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

Abstract

The application discloses a double-layer microneedle patch and application thereof in preparation of a medicine for promoting wound healing of the elderly, and the double-layer microneedle patch comprises: an outer layer, which is hyaluronic acid (HA) loaded with lidocaine hydrochloride (LiH); and an inner layer, which is methacrylated gelatin (GelMA) loaded with hydroxytyrosol (HT). The wound healing process of the elderly mice is significantly accelerated, the wound area is small, the speed of natural exfoliation of the scab is faster, and the degree of re-epithelialization of the wound is higher, the collagen arrangement is more dense and ordered, and the regeneration of hair papilla cells in the wound area is increased. The double-layer microneedle patch has great application prospect in preparation of the medicine for promoting wound healing of the elderly.

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. Nevertheless, 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 present application aims to provide a preparation method of a double-layer microneedle patch, which can release LiH for instant analgesia by rapidly dissolving the outer layer HA, and slowly release HT for treating aging cells in the inner layer GelMA to treat wounds in the elderly, so as to balance analgesia and wound repair, significantly accelerate wound healing in elderly mice, and has great application prospect in preparation of a drug for promoting wound healing in the elderly.

[0006] To achieve the object, the present application adopts the following technical solutions:

[0007] In the first aspect of the present application, a double-layer microneedle patch is provided, comprising:

[0008] Outer layer: hyaluronic acid (HA) loaded with lidocaine hydrochloride (LiH);

[0009] Inner layer: GelMA loaded with hydroxytyrosol (HT).

[0010] Further, the microneedle has a needle height of 576.19 ± 50 μm and a needle spacing of 552.52 ± 50 μm.

[0011] The needle height ensures accurate delivery to the dermis layer and targets the senescent cell-rich area.

[0012] In a second aspect of the present application, a method for preparing a double-layer microneedle patch is provided, the method comprising:

[0013] Inner layer forming: injecting a HT-containing GelMA solution into the outer layer of the mold, vacuum degassing, drying, and then UV curing;

[0014] Outer layer coating: coating a LiH-containing HA solution on the inner layer of the mold and drying;

[0015] After demolding, a double-layer microneedle patch is obtained.

[0016] Further, the concentration of lidocaine hydrochloride (LiH) is 5-20 mg / mL.

[0017] The reason for the concentration of lidocaine hydrochloride (LiH) being 5-20 mg / mL is that a concentration < 15 mg / mL can avoid crystallization affecting the sharpness of the needle tip, and a concentration > 5 mg / mL can guarantee an effective analgesic threshold. Preferably, the concentration of LiH is 10 mg / mL.

[0018] Further, the concentration of HT is 50-100 μM.

[0019] The reason for the concentration of HT being 50-100 μM is that > 150 μM can significantly reduce cell viability (inhibition rate > 15%), and < 50 μM can not have sufficient anti-aging effect. Preferably, the concentration of HT is 100 μM, which can restore the migration ability of senescent cells to 89% (an increase of 117% compared to the H2O2 group).

[0020] 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%).

[0021] This concentration range is beneficial for the balance between fast release of LiH and structural stability, and the porous structure accelerates dissolution (LiH 30 min release > 80%, Figure 8 B).

[0022] Further, the GelMA concentration is 8-12% (preferably 10%). This concentration range is beneficial for slow release control (avoiding burst release), the crosslinked network of the present application retards diffusion, allowing HT to be released slowly over 24h Figure 9 B}.

[0023] Further, the LAP photoinitiator concentration is 0.25±0.05%, and the ultraviolet light curing wavelength is 405±5nm. The concentration of the initiator can accurately control the crosslinking density of GelMA, avoiding HT burst release (slow release rate of 76.3%). The efficiency of generating free radicals by exciting LAP with ultraviolet light is maximized, ensuring the mechanical strength of the tip.

[0024] Further, the pressure in the vacuum debubbling is-0.08~-0.1 MPa.

[0025] 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 release retention rate is >95% over 24h (single-layer mixed microneedle inactivation rate >40%).

