Microneedle-tranexamic acid nanofiber membrane and preparation method thereof

Abstract

The invention discloses a microneedle-tranexamic acid nanofiber membrane and a preparation method thereof, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: preparing a soluble microneedle (DMN) by utilizing a hydrophilic polymer polyvinylpyrrolidone (PVP), fixing the microneedle on an electrostatic spinning receiver, and carrying out electrostatic spinning by using an electrostatic spinning solution containing tranexamic acid TA to prepare a microneedle-tranexamic acid nanofiber membrane, namely TA-PVP-DMN. The prepared microneedle-tranexamic acid nanofiber membrane is simple in process and high in epidermal permeability, meanwhile, the safety problem caused by systemic administration of tranexamic acid can be avoided, and finally the purposes of safely and effectively improving facial pigmentation and treating chloasma can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a microneedle-tranexamic acid nanofiber membrane and a preparation method thereof. Background Art

[0002] Melasma is a common acquired pigmentary disorder characterized by irregular, hyperpigmented macules or plaques, most commonly seen in women with darker skin. Current oral and injectable treatments for melasma suffer from poor clinical safety, necessitating the development of new, effective therapies.

[0003] Tranexamic acid, a drug used to treat melasma, primarily inhibits melanin synthesis by reducing the expression of vascular endothelial growth factor and endothelin-1,3, inhibiting the plasminogen / plasmin pathway, and competitively inhibiting tyrosinase. Tranexamic acid is typically used for the treatment of melasma via oral, intradermal, topical, and transdermal routes. However, oral and intradermal administration are prone to side effects such as thrombosis and pulmonary embolism. Therefore, topical application of tranexamic acid to affected areas of melasma via a transdermal patch is safer and more effective.

[0004] Electrospinning technology is an efficient, simple, and continuous method for producing nanofibers. The resulting electrospun fibers possess high surface area and porosity. The resulting electrospun membranes containing tranexamic acid can be tailored to any size, making them suitable for topical treatment. However, due to the high solubility of tranexamic acid, while it dissolves and releases rapidly, it has difficulty penetrating the fat-soluble skin, making skin penetration difficult.

[0005] Therefore, a new transdermal patch needs to be developed to address the problems of low skin permeability and systemic drug safety of tranexamic acid. Summary of the Invention

[0006] Purpose of the Invention: This invention addresses the shortcomings of existing technologies and provides a microneedle-tranexamic acid nanofiber membrane and its preparation method. This invention combines an electrospinning solution containing tranexamic acid (TA) with soluble microneedles to create a microneedle-tranexamic acid nanofiber membrane transdermal patch system. This system allows for quick penetration while simultaneously influencing melanin production and transport over a long period of time, ultimately achieving the goal of effectively treating epidermal chloasma.

[0007] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0008] The present invention provides a method for preparing a microneedle-tranexamic acid nanofiber membrane. The hydrophilic polymer polyvinylpyrrolidone (PVP) is used to prepare soluble microneedles (DMN). The soluble microneedles are fixed on an electrospinning receiver. An electrospinning solution containing tranexamic acid (TA) is used for electrospinning to prepare the microneedle-tranexamic acid nanofiber membrane, i.e., TA-PVP-DMN.

[0009] PVP, an N-vinylamide polymer, is a water-soluble polymer with excellent properties. PVP also has high mechanical strength and biocompatibility, making it commonly used in the manufacture of DMNs. When a DMN is inserted into the skin, the needle tip is dissolved by the interstitial fluid, allowing the encapsulated drug to be naturally released, achieving transdermal drug delivery.

[0010] A preferred embodiment of the present invention is that the method for preparing the microneedle-tranexamic acid nanofiber membrane comprises the following steps:

[0011] (1) Pour a polyvinylpyrrolidone (PVP) aqueous solution into a microneedle mold, solidify the microneedles, dry them, and demold them to obtain polyvinylpyrrolidone-soluble microneedles, i.e., PVP-DMN;

[0012] (2) dissolving a mixture of PVP and polycaprolactone (PCL) and tranexamic acid in hexafluoroisopropanol to obtain a uniform spinning solution;

[0013] (3) Fixing the PVP-DMN obtained in step (1) on an electrospinning receiver, using the spinning solution obtained in step (2), setting spinning parameters, performing uniaxial electrospinning, and drying to obtain the microneedle-tranexamic acid nanofiber membrane.

[0014] Further preferably, in step (1), the volume fraction of the polyvinyl pyrrolidone (PVP) aqueous solution is 30%.

[0015] The concentration of the PVP aqueous solution affects the mechanical properties of the microneedles. When the concentration is lower than 30%, the mechanical strength is weak, and when it is higher than 30%, the viscosity is high. Therefore, the present invention selects a 30% volume fraction of PVP aqueous solution to prepare the microneedles.

[0016] Further preferably, in step (1), the curing treatment is carried out by vacuum drying at 55° C. for 3 minutes under -0.095 MPa.

[0017] The mold used in the specific embodiment of the present invention has a cylindrical shape with a diameter of 2.4 cm, a height of 0.8 cm, a groove area of 16.7×16.7 cm, and a groove depth of 1.5 mm. The mold can produce microneedles in the shape of a cone with a height of 600 μm and a diameter of 300 μm. The distance between the needle tips is 700 μm, and the array is 20×20.

[0018] Furthermore, in step (2), the mass ratio of PVP to polycaprolactone (PCL) is 1-4:4-1.

[0019] The mass ratio of PVP to PCL affects the transdermal effect of the nanofiber membrane. More preferably, when the mass ratio of PVP to PCL is 1:1, the transdermal effect of the nanofiber membrane is optimal.

[0020] Furthermore, the mass volume of the PVP in hexafluoroisopropanol is 2% to 10%.

