A chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention and its preparation method

Abstract

This invention relates to the field of transdermal microneedle formulation technology, specifically to a chitosan-finasteride nanocrystal microneedle formulation and its preparation method that enhances skin retention. The microneedle formulation includes a needle tip, an extension, and a root. The needle tip and the extension are both composed of chitosan-finasteride nanocrystals, and the root is hyaluronic acid. After being coated with chitosan, the finasteride nanocrystals acquire a positive charge on their surface, forming an electrostatic interaction with the negative charge in the skin's microenvironment. This leads to the adsorption of the chitosan-finasteride nanocrystals onto the skin surface. Simultaneously, the positively charged surface of the chitosan-finasteride nanocrystals increases endocytosis, effectively improving drug entry efficiency and achieving drug accumulation within scalp cells. This dual effect enhances the retention of finasteride nanocrystals on the skin surface and prevents finasteride from entering the bloodstream.

Description

Technical Field

[0001] This invention relates to the field of transdermal microneedle formulation technology, specifically to a chitosan-finasteride nanocrystal microneedle formulation and its preparation method that enhances skin retention. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Androgenic alopecia (AGA) is the most common hair loss disease in men, with an incidence rate as high as 20%. AGA typically begins in or after puberty and can last for decades. It is characterized by a shortened anagen phase (growth phase) and progressive miniaturization of hair follicles. In AGA, the hair follicles do not disappear; instead, terminal hairs transform into vellus hairs. The mainstream theory of AGA pathogenesis is the androgen sensitivity theory, which posits that AGA is induced by the metabolism of testosterone to dihydrotestosterone (DHT) by 5-α-reductase (SRD5A2). DHT activates androgen receptors (ARs) in hair follicles. After binding to the androgen receptor, DHT enters the cell nucleus, activates androgen receptor coactivator, and initiates a multi-step molecular pathway involving target gene transcription and protein translation, thereby exerting its biological effects.

[0004] While alopecia areata (AGA) does not affect physical health, its adverse effects on appearance place a significant psychological burden on individuals experiencing hair loss, prompting them to actively seek treatment. Finasteride is a selective SRD5A2 inhibitor that reverses AGA by inhibiting the action of SRD5A2. Due to its hydrophobic nature and poor transdermal efficiency, current technologies for treating AGA require oral administration of finasteride. However, some male AGA patients who take oral finasteride experience adverse sexual reactions such as decreased libido, erectile dysfunction, and ejaculatory dysfunction. Since SRD5A2 is primarily distributed in the hair follicles of the scalp, topical administration of finasteride can minimize systemic adverse reactions and improve efficacy compared to oral administration. Nanocarriers, due to their nanoscale advantage and skin interaction that facilitates transdermal drug delivery, have been shown to be useful for drug delivery. Existing technologies disclose several nanocarrier formulations for transdermal delivery of finasteride, including liposomes, liposomes, and delivery systems; however, the stratum corneum barrier remains a major challenge for topical formulations in treating AGA, limiting the effectiveness of nanomedicines in penetrating the dermis. Meanwhile, although some transdermal finasteride nanocarrier formulations overcome the stratum corneum barrier, some of the drug still enters the bloodstream. Therefore, providing a nanocarrier formulation that can both overcome the stratum corneum barrier and remain in the skin for a long time to prevent the drug from entering the bloodstream is of great significance for achieving transdermal finasteride delivery. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a chitosan-finasteride nanocrystal microneedle formulation and its preparation method that enhances skin retention.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a chitosan-finasteride nanocrystal microneedle formulation that enhances skin retention. The microneedle formulation includes a needle tip, an extension, and a root. The needle tip and the extension are both composed of chitosan-finasteride nanocrystals, and the root is hyaluronic acid.

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned chitosan-finasteride nanocrystal microneedle formulation that enhances skin retention, comprising the following steps:

[0009] (1) Under ultrasonic conditions, finasteride ethanol solution was added to sodium dodecyl sulfonate aqueous solution, and then purified by ultrafiltration to obtain a suspension containing finasteride nanocrystals.

[0010] (2) Add chitosan to an aqueous acetic acid solution, let it stand, and adjust the pH of the chitosan mixture to 5.5-6.5. Place the entire reaction system in an ice bath and inject the suspension containing finasteride nanocrystals obtained in step (1) into the chitosan mixture using a syringe under ultrasonic conditions. Then, purify by ultrafiltration to obtain a chitosan-finasteride nanocrystal suspension.