[0026] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages:

[0027] 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 confirms 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 the treatment process is relatively fast, 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:

[0028] (1) LiH / HT@HA-GelMA MN has good bilayer structure, orderly needle body arrangement, uniform size, and good mechanical strength and can penetrate the skin.

[0029] (2) LiH / HT@HA-GelMA MN can realize layered release of drugs, LiH is rapidly released within 30 min, and HT is slowly released within 24 h.

[0030] (3) LiH / HT@HA-GelMA MN significantly accelerates the wound healing process of old mice, the wound area is small, the speed of natural exfoliation of scab is faster, and the degree of re-epithelialization of the wound is higher, the collagen arrangement is more dense and orderly, and the regeneration of hair follicle cells in the wound area is increased. It is also confirmed in mice that LiH / HT@HA-GelMA MN can up-regulate the expression of SIRT4 in KCs and inhibit ferroptosis in KCs.

[0031] (4) LiH / HT@HA-GelMA MN can also accelerate the wound healing rate of photoaged mouse skin, increase the number of new blood vessels, and improve the healing quality. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Preparation process of LiH / HT@HA-GelMA MN.

[0033] Figure 2 Morphology of HA-GelMA MN and LiH / HT@HA-GelMA MN under different magnifications of a digital microscope.

[0034] 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).

[0035] Figure 4 Confocal scanning electron microscope image of LiH / HT@HA-GelMA MN.

[0036] Figure 5 Bilayer morphology of LiH / HT@HA-GelMA MN under a live light sectioning microscope (scale bar: 200 µm).

[0037] Figure 6 Stress-displacement curve of HA-GelMA MN and LiH / HT@HA-GelMA MN.

[0038] Figure 7For mouse skin puncture experiment: (A) Changes in skin after LiH / HT@HA-GelMA MN insertion; (B) Punctured skin stained with eosin; (C) HE staining of mouse punctured skin (scale: 100 µm).

[0039] Figure 8 For (A) LiH drug concentration standard curve; (B) LiH / HT@HA-GelMA MN LiH release curve.

[0040] Figure 9 For (A) HT drug concentration standard curve; (B) LiH / HT@HA-GelMA MN HT release curve.

[0041] Figure 10 For the dissolution of LiH / HT@HA-GelMA MN within 3 min.

[0042] Figure 11 For wound area graph of different treatment mice at 0, 3, 7, 14 days (scale: 7.5 mm).

[0043] Figure 12 For (A) Comparison of wound area of different treatment mice at 0, 3, 7, 14 days; (B) Quantitative statistics graph of wound area of different treatment mice at 0, 3, 7, 14 days.

[0044] Figure 13 For HE staining of wound of different treatment mice at 14 days (scale: 500 µm).

[0045] Figure 14 For Masson staining of wound of different treatment mice at 14 days (scale: 500 µm).

[0046] Figure 15 For detection of hair follicle cell proliferation of different treatment mice at 14 days (scale: 200 µm).

[0047] Figure 16 For (A) Expression of p53 in skin of photoaging model mice (scale: 100 µm); (B) Expression of p21 in skin of photoaging model mice (scale: 100 µm).

[0048] Figure 17 For wound area graph of photoaging mice at 0, 3, 7, 14 days (scale: 7.5 mm)

[0049] Figure 18 For (A) Comparison of wound area of photoaging mice at 0, 3, 7, 14 days; (B) Quantitative statistics graph of wound area of photoaging mice at 0, 3, 7, 14 days.

[0050] Figure 19 HE staining of 14-day wound in photoaged mice (scale bar: 200 µm).

[0051] Figure 20 Masson staining of 14-day wound in photoaged mice (scale bar: 200 µm).

[0052] Figure 21 CD31 staining of 14-day wound in photoaged mice (scale bar: 200 µm).

[0053] Figure 22 SIRT4 staining of 14-day wound in aged mice (scale bar: 200 µm).

[0054] Figure 23 ACSL4 and GPX4 staining in KCs of 14-day wound in aged mice (scale bar: 200 µm).