[0021] Furthermore, the mass volume of the tranexamic acid in hexafluoroisopropanol is 2% to 10%.

[0022] Furthermore, the mass volume of the tranexamic acid in hexafluoroisopropanol is 6%.

[0023] Furthermore, the spinning solution is allowed to stand and degas for 10 minutes before spinning.

[0024] Furthermore, in step (3), the electrospinning parameters are: applied voltage of 15-22 kV, receiving distance of 10-20 cm, flow rate of spinning solution of 0.3-1.2 mL / h, temperature of 20-25° C., and relative humidity of 40%-60%.

[0025] Furthermore, in step (3), the drying method is: vacuum drying at 40-60° C. for 12-24 hours.

[0026] The present invention also provides a microneedle-tranexamic acid nanofiber membrane prepared by the above preparation method.

[0027] The present invention is based on the characteristics that DMN can directionally penetrate the stratum corneum, effectively reach the dermis, and generate micron-sized mechanical channels. At the same time, the hydrophilic polymer PVP in DMN dissolves in the skin and forms a gel state at the micro-nano channels in the dermis. An electrospinning solution containing tranexamic acid TA is combined with DMN to prepare a microneedle-tranexamic acid nanofiber membrane, namely TA-PVP-DMN, which allows it to penetrate for a short time while affecting the production and transport of melanin for a long time, ultimately achieving the purpose of effectively treating epidermal chloasma.

[0028] Beneficial effects:

[0029] (1) The present invention utilizes the advantages of nanofiber membranes that can rapidly release tranexamic acid drugs and the excellent mechanical properties of DMN, combines an electrospinning solution containing tranexamic acid TA with DMN, and prepares a microneedle-tranexamic acid nanofiber membrane rapid drug release system, which solves the problem of systemic drug safety and improves the permeability of the water-soluble drug tranexamic acid in the skin.

[0030] (2) Compared with the transdermal release of a single tranexamic acid nanofiber membrane, the cumulative release rate of the microneedle-tranexamic acid nanofiber membrane formed by the electrospinning solution containing tranexamic acid TA and DMN is much higher than the cumulative release rate of the single tranexamic acid fiber membrane when it reaches equilibrium, thereby increasing the amount of drug permeable in the skin.

[0031] (3) After the DMN needle dissolves in the skin, it forms a gel state in the micro-nano drug channel, which can act as a drug reservoir, prolong the drug's action time to a certain extent, and increase the total drug penetration rate.

[0032] (4) In vitro experiments showed that all materials of the microneedle-tranexamic acid nanofiber membrane synthesized by the present invention were non-cytotoxic and had good biocompatibility.

[0033] (5) In vivo experiments showed that the microneedle-tranexamic acid nanofiber membrane system constructed by the present invention can more effectively inhibit the activity of tyrosinase, thereby inhibiting the production of melanin, and is expected to provide a new option for the clinical treatment of epidermal melasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the preparation process of the microneedle-tranexamic acid nanofiber membrane of the present invention.

[0035] Figure 2 The SEM results and diameter distribution diagram of the TA nanofiber membranes with different polymer ratios prepared in Example 5; wherein, Figure 2 a is the result of the fiber membrane with a ratio of PVP:PCL=1:4; Figure 2 b is the result graph of the fiber membrane with a ratio of PVP:PCL=1:1; Figure 2 c is the result diagram of the fiber membrane with a ratio of PVP:PCL=4:1.

[0036] Figure 3 The following are the Fourier transform infrared spectroscopy (FT-IR) results of each analyzed sample.

[0037] Figure 4 The X-ray diffraction (XRD) results of each analyzed sample are shown.

[0038] Figure 5 The water contact angle results of TA nanofiber membranes with different polymer ratios.

[0039] Figure 6 The optical microscopic image and size annotation of PVP-DMN prepared in Example 1; wherein, Figure 6 a is a single needle microscopic image of PVP-DMN; Figure 6 b is an optical microscopic image of the top view of PVP-DMN.

[0040] Figure 7This is a microscopic image of the PVP-DMN needle prepared in Example 1; wherein, Figure 7 a is a side microscopic image of the PVP-DMN needle body; Figure 7 b is a microscopic image of the overall appearance of PVP-DMN.

[0041] Figure 8 This is a microscopic image of the in vitro penetration performance of PVP-DMN prepared in Example 1; wherein, Figure 8 a is a methylene blue-stained image of the PF membrane punctured by the microneedle patch; Figure 8 b is the microscopic image of the microneedle after penetrating the PF membrane.

[0042] Figure 9 This is an optical microscopic image of TA-PVP-DMN prepared in Example 1; wherein, Figure 9 a is a top-view microscopic image of TA-PVP-DMN; Figure 9 b is the overall microscopic image of TA-PVP-DMN.

[0043] Figure 10 This is a microscopic image of the in vitro skin penetration performance of TA-PVP-DMN prepared in Example 1; wherein, Figure 10 a is a methylene blue-stained membrane image of PF punctured by TA-PVP-DMN patch; Figure 10 b is a microscopic image of the membrane-carrying microneedle after it penetrated the membrane.

[0044] Figure 11 This is a graph showing the in vitro release results of polymer TA nanofiber membranes of various proportions prepared in Example 5.

[0045] Figure 12 This is a graph showing the transdermal release results of polymer TA nanofiber membranes of various proportions prepared in Example 5.

[0046] Figure 13 Comparison of drug release results between TA / PVP / PCL and TA-PVP-DMN.

[0047] Figure 14 Figure 2 shows the cytotoxicity analysis results of each analyzed sample.

[0048] Figure 15 Comparison of mouse ear skin before and after two weeks of UVB irradiation.

[0049] Figure 16 This is a comparison of the ear skin color of C57BL / 6 mice after one week of treatment; Figure 16 a: UV irradiation without treatment group; Figure 16 b: TA nanofiber membrane treated group; Figure 16 c: TA-PVP-DMN treatment group.

