Autologous dermal nitroglycerin sustained-release microneedle patch as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to treatment of skin flap avascular necrosis. The invention discloses a preparation method of an autologous dermal nitroglycerin sustained-release microneedle patch and application of the autologous dermal nitroglycerin sustained-release microneedle patch in prevention and treatment of skin flap avascular necrosis. The autologous dermal nitroglycerin sustained-release microneedle patch comprises a polyacrylic acid polymer base and a conical microneedle arranged on one side of the base, the conical microneedle is prepared from a mixture containing nitroglycerin, sodium hyaluronate and autologous nano dermis through a freeze-drying process. According to the autologous dermal nitroglycerin sustained-release microneedle, the histocompatibility of the microneedle is improved through application of autologous dermis, targeted and sustained release of nitroglycerin in a skin flap area is achieved through the microneedle, and skin flap avascular necrosis can be effectively prevented and treated; the method has remarkable advantages and good application prospects in the aspects of improving local drug concentration, improving the survival rate of skin flaps, reducing side effects and simplifying the treatment process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a microneedle patch for nitroglycerin sustained release prepared from autologous dermis tissue, a preparation method thereof and application thereof in preventing and treating skin flap ischemic necrosis. BACKGROUND

[0002] Skin flap transplantation is a common technical means for repairing large-area tissue defects and organ reconstruction in surgical operations. However, in the process of skin flap design and transfer, the distal end of the skin flap is prone to microcirculation disorder, and then tissue ischemia and necrosis due to the influence of various factors such as the length-width ratio of the skin flap, blood supply source, postoperative infection or hematoma. Skin flap ischemic necrosis is one of the most common complications after skin flap transplantation, which seriously affects the operation effect and brings great pain to the patient.

[0003] In order to prevent and treat skin flap ischemic necrosis, blood vessel dilating drugs such as nitroglycerin are often used locally to improve the blood perfusion of the skin flap. In the prior art, nitroglycerin is often prepared into an ointment or a gel for local application. This kind of drug delivery mode has significant technical defects: because the stratum corneum of human skin is a dense physical barrier, it will seriously hinder the penetration of macromolecular drugs such as nitroglycerin, resulting in low transdermal absorption efficiency and low bioavailability.

[0004] In order to solve the problem of low efficiency of traditional transdermal drug delivery, a microneedle drug delivery system has been developed. The microneedle array can penetrate the stratum corneum of the skin in a non-invasive or minimally invasive way, build a drug delivery channel in the skin, and directly deliver the active ingredients to the epidermis or dermis layer, thereby bypassing the stratum corneum barrier, significantly improving the local bioavailability of the drug, and reducing the risk of systemic side effects.

[0005] At present, the materials used to prepare soluble microneedles are mainly biodegradable polymer materials such as hyaluronic acid (HA) and polyvinyl alcohol (PVA). However, these materials are essentially exogenous materials, which are different from human tissues in composition and structure, and cannot completely avoid potential immune reactions or biological incompatibility problems. In addition, in surgical operations such as skin flap transplantation, a part of the healthy autologous skin tissue removed will inevitably be generated, which is usually treated as medical waste; the dermis layer of which is rich in collagen and elastin, has perfect biocompatibility and excellent mechanical properties, and is an ideal natural biological material. The prior art fails to effectively utilize this valuable autologous biological resource, resulting in waste. SUMMARY

[0006] To address the problems existing in the prior art, this invention provides an autologous dermal nitroglycerin sustained-release microneedle patch, its preparation method, and its application in the prevention and treatment of flap ischemia and necrosis. This microneedle patch can overcome the barrier effect of the stratum corneum of the skin and achieve efficient local drug delivery. It can also utilize autologous tissue physical modification as a carrier material to ensure optimal biocompatibility, thus providing a safer and more effective technical solution for the prevention and treatment of flap ischemia and necrosis.

