid, and then put the mold back into the defoaming machine.

[0067] (5) Repeat steps (3) and (4) until there are no obvious bubbles on the surface of the liquid after vacuum suction.

[0068] (6) Scrape off the GelMA solution on the surface of the mold, leaving only the GelMA solution in the needle tip cavity.

[0069] (7) Place the mold in a constant temperature drying oven at 37 °C for 2 h, and photocure for 15 s.

[0070] (8) Use a pipette to suck 500 μL of HA solution and slowly add it along the side wall of the mold until the liquid evenly covers the entire surface of the mold.

[0071] (9) Repeat steps (3) and (4) until there are no obvious bubbles on the surface.

[0072] (10) Place the mold in a constant temperature drying oven at 37 °C for 1 h, and when the liquid surface is slightly concave, add HA solution to the surface of the mold again.

[0073] (11) Blow dry again for 2 h, and then demold after the microneedles are completely dried.

[0074] Experimental Example 1, LiH / HT@HA-GelMA MN Morphology Characteristics After successfully preparing LiH / HT@HA-GelMA MN by a two-step method, Figure 1 , the microneedles were demolded to observe the overall morphology. Both groups of microneedle patches were 1×1 cm 2 square, and the HA-GelMA MN presented a transparent appearance, while the LiH / HT@HA-GelMA MN presented a light yellow appearance after drug loading. After magnification by a digital microscope, it was found that the needle bodies were evenly and uniformly distributed, and the sizes were consistent. Figure 2 In the SEM results, the conical needle bodies were observed, and the needle tips were sharp. Compared with the blank MN, drug loading did not change the morphology of the microneedles. Figure 3 The confocal scanning results showed that the LiH / HT@HA-GelMA MN needle spacing was 552.52 μm, and the needle tip height was 576.19 μm. Figure 4

[0075] ​The above results show that we successfully prepared LiH / HT@HA-GelMA MN patches with sharp needle tips and sufficient needle body length, providing the necessary microneedle structure for transdermal drug delivery. To further confirm the double-layer morphology of LiH / HT@HA-GelMA MN, the green FITC-labeled GelMA layer and the red rhodamine B-labeled HA layer were used, and the results showed that LiH / HT@HA-GelMA MN had a uniform distribution of fluorescent signals, which indicated that the drug could be uniformly distributed in the needle body, and the inner layer of the needle body was green and the outer layer was red, indicating that LiH / HT@HA-GelMA MN had a double-layer microneedle morphology. Figure 5 ).

[0076] Experimental Example 2, Performance Detection of LiH / HT@HA-GelMA MN I. Mechanical Properties of LiH / HT@HA-GelMA MN In order to detect whether LiH / HT@HA-GelMA MN has the ability to penetrate the skin, first, the compression experiment was performed to detect its mechanical properties, and the needle body displacement curve of HA-GelMA MN and LiH / HT@HA-GelMA MN with increasing load stress was drawn. Figure 6 ).

[0077] The results show that within the deformation range of 0-600 mm, the stress-displacement curves of the two groups show a continuous smooth feature, which indicates that the needle body has not been broken during this period. Under the same stress, the needle tip displacement of LiH / HT@HA-GelMA MN is smaller than that of HA-GelMA MN, indicating that the mechanical properties of LiH / HT@HA-GelMA MN are better. At the same time, the results show that the needle tips of HA-GelMA MN and LiH / HT@HA-GelMA MN can withstand strong compression force, and both have mechanical properties to pierce the skin. The needle tip of LiH / HT@HA-GelMA MN of the present application can withstand 0.35N / needle, which can guarantee effective drug delivery. II. Evaluation of Skin Penetration Ability of LiH / HT@HA-GelMA MN In order to further verify the ability of LiH / HT@HA-GelMA MN to deliver drugs to the deep part, a puncture experiment was performed on the skin of mice. Figure 7). The skin surface presented a neat array of microneedle array after LiH / HT@HA-GelMA MN insertion, and the combination rate of the broken skin with the eosin solution was more than 80%, indicating that LiH / HT@HA-GelMA MN could effectively penetrate the skin. At the same time, the HE staining results showed that a microchannel was left at the puncture site, reaching the dermis layer, which indicated that LiH / HT@HA-GelMA MN could effectively perform deep drug delivery, and the microneedle reached the dermis layer, precisely targeting senescent cells.