[0050] Figure 17 This is a comparison of the ear skin color of C57BL / 6 mice after two weeks of treatment; Figure 17 a: UV irradiation without treatment group; Figure 17 b: TA nanofiber membrane treated group; Figure 17 c: TA-PVP-DMN treatment group.

[0051] Figure 18 This is the HE staining result; Figure 18 a is the blank control group mice; Figure 18 b is UVB irradiation group; Figure 18 c: TA nanofiber membrane treated group; Figure 18 d is the TA-PVP-DMN treatment group.

[0052] Figure 19 This figure shows the effects of TA / PVP / PCL and TA-PVP-DMN on UVB radiation-induced epidermal thickness of mouse ear skin.

[0053] Figure 20 This is the result of Fontana Masson staining; Figure 20 a is the blank control group mice; Figure 20 b is UVB irradiation group; Figure 20 c: TA nanofiber membrane treated group; Figure 20 d is the TA-PVP-DMN treatment group.

[0054] Figure 21 This figure shows the effects of TA / PVP / PCL and TA-PVP-DMN on the melanin intensity of mouse ear skin tissue induced by UVB radiation.

[0055] Figure 22 This is the result of anti-tyrosinase antibody staining; Figure 22 a is the blank control group mice; Figure 22 b is UVB irradiation group; Figure 22 c: TA nanofiber membrane treated group; Figure 22 d is the TA-PVP-DMN treatment group.

[0056] Figure 23 This is a graph showing the effects of TA / PVP / PCL and TA-PVP-DMN on the TYR intensity of mouse ear skin tissue induced by UVB radiation. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0058] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions shall be followed. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0059] Polyvinylpyrrolidone PVP: Specification Mn = 22 × 10 4 ; Manufacturer: Shanghai McLean Biochemical Technology

[0060] Polycaprolactone PCL: Specification Mn = 8 × 10 4 ; Manufacturer: Shanghai McLean Biochemical Technology

[0061] Tranexamic acid TA: Purity 98%; Manufacturer: Shanghai MacLean Biochemical Technology

[0062] The microneedle mold used in the embodiment has a cylindrical shape with a diameter of 2.4 cm, a height of 0.8 cm, a groove area of 16.7×16.7 cm, and a groove depth of 1.5 mm. The mold can produce microneedles in the shape of a cone with a height of 600 μm and a diameter of 300 μm. The distance between the needle tips is 700 μm, and the array is 20×20.

[0063] Room temperature has a well-known meaning in the art, generally referring to 25±2°C.

[0064] The present invention is based on Figure 1 The schematic diagram of the preparation process of the microneedle-tranexamic acid nanofiber membrane shown in the figure is used to prepare the microneedle-tranexamic acid nanofiber membrane, namely the TA-PVP-DMN transdermal patch system.

[0065] Example 1 Preparation of TA-PVP-DMN Transdermal Patch System

[0066] (1) Preparation of PVP soluble microneedles

[0067] PVP was placed in a 10mL vial, and 5mL of ultrapure water was added. The solution was then magnetically stirred at room temperature for 4 hours to prepare a 30% by volume aqueous solution of PVP. The aqueous solution was then placed in a 5mL syringe and pushed into the groove of a poly(dimethylsiloxane) (PDMS) mold, flush with the mold surface. The mold was placed in a vacuum drying oven and evacuated at 55°C for 3 minutes at -0.095MPa. The microneedles were removed and the bubbles in the mold were stirred and scraped with a syringe needle to remove any bubbles adhering to the inner wall of the groove. The microneedles were then dried in a 55°C constant temperature drying oven for 24 hours. After complete drying, the microneedles were demolded from the mold using tweezers to obtain PVP-soluble microneedles (PVP-DMN). The height of a single PVP-DMN needle was approximately 599.1±0.3μm, the base diameter was approximately 300μm, and the molding rate was over 98%.

[0068] (2) Preparation of electrospinning solution

[0069] According to the requirement of a PVP to PCL mass ratio of 1:1, 0.3 g of PVP and 0.3 g of PCL were weighed and dissolved in 5 mL of hexafluoroisopropanol with 6% (w / v) TA. The solution was stirred magnetically at 40 °C and 300 rpm for 4 h to completely dissolve the polymer and obtain a uniform spinning solution. The solution was allowed to stand for degassing for 10 min before spinning.

[0070] (3) Preparation of TA-PVP-DMN transdermal patch system

[0071] The spinning solution prepared in step (2) was loaded into a 5 mL medical syringe connected to a 22-gauge needle, and the syringe was fixed to a syringe pump. The PVP-DMN prepared in step (1) was fixed to an electrospinning receiver, and the voltage was set to 20 kV, the receiving distance was 15 cm, and the flow rate of the spinning solution was 0.6 mL / h. Electrospinning was performed at a temperature of 25°C and a relative humidity of 55% to obtain spun fibers. The spun fibers were placed in a vacuum drying oven at 45°C and -0.95 kPa for drying for 24 h to finally obtain TA-PVP-DMN.

[0072] Example 2 Preparation of TA-PVP-DMN Transdermal Patch System

[0073] (1) Preparation of PVP soluble microneedles

[0074] PVP was placed in a 10mL vial, and 5mL of ultrapure water was added. The solution was then magnetically stirred at room temperature for 4 hours to prepare a 30% by volume aqueous solution of PVP. The aqueous solution was then placed in a 5mL syringe and pushed into the groove of a poly(dimethylsiloxane) (PDMS) mold, flush with the mold surface. The mold was placed in a vacuum drying oven and evacuated at 55°C for 3 minutes at -0.095MPa. The microneedles were removed and the bubbles in the mold were stirred and scraped with a syringe needle to remove any bubbles adhering to the inner wall of the groove. The microneedles were then placed in a 55°C constant temperature drying oven for 16 hours. After complete drying, the microneedles were demolded from the mold using tweezers to obtain PVP-soluble microneedles (PVP-DMN). The height of a single PVP-DMN needle was approximately 599.1±0.3μm, the base diameter was approximately 300μm, and the molding rate was over 98%.