[0007] This invention provides an autologous dermal nitroglycerin sustained-release microneedle patch, comprising a microneedle array and an array base, wherein the array base is a polyacrylic polymer backing layer, and the microneedle array is integrally formed on the polyacrylic polymer backing layer; the raw material of the microneedle array is a mixture of nano-dermal, sodium hyaluronate and nitroglycerin in a mass ratio of 1000:50:1.

[0008] The nano-dermis is a precipitate obtained by repeatedly emulsifying fresh autologous soft tissue after rinsing it with sterile PBS solution, cutting it into small pieces, and then centrifuging it.

[0009] The preferred technical solution of the present invention is as follows: the height of the microneedles in the microneedle array is 700-750 μm, the bottom side length is 320-340 μm, and the needle tip spacing is 680 μm.

[0010] The preferred technical solution of the present invention is that the fresh autologous soft tissue is derived from the patient's own skin, fat or any other tissue rich in extracellular matrix.

[0011] The preferred technical solution of the present invention is that the microneedle array is a 12x12 array.

[0012] This invention also provides a method for preparing an autologous dermal nitroglycerin sustained-release microneedle patch, specifically including the following steps:

[0013] S1. Fresh autologous soft tissue was rinsed with sterile PBS solution, cut into small pieces, and emulsified by repeatedly pushing through filters with pore sizes of 120μm, 100μm and 80μm 20 to 30 times. Finally, it was centrifuged to obtain nano-dermal sebum.

[0014] S2. Take the nano-dermis prepared in step S1, sodium hyaluronate powder, and tablet powder containing nitroglycerin active ingredient and mix them evenly at a mass ratio of 1000:50:100 to obtain microneedle array raw material; wherein, the tablet powder containing nitroglycerin active ingredient is obtained by crushing tablets containing 1% nitroglycerin, and 50mg of tablet powder is added to every 0.5g of nano-dermis;

[0015] S3 Place the microneedle array material from step S2 into a PDMS microneedle mold, the bottom of which is provided with a metal pad to control the thickness of the array base;

[0016] S4. Cover the top of the mold with a polyacrylic acid polymer backing layer, and then centrifuge the assembled mold at a speed of 3000-3500 rpm for 3-5 minutes to ensure that the mixture fully fills the mold cavity and is tightly bonded to the backing layer;

[0017] S5. After centrifugation, freeze-dry the mold under vacuum at -40℃ to -50℃ for 8 to 10 hours, then demold to obtain autologous dermal nitroglycerin sustained-release microneedle patch.

[0018] The preferred technical solution of the present invention is as follows: the autologous soft tissue in step S1 is derived from healthy autologous soft tissue removed during the patient's surgery, including skin, fat or any other tissue rich in extracellular matrix;

[0019] Fresh autologous soft tissue is rinsed 3-5 times with sterile PBS solution to remove residual blood and impurities. After rinsing, excess liquid is filtered out through sterile gauze, and the tissue is cut into fragments suitable for processing.

[0020] The emulsion obtained after emulsifying autologous dermal tissue is centrifuged at 10,000–12,000 rpm for 5–8 minutes to remove the upper layer of oil and liquid. The collected precipitate is the nano-dermis.

[0021] The preferred technical solution of the present invention is as follows: In step S2, the tablet containing 1% nitroglycerin contains growth factors, antimicrobial peptides, anti-inflammatory drugs or local anesthetics in addition to nitroglycerin; in step S2, the thickness of the metal pad is precisely 2.0 ± 0.1 mm; the tip of the PDMS microneedle mold is designed to be conical or pyramidal, with a height of 700-750 μm and a bottom diameter of 320-340 μm.

[0022] The preferred technical solution of the present invention is as follows: In step S5, before vacuum freeze drying, the mold is pre-frozen at 4°C for 2-4 hours; the vacuum freeze drying adopts a programmed gradient cooling: first, the temperature is lowered to -40°C at a rate of 1°C / min and held for 2 hours; then, the temperature is lowered to -50°C at a rate of 0.5°C / min and held for 1 hour; finally, the main drying is carried out at -50°C for 10 hours under a vacuum of <20 Pa.