[0011] (3) Add the chitosan-finasteride nanocrystal suspension obtained in step (2) to the polyvinylpyrrolidone solution, mix evenly, add it to the microneedle cavity, centrifuge and dry multiple times to prepare the needle tip and extension, then add hyaluronic acid solution, centrifuge and dry to obtain the chitosan-finasteride nanocrystal microneedle preparation that enhances skin retention.

[0012] The beneficial effects of this invention are as follows:

[0013] Microneedle formulations have been disclosed to overcome the barrier of the stratum corneum. Secondly, after finasteride nanocrystals are coated with chitosan, the surface of the chitosan-finasteride nanocrystals carries a positive charge, forming an electrostatic interaction with the negative charge in the human skin microenvironment. This leads to the adsorption of the chitosan-finasteride nanocrystals onto the skin surface. Simultaneously, the positively charged surface of the chitosan-finasteride nanocrystals increases endocytosis, effectively improving drug entry efficiency and achieving drug accumulation within scalp cells. This dual effect enhances the retention of finasteride nanocrystals on the skin surface, preventing finasteride from entering the bloodstream. The chitosan-finasteride nanocrystal microneedle formulation prepared in this invention shows promise for transdermal delivery of finasteride. Attached Figure Description

[0014] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0015] Figure 1 Scanning electron microscope image of finasteride nanocrystals;

[0016] Figure 2 DSC curves of finasteride technical, finasteride nanocrystals, polyvinylpyrrolidone, and physical mixtures of finasteride nanocrystals and polyvinylpyrrolidone, as well as chitosan-finasteride nanocrystals prepared in Example 1.

[0017] Figure 3 The graph shows the particle size, PDI, and zeta potential of the nanocrystals in the finasteride nanocrystal suspension and the chitosan-finasteride nanocrystal suspension obtained in Examples 1-3.

[0018] Figure 4These are images of the chitosan-finasteride nanocrystal microneedles prepared in Examples 1-3 under a stereomicroscope.

[0019] Figure 5 This is a schematic diagram of the structure of the chitosan-finasteride nanocrystal microneedles prepared in Examples 1-3;

[0020] Figure 6 This is a bright-field microscope image of the chitosan-finasteride nanocrystal microneedles prepared in Example 1;

[0021] Figure 7 Figures showing the results of external release experiments of finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3;

[0022] Figure 8 The mechanical properties and insertion properties of finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3 are evaluated. a is the force-displacement curve, and b is the insertion rate in Parafilm.

[0023] Figure 9 Figures showing the skin retention effect of finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3;

[0024] Figure 10 The images show the passive penetration effect of finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3 on the skin diffusion. (a) shows the passive skin penetration, (b) shows the skin diffusion area, and (c) shows the average gray value.

[0025] Figure 11 The images show the skin diffusion and hair follicle accumulation effects of finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3. (a) shows the skin hair follicle accumulation, (b) shows the average gray value, and (c) shows the hair follicle accumulation depth. Detailed Implementation

[0026] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, unless otherwise expressly indicated by the invention, the singular form is intended to include the plural form as well. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] A first typical embodiment of the present invention provides a chitosan-finasteride nanocrystal microneedle formulation that enhances skin retention. The microneedle formulation includes a needle tip, an extension, and a root. The needle tip and the extension are both composed of chitosan-finasteride nanocrystals, and the root is hyaluronic acid.

[0029] A second typical embodiment of the present invention provides a method for preparing the above-mentioned chitosan-finasteride nanocrystal microneedle formulation that enhances skin retention, comprising the following steps:

[0030] (1) Under ultrasonic conditions, finasteride ethanol solution was added to sodium dodecyl sulfonate aqueous solution, and then purified by ultrafiltration to obtain a suspension containing finasteride nanocrystals.

[0031] (2) Add chitosan to an aqueous acetic acid solution, let it stand, and adjust the pH of the chitosan mixture to 5.5-6.5. Place the entire reaction system in an ice bath and inject the suspension containing finasteride nanocrystals obtained in step (1) into the chitosan mixture using a syringe under ultrasonic conditions. Then, purify by ultrafiltration to obtain a chitosan-finasteride nanocrystal suspension.