[0075] (2) Preparation of electrospinning solution

[0076] According to the requirement of PVP to PCL mass ratio of 1:4, 0.12g of PVP and 0.48g of PCL were weighed and dissolved in 5mL of hexafluoroisopropanol with 6% (w / v) TA. The solution was stirred magnetically at 40°C and 300rpm for 4h to completely dissolve the polymer and obtain a uniform spinning solution. The solution was allowed to stand and degas for 10min before spinning.

[0077] (3) Preparation of TA-PVP-DMN transdermal patch system

[0078] The spinning solution prepared in step (2) was loaded into a 5 mL medical syringe connected to a 22-gauge needle, and the syringe was fixed to a syringe pump. The PVP-DMN prepared in step (1) was fixed to an electrospinning receiver, and the voltage was set to 20 kV, the receiving distance was 15 cm, and the flow rate of the spinning solution was 0.6 mL / h. Electrospinning was performed at a temperature of 25°C and a relative humidity of 55% to obtain spun fibers. The spun fibers were placed in a vacuum drying oven at 45°C and -0.95 kPa for drying for 24 h to finally obtain TA-PVP-DMN.

[0079] Example 3 Preparation of TA-PVP-DMN Transdermal Patch System

[0080] (1) Preparation of PVP soluble microneedles

[0081] PVP was placed in a 10mL vial, and 5mL of ultrapure water was added. The solution was then magnetically stirred at room temperature for 4 hours to prepare a 30% by volume aqueous solution of PVP. The aqueous solution was then placed in a 5mL syringe and pushed into the groove of a poly(dimethylsiloxane) (PDMS) mold, flush with the mold surface. The mold was placed in a vacuum drying oven and evacuated at 55°C for 3 minutes at -0.095MPa. The microneedles were removed and the bubbles in the mold were stirred and scraped with a syringe needle to remove any bubbles adhering to the inner wall of the groove. The microneedles were then dried in a 55°C constant temperature drying oven for 20 hours. After complete drying, the microneedles were demolded from the mold using tweezers to obtain PVP-soluble microneedles (PVP-DMN). The height of a single PVP-DMN needle was approximately 599.1±0.3μm, the base diameter was approximately 300μm, and the molding rate was over 98%.

[0082] (2) Preparation of electrospinning solution

[0083] According to the requirement of a PVP to PCL mass ratio of 4:1, 0.48 g of PVP and 0.12 g of PCL were weighed and dissolved in 5 mL of hexafluoroisopropanol with 6% (w / v) TA. The solution was stirred magnetically at 40 °C and 300 rpm for 4 h to completely dissolve the polymer and obtain a uniform spinning solution. The solution was allowed to stand for degassing for 10 min before spinning.

[0084] (3) Preparation of TA-PVP-DMN transdermal patch system

[0085] The spinning solution prepared in step (2) was loaded into a 5 mL medical syringe connected to a 22-gauge needle, and the syringe was fixed to a syringe pump. The PVP-DMN prepared in step (1) was fixed to an electrospinning receiver, and the voltage was set to 20 kV, the receiving distance was 15 cm, and the flow rate of the spinning solution was 0.8 mL / h. Electrospinning was performed at a temperature of 25°C and a relative humidity of 55% to obtain spun fibers. The spun fibers were placed in a vacuum drying oven at 45°C and -0.95 kPa for drying for 24 h to finally obtain TA-PVP-DMN.

[0086] Example 4 Preparation of PVP-PCL Nanofiber Membrane

[0087] Three polymers with different mass ratios of PVP:PCL = 1:4 (PVP 0.12 g), PVP:PCL = 1:1 (PVP 0.3 g), and PVP:PCL = 4:1 (PVP 0.48 g) were dissolved in 5 mL of hexafluoroisopropanol and magnetically stirred at a constant temperature of 40°C and 300 rpm for 4 hours to completely dissolve the polymer and obtain a uniform spinning solution. The solution was allowed to stand and degas for 10 minutes before spinning.

[0088] The spinning solution was loaded into a 5mL medical syringe connected to a 22-gauge needle, which was fixed to the injection pump. A metal roller receiver was used to collect the spinning, and gauze was attached to the surface of the metal roller. The voltage was set to 20kV, the receiving distance was 15cm, and the flow rate of the spinning solution was 0.6mL / h. Electrospinning was performed at a temperature of 25°C and a relative humidity of 55% to obtain spun fibers. The spun fibers were placed in a vacuum drying oven at 45°C and -0.95kPa for drying for 24h to finally obtain PVP-PCL nanofiber membranes.

[0089] Example 5 Preparation and characterization of TA nanofiber membrane

[0090] Three polymers with different mass ratios of PVP:PCL = 1:4 (PVP 0.12 g), PVP:PCL = 1:1 (PVP 0.3 g), and PVP:PCL = 4:1 (PVP 0.48 g) and 6% (w / v) TA were dissolved in 5 mL of hexafluoroisopropanol and magnetically stirred at a constant temperature of 40°C and 300 rpm for 4 hours to completely dissolve the polymer and obtain a uniform spinning solution. The solution was allowed to stand and degas for 10 minutes before spinning.

[0091] The spinning solution was loaded into a 5mL medical syringe connected to a 22-gauge needle, which was fixed to a syringe pump. A metal roller receiver was used to collect the spinning, and gauze was attached to the surface of the metal roller. The voltage was set to 20kV, the receiving distance was 15cm, and the flow rate of the spinning solution was 0.6mL / h. Electrospinning was performed at a temperature of 25°C and a relative humidity of 55% to obtain spun fibers. The spun fibers were placed in a vacuum drying oven at 45°C and -0.95kPa for drying for 24h to finally obtain TA nanofiber membranes.