[0023] The present invention also provides the application of autologous dermal nitroglycerin sustained-release microneedle patch in the preparation of materials for preventing ischemic necrosis during flap design and repair.

[0024] A further technical solution of the present invention is to use the autologous dermal nitroglycerin sustained-release microneedle patch as a local drug delivery system for skin flap ischemia and necrosis.

[0025] Regarding the raw materials in the conical microneedles of this application, the core pathophysiological process of flap ischemia and necrosis is local microcirculatory disturbance, leading to tissue ischemia and hypoxia. Nitroglycerin, used in this invention, is a potent vasodilator. Its mechanism of action involves releasing nitric oxide (NO) in vivo, relaxing vascular smooth muscle, thereby dilating capillaries in the flap area, significantly increasing blood flow and oxygen supply, and improving tissue perfusion. This invention selects nitroglycerin as the core active pharmaceutical ingredient to directly intervene in the root cause of flap ischemia. In a preferred embodiment of this invention, the effective dose of nitroglycerin per microneedle patch is 0.5 mg. This dose is based on clinically effective concentration considerations, and the microneedle delivery system can achieve a highly effective local therapeutic effect with a total dose far lower than traditional drug delivery methods.

[0026] The nano-dermis in this invention is a functionalized biomaterial made from autologous skin tissue, which was originally considered medical waste during surgery, through physical methods; using autologous dermal tissue has dual advantages:

[0027] (1) As a structural material: The dermis is rich in collagen and elastin, and has excellent natural mechanical strength and toughness. It is an ideal matrix material for constructing microneedle arrays, which can ensure that the microneedles have sufficient hardness to penetrate the stratum corneum of the skin.

[0028] (2) As a bioactive scaffold: Because it originates from the patient's own tissue, the nanodermal matrix has perfect biocompatibility, fundamentally eliminating the risk of immune rejection and foreign body reaction; more importantly, the dermal matrix itself can serve as an active scaffold that promotes tissue repair, providing a favorable microenvironment for the adhesion, proliferation, and migration of fibroblasts, thereby actively participating in and accelerating the healing and integration process of the flap. In a preferred embodiment of the present invention, the amount of nanodermal matrix used is 0.5g, which is sufficient to construct a complete microneedle array with the required mechanical properties.

[0029] Sodium hyaluronate, used in this invention, is a natural polysaccharide widely found in human connective tissue, possessing excellent biocompatibility, biodegradability, and moisturizing properties. In this invention, sodium hyaluronate primarily functions as follows:

[0030] (1) As an adhesive and molding agent: After mixing its powder with nano-dermal and nitroglycerin powder, it can play a role in bonding and filling in the subsequent centrifugal molding step, ensuring that the mixture can be uniformly filled into the tiny cavity of the microneedle mold to form a regular microneedle.

[0031] (2) As a soluble matrix: After the microneedles are inserted into the skin, sodium hyaluronate will gradually dissolve, thereby smoothly releasing the loaded nitroglycerin and achieving sustained drug release. In a preferred embodiment of the present invention, the amount of sodium hyaluronate used is 50 mg, which is preferred to obtain the best physical properties suitable for mold filling and centrifugal molding.

[0032] The present invention has the following beneficial effects:

[0033] 1. This invention combines the vasodilatory effect of nitroglycerin with the tissue repair-promoting effect of autologous dermis; microneedles directly deliver nitroglycerin to the subcutaneous layer, effectively improving microcirculation; at the same time, the autologous dermal scaffold provides an ideal microenvironment for tissue regeneration, promoting the self-healing of the flap.

[0034] 2. This invention greatly improves the local bioavailability of nitroglycerin by bypassing the stratum corneum barrier with microneedles, and reduces the dosage and risk of systemic side effects; using autologous dermis as the core material completely avoids the immune rejection reaction that may be caused by exogenous materials, and has unparalleled safety and biocompatibility.

