Nanoparticle microneedle for treating alopecia and preparation method thereof

Abstract

The invention discloses a nanoparticle microneedle for treating alopecia and a preparation method thereof.The nanoparticle microneedle comprises a backing layer and a needle body arranged on the surface of the backing layer, the needle body comprises drug-loaded nanoparticles and a polymer matrix, and the backing layer comprises the polymer matrix; the drug-loaded nanoparticle is of a core-shell structure, a core material is a 5 alpha-reductase inhibitor, and a shell material is a ferulic acid polymer. Wherein the polyferulic acid not only has the capacity of loading drugs, but also can provide beneficial effects for treatment of alopecia compared with common high-molecular polymers, so that the advantages of combination of the two drugs can play a positive role in treatment of androgen-derived alopecia. The nanoparticles based on the ferulic acid polymer are further prepared into the soluble microneedle, so that the delivery efficiency of the medicine can be remarkably improved, the problem of high flowability of solution administration is avoided, the retention time of the medicine in the skin is prolonged, and the effect of treating androgen-derived alopecia is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a nanoparticle microneedle for treating hair loss and a preparation method thereof. Background Art

[0002] Androgenic alopecia (AGA) is a progressive hair follicle miniaturization disease that begins in adolescence or late adolescence. It includes male pattern baldness and female pattern baldness and is the most common form of hair loss in the world. The pathogenesis of androgenic alopecia has not yet been clearly studied. It is currently believed to be related to the local enrichment of dihydrotestosterone (DHT) caused by androgen disorders. Testosterone and DHT are types of endogenous androgens. Testosterone can be converted into DHT by binding to 5α-reductase (5α-reductase, SRD5A2). At present, the main clinical treatments for hair loss are drug therapy and surgical treatment. The first-line clinical hair loss treatment drugs are minoxidil and finasteride, which are often used in combination to treat hair loss. Surgical treatment mainly increases the patient's hair volume through hair follicle transplantation, but due to the difficulty of obtaining donor hair follicles and the technical difficulty of surgery, it is still limited in development.

[0003] Finasteride is a competitive type II 5α-reductase inhibitor, which is mainly used in the treatment of benign prostatic hyperplasia in clinical practice and has been widely used to treat AGA. Finasteride is mainly used to treat androgen-induced hair loss. Its mechanism of action is mainly that finasteride, as a 5α-reductase inhibitor, inhibits 5α-reductase at the hair follicles, prevents the increase of dihydrotestosterone at the hair follicles, and reduces the effect of dihydrotestosterone on hair follicle atrophy. In 1997, the FDA approved an oral dose of 1 mg / day of finasteride for the treatment of mild to moderate AGA in adult men. However, the androgen imbalance side effects caused by oral finasteride tablets and the need for long-term use make patient compliance poor. In addition, oral finasteride also has side effects such as decreased libido, male breast development, and erectile dysfunction. At present, studies have been conducted on the topical application of finasteride. A phase III randomized controlled clinical trial found that the topical use of 0.25% finasteride alone can significantly improve the number of hair in patients with male pattern hair loss compared with the placebo group. Therefore, topical finasteride is promising for the treatment of AGA, but research on the dosage form is still needed to develop formulations more suitable for topical application. Summary of the invention

[0004] In order to overcome the problems existing in the above-mentioned prior art, one of the purposes of the present invention is to provide a nanoparticle microneedle. The second purpose of the present invention is to provide a method for preparing the above-mentioned nanoparticle microneedle. The third purpose of the present invention is to provide an application of the above-mentioned nanoparticle microneedle.

[0005] Ferulic acid is one of the effective ingredients of Chinese medicinal materials such as Ferula, Angelica, Chuanxiong, Cimicifuga, and Ziziphus jujuba seeds, and is often used as one of the quality indicators of Chinese patent medicines. Yangxue Shengfa Capsule has been used for many years to improve the symptoms of androgenic alopecia, and its prescription composition includes ferulic acid as an effective ingredient. Ferulic acid has a certain proliferation-promoting effect on human hair papilla cells cultured in vitro, thereby affecting hair growth. Polyferulic acid is polymerized by esterification reaction of ferulic acid. Studies have shown that polyferulic acid can self-assemble to form nanoparticles and has a certain drug loading capacity.