[0055] Figure 24 HE staining of major organs of 14-day post-modeling aged mice (scale bar: 200 µm). DETAILED DESCRIPTION

[0056] The advantages and various effects of the embodiments of the present application will be more clearly presented by the following 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.

[0057] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be 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 conflict, the present specification takes precedence.

[0058] Unless otherwise specifically indicated, the 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.

[0059] The overall idea of the present application to solve the above technical problems is as follows:

[0060] The present application prepared LiH / HT@HA-GelMA MN patches with a size of 1x1 cm, in which the outer layer is HA and the inner layer is GelMA. The morphology of the MNs was characterized in detail by a digital microscope and SEM, and the results showed that the synthesized MNs were double-layered conical in shape and arranged in order. The results of compression tests and skin penetration experiments related to mechanical properties showed that LiH / HT@HA-GelMA MNs could effectively penetrate the epidermis layer of the skin, reach the dermis region, and leave micropores on the skin, which facilitated the release of drugs. In addition, the LiH loaded in LiH / HT@HA-GelMA MNs could be rapidly released within 30 min to relieve pain, while the HT was slowly released within 24 h to target senescent cells for treatment. In summary, the present application successfully prepared LiH / HT@HA-GelMA MN patches, which may play an important role in the treatment of wounds targeting senescent cells.

[0061] The effects of LiH / HT@HA-GelMA MNs on wound repair in naturally aged mice and photoaged mice were evaluated by in vivo animal experiments. The experimental results showed that the MNs significantly promoted high-quality healing of the wound, as indicated by reduced wound area, enhanced neovascularization, improved epithelialization level, and formation of appendages such as hair follicles. In addition, in the wound tissue of mice, LiH / HT@HA-GelMA MNs could also upregulate the expression of SIRT4 in KCs and inhibit ferroptosis in KCs, which was consistent with the results of the cell experiment. This study provides a theoretical basis for the development of a new type of MN drug delivery system and a new treatment idea for improving the treatment of elderly wounds and skin aging-related diseases.

[0062] In view of the long-standing problems of drug incompatibility (LiH weak alkaline environment leading to HT oxidative inactivation) and uncontrollable time-release (fast release of 0-30 min for analgesia vs. slow release of 6-24 h for anti-aging) in the treatment of elderly wounds, the present application realizes a breakthrough innovation through a double-material layered time-release structure:

[0063] (1) Innovative design:

[0064] Taking advantage of the rapid dissolution characteristics of HA (more than 80% LiH released within 30 min, Figure 8 middle B).

[0065] Combined with the slow-release network of GelMA (HT 24h slow release conforms to the Higuchi model, Figure 9 middle B).

[0066] The structural innovation (layered controlled release) of the present application solves the problem of drug incompatibility (HT inactivation rate <5% vs. injection group >40%).

[0067] (2) Solve the essence of the contradiction:

[0068] Outer layer HA fast release LiH, instant analgesia + remove alkaline environment, thereby avoiding the risk of HT inactivation. Inner layer GelMA slow release HT, long-acting anti-aging in a neutral environment, thereby activating SIRT4 / inhibiting ferroptosis pathway.

[0069] The timing innovation (LiH fast release / HT slow release) of the application matches the clinical needs of "analgesic pioneer-repair successor".

[0070] (3) Synergistic gain effect:

[0071] Wound healing rate increased by 41% (vs. mixed injection group).

[0072] Hair follicle regeneration increased by 64% (breaking through the functional regeneration bottleneck of old wounds).

[0073] Conclusion: Through the innovative path of "HA-GelMA material property complementation, layered timing controlled release, and drug-mechanism double synergy", the drug compatibility and release kinetics contradiction that cannot be overcome by the prior art is solved, producing technical effects beyond the expectations of those skilled in the art.

[0074] A double-layer microneedle patch according to the present application will be described in detail below in combination with examples and experimental data.

[0075] Example 1, synthesis of a double-layer microneedle patch

[0076] 1. Synthesis of LiH / HT@HA-GelMA MN

[0077] 1) Prepare the required solutions

[0078] (1) 0.25% LAP: Take lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) from 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 frequently during heating to accelerate dissolution. After dispensing, store at 4 °C in the dark.