[0035] 3. This invention transforms discarded autologous tissue during surgery into a high-performance drug delivery system, realizing the regeneration and utilization of medical resources. Its preparation method mainly relies on physical processes, which is expected to enable immediate preparation and use during surgery, providing a new, efficient and convenient solution for the prevention and treatment of flap ischemia and necrosis in clinical practice. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 Schematic diagram of transdermal microneedle design;

[0038] Figure 2 This is a flowchart of the preparation method of the autologous dermal nitroglycerin sustained-release microneedle patch in Example 1 of the present invention;

[0039] Figure 3 The image shows a scanning electron microscope (SEM) image of the backing layer and the autologous dermal nitroglycerin sustained-release microneedle patch in the embodiment.

[0040] Figure 4 This is a microscope image of the polyacrylic acid polymer backing layer in the example;

[0041] Figure 5This is a microscope image of the autologous dermal nitroglycerin sustained-release microneedle patch in the example;

[0042] Figure 6 These are experimental images of autologous dermal nitroglycerin sustained-release microneedle patch puncture. The image numbers correspond to the layer numbers.

[0043] Figure 7 Drug release curve of autologous dermal nitroglycerin sustained-release microneedle patch;

[0044] Figure 8 A curve comparing the survival rates of random skin flaps from different groups of SD rats;

[0045] Figure 9 A comparison image of skin flap survival from random skin flaps in different groups of SD rats;

[0046] Figure 10 Image showing HE staining results of random skin flaps from SD rats;

[0047] Figure 11 This image shows the results of immunostaining of random skin flaps from SD rats. Detailed Implementation

[0048] The present invention will now be described in detail with reference to embodiments, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1: A method for preparing autologous dermal nitroglycerin sustained-release microneedle patches using human-derived dermal skin as raw material, the process of which is as follows: Figure 2 As shown, the specific preparation process is as follows:

[0050] (1) Preparation of nano-dermis;

[0051] a. Take 50 mL of fresh autologous dermal tissue, aliquot it into 50 mL centrifuge tubes, and rinse with sterile PBS solution 3 to 5 times for 10 minutes each time to remove residual blood and impurities. After rinsing, filter out excess liquid through sterile gauze and cut the dermal tissue into appropriately processed fragments for later use.

[0052] b. Place the shredded dermal tissue from step a into a mechanical emulsification device and pass it through filters with pore sizes of 120μm, 100μm and 80μm in sequence. Repeat the pushing motion 20 to 30 times to perform mechanical emulsification and obtain a uniform emulsion.

[0053] c. Centrifuge the emulsion obtained in step b at 12,000 rpm for 5 minutes to remove the upper layer of grease and liquid, and collect the precipitate, which is the nano-dermis.

[0054] (2) Preparation of microneedle patches;

[0055] a. Place 0.5g of the nano-dermis prepared in step (1), 25mg of sodium hyaluronate powder and 50mg of the powder containing 1% nitroglycerin active ingredient in a sterile container and mix thoroughly.

[0056] b. Fill the PDMS microneedle mold cavity pre-placed with a 2 mm thick metal gasket into the mixture obtained in step a;

[0057] c. Cover the filled mixture surface with a polyacrylic acid polymer backing layer;

[0058] d. Centrifuge the assembled mold at 3500 rpm for 5 minutes to ensure the mixture fully fills the mold cavity and bonds tightly with the backing layer;

[0059] e. Place the mold centrifuged in step d at -50°C for vacuum freeze-drying for 10 hours;

[0060] f. After freeze-drying, carefully demold to obtain the finished autologous dermal nitroglycerin sustained-release microneedle patch; the microneedles are designed with a height of 720μm and a bottom diameter of 330μm, aiming to effectively penetrate the stratum corneum without causing significant pain.

[0061] Comparative Example 1: Compared with Example 1, the difference is that no powder containing 1% nitroglycerin was added in step (2)a; specifically, 0.5g of the nano-dermis prepared in step (1) and 25mg of sodium hyaluronate powder were placed in a sterile container and mixed thoroughly. The other steps in Comparative Example 1 were the same as in Example 1, and an autologous dermal microneedle patch without nitroglycerin was finally prepared.