[0006] Microneedles can deliver therapeutic drugs through the stratum corneum of the skin by forming microchannels in the skin. Due to the advantages of easy use and almost no pain, microneedles have been widely studied for skin diseases such as androgenic alopecia, acne and melanoma. In a randomized double-blind clinical trial, it was found that patients who used microneedle treatment once a week and applied 5% minoxidil solution externally had earlier new hair growth and longer-lasting effects compared to patients who used 5% minoxidil solution alone. Dissolving microneedles are a type of microneedle that combines the dual characteristics of subcutaneous injection and skin patch. It has unique advantages in the treatment of hair loss. Microneedles can improve the hair follicle microenvironment and promote hair regeneration by stimulating dermal\hair follicle stem cells. It increases the expression of hair follicle-related cytokines and enhances the expression of hair growth-related Wnt / β-catenin pathway proteins. In addition, microneedles improve the efficiency of drug percutaneous penetration, release drugs through needle tip swelling or dissolution, and avoid the defects of inaccurate dosage of solid microneedles.

[0007] The present invention loads 5α-reductase inhibitors onto naturally derived polyferulic acid nanoparticles to prepare nanoparticle microneedles, which are topically applied to treat androgenic alopecia. This avoids systemic side effects caused by oral administration of finasteride, and promotes the treatment of androgenic alopecia through multiple mechanisms by combining ferulic acid with finasteride. After the nanoparticle microneedles are prepared, the dosage of finasteride is effectively reduced, achieving a better effect.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a nanoparticle microneedle, comprising a backing layer and a needle body arranged on the surface of the backing layer, wherein the backing layer comprises a polymer matrix; the needle body comprises drug-loaded nanoparticles and the polymer matrix; the polymer matrix wraps the drug-loaded nanoparticles; the drug-loaded nanoparticles are a core-shell structure, wherein the core material is a 5α-reductase inhibitor and the shell material is a ferulic acid polymer.

[0010] Preferably, the polymer matrix includes at least one of hyaluronic acid, sodium hyaluronate, chitosan, dextran, sodium carboxymethyl cellulose, sodium alginate, polyvinyl alcohol, polyvinyl pyrrolidone and polyethylene glycol.

[0011] More preferably, the molecular weight of the hyaluronic acid is 50-400 kDa.

[0012] Preferably, the needles form a needle array on the backing layer.

[0013] More preferably, the top of the needle body is 500-800 μm away from the backing layer.

[0014] More preferably, the distance between the tops of the needle bodies is 500-800 μm.

[0015] More preferably, the bottom size of the microneedle is (150-250) μm×(150-250) μm.

[0016] Further preferably, the bottom of the microneedle is square.

[0017] More preferably, the microneedle is in the shape of a quadrangular pyramid.

[0018] Preferably, the 5α-reductase inhibitor is selected from finasteride or dutasteride.

[0019] Preferably, the particle size of the drug-loaded nanoparticles is 100 nm.

[0020] Preferably, the drug-loaded nanoparticles are prepared by a preparation method comprising the following steps: adding an oil phase containing ferulic acid polymer and finasteride to water, and preparing the drug-loaded nanoparticles by a nanoprecipitation method.

[0021] More preferably, the mass ratio of the ferulic acid polymer to finasteride is 1:(0.3-1).

[0022] More preferably, the oil phase also includes the use of a stabilizer.

[0023] More preferably, the stabilizer is selected from polysorbate derivatives, polyoxyethylene castor oil derivatives, and vitamin E derivatives.

[0024] More preferably, the mass ratio of the ferulic acid polymer to the stabilizer is 1:(0.5-1).