[0032] (3) Add the chitosan-finasteride nanocrystal suspension obtained in step (2) to the polyvinylpyrrolidone solution, mix evenly, add it to the microneedle cavity, centrifuge and dry multiple times to prepare the needle tip and extension, then add hyaluronic acid solution, centrifuge and dry to obtain the chitosan-finasteride nanocrystal microneedle preparation that enhances skin retention.

[0033] In one or more embodiments, in step (1), the power of the ultrasound is 380 to 420 W.

[0034] In one or more embodiments, in step (1), the concentration of the finasteride ethanol solution is 1 mg / mL to 30 mg / mL, preferably 20 mg / mL.

[0035] In one or more embodiments, in step (1), the concentration of the sodium dodecyl sulfonate aqueous solution is 4 mg / mL to 5 mg / mL.

[0036] In one or more embodiments, in step (1), the mass ratio of finasteride ethanol to sodium dodecyl sulfonate is 1 to 30:40, preferably 20:40.

[0037] In one or more embodiments, in step (1), the molecular weight cutoff by ultrafiltration purification is 3kDa to 30kDa.

[0038] In one or more embodiments, in step (2), the viscosity of chitosan includes 100-200 mpa·s, 200-400 mpa·s and greater than 400 mpa·s, preferably 100-200 mpa·s.

[0039] In one or more embodiments, in step (2), the mass fraction of acetic acid in the aqueous acetic acid solution is 0.1-10%, preferably 1%.

[0040] In one or more embodiments, in step (2), the concentration of chitosan in the aqueous acetic acid solution is 4.5 mg / mL to 5.5 mg / mL, preferably 5 mg / mL.

[0041] In one or more embodiments, in step (2), the settling time is 2 to 12 hours.

[0042] In one or more embodiments, in step (2), the power of the ultrasound is 180 to 220 W.

[0043] In one or more embodiments, in step (2), the mass ratio of chitosan to finasteride nanocrystals is 1:10 to 10:1.

[0044] In one or more embodiments, in step (2), the injection rate using a syringe is 1.5 to 2.5 mL / min.

[0045] In one or more embodiments, in step (2), the molecular weight cutoff by ultrafiltration purification is 3kDa to 30kDa, preferably 10kDa.

[0046] In one or more embodiments, in step (3), the mass fraction of polyvinylpyrrolidone in the polyvinylpyrrolidone solution is 25-35%, preferably 30%.

[0047] In one or more embodiments, in step (3), after the chitosan-finasteride nanocrystal suspension is added to the polyvinylpyrrolidone solution, the concentration of the chitosan-finasteride nanocrystals is 7 mg / mL to 9 mg / mL, preferably 8 mg / mL.

[0048] In one or more embodiments, in step (3), the hyaluronic acid solution includes a 5% by mass of a 5kDa hyaluronic acid solution and a 10% by mass of a 30kDa hyaluronic acid solution, with a mass ratio of 1:1.5 to 2.5, preferably 1:2.

[0049] In one or more embodiments, in step (3), the total amount of hyaluronic acid solution added is 1 to 1.5 mL.

[0050] Hyaluronic acid is a naturally derived gel material with high biocompatibility and excellent hygroscopicity and solubility. Small molecule hyaluronic acid (5kDa) plays a role in absorbing and moisturizing water, while large molecule hyaluronic acid (30kDa) plays a role in shaping and supporting. In this invention, hyaluronic acid is used as the base layer material at the root of the microneedle, which can not only support the formation of the microneedle patch, but also avoid related safety issues due to its good biocompatibility.

[0051] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

[0052] Solution preparation:

[0053] Dissolve finasteride in ethanol to prepare a finasteride ethanol solution with a concentration of 20 mg / mL.

[0054] Dissolve 400 mg of sodium dodecyl sulfonate in 90 mL of ultrapure water to prepare an aqueous solution of sodium dodecyl sulfonate with a concentration of 4.4 mg / mL.

[0055] Dissolve 30 mg of polyvinylpyrrolidone in 70 mg of ultrapure water to prepare a 30% polyvinylpyrrolidone solution.