[0062] Example 2: In vitro puncture experiment of autologous dermal nitroglycerin sustained-release microneedle patch;

[0063] To evaluate the performance of autologous dermal nitroglycerin sustained-release microneedle patches during skin puncture, the Parafilm M® insertion model developed by Larrañeta et al. was used.

[0064] (1) The autologous dermal nitroglycerin sustained-release microneedle array prepared in Example 1 was inserted into the eight-layer folded Parafilm M® film using a force of approximately 40 N; the insertion process ensured that the microneedle array penetrated the film uniformly, simulating the puncture of microneedles on actual skin.

[0065] (2) After insertion, carefully remove the microneedle array from the folded Parafilm M® film and unfold the film for subsequent analysis. Use an upright microscope to evaluate the pores in each layer of Parafilm M® and record the number of pores and their uniformity in each layer to ensure that the insertion depth and distribution uniformity of the microneedles meet expectations.

[0066] The experimental results are as follows Figure 5 As shown, Figure 5 This shows the percentage of pores formed by the autologous dermal nitroglycerin sustained-release microneedle patch on the first five layers of Parafilm M®. Figure 5 The image numbers correspond to the layer numbers; the average thickness of the Parafilm M® layer is 126 ± 7 μm, and the insertion depth of the microneedles reaches 504 μm out of the total height of 720 μm, accounting for approximately 70% of the microneedle tip height. This result indicates that autologous dermal nitroglycerin sustained-release microneedle patches can effectively penetrate the stratum corneum, reach the dermis, and exert their therapeutic effects.

[0067] Example 3: Test of nitroglycerin release from autologous dermal nitroglycerin sustained-release microneedle patch;

[0068] (1) Take the autologous dermal nitroglycerin sustained-release microneedle patch prepared in Example 1, transfer it to a centrifuge tube containing 10 mL PBS, and incubate it at a constant temperature of 37°C.

[0069] (2) At different time points (1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours), 1 mL of PBS solution was taken from each centrifuge tube and immediately replenished with an equal amount of 1 mL of fresh PBS solution;

[0070] (3) The PBS solution taken at each time point was analyzed by high performance liquid chromatography (HPLC) to test its nitroglycerin concentration. The specific parameters of HPLC analysis are as follows: mobile phase: acetonitrile:water (50:50, volume ratio), detection wavelength: 215nm, injection volume: 10 μL;

[0071] (4) The average concentration of nitroglycerin at each time point is calculated by averaging the data from the three parallel groups at each time point.

[0072] Test results are as follows Figure 6 As shown, Figure 6 The nitroglycerin release curve of the autologous dermal nitroglycerin sustained-release microneedle patch, plotted by HPLC analysis, is shown, and its release rate and sustained-release effect are evaluated. The test results indicate that the autologous dermal nitroglycerin sustained-release microneedle patch can continuously release nitroglycerin within a specified time, exhibiting good sustained-release characteristics.

[0073] Example 4: Application of autologous dermal nitroglycerin sustained-release microneedle patch in an ischemic flap animal model;

[0074] (1) Twelve 8-week-old female SD rats were selected, with a weight ranging from 220g to 250g.

[0075] (2) After successful anesthesia with 500 μL / rat of 3% sodium pentobarbital solution, the hair on the back was removed, and a rectangular skin flap of about 1 cm × 3 cm was designed on the back. Strict disinfection was performed, and the pedicle was on the side of the rat's head. The rat skin was cut along the designed line and separated to the deep fascia. The flap was sutured in situ with 5-0 silk thread. After the operation, the flap was fixed with a transparent dressing. The aseptic principle was strictly followed during the operation.