[0025] More preferably, the ferulic acid polymer is prepared by a preparation method comprising the following steps: thionyl chloride and ferulic acid are reacted in pyridine to prepare the ferulic acid polymer.

[0026] The second aspect of the present invention provides a method for preparing the nanoparticle microneedle according to the first aspect, comprising the following steps:

[0027] S1, dispersing the drug-loaded nanoparticles and the polymer matrix into water to obtain a needle body solution; dispersing the polymer matrix into pure water to obtain a backing layer solution;

[0028] S2. Take the needle body solution and add it to the microneedle mold, centrifuge it, so that the needle body solution fills the cavity of the microneedle mold; then add the backing layer solution to the microneedle mold filled with the needle body solution, centrifuge it again, so that the backing layer solution fills the base of the microneedle mold; dry the microneedle mold filled with the needle body solution and the backing layer solution, and demold it to obtain the nanoparticle microneedle.

[0029] Preferably, the mass ratio of the drug-loaded nanoparticles to the polymer matrix is ​​(0.1-0.5):1, and the dosage ratio of the drug-loaded nanoparticles to water is (0.01-0.3) g:1 mL;

[0030] Preferably, in the backing layer solution, the usage ratio of the polymer matrix to water is (0.1-1) g:1 mL.

[0031] Preferably, step S1 also includes the following steps: dispersing the drug-loaded nanoparticles and the polymer matrix into water to obtain a needle body solution, refrigerating the needle body solution for 8-16 hours to allow it to fully swell, and centrifuging to remove bubbles before use; dispersing the polymer matrix into water to obtain a backing layer solution, refrigerating the backing layer solution for 8-16 hours to allow it to fully swell, and centrifuging to remove bubbles before use.

[0032] Preferably, the centrifugation is performed at 1-5°C.

[0033] Preferably, the drying is carried out at 25-40°C.

[0034] The third aspect of the present invention provides the use of the nanoparticle microneedle described in the first aspect in the preparation of a product for treating androgen-dependent diseases / hair loss diseases.

[0035] Preferably, the nanoparticle microneedle is used in the preparation of a product for treating androgenic alopecia.

[0036] The beneficial effects of the present invention are:

[0037] (1) The present invention provides a nanoparticle microneedle, which comprises a backing layer and a needle body arranged on the surface of the backing layer, wherein the needle body comprises drug-loaded nanoparticles and a polymer matrix, wherein the drug-loaded nanoparticles are prepared based on ferulic acid polymers, wherein the polyferulic acid not only has the ability to load drugs, but also can load 5α-reductase inhibitors. Compared with commonly used polymers, it can also provide beneficial effects for the treatment of hair loss. Therefore, the advantages of the combination of the two drugs can play a positive role in the treatment of androgenic alopecia.

[0038] (2) The present invention further prepares nanoparticles based on ferulic acid polymer into soluble microneedles, which can significantly improve the delivery efficiency of drugs, avoid the problem of high fluidity of solution administration, increase the retention time of drugs in the skin, and significantly improve the effect of androgenic alopecia treatment. In addition, nanoparticle microneedles can upregulate the Wnt / β-catenin pathway, improve the microenvironment of insufficient angiogenesis around hair follicles, and have an anti-oxidative stress effect. The physical stimulation brought by microneedle administration can further promote the treatment of hair loss.

[0039] (3) The present invention reduces the total amount and frequency of finasteride administration while achieving a better hair growth promoting effect than the commercial finasteride preparation. Compared with the commercial preparation with a concentration of 0.25%, the dosage volume is 0.1 mL, 250 μg / time, 1 time / day for 14 consecutive days, and the total amount of administration is 3 mg, while the microneedle administration is 150 μg / tablet, 1 tablet / 2 days, 7 times in 14 days, and the total amount of administration is 900 μg. Nanoparticle microneedles achieve better therapeutic effects while reducing the dosage and frequency of administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the infrared spectrum of the polyferulic acid of Example 1;

[0041] Figure 2 is the HNMR spectrum of the polyferulic acid of Example 1;

[0042] Figure 3 is a particle size distribution diagram of the drug-loaded nanoparticles of Example 2;

[0043] Figure 4 These are the SEM scanning electron microscope images and actual images of the nanoparticle microneedle of Example 3; a and b are SEM scanning electron microscope images at different angles; c and d are the actual image and corresponding enlarged image of the nanoparticle microneedle, respectively.