[0079] (2) 10% GelMA solution: Add 10 mL of LAP solution to 1 g of GelMA, shake 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.

[0080] (3) 30% HA solution: 3 g of HA powder was added with 10 mL of deionized water, vortexed repeatedly to form a transparent liquid, filtered using a 0.22 μm filter, and stored at -20 °C in the dark after aliquoting.

[0081] (4) GelMA solution containing 100 μmol / L HT: 1 μL of sterile 100 mmol / L HT was added to 10 mL of 10% GelMA, and mixed to prepare GelMA with a final concentration of 100 μmol / L HT.

[0082] (5) HA solution containing 10 mg / mL LiH: 20 mg / mL LiH was added to HA to a final concentration of 10 mg / mL.

[0083] 2) LiH / HT@HA-GelMA MN synthesis

[0084] (1) The microneedle mold was placed in a biological safety cabinet and sterilized with ultraviolet and ozone for 1 h.

[0085] (2) 500 μL of GelMA solution was slowly added along the side wall of the mold using a pipette until the entire liquid evenly covered the surface of the mold.

[0086] (3) The mold was clamped and placed in a vacuum debubbling machine, and the liquid was filled into the needle tip by vacuum negative pressure, while the gas floated on the surface to form bubbles.

[0087] (4) The surface bubbles were removed using a 200 μL pipette, and the liquid was filled again. The mold was then placed back in the debubbling machine.

[0088] (5) Steps (3) and (4) were repeated until there were no obvious bubbles on the surface of the liquid after vacuum suction.

[0089] (6) The surface GelMA solution was scraped off the mold, leaving only the GelMA solution in the needle tip cavity.

[0090] (7) The mold was placed in a constant-temperature drying oven at 37 °C for 2 h, and photocured for 15 s.

[0091] (8) 500 μL of HA solution was slowly added along the side wall of the mold using a pipette until the liquid evenly covered the entire surface of the mold.

[0092] (9) Steps (3) and (4) were repeated until there were no obvious bubbles on the surface.

[0093] (10) The mold was placed in a constant-temperature drying oven at 37 °C for 1 h, and when the liquid surface was slightly concave, the HA solution was added again to level the surface of the mold.

[0094] (11) Again, blow-drying for 2 h, and then demolding after the microneedles are completely dried for standby.

[0095] Experimental Example 1, Morphological Characteristics of LiH / HT@HA-GelMA MN

[0096] After successfully preparing LiH / HT@HA-GelMA MN by 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, and the LiH / HT@HA-GelMA MN presented a light yellow appearance after drug loading. After magnification by a digital microscope, it can be seen that the needle bodies are uniformly and neatly distributed, and the sizes are consistent Figure 2 . In the SEM results, the conical needle bodies can be seen, and the needle tips are sharp. Compared with the blank MN, drug loading does not change the morphology of the microneedles Figure 3 . The confocal scanning results show that the LiH / HT@HA-GelMA MN needle spacing is 552.52 μm, and the needle tip height is 576.19 μm Figure 4 .

[0097] The above results show that we successfully prepared LiH / HT@HA-GelMA MN patches with sharp needle tips and sufficient length of needle bodies, providing the necessary microneedle structure for transdermal drug delivery. In order to further confirm the double-layer morphology of LiH / HT@HA-GelMA MN, FITC was used to label the GelMA layer with green fluorescence, and rhodamine B was used to label the HA layer with red fluorescence. The results show that LiH / HT@HA-GelMA MN has uniformly distributed fluorescence signals, which indicates that the drug can be uniformly distributed in the needle body, and the inner layer of the needle body presents green color and the outer layer presents red color, indicating that LiH / HT@HA-GelMA MN has a double-layer microneedle morphology Figure 5 .

[0098] Experimental Example 2, Performance Detection of LiH / HT@HA-GelMA MN

[0099] I. Mechanical Properties of LiH / HT@HA-GelMA MN

[0100] In order to detect whether LiH / HT@HA-GelMA MN has the ability to penetrate the skin, compression experiments were first conducted 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 .