[0092] Characterization of TA nanofiber membrane:

[0093] The diameter and morphology of the obtained TA nanofiber membrane were observed by field emission scanning electron microscopy (FE-SEM). The TA nanofiber membranes of each ratio were gold-sprayed and observed using a scanning electron microscope with a resolution of 1.0 nm and an acceleration voltage of 0.02-30 kV, with an acceleration voltage of 10 kV. In each image, at least 100 different fiber filaments were randomly selected, and the diameter and size distribution were measured using Image J software. The fiber diameter distribution range was statistically analyzed using Origin 2021 software. The results are shown in Figure 2 .

[0094] Depend on Figure 2 It can be seen that the diameter of the drug-loaded electrospun fibers with different polymer ratios is uniform, no liquid beads are formed on the fiber surface, and the morphology is good. It is speculated that the PVP / PCL ratio is not an important factor affecting the diameter of the nanofiber membrane.

[0095] TA nanofiber membrane, PVP-PCL nanofiber membrane (blank membrane, i.e. Figure 3 Fourier transform infrared spectroscopy (FT-IR) scanning was performed on PCL / PVP), PCL, PVP, and TA, and the results were plotted using Origin 2021. Figure 3 The positional changes of characteristic functional groups in the spectra were used to analyze whether the drug was successfully incorporated into the fiber, and to analyze the interaction between the drug and each polymer.

[0096] Depend on Figure 3 It can be seen that the spectrum of TA nanofiber membrane shows the presence of separate characteristic peaks of PCL, PVP and TA, respectively, and the characteristic peaks of PCL and PVP overlap with the absorption bands of the drug. Therefore, after adding the drug to the PVP-PCL solution, the spectral distribution is similar to the spectrum without drug loading, which also indirectly reflects the good compatibility between the drug and the carrier.

[0097] TA nanofiber membrane (i.e. Figure 4PVP / PCLTA), PVP-PCL nanofiber membrane (blank membrane, i.e. Figure 4 The X-ray diffraction (XRD) of PVP / PCL and TA was carried out. The results are as follows: Figure 4 shown.

[0098] Depend on Figure 4 It can be seen that the XRD spectrum of TA alone shows sharp and numerous obvious peaks, indicating that the drug exists in a crystalline state with characteristic diffraction peaks; these peaks completely disappear in the XRD spectrum of TA nanofiber membrane; at the same time, an amorphous form with a small hump is observed, which is the same as the spectrum of PVP-PCL nanofiber membrane. Therefore, the XRD of TA nanofiber membrane has no diffraction peaks, indicating that the drug is completely converted into an amorphous physical state after being wrapped by nanofibers.

[0099] Mechanical properties of each TA nanofiber membrane sample were tested according to GB / T 1040.3-2006, "Determination of Tensile Properties of Plastics," Part 3 - Test Conditions for Films and Sheets. Each nanofiber membrane sample was cut into 1.5 cm x 10 cm rectangular strips. Thickness measurements were taken at three randomly selected locations on each sample, and the average thickness was calculated as the sample thickness. The strips were secured in a clamp with a 40 mm gap between the clamps. Tensile tests were performed at room temperature at a rate of 0.05 N / min. Each sample was tested three times, and the average results were calculated. The results are shown in Table 1.

[0100] Table 1 Mechanical strength of polymer nanofiber membranes with different ratios

[0101]

[0102] As shown in Table 1, the greater the amount of PCL added, the lower the elastic modulus value, which proves that the flexibility of the fiber membrane is better, while the less the amount of PCL added, the more brittle the fiber membrane is. Therefore, the TA nanofiber membrane with PVP:PCL=1:4 has the best flexibility.

[0103] The hydrophilicity of the prepared TA nanofiber membrane was measured and characterized by WCA. Ultrapure water was dropped on the surface of the TA nanofiber membrane (1.5 cm × 1.5 cm). After 5 seconds, the sample was photographed under a microscope and the results were analyzed by Image J software to determine the contact angle between water and the TA nanofiber membrane. The results are shown in Figure 5 .

[0104] Depend on Figure 5 It can be seen that with the increase of the content of hydrophilic polymer PVP, the water contact angles of TA nanofiber membranes with three polymer ratios are 104.2°, 80.3°, and 73.7°, respectively, showing a gradually decreasing trend, indicating that PVP can improve the hydrophilic properties of the fiber membrane.

[0105] Example 6 Characterization of PVP-DMN

[0106] The structure and morphology of the PVP-DMN prepared in Example 1 were observed using a stereo microscope. The height, distance between needle tips, bottom diameter, etc. of the PVP-DMN were measured using Image J software to observe whether the prepared microneedles were well formed. The results are shown in FIG. Figure 6 、 Figure 7 .

[0107] Depend on Figure 6 、 Figure 7 The PVP-DMN substrate is flat, the microneedles are neatly arranged and evenly distributed, smooth and transparent, with sharp tips and uniform length. The microneedles are well-formed, with no visible cracks and a conical shape. The height of a single microneedle was measured to be approximately 599.1 ± 0.3 μm, the base diameter was approximately 300 μm, and the microneedle formation rate was over 98%, indicating that the microneedles were well-formed.

[0108] The penetration performance of the prepared PVP-DMN was investigated.

[0109] PF membrane was selected to simulate pig skin for penetration test. Eight layers of PF membrane were stacked and pressed tightly with tools to achieve the effect. The total thickness was 1.2-1.5mm. The microneedle was pressed into the membrane with force and held for 30s. It was then stained with methylene blue solution and observed and photographed under a microscope. The results are as follows Figure 8 .