[0044] Figure 5 is the force and displacement curve of the nanoparticle microneedle of Example 3;

[0045] Figure 6 is the pore-forming rate of the nanoparticle microneedle on the sealing membrane of Example 3;

[0046] Figure 7 The drug-loaded nanoparticles in Example 2 promote the proliferation of human dermal papilla cells; Note: * P<0.05, *** P<0.001, compared with the control group, ### P < 0.001 compared with nanoparticles;

[0047] Figure 8 The results of the cellular uptake experiment of coumarin 6 nanoparticles;

[0048] Fig. 9 Release curves for suspension, nanoparticles, and nanoparticle-microneedle;

[0049] Fig.10 The results are for the in vitro permeation of suspension, nanoparticles and nanoparticle microneedles; Note: ** P<0.01, compared with the suspension group, # P<0.05 compared with the nanoparticle microneedle group; a is the retention amount result, b is the penetration amount result;

[0050] Fig.11 Relative expression of CTNNBIP1 mRNA in human hair papilla cells; Note: ** P<0.01, *** P<0.001, compared with the model group, && P<0.01, compared with the control group, ### P < 0.001, compared with the nanoparticle group;

[0051] Fig.12 The relative expression of DKK1 mRNA in human hair papilla cells; Note: ** P<0.01, *** P<0.001, compared with the model group, && P<0.01, compared with the control group, ### P < 0.001, compared with the nanoparticle group;

[0052] Fig.13 The relative expression level of SRD5A2 mRNA in human hair papilla cells; Note: *** P<0.001, compared with the model group, &&& P<0.001, compared with the control group, ## P<0.01, ### P < 0.001, compared with the nanoparticle group;

[0053] Fig.14 Relative expression of VEGF mRNA in human hair papilla cells; Note: * P<0.05, compared with the model group, &&& P<0.001, compared with the control group, # P < 0.05, compared with the nanoparticle group;

[0054] Fig.15 is the survival rate of human dermal papilla cells in each treatment group under hydrogen peroxide conditions; Note: *** P < 0.001, compared with the model group;

[0055] Fig.16 Fluorescence detection of ROS produced by human dermal papilla cells in each treatment group under hydrogen peroxide conditions; Note:*** P < 0.001, compared with the model group;

[0056] Fig.17 Nanoparticles promote the proliferation and migration of human umbilical vein endothelial cells;

[0057] Fig.18 The promoting effect of each treatment group on the migration of human umbilical vein endothelial cells;

[0058] Fig.19 The migration area of ​​human umbilical vein endothelial cells in each group is statistically shown; Note: *** P<0.001, compared with the control group, ## P<0.01, ### P<0.001, compared with the blank nanoparticle group, &&& P < 0.001, compared with the finasteride group;

[0059] Fig. 20 Visualization results of coumarin 6 enrichment in hair follicles in vivo;

[0060] Fig.21 The changes in hair growth of mice in each group after drug administration;

[0061] Fig. 22 This is the statistics of the regenerated hair coverage rate of each group of mice after drug administration; Note: ** P<0.01, *** P<0.001, compared with the model group, ## P<0.01, ### P<0.001, compared with the nanoparticle microneedle group

[0062] Fig.23 This is the statistics of pigmentation time of mice in each group after drug administration; Note: *** P<0.001, compared with the model group, # P<0.05, ### P < 0.001, compared with the nanoparticle-microneedle group;

[0063] Fig.24 The length of regenerated hair of each group of mice after drug administration; Note: *** P<0.001, compared with the model group, # P<0.05, ### P<0.001, compared with the nanoparticle-microneedle group. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below through specific examples. The raw materials used in the following examples, unless otherwise specified, can be obtained from conventional commercial sources or prepared and separated by simple synthesis; the processes used, unless otherwise specified, are conventional processes in the art.