[0076] (3) Twelve SD rats were divided into four groups of three each: Group A was the control group, Group B was the autologous dermal microneedle patch group containing nitroglycerin, Group C was the nitroglycerin cream treatment group, and Group D was the autologous dermal microneedle patch group without nitroglycerin. Group B received the autologous dermal microneedle patch containing nitroglycerin prepared in Example 1 immediately after the operation, Group C received 1% nitroglycerin cream immediately after the operation, and Group D received the autologous dermal microneedle patch without nitroglycerin prepared in Comparative Example 1 immediately after the operation. The cream coverage area was the same as that of the microneedle patch.

[0077] (4) On postoperative days 1, 3, 7, and 14, the survival of the dorsal skin flaps in SD rats was observed. The survival status was determined based on the flap's color and temperature. Photos were taken to differentiate necrotic areas, and ImageJ software was used to calculate the area of ​​ischemic necrosis and survival of the flaps. The calculation was based on the formula (area of ​​ischemic necrosis / total area × 100%) = flap ischemic necrosis rate. The results are as follows: Figure 8 and Figure 9 As shown, all groups of flaps had varying degrees of ischemia, with a clear boundary between the ischemic and viable areas. The microneedle group showed a significant improvement in ischemia compared to the control group. On days 7 and 14, except for the microneedle patch group, the flaps in the other groups developed scabs and necrosis. The necrotic flaps turned black, with a clear boundary between them and the viable areas.

[0078] (5) On the 14th day after surgery, the rats were euthanized by vertebral dislocation. A 0.2cm × 0.5cm full-thickness skin flap was taken from the center of the junction between the ischemic necrosis and the surviving flap. The skin flap was fixed with 4% paraformaldehyde and then dehydrated and embedded. The paraffin block was cut into 5μm sections. Masson staining and immunohistochemical staining were performed. Masson staining was used to observe the skin tissue structure, collagen fiber proliferation and microneedle puncture depth. Immunohistochemical staining was used to capture the immunofluorescence images of CD31 and Collagen I under a fluorescence microscope and the relative fluorescence intensity was calculated.

[0079] HE staining results of the skin flap tissue are as follows Figure 9 As shown,Figure 9 In Figure a, the microneedles have been inserted into the dermis. Figures b and c show that the tissue structure of the control group is more disordered, with reduced vascular occlusion and thinner tissue thickness. In the microneedle group, collagen fibers grow neatly and the striations are clearer.

[0080] Immunohistochemical staining results of skin flap tissue are as follows Figure 10 As shown, Figure 10 In Figures a and c, the control group showed a significant reduction in blood vessels and thinner collagen at the flap site; in Figures b and d, after using autologous dermal microneedle patches containing nitroglycerin, the number of blood vessels in the random flaps was similar to that in normal skin, and there was no significant difference in collagen thickness.

[0081] Based on the summary of different experimental results, the following conclusions were drawn: In ordinary flap transplantation, the flap survival rate and angiogenesis ability are low. However, flaps treated with autologous dermal nitroglycerin sustained-release microneedle patches have improved angiogenesis ability and significantly improved flap survival rate. This indicates that autologous dermal nitroglycerin sustained-release microneedle patches can treat flap death caused by ischemia and hypoxia in flaps / long flaps.

Claims

1. An autologous dermal nitroglycerin sustained-release microneedle patch, comprising a microneedle array and an array base, characterized in that: The array base is a polyacrylic acid polymer backing layer, and the microneedle array is integrally formed on the polyacrylic acid polymer backing layer; the raw material of the microneedle array is made by mixing nano-dermis, sodium hyaluronate and nitroglycerin in a mass ratio of 1000:50:

1. The nano-dermis is a precipitate obtained by repeatedly emulsifying fresh autologous soft tissue after rinsing it with sterile PBS solution, cutting it into small pieces, and then centrifuging it.

2. The autologous dermal nitroglycerin sustained-release microneedle patch according to claim 1, characterized in that: The microneedles of the microneedle array have a height of 700–750 μm, a bottom side length of 320–340 μm, and a tip-to-tip spacing of 680 μm.