[0110] Depend on Figure 8 It can be seen from a that the surface of the PF membrane pierced by the microneedle leaves evenly arranged holes. At the same time, the PF membrane after the microneedle is pierced can be observed. The microneedle can smoothly penetrate the three layers of PF membrane. This shows that the prepared microneedle has good penetration performance and can completely and smoothly penetrate the skin. Figure 8 b It can be seen that after the microneedle penetrates the PF membrane, the shape of the microneedle tip changes differently due to the influence of resistance. Some microneedle tips remain upright, some are slightly bent, and some are more bent. However, the tips all exist after penetration, indicating that the microneedle can maintain the integrity of the tip during the process of penetrating the PF membrane, and the overall needle body has not undergone obvious changes, and the overall morphology is still neatly arranged.

[0111] Example 7 Characterization of TA-PVP-DMN Transdermal Patch System

[0112] The structure and morphology of the TA-PVP-DMN prepared in Example 2 were observed using a stereo microscope. The surface morphology of the microneedles after loading the nanofiber membrane was observed. The process of fiber formation on the microneedle surface was explored, and the similarities and differences in the morphology of the microneedles after loading the nanofiber membrane were examined. The results are shown in Figure 2. Figure 9 .

[0113] Depend on Figure 9 It can be seen that the fiber filaments are first electrospun between the needle bodies, and then a "bridge" is formed between the needle tips. The electrospun filaments are gradually deposited into a fiber membrane surface supported by the needle tips. Finally, the fiber filaments gradually settle and thicken on the surface and are not easy to fall off.

[0114] The penetration performance of the prepared TA-PVP-DMN was investigated by referring to the relevant method in Example 6. The results are as follows: Figure 10 .

[0115] Depend on Figure 10 It can be seen that TA-PVP-DMN can also penetrate the three-layer PF membrane, which shows that it can still effectively penetrate the skin. Although the nanofiber membrane loaded on the surface has a certain influence on the mechanical properties of the microneedle tip, the overall needle body has strong enough mechanical properties, so it does not affect the final effect after penetration into the skin.

[0116] Example 8 Drug release experiment of TA nanofiber membrane

[0117] A drug release experiment was performed on the TA nanofiber membrane prepared in Example 5.

[0118] Take 10 mg of each fiber membrane with each polymer ratio, cut three parallel samples of each membrane, place them in 5 mL of PBS solution, and put them in a 37°C constant temperature shaker for drug release test. Take out 1.0 mL of solution at 1 min, 3 min, 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h, and absorb an equal amount of PBS solution for replenishment. The receiving solution is filtered with a 0.22 μm microporous filter membrane, and then the TA concentration in the receiving solution is determined by HPLC. The drug release curve is drawn with the release time as the horizontal axis and the cumulative release rate as the vertical axis. The results are shown in Figure 2. Figure 11 shown.

[0119] It can be seen from the figure that as the proportion of PCL in the TA nanofiber membrane continues to increase, the drug release rate in the PBS buffer solution will slow down relatively, but the drug can still be completely released within 40 minutes. This shows that the polymer ratio does not affect the overall drug release, but only affects the time it takes for the drug to reach equilibrium.

[0120] Example 9 In vitro transdermal release experiment of TA nanofiber membrane

[0121] The TA nanofiber membrane prepared in Example 5 was subjected to an in vitro transdermal release experiment.

[0122] Take the pig ear skin, remove the hair on the surface, cut it into a circle with a diameter of about 30mm, rinse it with physiological saline, and fix it on the receiving chamber of the transdermal diffusion tester, with the dermis layer facing down and the stratum corneum facing up. Inject ultrasonically degassed PBS buffer (pH 7.4) into the receiving chamber as the receiving liquid. At the same time, place the magnetic stirrer in the diffusion cell with a speed of 250r / min and a constant temperature water bath at 32°C for 30 minutes. After the temperature is maintained at a constant temperature, use tweezers to attach the TA nanofiber membrane to the stratum corneum and dermis of the skin. At 1h, 2h, 4h, 6h, 8h and 10h, draw 1.0mL of transdermal receiving liquid and place it in a centrifuge tube. Add an equal amount of isothermal PBS buffer solution in time and remove all bubbles. The receiving liquid was filtered with a 0.45μm filter membrane, and the content of TA in the receiving liquid was determined by HPLC. The results are as follows: Figure 12 shown.

[0123] It can be seen from the figure that the in vitro transdermal release results of TA nanofiber membrane are different from the in vitro drug release results. The transdermal release rates and trends of the three polymer ratios are almost the same in the first 1 hour. After that, due to the different ratios of each polymer, the drug transdermal rate begins to change. Overall, PVP:PCL=1:1 drug-loaded nanofiber membrane has the best drug transdermal effect, but the cumulative release rate is only 57% at 10 hours. The bioavailability of the drug is low and it cannot achieve the ideal transdermal effect.

[0124] Example 10 Investigating the in vitro transdermal effect of TA-PVP-DMN

[0125] An in vitro transdermal release experiment was conducted on the TA-PVP-DMN (drug-loaded nanofiber membrane with polymers having the optimal ratio for transdermal release) prepared in Example 1. The experimental scheme is shown in Example 9.

[0126] After the temperature was maintained at a constant temperature, TA-PVP-DMN was pressed into the epidermis of the pig skin and held for 30 seconds. The TA nanofiber membrane was placed on the surface of the pig skin using tweezers. The action time was 30 minutes. The subsequent operations were the same as in Example 8. Whether the transdermal release effect of TA-PVP-DMN was better than that of TA nanofiber membrane (ie, TA / PVP / PCL) was investigated. The results are as follows: Figure 13 shown.