[0065] Example 1

[0066] This embodiment provides a method for preparing polyferulic acid, and the preparation method is as follows:

[0067] Add 10 mL of pyridine to a round-bottom flask under ice bath and stir for 5 min. Add a certain amount of thionyl chloride quickly under stirring and stir for 15 min. Add ferulic acid so that the molar ratio of thionyl chloride to ferulic acid is 1:1, and stir to react for 4 h. After the reaction is completed, add ultrapure water to obtain a yellow insoluble substance. Wash with 1% dilute hydrochloric acid and ultrapure water, filter and dry to obtain polyferulic acid powder. Figure 1 and Figure 2 The FTIR and HNMR spectra shown confirm the successful preparation of polyferulic acid.

[0068] Example 2

[0069] This embodiment provides a polyferulic acid nanoparticle and a preparation method thereof, and the preparation method is specifically as follows:

[0070] (1) Preparation of blank nanoparticles: Raw materials: 2 mg polyferulic acid, 1.2 mg vitamin E polyethylene glycol succinate (TPGS) and 5 mL water.

[0071] Preparation method of blank nanoparticles: polyferulic acid and vitamin E polyethylene glycol succinate are dissolved in dimethyl sulfoxide as the oil phase, the oil phase solution is added dropwise into 5 mL of water at a rotation speed of 1200 rpm, stirred for 30 seconds to obtain a nanoparticle solution, and washed by ultrafiltration.

[0072] (2) Preparation of drug-loaded nanoparticles: Raw materials: 2 mg polyferulic acid, 1.2 mg vitamin E polyethylene glycol succinate (TPGS), 1 mg finasteride and 5 mL water.

[0073] Preparation method of drug-loaded nanoparticles: polyferulic acid, finasteride and vitamin E polyethylene glycol succinate are dissolved in dimethyl sulfoxide as an oil phase, and the above oil phase solution is added dropwise into 5 mL of water at a rotation speed of 1200 rpm, stirred for 30 seconds to obtain a nanoparticle solution, and then ultrafiltered and washed.

[0074] The particle size distribution of drug-loaded nanoparticles is shown in Figure 3 , particle size distribution is 30~120nm, D 50 Around 100nm.

[0075] Example 3

[0076] This embodiment provides a soluble microneedle and a preparation method thereof, and the preparation method is specifically as follows:

[0077] (1) Preparation of soluble microneedle tip matrix: 0.25 g hyaluronic acid (52 kDa) was added to 1 mL of 6.25% (w / v) nanoparticle aqueous solution, stirred evenly and placed in a refrigerator overnight to allow it to fully swell. Bubbles were removed by centrifugation before use.

[0078] (2) Preparation of soluble microneedle backing layer matrix

[0079] Preparation Materials: 0.1 g hyaluronic acid (52 kDa), 0.1 g hyaluronic acid (200-400 kDa), 0.1 g dextran 80, 1 mL water;

[0080] Preparation method: Add hyaluronic acid (52kDa), hyaluronic acid (200-400kDa) and dextran into water, stir well, put in the refrigerator overnight to allow it to fully swell, and remove bubbles by centrifugation before use.

[0081] (3) Preparation of soluble microneedles:

[0082] The soluble microneedle consists of two parts, namely, the backing layer and the needle tip from top to bottom, and is prepared using a step-by-step centrifugal drying method. Preparation of the needle tip: Add 0.05 g of the needle tip matrix of step (1) to the PDMS mold, centrifuge it 4 times at 3750 rpm at 4°C for 3 minutes each time, and change the direction of the mold so that the matrix fills the needle tip cavity of the mold. Preparation of the backing layer: Add 0.1 g of the backing layer matrix of step (2) to the microneedle mold, centrifuge it 4 times at 3750 rpm at 4°C for 2 minutes each time, and change the direction of the mold so that the base matrix is ​​flattened in the mold. Drying of the microneedle: Place the microneedle in a dryer and dry it at room temperature for 48 hours. The target microneedle can be obtained after demolding.