[0078] III. Drug release evaluation of LiH / HT@HA-GelMA MN Next, we detected the drug release performance. First, we quantitatively evaluated the release of LiH in LiH / HT@HA-GelMA MN. According to the OD value of LiH standard solution at λ230, the LiH standard curve was drawn (Fig. 2A). According to the standard curve, the LiH release curve at different time points was drawn, and the results showed that LiH / HT@HA-GelMA MN could release 80% of LiH within 20 min, and all was released within 30 min (Fig. 2B). Figure 8 Figure 8 This indicates that LiH can be quickly released from LiH / HT@HA-GelMA MN to achieve rapid analgesic effect. The release of >80% in 30 min indicates that it meets the analgesic time requirement. According to the OD value of HT standard solution at λ280, the HT standard curve was drawn (Fig. 2A). The release curve results of HT showed that the release rate of HT in LiH / HT@HA-GelMA MN was faster in the first 12 h, and basically reached a stable state after 24 h (Fig. 2B), and the sustained release of HT in 24 h conforms to the Higuchi model (diffusion-controlled release), thereby maintaining long-acting anti-aging. Figure 9 Figure 9

[0079] IV. Dissolution performance evaluation of LiH / HT@HA-GelMA MN To explore the dissolution performance of LiH / HT@HA-GelMA MN, 3% agarose gel was used for simulation. LiH / HT@HA-GelMA MN was inserted into the gel, and the changes of the needle body at different time points were photographed (Fig. 3). Figure 10

[0080] ​​​​Results show that after 15 s of contact with the gel, the tip of the needle begins to blunt, the body of the needle widens, and at 30 s the body of the needle shortens due to the dissolution of the outer layer of HA, but the body of the needle and the base further widen due to the swelling of the GelMA, which has a round and blunt appearance. At 1 min, the normal shape of the needle body is lost, and part of the swollen needle body can be seen on the base surface. At 2 min and 3 min, most of the needle body of the microneedle is not visible. The outer layer of HA dissolves at 30 s, and the inner layer of GelMA slowly swells at 3 min, proving the visualization of the time and space sequential release.

[0081] Experimental Example 3, Effect and mechanism of LiH / HT@HA-GelMA MN in promoting wound healing in the elderly I. LiH / HT@HA-GelMA MN improves wound healing in old mice In order to detect the effect of LiH / HT@HA-GelMA MN in promoting wound healing in animals, a full-thickness skin defect wound with a diameter of 10 mm was constructed on the back of the old mice, and the wound was photographed and recorded at 0 days, 3 days, 7 days and 14 days after the operation, and the wound area was calculated.

[0082] Among them, the method for establishing a full-thickness skin defect model is as follows: 1) Anesthetize the mice using a gas anesthesia machine, and perform the hair removal operation on the mice using the above method.

[0083] 2) After disinfecting the back skin, remove the full-thickness skin on the back using a biopsy needle.

[0084] 3) After fixing the photographing height, record the initial area of the wound of each mouse.

[0085] 4) After modeling, the mice were randomly divided into four groups: Control group, HT+LiH group, HA-GelMA MN group and LiH / HT@HA-GelMA MN group. The Control group was given 100 μL PBS, the HT / LiH injection group was given 100 μL HT / LiH mixed solution, the HA-GelMA MN group was given blank MN (double-layer microneedle patch without drug), and the LiH / HT@HA-GelMA MN group was given the double-layer MN of the application.

[0086] 5) After anesthesia, the mice were raised in individual cages, and the wound area was photographed and recorded at 3, 7 and 14 days after the operation, and the wound area was calculated using the formula Wwound area / W0 day area x 100%.

[0087] 6) At 14 days after the operation, the wound and the surrounding normal tissue were collected and immersed in a fixing solution for subsequent pathological evaluation.

[0088] The results are as follows Figure 11As shown, the wound contraction rate of the LiH / HT@HA-GelMA MN group was the fastest, and the scab naturally fell off the earliest. The wound area of the HT / LiH injection group and the HA-GelMA MN group contracted slower than that of the LiH / HT@HA-GelMA MN group, but the wound healing rate was also improved compared with the Control group, which indicated that HA-GelMA MN and HT / LiH themselves also had a certain degree of effect on promoting wound healing.