[0127] As can be seen from the figure, the cumulative release rate of TA-PVP-DMN is much higher than that of TA nanofiber membrane. Although the time for the two forms of drug release to reach equilibrium varies, the total cumulative release rate of TA-PVP-DMN ultimately reaches 85%, while the cumulative release rate of TA nanofiber membrane reaches only 48%. This also shows that the prepared TA-PVP-DMN has better transdermal release than TA nanofiber membrane, which can effectively improve the low permeability of TA in the skin and improve drug bioavailability.

[0128] Example 11 Cytotoxicity Experiment of TA Nanofiber Membrane (TA / PVP / PCL) and Soluble Microneedles (PVP-DMN)

[0129] PVP-PCL (blank membrane, i.e. Figure 14 The PVP / PCL and TA nanofiber membranes (PVP:PCL=4:1, prepared in Example 5) were cut into 3×4 cm 2 The membrane was irradiated with ultraviolet light for 30 minutes for disinfection. The ratio of the membrane area to the complete culture medium (made by mixing 90% high glucose medium (DMEM), 9% fetal bovine serum albumin (FBS) and 1% double antibody) was 6 cm 2 / mL, place the cut nanofiber membrane in a sterile culture dish, add the calculated complete culture medium, and extract at 37°C for 24 hours. After the extraction is complete, carefully remove the nanofiber membrane and transfer the extract to a sterile centrifuge tube to obtain the nanofiber membrane extract. Take a piece of soluble microneedle (PVP-DMN) and add 2mL of complete culture medium to dissolve it. Extract at 37°C for 24 hours to obtain the microneedle extract.

[0130] Mouse fibroblasts (L929) were cultured and the cells of L929 cultured to the third generation were prepared into 1×10 5 Add L929 cell suspension to a 96-well plate at a concentration of 1×10 cells / mL per well. 4 Cells were seeded at a specific density and incubated in a 96-well plate at 37°C and 5% CO2 in a constant-temperature incubator. When the cells reached 80% growth, the culture medium was aspirated and the material extracts were added. A blank control group and three experimental groups were set up. The blank control group received complete culture medium, while the experimental groups received nanofiber membrane extracts and microneedle extracts, respectively. The cytotoxicity experimental settings are shown in Table 2.

[0131] Table 2 Cytotoxicity experiment settings

[0132]

[0133] The 96-well plate was then placed in a constant temperature incubator at 37°C and 5% CO2 for incubation. After 24 hours of incubation, the culture medium was removed with a pipette, and then MTT (0.5 mg / mL, 20 μL) solution was added to each well. After incubation for 4 hours, the waste liquid was discarded, and 200 μL of dimethyl sulfoxide was added to each well. The plate was shaken at low speed for 10 minutes to fully dissolve the crystals. The optical density (OD) value of the 96-well plate was then measured at 492 nm using a microplate reader. The cell survival rate (%) was calculated according to the formula "OD = OD". 实验组 / OD 对照组 ×100” formula to calculate cell survival rate, the result is as follows Figure 14 .

[0134] As can be seen from the figure, the cell viability of all three samples was above 95%. According to GB / T 16886.5-2017, "Biological Evaluation of Medical Devices - Part 5: In Vitro Cytotoxicity Tests," a cell viability of 75% or higher demonstrates the non-toxicity of the biomaterial used. Therefore, the TA nanofiber membrane and microneedle extract exhibit excellent biocompatibility and non-toxicity for fibroblasts, which have high growth and proliferation rates.

[0135] Example 12 In vivo study of TA nanofiber membrane and TA-PVP-DMN

[0136] In vivo studies were conducted on TA nanofiber membranes (PVP:PCL=1:1, prepared in Example 5) and TA-PVP-DMN (PVP:PCL=1:1, prepared in Example 1) to verify their therapeutic effects on melasma. 16 female C57BL / 6 mice aged 6-8 weeks and weighing 16-18 g were selected from Nanjing Qinglongshan Animal Farm and raised in a temperature-controlled room (25±2°C) for 7 days to adapt to the laboratory environment. After one week of adaptation, the skin of the mice's ears was selected for UVB irradiation. Ultraviolet irradiation was performed every two days for two weeks. During the experiment, the skin changes of the exposed parts of the experimental mice were carefully observed, and more attention was paid to the mice's diet, urination, and defecation.

[0137] After successful model establishment, mice were randomly divided into three groups: Group A was irradiated with UVB, Group B was treated with TA nanofiber membranes, and Group C was treated with TA-PVP-DMN. Group D served as a non-irradiated blank control group, with four mice in each group. For each treatment, ultrapure water was applied topically to the pigmented areas of the ears of mice in Group A. For mice in Group B, the ears were moistened with ultrapure water before treatment, and then the excised TA nanofiber membranes were applied to the ears while the timer was recorded. Each treatment lasted 30 minutes. For mice in Group C, the ears were similarly moistened with ultrapure water before treatment, and then the excised TA-PVP-DMNs were applied to the ears, pinched and held for 10 seconds, with the timer starting. Each treatment lasted 30 minutes. Mice in Group D received no treatment. Treatment was conducted once daily, six days a week, for two weeks. After each treatment, photographs were taken to document improvements in pigmentation. During the experimental treatment, the mice were closely observed for any abnormalities in diet and excretion. After the treatment, the mice were killed and HE staining, Fontana Masson staining and immunohistochemical staining using monoclonal anti-mouse tyrosinase (TYR) antibody were performed to evaluate the therapeutic effect of TA-PVP-DMN on melasma.

[0138] Figure 15This image compares the ear skin color of mice in the normal control group (i.e., the unexposed blank control group) and mice irradiated with UVB for two weeks. The image shows that the ear skin of UVB-irradiated mice is darker, darker, and less shiny than that of the unexposed blank control group. Slight desquamation can also be observed on the surface of the ear skin. This suggests that UVB irradiation stimulates the activity of melanocytes in the ear skin of C57BL / 6 mice, causing hyperpigmentation and demonstrating the successful establishment of an animal model for melasma.