[0083] The prepared microneedles are Figure 4 As shown, under a digital photo microscope and a scanning electron microscope, a microneedle is square in shape, containing 144 microneedles distributed in a 12×12 pattern. The microneedles are regular quadrangular pyramids with a height of 600 μm and a bottom width of 200 μm. The distance between the microneedle tips is about 660 μm.

[0084] The prepared microneedles were characterized. The mechanical strength of the microneedles was measured using a texture analyzer. The microneedle tip was placed on the stage with the tip facing upwards, the probe was moved to the tip of the microneedle, and the initial position was recorded. The probe was set to move downward at a speed of 1 mm / s from the initial position, and the force and displacement curve of the probe from the initial position to the complete breaking of the microneedle body was recorded. The results are shown in Figure 2. Figure 5As shown, it shows that the microneedle did not break during the force process and has good mechanical properties. The penetration depth of the microneedle was measured using Parafilm sealing film. Each layer of sealing film is equivalent to the thickness of 100μm skin. Eight layers of sealing film were superimposed to simulate human skin. The microneedle tip was placed on the sealing film with the tip facing down. A digital push-pull force gauge was used to apply a force of 30N vertically downward on the microneedle backing layer. After maintaining for 1 minute, the microneedle was removed and the porosity of each layer of sealing film was calculated. Figure 6 The results showed that the microneedles could penetrate into the subcutaneous tissue to a depth of about 400 μm.

[0085] Experimental Example 1 Cellular Efficacy Experiment of Nanoparticles

[0086] 1. Cell proliferation assay

[0087] Since the nanoparticles will be used to treat androgenic alopecia, the cell proliferation of human dermal follicle papilla cells (HDPCs) which play an important role in the process of hair loss was investigated. The proliferation of HDPC cells is beneficial to the treatment of androgenic alopecia.

[0088] HDPC in the logarithmic growth phase was digested and inoculated into a 96-well plate to ensure that the cell inoculation density was 8000 cells / well. Subsequently, the well plate was placed in an incubator for overnight culture to allow the cells to adhere to the wall. The nanoparticle suspension, ferulic acid and vitamin E polyethylene glycol succinate were diluted to the corresponding concentrations with complete DMEM culture medium and incubated with the cells for 24 hours. Then, the 96-well plate was taken out of the incubator, the original culture medium was removed, 120 μL of MTT dilution (containing MTT 0.83 mg / mL) was added to each well, and the plate was placed in an incubator to be cultured in the dark. After 4 hours, the culture medium in the well was removed, 150 μL of DMSO was added to each well, and the plate was placed on a shaker for 10 minutes to allow the crystals to fully develop color. The above process was kept in the dark. Finally, the absorbance value (OD) of each well in the 96-well plate was measured at 490 nm using an ELISA reader, and the cell survival rate was calculated according to formula (1).

[0089]

[0090] Among them, the drug administration group is a group that is both implanted with cells and administered with drugs, the control group is a group that is implanted with cells but not administered with drugs, and the zero adjustment group is a group that is neither implanted with cells nor administered with drugs.

[0091] The experimental results are as follows Figure 7 As shown, both nanoparticles and ferulic acid have the effect of promoting HDPC cell proliferation below 20 μM concentration, and the effect of nanoparticles is stronger than that of ferulic acid alone, which may be due to the synergistic effect of TPGS in the nanoparticle composition.