[0089] By visualizing the wound area of each group at different time periods, it was found that the wound healing of the LiH / HT@HA-GelMA MN group was better than that of the other groups (Fig. 6A), and there were significant statistical differences between the wound area of each group and that of the LiH / HT@HA-GelMA MN group (Fig. 6B). Figure 12 Figure 12

[0090] On the 14th day after modeling, the skin in the wound area was taken for HE staining, and the results showed that the LiH / HT@HA-GelMA MN group could see a clear and complete epidermis layer, and the area of the tissue in the scar stage was smaller, and the area with skin appendages such as hair follicles was larger. Although the wounds in the other groups had also closed, the area of the tissue in the repair stage was larger, indicating that the functional reconstruction had not yet been completed. Figure 13

[0091] The deposition of collagen in the wound healing area is closely related to the quality of skin healing, and the deposition of collagen is beneficial to remodeling the structure of the dermis layer of the skin and restoring the normal tension and elasticity of the skin. Masson staining showed deep blue, indicating that the collagen fibers had high density, and the collagen fibers in the LiH / HT@HA-GelMA MN group were arranged in order, and the blue staining degree was deeper, while the staining color of the Control group was lighter, and the collagen fibers were arranged loosely and irregularly. Figure 14 Based on the above results, LiH / HT@HA-GelMA MN treatment can effectively promote wound contraction in mice and achieve high-quality healing effect.

[0092] II. LiH / HT@HA-GelMA MN promotes hair regeneration in the wound area ​​​Hair follicle as a part of skin appendage exists in the dermis and even deeper subcutaneous tissue. The regeneration of hair follicle in wound area indicates that the wound repair not only completes re-epithelialization, but also restores and rebuilds the dermis and even subcutaneous tissue. This can fully explain that the repair process of the wound is complete and of high quality. Based on this, we detected the proliferation ability of hair papilla cells in each group. SRY-box transcription factor 2 (SRY-related HMG-box 2, SOX2) is a marker of hair follicle papilla cells. Alpha-SMA is not only a marker of fibroblasts, but also a temporary expression of hair papilla cells when the hair follicle is in the growth period, wound repair, hair follicle regeneration or external mechanical stimulation. Therefore, SOX2 and alpha-SMA double staining positive represents that the hair papilla cells are in the repair period. Ki-67 is a cell proliferation marker.

[0093] The results, as shown in Figure 15 , SOX2, alpha-SMA, Ki-67 positive cells in the LiH / HT@HA-GelMA MN group were significantly more than in the other three groups, and the distribution area was in the deep layer of the skin. This proves that LiH / HT@HA-GelMA MN not only promotes wound healing, but also promotes the reconstruction of damaged skin function.

[0094] III. LiH / HT@HA-GelMA MN improves wound healing in photoaging mice In order to verify the effectiveness of LiH / HT@HA-GelMA MN under different aging mechanisms, a photoaging mouse model was constructed. After the mice received 8 weeks of UVA and UVB irradiation, the mouse back skin was taken for IF staining.

[0095] The results showed that the expression of p21 and p53 proteins in photoaging mice increased, proving that the induced mouse skin presented the characteristics of aged skin (Fig. Figure 16 A-B). Subsequently, a full-thickness skin defect model was established on the back of the mice, and the wound area was photographed on days 0, 3, 7 and 14. The results showed that from day 7, the wound area in each group showed a gap, and the LiH / HT@HA-GelMA MN group had the smallest wound area, and the scab in the LiH / HT@HA-GelMA MN group fell off faster on day 14 (Fig. Figure 17 ).

[0096] In Figure 18 , it can be seen that the wound area in each group has decreased over time, but in the Control group, there is still a thick scab with a large area, and in the LiH / HT@HA-GelMA MN group, the area of the scab that naturally falls off is large, and the area of the scab left is the smallest, and the statistical data shows a significant difference from the other groups.

[0097] Next, HE staining results showed that the epidermis continuity of LiH / HT@HA-GelMA MN group was basically restored, and the re-epithelialization was relatively complete, with multiple layers of KCs covering the wound. In the Control group, the epithelial integrity had not yet fully recovered, and there were unhealed areas under the scab. Figure 19 In the Masson staining results, the blue collagen fibers in the dermis layer of the LiH / HT@HA-GelMA MN group increased, and the arrangement of the collagen fibers was parallel, regular, and high in density, gradually approaching normal skin tissue. In contrast, in the Control group, there were still uncolored areas in the central region of the wound, and the arrangement of collagen fibers around the wound was also relatively loose. Figure 20

[0098] In addition, the CD31 IHC staining results showed that the number of CD31 positive cells, as a blood vessel marker, in the HT / LiH injection group and the LiH / HT@HA-GelMA MN group was more than that in the Control group and the HA-GelMA MN group, which indicated that HT / LiH had a role in promoting blood vessel formation. Moreover, due to the advantages of microneedle administration, the number of blood vessels in the LiH / HT@HA-GelMA MN group was superior to that in the other three groups. Figure 21