[0056] Example 1

[0057] (1) Under ultrasonic conditions, 1 mL of finasteride ethanol solution was added to sodium dodecyl sulfonate aqueous solution, and then ultrafiltration was performed using an ultrafiltration tube with a molecular weight of 10 kDa to obtain a suspension containing finasteride nanocrystals.

[0058] (2) 0.25 g chitosan (100-200 mPa·s) was added to 50 mL of aqueous solution containing 1% acetic acid. After standing for at least 3 h, 2 mL of chitosan mixed solution was taken and 8 mL of ultrapure water was added. The pH of the chitosan mixed solution was adjusted to 6 using 10 mg / mL NaOH. The entire reaction system was placed in an ice bath. Under ultrasonic conditions, 100 mg of the suspension containing finasteride nanocrystals obtained in step (1) was injected into 200 mg of chitosan mixed solution using a syringe. Then, the chitosan-finasteride nanocrystal suspension was obtained by ultrafiltration using an ultrafiltration tube with a molecular weight of 10 kDa.

[0059] (3) Add the chitosan-finasteride nanocrystal suspension obtained in step (2) to the polyvinylpyrrolidone solution to make the final concentration of chitosan-finasteride nanocrystals 8 mg / mL, and mix well. Add it to the microneedle chamber, centrifuge at 4000 rpm to remove excess liquid, place the mold in a vacuum oven at 38℃ and dry for 1 h, repeat the above centrifugation and drying operation three times, then add 1.5 mL of hyaluronic acid solution (5% 5 kDa HA + 10% 30 kDa HA), centrifuge at 4000 rpm to remove excess liquid, place the mold in a vacuum oven at 38℃ and dry overnight, after drying, use tweezers to remove the chitosan-finasteride nanocrystal microneedle patch and store it in a desiccator.

[0060] Example 2

[0061] (1) The preparation method of the suspension of finasteride nanocrystals is the same as in Example 1.

[0062] (2) 0.25 g chitosan (200-400 mPa·s) was added to 50 mL of aqueous solution containing 1% acetic acid. After standing for at least 3 h, 2 mL of chitosan mixed solution was taken and 8 mL of ultrapure water was added. The pH of the chitosan mixed solution was adjusted to 6 using 10 mg / mL NaOH. The entire reaction system was placed in an ice bath. Under ultrasonic conditions, 100 mg of the suspension containing finasteride nanocrystals obtained in step (1) was injected into 200 mg of chitosan mixed solution using a syringe. Then, the chitosan-finasteride nanocrystal suspension was obtained by ultrafiltration using an ultrafiltration tube with a molecular weight of 10 kDa.

[0063] (3) Add the chitosan-finasteride nanocrystal suspension obtained in step (2) to the polyvinylpyrrolidone solution to make the final concentration of chitosan-finasteride nanocrystals 8 mg / mL, and mix well. Add it to the microneedle chamber, centrifuge at 4000 rpm to remove excess liquid, place the mold in a vacuum oven at 38℃ and dry for 1 h, repeat the above centrifugation and drying operation three times, then add 1.5 mL of hyaluronic acid solution (5% 5 kDa HA + 10% 30 kDa HA), centrifuge at 4000 rpm to remove excess liquid, place the mold in a vacuum oven at 38℃ and dry overnight, after drying, use tweezers to remove the chitosan-finasteride nanocrystal microneedle patch and store it in a desiccator.

[0064] Example 3

[0065] (1) The preparation method of the suspension of finasteride nanocrystals is the same as in Example 1.

[0066] (2) 0.25 g chitosan (greater than 400 mPa·s) was added to 50 mL of aqueous solution containing 1% acetic acid. After standing for at least 3 h, 2 mL of chitosan mixed solution was taken and 8 mL of ultrapure water was added. The pH of the chitosan mixed solution was adjusted to 6 using 10 mg / mL NaOH. The entire reaction system was placed in an ice bath. Under ultrasonic conditions, 100 mg of the suspension containing finasteride nanocrystals obtained in step (1) was injected into 200 mg of chitosan mixed solution using a syringe. Then, the chitosan-finasteride nanocrystal suspension was obtained by ultrafiltration using an ultrafiltration tube with a molecular weight of 10 kDa.