[0092] 2. Cellular uptake experiment

[0093] HDPC cells in the logarithmic growth phase were taken and digested to a concentration of about 1×10 5 The cells were inoculated in the confocal dish at a concentration of 100 cells / dish. The confocal dish was placed in an incubator and cultured overnight to allow it to adhere completely to the wall. After the cells adhered to the wall, the culture medium was removed, and the cells were washed three times with PBS. 1 mL of culture medium, diluted coumarin 6 suspension, and coumarin 6 nanoparticles were added to each dish. The confocal dish was placed in a 37°C incubator and incubated for 4 hours. The culture medium was removed, and the cells were washed three times with pre-cooled PBS. A 4% paraformaldehyde solution was added to fix the cells for 15 minutes, and the cells were washed three times with pre-cooled PBS. DAPI solution was added and removed after 25 minutes to stain the cell nuclei blue, and the cells were washed three times with pre-cooled PBS. The whole process was kept away from light. Observe and take pictures with a laser confocal microscope.

[0094] The experimental results are as follows Figure 8 As shown, in the control group, DAPI can be seen to dye the cell nucleus blue, and no C6 green fluorescence is observed. In the coumarin 6 suspension group, partial green fluorescence is observed. HDPC cells take up less coumarin 6 in the suspension, which may be due to the low solubility of coumarin 6 in aqueous solution. In the coumarin 6 nanoparticle group, the green fluorescence is stronger than that in the suspension group, indicating that the preparation of nanoparticles promotes the cell uptake of coumarin 6.

[0095] Comparative Example 1

[0096] This comparative example provides a finasteride suspension, and the preparation method thereof is as follows: weigh 0.5 mg of finasteride, add it into 1 mL of water and stir evenly to prepare a suspension.

[0097] Comparative Example 2

[0098] This comparative example provides a nanoparticle suspension, and the preparation method thereof is as follows: the nanoparticles of Example 2 are diluted to a finasteride concentration of 0.5 mg / mL to obtain a nanoparticle suspension.

[0099] Comparative Example 3

[0100] This comparative example provides a finasteride solution, and the preparation method thereof is as follows: weigh 0.5 g of hydroxypropyl chitosan and dissolve it in 27.5 mL of anhydrous ethanol and 2.5 mL of propylene glycol, add water to 50 mL, weigh 125 mg of finasteride and dissolve it in the above-mentioned composite solvent to prepare a finasteride solution with a concentration of 2.5 mg / mL. This comparative example is prepared with reference to a commercially available prescription.

[0101] Comparative Example 4

[0102] This comparative example provides a minoxidil tincture, which is a commercially available minoxidil tincture with a concentration of 5%.

[0103] Experimental Example 2 In vitro drug release experiment of microneedles

[0104] The microneedles prepared in Example 3 were used to examine the release behavior of finasteride from the microneedles.

[0105] In this experiment, the dialysis bag method was used to investigate the in vitro release behavior of microneedles. The dialysis bag with a molecular weight cutoff of 500Da was used for the experiment. The dialysis bag was taken out of the 4°C refrigerator, washed with ultrapure water, and the filter paper was used to absorb the water. The microneedles were placed in the dialysis bag, and the nanoparticle suspension and finasteride suspension were also added to the dialysis bag. The two ends of the dialysis bag were tied with nylon tie. The dialysis bag was placed in a 50mL centrifuge tube, and 7mL of 30% (v / v) PEG 400 saline was added to each centrifuge tube as the release medium. In a constant temperature air bath shaker, the temperature was set to 32°C and the speed was set to 250rpm. 1mL of release medium was taken out from the centrifuge tube at 30min, 1h, 2h, 4h, 6h, 9h, 12h, and 24h, and 1mL of blank release medium was added. After the sample was filtered through a 0.22μm microporous filter membrane, the content of FIN was determined by HPLC. The cumulative release amount at each time point was calculated according to formula (2), and the cumulative release degree (%) was calculated according to the ratio of the cumulative release amount to the total content of finasteride in the microneedles.

[0106]

[0107] Wherein, Cn is the drug concentration measured at the nth sampling, Ci is the drug concentration measured at each sampling, V represents the volume of the release medium, Vi represents the volume of each sampling, and Qn (μg) is the cumulative drug release.

[0108] Finasteride content determination method: Chromatographic column: XB-C18 column ( 250×4.6mm,5μm). Mobile phase: water:acetonitrile=50:50 (v / v); flow rate: 1mL / min; detection wavelength: 210nm; injection volume: 20μL.