[0099] Four, Mechanism of LiH / HT@HA-GelMA MN in promoting wound healing IF staining was performed on the 14th day specimens of the old mouse wounds, and CK15 is a KCs marker. The results showed that compared with the Control group and the HA-GelMA MN group, the number of SIRT4 positive cells in KCs in the HT / LiH injection group and the LiH / HT@HA-GelMA MN group increased, which indicated that at the mouse level, HT could also increase the expression of SIRT4 in KCs, and microneedle administration was better than simple injection. Figure 22

[0100] To detect the ferroptosis phenomenon in the old wound tissue, the ferroptosis protective factor GPX4 and the ferroptosis promoting factor ACSL4 were selected for detection. The results showed that in the Control group, the expression of ACSL4, which could enhance the generation of lipid peroxides, was more in the CK15 positive KCs, while the expression of antioxidant enzyme GPX4, which could reduce the occurrence of lipid peroxidation reaction, was less. In the LiH / HT@HA-GelMA MN group, the opposite trend was observed, with decreased green ACSL4 expression and increased pink GPX4 expression. Figure 23 The above detection results were consistent with the results in the cell experiment.

[0101] ​​​V. Bio-safety evaluation of LiH / HT@HA-GelMA MN HA itself is a component of living organisms, and has high bio-safety. GelMA is also widely used in transdermal drug delivery systems due to its good biocompatibility and bio-safety. In order to verify the bio-safety of the microneedle, the main organs of the control group and the LiH / HT@HA-GelMA MN group mice after modeling for 14 days were taken, and HE staining was performed.

[0102] The results show that there is no significant difference in the HE staining results of the main organs of the mice in the two groups, which proves that LiH / HT@HA-GelMA MN has no obvious toxicity to mice and has high bio-safety. Figure 24 ).

[0103] In summary, the above data are shown in the following table: Table 1

[0104] The above data show that: (1) The healing rate of the LiH / HT@HA-GelMA MN group is 89% vs. the HT / LiH injection group of 63%, and the microneedle delivery system breaks through the transdermal efficiency bottleneck (<5%) of traditional injection therapy, achieving efficient drug delivery; (2) The number of hair follicle regeneration is 8.7±1.3 vs. the injection group of 5.3±0.9, which is the first time to achieve dermal-epidermal synergistic regeneration of old wound surface (natural healing is only 3.2±0.8), and produces an unexpected effect of 64% increase in hair follicle regeneration (exceeding the theoretical additive value), which proves that the synergistic effect is not caused by simple dosage form change.

[0105] Finally, it should be noted that the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such a process, method, article or equipment.

[0106] Although the preferred embodiments of the embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present application.

[0107] It will be apparent to those skilled in the art that various modifications and variations can be made to the present embodiments without departing from the spirit or scope of the present embodiments. Thus, it is intended that the present embodiments cover the modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A dual-layer microneedle patch, characterized by, The double-layer microneedle patch comprises: Outer layer: hyaluronic acid (HA) loaded with lidocaine hydrochloride (LiH); Inner layer: methacrylated gelatin (GelMA) loaded with hydroxytyrosol (HT); The needle height of the microneedle is 576.19±50 μm; the needle spacing is 552.52±50 μm; The preparation method of the double-layer microneedle patch comprises: Dissolve methacrylated gelatin (GelMA) in a PBS solution containing LAP photoinitiator to prepare a GelMA solution; add hydroxytyrosol (HT) and mix to obtain an inner layer solution; inject the inner layer solution into the inner layer of the mold, vacuum degassing, dry, and then ultraviolet curing to obtain a formed inner layer; Dissolve lidocaine hydrochloride (LiH) in a 30% hyaluronic acid (HA) solution to obtain an outer layer solution; cover the outer layer solution on the inner layer of the mold and dry to obtain a formed outer layer; After demolding, a double-layer microneedle patch is obtained; The concentration of HT is 50-100 μM; The concentration of lidocaine hydrochloride (LiH) is 5-20 mg / mL; The concentration of hyaluronic acid (HA) is 25-35%; The concentration of GelMA is 8-12%.

2. The double-layer microneedle patch of claim 1, wherein, The crosslinking density of methacrylated gelatin (GelMA) is 15-20%, formed by ultraviolet curing of 0.2-0.3% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator.

3. The double-layer microneedle patch of claim 1, wherein The pressure in the vacuum degassing is -0.08~-0.1 MPa.

4. Use of the double-layer microneedle patch of any one of claims 1-3 in the preparation of a drug for promoting the healing of old wounds.

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