[0067] (3) Add the chitosan-finasteride nanocrystal suspension obtained in step (2) to the polyvinylpyrrolidone solution to make the final concentration of chitosan-finasteride nanocrystals 8 mg / mL, and mix well. Add it to the microneedle chamber, centrifuge at 4000 rpm to remove excess liquid, place the mold in a vacuum oven at 38℃ and dry for 1 h, repeat the above centrifugation and drying operation three times, then add 1.5 mL of hyaluronic acid solution (5% 5 kDa HA + 10% 30 kDa HA), centrifuge at 4000 rpm to remove excess liquid, place the mold in a vacuum oven at 38℃ and dry overnight, after drying, use tweezers to remove the chitosan-finasteride nanocrystal microneedle patch and store it in a desiccator.

[0068] Example 4

[0069] This embodiment characterizes the finasteride nanocrystals, chitosan-finasteride nanocrystal suspension, and chitosan-finasteride nanocrystal microneedles of Examples 1-3.

[0070] (1) Scanning electron microscopy images of finasteride nanocrystals in finasteride nanocrystal suspension are shown below. Figure 1 As shown, from Figure 1 It can be seen that finasteride nanocrystals have a spherical structure.

[0071] (2) DSC curves of finasteride technical, finasteride nanocrystals, polyvinylpyrrolidone, and physical mixtures of finasteride nanocrystals and polyvinylpyrrolidone, as well as the chitosan-finasteride nanocrystals prepared in Example 1, are shown below. Figure 2 As shown, both the finasteride nanocrystals and the chitosan-finasteride nanocrystals prepared in Example 1 are in an amorphous state, and the characteristic endothermic peaks of finasteride technical material (252℃) and polyvinylpyrrolidone (203℃) disappear, which also proves that the finasteride nanocrystals and the chitosan-finasteride nanocrystals prepared in Example 1 were successfully prepared.

[0072] (3) The particle size, PDI, and zeta potential of the nanocrystals in the finasteride nanocrystal suspension and the chitosan-finasteride nanocrystal suspensions obtained in Examples 1-3 were characterized, and the results are as follows: Figure 3 As shown, from Figure 3 As can be seen, the particle size of finasteride nanocrystals increases after being coated with chitosan, and the particle size of chitosan-finasteride nanocrystals increases with the increase of chitosan viscosity. Simultaneously, the zeta potential of finasteride nanocrystals shows that they are positively charged, but after being coated with chitosan, the zeta potential of chitosan-finasteride nanocrystals shows that they are negatively charged. This indicates that chitosan alters the surface potential of finasteride nanocrystals. Furthermore, the viscosity of chitosan affects the surface charge of finasteride nanocrystals; chitosan with a viscosity of 100-200 mPa·s exhibits the highest zeta potential after coating finasteride nanocrystals. The negative charge in the human skin microenvironment and the positively charged surface of chitosan-finasteride nanocrystals can lead to electrostatic adsorption onto the skin surface; the higher the zeta potential, the stronger the adsorption.

[0073] (4) Figure 4 These are images of the chitosan-finasteride nanocrystal microneedles prepared in Examples 1-3, under a stereomicroscope. Figure 5 The diagram shows the structure of the chitosan-finasteride nanocrystal microneedles prepared in Examples 1-3. Figure 6 This is a bright-field microscope image of the chitosan-finasteride nanocrystal microneedles prepared in Example 1. Figures 4-6 It can be seen that the chitosan-finasteride nanocrystal microneedles prepared by this invention are bullet-shaped.

[0074] Experimental Example 1

[0075] In vitro release experiments were conducted on finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3.

[0076] Appropriate amounts of finasteride nanocrystal suspension and the chitosan-finasteride nanocrystal suspension obtained in Examples 1-3 were placed in dialysis bags, ultimately achieving a finasteride drug concentration of 3 mg / 10 mL. The dialysis medium was 20 mL of physiological saline containing 30% ethylene glycol, and the mixture was shaken at 120 rpm at 37°C. Samples were taken at 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, and 120 h, with 1 mL of sample taken each time and 1 mL of fresh release medium added immediately. The collected samples were centrifuged at 12000 rpm for 20 min and analyzed by high-performance liquid chromatography (HPLC).

[0077] Results of in vitro release experiments are as follows Figure 7 As shown, from Figure 7As can be seen, the chitosan-finasteride nanocrystals obtained in Examples 1 to 3 have different release rates in vitro. At 120 h, the cumulative release rate of chitosan with a viscosity of 100-200 mpa·s is the highest, followed by chitosan with a viscosity of 200-400 mpa·s and greater than 400 mpa·s.