[0101] The results show that the stress-displacement curves of the two groups present continuous and smooth characteristics in the deformation range of 0-600 mm, which indicates that the needle body has not been broken during this period. Under the same stress, the displacement amount of the needle tip of LiH / HT@HA-GelMA MN is smaller than that of HA-GelMA MN, indicating that the mechanical property of LiH / HT@HA-GelMA MN is better. At the same time, the results show that the needle tip of both HA-GelMA MN and LiH / HT@HA-GelMA MN can withstand strong compression force, and both have the mechanical properties of piercing the skin. The needle tip of LiH / HT@HA-GelMA MN of the application can withstand 0.35 N / needle, which can ensure effective drug delivery.

[0102] II. Evaluation of skin penetration ability of LiH / HT@HA-GelMA MN

[0103] 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 After the insertion of LiH / HT@HA-GelMA MN, the skin surface presented a neat array of microneedle array morphology, and the combination rate of the ponceau dye with the damaged skin 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 deliver drugs to the deep part, and the microneedle reached the dermis layer, accurately targeting the aging cells.

[0104] III. Evaluation of drug release of LiH / HT@HA-GelMA MN

[0105] Next, we detected the drug release performance. First, the release of LiH in LiH / HT@HA-GelMA MN was quantitatively evaluated. 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 of it 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 the effect of rapid analgesia. The release of >80% in 30 min indicates that the analgesia time requirement is met. According to the OD value of HT standard solution at λ280, the HT standard curve was drawn (Fig. 3A). According to the standard curve, the HT release curve at different time points was drawn, and the results showed that LiH / HT@HA-GelMA MN could release 80% of HT within 20 min, and all of it was released within 30 min (Fig. 3B). Figure 9 ​The release curve results of HT show that the release rate of HT in LiH / HT@HA-GelMA MN is faster in the first 12 h, and basically reaches stability after 24 h Figure 9 The 24 h sustained release of HT conforms to the Higuchi model (diffusion-controlled release), thereby maintaining long-term anti-aging effects.

[0106] IV. Evaluation of the dissolution performance of LiH / HT@HA-GelMA MN

[0107] 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 in the needle body at different time points were photographed Figure 10 ).

[0108] The results show that the needle tip began to blunt after 15 s of contact with the gel, and the needle body became wider. At 30 s, the needle body became shorter due to the dissolution of the outer HA, but the needle body and base became further widened due to the swelling of GelMA. At 1 min, the normal shape of the needle body was lost, and part of the swollen needle body could be seen on the base surface. At 2 min and 3 min, most of the microneedle bodies were not visible. The outer HA dissolved at 30 s, and the inner GelMA slowly swelled at 3 min, proving the spatiotemporal sequential release visualization.

[0109] Example 3: Effect and mechanism of LiH / HT@HA-GelMA MN in promoting wound healing in elderly

[0110] I. LiH / HT@HA-GelMA MN improves wound healing in elderly mice

[0111] 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 elderly mice. 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.

[0112] The method for establishing a full-thickness skin defect model is as follows:

[0113] 1) Anesthetize the mice using a gas anesthesia machine, and perform the hair removal operation on the mice using the above method.

[0114] 2) After disinfecting the back skin, use a biopsy needle to remove the full-thickness skin on the back.

[0115] 3) After fixing the photographing height, record the initial area of the wound for each mouse.

[0116] 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 loading), and the LiH / HT@HA-GelMA MN group was given the double-layer MN of the application.

[0117] 5) After anesthesia, the mice were individually caged and photographed at 3, 7 and 14 days after surgery, and the wound area was calculated using the formula Wwound area / Wday 0 area x 100%.

[0118] 6) At 14 days after surgery, the wound and surrounding normal tissue were collected and immersed in fixative for subsequent pathological evaluation.

[0119] The results are shown in Figure 11 , the wound contraction rate of the LiH / HT@HA-GelMA MN group was the fastest, and the scab natural shedding time was the earliest. Although the wound area of the HT / LiH injection group and the HA-GelMA MN group was smaller than that of the LiH / HT@HA-GelMA MN group, the wound healing rate was also improved compared with the Control group, indicating that HA-GelMA MN and HT / LiH themselves also had a certain degree of effect on promoting wound healing.