[0139] Figure 16 The depigmentation effect of mice after one week of treatment was compared with the UVB-irradiated mice. TA nanofiber membranes (TA / PVP / PCL) and TA-PVP-DMN brightened the pigmentation of the mouse ear skin to varying degrees, effectively improving the dull effect. However, the difference in treatment effect between the two was not significant. Figure 17 This is the depigmentation effect of mice after two weeks of treatment. It can be observed with the naked eye that the color of the ear skin of the mice in the TA-PVP-DMN treatment group is more obviously brightened, which greatly improves the pigmentation of the mice, proving the effectiveness of TA-PVP-DMN in treating melasma.

[0140] HE staining results are shown in Figure 18 、 19 When the skin is exposed to ultraviolet rays, it can cause skin growth. Figure 18 、 19 It can be seen that the epidermis of the mice in the blank control group was very thin, with normal sebaceous glands and hair follicles, no hyperplasia, and no obvious inflammatory cells. However, the skin epithelial cells in the UVB irradiation group significantly proliferated, resulting in significant thickening of the epidermis, hyperplasia of sebaceous glands and hair follicles, and the appearance of verrucous hyperplasia. Some inflammatory cells appeared locally. Compared with the UVB irradiation group, the epidermal hyperplasia in both treatment groups was milder, the epidermal thickness was significantly reduced, the verrucous hyperplasia was not obvious, and the number of sebaceous glands and inflammatory cells decreased. At the same time, compared with the TA nanofiber membrane treatment, the TA-PVP-DMN treatment showed that the epidermal verrucous hyperplasia and epidermal thickness were closer to normal skin, and the treatment effect was better.

[0141] Fontana Masson staining results are shown in Figure 20 、 21 .Depend on Figure 20 、 21It can be seen that slight melanocyte aggregation with melanin can be found in the subcutaneous tissue of the ears of mice in the blank control group (because C57BL / 6 mice themselves are melanin, there will be a small amount of melanin in the skin epidermis). Moderate / severe melanocyte aggregation and melanin pigment and inflammatory cell infiltration were found in the subcutaneous tissue of mice in the UVB irradiation group. The amount of melanin in the subcutaneous tissue of the ears of mice treated with TA nanofiber membrane (ie TA / PVP / PCL) and TA-PVP-DMN was reduced compared with the UVB group, and the melanocyte aggregation was alleviated. However, the melanin content of mice in the TA-PVP-DMN treatment group decreased more significantly, indicating that this treatment method has better effect.

[0142] Immunohistochemistry results are shown in Figure 22 、 23 .Depend on Figure 22 、 23 It can be seen that compared with the blank control group, the TYR level in the ear skin of mice in the UVB irradiation group was significantly increased. Although the expression level of TYR was reduced after treatment with TA nanofiber membrane (i.e., TA / PVP / PCL) and TA-PVP-DMN, the TA-PVP-DMN treatment group showed a more obvious inhibitory effect, indicating that the treatment effect of TA-PVP-DMN was better than that of TA nanofiber membrane.

[0143] In summary, compared with TA nanofiber membrane, the TA-PVP-DMN transdermal patch system can effectively change the permeability of TA in the mouse epidermis and increase the drug permeation rate due to the load of microneedles, thereby achieving the purpose of safe and efficient treatment of melasma.

[0144] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A method for preparing a microneedle-tranexamic acid nanofiber membrane, characterized in that: Soluble microneedles DMN are prepared using the hydrophilic polymer polyvinylpyrrolidone (PVP), the soluble microneedles are fixed on an electrospinning receiver, and electrospinning is performed using an electrospinning solution containing tranexamic acid (TA) to prepare the microneedle-tranexamic acid nanofiber membrane, namely TA-PVP-DMN.

2. The preparation method according to claim 1, characterized in that The following steps are involved: (1) Pour a polyvinylpyrrolidone (PVP) aqueous solution into a microneedle mold, solidify the microneedles, dry them, and demold them to obtain polyvinylpyrrolidone-soluble microneedles, namely PVP-DMN; (2) dissolving a mixture of PVP and polycaprolactone (PCL) and tranexamic acid in hexafluoroisopropanol to obtain a uniform spinning solution; (3) Fixing the PVP-DMN obtained in step (1) on an electrospinning receiver, using the spinning solution obtained in step (2), setting spinning parameters, performing uniaxial electrospinning, and drying to obtain the microneedle-tranexamic acid nanofiber membrane.

3. The preparation method according to claim 2, characterized in that In step (1), the volume fraction of the PVP aqueous solution is 30%.

4. The preparation method according to claim 2, characterized in that In step (1), the curing treatment is performed by vacuum drying at 55° C. for 3 minutes at -0.095 MPa.

5. The preparation method according to claim 2, characterized in that In step (2), the mass ratio of PVP to PCL is 1-4:4-1.

6. The preparation method according to claim 2, characterized in that In step (2), the mass volume of the PVP in hexafluoroisopropanol is 2% to 10%.

7. The preparation method according to claim 2, characterized in that In step (2), the mass volume of tranexamic acid in hexafluoroisopropanol is 2% to 10%.

8. The preparation method according to claim 2, characterized in that In step (3), the electrospinning parameters are: applied voltage of 15-22 kV, receiving distance of 10-20 cm, flow rate of spinning solution of 0.3-1.2 mL / h, temperature of 20-25° C., and relative humidity of 40%-60%.

9. The preparation method according to claim 2, characterized in that In step (3), the drying method is: vacuum drying at 40-60° C. for 12-24 hours.

10. The microneedle-tranexamic acid nanofiber membrane prepared by the preparation method according to any one of claims 1 to 9.

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