[0078] Experiment Example 2

[0079] The mechanical properties and insertion properties of finasteride nanocrystals and the chitosan-finasteride nanocrystals obtained in Examples 1-3 were evaluated.

[0080] Mechanical force was tested using a texture analyzer, starting from the moment the sensor first contacted the tip of the nanocrystal needle and continuing until the sensor moved 0.8 mm from the nanocrystal tip towards the backing layer. Parafilm films were stacked into 8 layers, and the nanocrystals were inserted into the Parafilm film by pressing with the thumb for 30 seconds. The number of pores pierced in each layer of the Parafilm film was observed under a stereomicroscope; the ratio of the number of pores to the number of nanocrystal needle tips represented the insertion performance.

[0081] The results are as follows Figure 8 As shown, from Figure 8 As can be seen, both the finasteride nanocrystals and the chitosan-finasteride nanocrystals obtained in Examples 1-3 can withstand a compressive force of ≥0.14 N / needle, indicating that they have sufficient strength to penetrate the skin and effectively deliver the drug. The tips of the finasteride nanocrystals and the chitosan-finasteride nanocrystals obtained in Examples 1-3 can be inserted into 6 layers of Parafilm membrane, with a theoretical insertion depth of up to 750 μm.

[0082] Experimental Example 3

[0083] The in vitro skin retention capacity of finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3 was evaluated.

[0084] The experiment was conducted using a vertical Franz diffusion cell, with the diffusion area being a circular region with a diameter of 1.5 cm. Pig skin was soaked in physiological saline for 1 hour, and then finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3 were inserted into it. The skin was fixed between the donor and recipient chambers with strong adhesive, with the skin side of the nanocrystals facing upwards. The recipient medium was a 30% (v / v) PEG 400 physiological saline solution, the experimental temperature was 37±1℃, and the rotation speed was 100 rpm. After 24 hours, the skin was removed, and a 1.5 cm diameter section of the diffusion area was cut off, dissolved in 0.5 mL of acetonitrile solution, homogenized, ground with grinding beads, and then ultrasonically disrupted (ultrasonic conditions: 3s on, 2s off, 5 min, 400 W power). The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was used to determine the skin retention volume by HPLC.

[0085] The results of in vitro skin retention are as follows Figure 9 As shown, chitosan-finasteride nanocrystals with different viscosities exhibit different penetration and retention effects. With increasing chitosan viscosity, the amount of finasteride retained in the skin from the chitosan-finasteride nanocrystals gradually decreases, with chitosan-finasteride nanocrystals of 100-200 mPa·s showing the best skin retention effect. Chitosan-finasteride nanocrystals with viscosities greater than 400 mPa·s have lower skin retention than finasteride nanocrystals, indicating that increased viscosity affects the retention effect of the finasteride nanocrystals themselves.

[0086] Experiment Example 4

[0087] The passive penetration and hair follicle accumulation effects of finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1-3 were investigated.

[0088] The specific method is as follows: Take pig skin, soak it in physiological saline for 4 hours, and then insert finasteride nanocrystals and chitosan-finasteride nanocrystals obtained in Examples 1 to 3 into it respectively. After incubation at 37°C for 30 minutes, slice it and observe it under an inverted fluorescence microscope.

[0089] The results of the passive penetration effect evaluation are as follows: Figure 10 As shown, the skin diffusion area was measured using ImageJ. With the increase of chitosan viscosity, the skin diffusion area of ​​chitosan-finasteride nanocrystals passively penetrated gradually decreased (100-200 mPa·s > 200-400 mPa·s > greater than 400 mPa·s), all of which were significantly different from those of finasteride nanocrystals.

[0090] The results of the study on the effect of hair follicle accumulation are as follows: Figure 11 As shown, the skin diffusion area was measured using ImageJ. With increasing chitosan viscosity, the follicular accumulation effect of chitosan-finasteride nanocrystals weakened, and the average gray value decreased (100-200 mPa·s > 200-400 mPa·s > greater than 400 mPa·s). Among these, chitosan with a molecular weight and viscosity greater than 400 mPa·s had a similar accumulation effect to the finasteride nanocrystal group, without a significant enhancement effect.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention, characterized in that, The microneedle formulation includes a needle tip, an extension, and a root. The needle tip and the extension are both composed of chitosan-finasteride nanocrystals, and the root is hyaluronic acid. The viscosity of the chitosan is 100-200 mPa•s or 200-400 mPa•s.

2. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 1, characterized in that, Includes the following steps: (1) Under ultrasonic conditions, finasteride ethanol solution was added to sodium dodecyl sulfonate aqueous solution, and then purified by ultrafiltration to obtain a suspension containing finasteride nanocrystals. (2) Add chitosan to an aqueous acetic acid solution, let it stand, and adjust the pH of the chitosan mixture to 5.5~6.

5. Place the entire reaction system in an ice bath, and under ultrasonic conditions, inject the suspension containing finasteride nanocrystals obtained in step (1) into the chitosan mixture using a syringe, and then purify by ultrafiltration to obtain a chitosan-finasteride nanocrystal suspension. (3) Add the chitosan-finasteride nanocrystal suspension obtained in step (2) to the polyvinylpyrrolidone solution, mix evenly, add it to the microneedle cavity, centrifuge and dry multiple times to prepare the needle tip and extension, then add hyaluronic acid solution, centrifuge and dry to obtain the chitosan-finasteride nanocrystal microneedle preparation that enhances skin retention.

3. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (1), the concentration of the finasteride ethanol solution is 1 mg / mL to 30 mg / mL; Alternatively, in step (1), the concentration of the sodium dodecyl sulfonate aqueous solution is 4 mg / mL to 5 mg / mL.

4. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 3, characterized in that, The concentration of the finasteride ethanol solution is 20 mg / mL.

5. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (1), the mass ratio of finasteride ethanol to sodium dodecyl sulfonate is 1~30:40; Alternatively, in step (1), the molecular weight cutoff by ultrafiltration purification is 3kDa~30kDa.

6. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 5, characterized in that, The mass ratio of finasteride ethanol to sodium dodecyl sulfonate is 20:

40.

7. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (2), the viscosity of chitosan is 100-200 mpa•s or 200-400 mpa•s.

8. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (2), the mass fraction of acetic acid in the acetic acid aqueous solution is 0.1% to 10%. Alternatively, in step (2), the concentration of chitosan in the aqueous acetic acid solution is 4.5 mg / mL to 5.5 mg / mL.

9. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 8, characterized in that, The mass fraction of acetic acid in the aqueous solution of acetic acid is 1%.

10. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 8, characterized in that, The concentration of chitosan in the aqueous acetic acid solution was 5 mg / mL.

11. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (2), the mass ratio of chitosan to finasteride nanocrystals is 1:10 to 10:1; Alternatively, in step (2), the injection rate using a syringe is 1.5~2.5 mL / min.

12. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (2), the molecular weight cutoff for ultrafiltration purification is 3 kDa to 30 kDa.

13. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 12, characterized in that, The molecular weight cutoff for ultrafiltration purification is 10 kDa.

14. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (3), the mass fraction of polyvinylpyrrolidone in the polyvinylpyrrolidone solution is 25-35%; Alternatively, in step (3), after adding the chitosan-finasteride nanocrystal suspension to the polyvinylpyrrolidone solution, the concentration of the chitosan-finasteride nanocrystals is 7 mg / mL to 9 mg / mL.

15. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 14, characterized in that, The polyvinylpyrrolidone solution contains 30% polyvinylpyrrolidone by mass.

16. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 14, characterized in that, The concentration of chitosan-finasteride nanocrystals was 8 mg / mL.

17. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 2, characterized in that, In step (3), the hyaluronic acid solution includes a 5% hyaluronic acid solution with a molecular weight of 5kDa and a 10% hyaluronic acid solution with a molecular weight of 30kDa, with a mass ratio of 1:1.5~2.5; the total amount of hyaluronic acid solution added is 1~1.5mL.

18. The method for preparing the chitosan-finasteride nanocrystal microneedle formulation for enhancing skin retention as described in claim 17, characterized in that, The hyaluronic acid solution contains 5% by mass of a 5kDa hyaluronic acid solution and 10% by mass of a 30kDa hyaluronic acid solution, with a mass ratio of 1:2.

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