[0120] 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 Figure 12 A), and there were significant statistical differences in the wound area of each group compared with the LiH / HT@HA-GelMA MN group Figure 12 B).

[0121] At 14 days 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 epidermal layer, and the area of scar tissue was smaller, and the area with skin appendages such as hair follicles was larger. Although the wounds in the other groups were also closed, the area of the tissue in the repair period was larger, indicating that the functional reconstruction was not yet complete Figure 13 .

[0122] The deposition of collagen in the wound healing area is closely related to the quality of skin healing. The deposition of collagen protein is beneficial to the remodeling of the dermal layer structure of the skin and the restoration of normal skin tension and elasticity. Masson staining presents a deep blue color, indicating that the collagen fibers have a high density. The collagen fibers in the LiH / HT@HA-GelMA MN group are arranged in an orderly and regular manner, and the blue staining degree is deeper. The staining color of the Control group is lighter, and the collagen fibers are 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 effects.

[0123] II. LiH / HT@HA-GelMA MN promotes hair regeneration in the wound area

[0124] Hair follicles, as part of the skin appendages, exist in the dermis and even deeper subcutaneous tissue. The regeneration of hair follicles in the wound area indicates that the wound repair not only completes reepithelialization, but also restores and rebuilds the dermis and even subcutaneous tissue. This fully demonstrates 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. α-SMA is not only a marker of fibroblasts, but also a temporary expression of hair papilla cells during the growth period of hair follicles, wound repair, hair follicle regeneration, or external mechanical stimulation. Therefore, SOX2 and α-SMA double staining positive represents that the hair papilla cells are in the regeneration and repair period. Ki-67 is a cell proliferation marker.

[0125] As shown in Figure 15 , the cells positive for SOX2, α-SMA, and Ki-67 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.

[0126] III. LiH / HT@HA-GelMA MN improves wound healing in photoaged mice

[0127] To verify the effectiveness of LiH / HT@HA-GelMA MN under different aging mechanisms, a photoaged mouse model was constructed. After the mice received 8 weeks of UVA and UVB irradiation, the skin on the back of the mice was taken for IF staining.

[0128] The results show that the expression of p21 and p53 proteins in photoaged mice increases, proving that the induced mouse skin presents the characteristics of aged skin. Figure 16In the middle A-B). Subsequently, the full-thickness skin defect model was established on the back of mice, and the wound area was recorded by taking pictures on day 0, 3, 7 and 14. The results showed that from the 7th day, the wound area showed a gap between each group, and the LiH / HT@HA-GelMA MN group had the smallest wound area, and the scab skin of the LiH / HT@HA-GelMA MN group fell off faster on the 14th day Figure 17 ].

[0129] In Figure 18 , it can be seen that the wound area of each group is reduced over time, but the Control group still has thick and heavy scab skin and a larger range, and the LiH / HT@HA-GelMA MN group has a large area of natural scab skin shedding, and the smallest area of residual scab skin, with significant differences in statistical data from other groups.

[0130] Next, the HE staining results show that the epidermal continuity of the LiH / HT@HA-GelMA MN group is basically restored, and the re-epithelialization is relatively complete, with multiple KCs covering the wound, while the epithelial integrity in the Control group has not fully recovered, with an unhealed area under the scab skin Figure 19 ). In the Masson staining results, the dermal layer of the LiH / HT@HA-GelMA MN group has an increase in blue collagen fibers, and the arrangement of collagen fibers is parallel, tending to be regular, with a higher density, gradually approaching normal skin tissue. In contrast, in the Control group, there is an uncolored area in the central region of the wound, and the arrangement of collagen fibers around the wound is also relatively loose Figure 20 ].

[0131] In addition, the CD31 IHC staining results show that the number of CD31 positive cells as a blood vessel marker is higher in the HT / LiHinjection group and the LiH / HT@HA-GelMA MN group than in the Control group and the HA-GelMA MN group, indicating that HT / LiH has 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 is superior to the other three groups Figure 21 ].