3. The autologous dermal nitroglycerin sustained-release microneedle patch according to claim 1, characterized in that: The fresh autologous soft tissue is derived from the patient's own skin, fat, or any other tissue rich in extracellular matrix.

4. The autologous dermal nitroglycerin sustained-release microneedle patch according to claim 2, characterized in that: The microneedle array is a 12x12 array.

5. A method for preparing an autologous dermal nitroglycerin sustained-release microneedle patch, characterized in that, Specifically, the following steps are included: S1. Fresh autologous soft tissue was rinsed with sterile PBS solution, cut into small pieces, and emulsified by repeatedly pushing through filters with pore sizes of 120μm, 100μm and 80μm 20 to 30 times. Finally, it was centrifuged to obtain nano-dermal sebum. S2. Take the nano-dermis prepared in step S1, sodium hyaluronate powder, and tablet powder containing nitroglycerin active ingredient and mix them evenly at a mass ratio of 1000:50:100 to obtain microneedle array raw material; wherein, the tablet powder containing nitroglycerin active ingredient is obtained by crushing tablets containing 1% nitroglycerin, and 50mg of tablet powder is added to every 0.5g of nano-dermis; S3 Place the microneedle array material from step S2 into a PDMS microneedle mold, the bottom of which is provided with a metal pad to control the thickness of the array base; S4. Cover the top of the mold with a polyacrylic acid polymer backing layer, and then centrifuge the assembled mold at a speed of 3000-3500 rpm for 3-5 minutes to ensure that the mixture fully fills the mold cavity and is tightly bonded to the backing layer; S5. After centrifugation, freeze-dry the mold under vacuum at -40℃ to -50℃ for 8 to 10 hours, then demold to obtain autologous dermal nitroglycerin sustained-release microneedle patch.

6. The method for preparing an autologous dermal nitroglycerin sustained-release microneedle patch according to claim 5, characterized in that: In step S1, the autologous soft tissue is derived from healthy autologous soft tissue removed during the patient's surgery, including skin, fat, or any other tissue rich in extracellular matrix. The fresh autologous soft tissue is rinsed 3-5 times with sterile PBS solution to remove residual blood and impurities. After rinsing, excess liquid is filtered out through sterile gauze, and the tissue is cut into appropriately processed fragments for later use. The emulsion obtained after emulsifying the autologous dermal tissue is centrifuged at 10,000-12,000 rpm for 5-8 minutes to remove the supernatant oil and liquid. The precipitate collected is the nano-dermal tissue.

7. The method for preparing an autologous dermal nitroglycerin sustained-release microneedle patch according to claim 5, characterized in that: In step S2, the tablet containing 1% nitroglycerin contains growth factors, antimicrobial peptides, anti-inflammatory drugs, or local anesthetics in addition to nitroglycerin; in step S2, the thickness of the metal pad is precisely 2.0 ± 0.1 mm; the tip of the PDMS microneedle mold is designed to be conical or pyramidal, with a height of 700–750 μm and a bottom diameter of 320–340 μm.

8. The method for preparing an autologous dermal nitroglycerin sustained-release microneedle patch according to claim 5, characterized in that: In step S5, before vacuum freeze drying, the mold is pre-frozen at 4°C for 2–4 hours; for vacuum freeze drying, a programmed gradient cooling method is used: first, the temperature is lowered to -40°C at a rate of 1°C / min and held for 2 hours; then, the temperature is lowered to -50°C at a rate of 0.5°C / min and held for 1 hour; finally, the main drying is carried out at -50°C under a vacuum of <20 Pa for 10 hours.

9. The use of an autologous dermal nitroglycerin sustained-release microneedle patch according to any one of claims 1 to 8 in the preparation of materials for preventing ischemic necrosis during flap design and repair.

10. The application of an autologous dermal nitroglycerin sustained-release microneedle patch according to claim 9, characterized in that: The autologous dermal nitroglycerin sustained-release microneedle patch was used as a drug delivery system for flap ischemia and necrosis and local tissue regeneration.

Images