[0132] Four, the mechanism of LiH / HT@HA-GelMA MN in promoting wound healing

[0133] IF staining was performed on the 14th day specimens of the aged mice wounds, CK15 is a marker of KCs. The results showed that compared with the Control group and the HA-GelMA MN group, the number of SIRT4 positive cells in the KCs of the HT / LiH injection group and the LiH / HT@HA-GelMA MN group increased, which indicated that HT could also increase the expression of SIRT4 in KCs at the mouse level, and microneedle administration was better than simple injection (Fig. 6). Figure 22 ).

[0134] To detect the ferroptosis phenomenon in the aged wound tissue, we selected the ferroptosis protective factor GPX4 and the ferroptosis promoting factor ACSL4 for detection. The results showed that in the CK15 positive KCs of the Control group, ACSL4, which could enhance the generation of lipid peroxide, was more expressed, while antioxidant enzyme GPX4, which could reduce the occurrence of lipid peroxide reaction, was less expressed. While in the LiH / HT@HA-GelMA MN group, the opposite trend could be observed, the green ACSL4 expression decreased, and the pink GPX4 expression increased (Fig. 7). Figure 23 The above detection results were consistent with our results in cell experiments.

[0135] V. Biological safety evaluation of LiH / HT@HA-GelMA MN

[0136] HA itself is a component of living organisms, and has high biological safety. GelMA is also widely used in transdermal drug delivery systems due to its good biocompatibility and biological safety. In order to verify the biological safety of microneedles, we took the main organs of the Control group and the LiH / HT@HA-GelMA MN group mice after modeling for 14 days, namely heart, liver, spleen, lung and kidney, and performed HE staining.

[0137] The results showed that there was no significant difference in the HE staining results of the main organs of the mice in the two groups, which proved that LiH / HT@HA-GelMA MN had no obvious toxicity to the mice and had high biological safety (Fig. 8). Figure 24 ).

[0138] In summary, the above part of the data list is shown as follows:

[0139] Table 1

[0140]

[0141] The above data show that:

[0142] (1) LiH / HT@HA-GelMA MN group healing rate 89% vs HT / LiH injection group 63%, microneedle delivery system breaks through the transdermal efficiency bottleneck (<5%) of traditional injection therapy, and realizes efficient drug delivery;

[0143] (2) Hair follicle regeneration number 8.7±1.3 vs injection group 5.3±0.9, first time to realize dermal-epidermal synergistic regeneration of old wound surface (natural healing only 3.2±0.8), unexpected effect of 64% increase in hair follicle regeneration (exceeding theoretical superposition value) is produced, proving that synergistic effect is not caused by simple change of dosage form.

[0144] Finally, it should be noted that the terms "comprising", "including", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements, but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0145] 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 cover all the preferred embodiments and all the changes and modifications falling within the scope of the embodiments of the present application.

[0146] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present application without departing from the spirit and scope of the embodiments of the present application. Thus, if these modifications and variations of the embodiments of the present application fall within the scope of the claims of the embodiments of the present application and their equivalent technologies, the embodiments of the present application also intend to include these modifications and variations.

Claims

1. A dual-layer microneedle patch, characterized by, The double-layer microneedle patch comprises: Outer layer: hyaluronic acid loaded with lidocaine hydrochloride; Inner layer: methacrylated gelatin loaded with hydroxytyrosol; 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 in a PBS solution containing LAP photoinitiator to prepare a GelMA solution; add hydroxytyrosol 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 in a 30% hyaluronic acid 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 hydroxytyrosol is 50-100 μM; The concentration of lidocaine hydrochloride is 5-20 mg / mL; The concentration of the hyaluronic acid solution is 25-35%; The concentration of the GelMA solution is 8-12%; The crosslinking density of the methacrylated gelatin is 15-20%, formed by 0.2-0.3% lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator through ultraviolet curing.

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

3. Use of the double-layer microneedle patch of any one of claims 1-2 in the preparation of a drug for promoting the healing of old wounds.

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