[0109] The experimental results are as follows Fig. 9 As shown in the figure, within 48 hours, finasteride was completely released from the microneedles, reaching more than 80%. The release rate of the microneedle group was slower than that of the suspension.

[0110] Experimental Example 3 In vitro microneedle permeation experiment

[0111] The rat skin was removed and thawed in physiological saline, and the surface moisture was dried with filter paper to check the skin integrity. The microneedle was pressed on the stratum corneum of the skin with a fixed force of 30N for 3 minutes, and then fixed on the skin with medical 3M tape.

[0112] Then assemble the transdermal device. First, add a stir bar to the receiving pool, then place the gasket, rat skin, and supply pool on top. Make sure the stratum corneum of the rat skin faces the supply pool. The effective area of ​​the diffusion pool is 3.14 cm 2 , the receiving pool capacity is 8mL, and the selection of the receiving medium is the same as the release experiment, which is 30% (v / v) PEG 400 saline. Add 8mL of receiving medium to the receiving pool, and be careful not to generate bubbles. Place the device in a transdermal diffusion tester, adjust the temperature to 32°C, and the speed to 250rpm. Example 6, Comparative Example 1 and Comparative Example 2 are selected as controls, added to the supply pool, and the dosage per unit area is kept consistent with the microneedle group. Take out 1mL of receiving solution at 1, 2, 4, 8, 12, and 24h, respectively, and add 1mL of blank receiving solution to ensure that the volume of the receiving solution in the receiving pool is consistent. After filtering the obtained sample with a 0.22μm microporous filter membrane, the content of finasteride therein is determined by high performance liquid chromatography. The cumulative permeation amount Qn of finasteride per unit area is calculated according to formula (3).

[0113]

[0114] Where A is the transdermal area, Cn is the measured drug concentration at the nth sampling, V is the diffusion medium volume, Vi is the sampling volume at each time, Ci is the concentration measured at each sampling, and Qn is the cumulative permeation per unit area of ​​FIN.

[0115] After sampling at the 24h time point, the diffusion cell was removed, the rat skin was taken out, and the skin at the administration site was wiped with medical cotton balls containing water, methanol, and water, three times each, in order to remove the remaining drugs on the surface. Then the administration part of the skin was cut off, the water was absorbed with filter paper, the skin was placed in an EP tube, the skin was cut into pieces with surgical scissors, and weighed. 1mL of methanol was added to the EP tubes with cut skin, and ultrasonicated for 30min to fully dissolve the drug in the skin in methanol, and then stood at room temperature for 2h. The supernatant was taken out, filtered with a 0.22μm microporous membrane, and the content of finasteride was determined by high performance liquid chromatography, and divided by the weight of the skin to obtain the retention amount per gram of skin.

[0116] The experimental results are shown in Fig.10 The 24h permeation amount of the microneedle group was similar to that of the suspension group and the nanoparticle group, and the retention amount in the skin was higher than that of the nanoparticle group and the suspension group. Loading nanoparticles into microneedles is conducive to the retention of drugs in the skin, thereby reducing systemic side effects.

[0117] Experimental Example 4 Cellular Pharmacodynamic Evaluation of Finasteride Nanoparticles

[0118] 1. Cell PCR experiment

[0119] In order to investigate the effects of nanoparticles on the expression of factors related to hair-related signaling pathways and angiogenesis in HDPC cells, HDPC cells were modeled using dihydrotestosterone. After administration, qRT-PCR experiments were performed to investigate the expression of mRNA of CTNNBIP1, DKK1, SRD5A2 and VEGF.

[0120] The specific method is as follows: HDPC cells in the logarithmic growth phase were digested and inoculated in a 12-well plate at a density of 3×10 5 / well. Overnight culture allowed the cells to adhere to the wall, then the original culture medium was removed, DMEM culture medium was added to the control group, 1 μM dihydrotestosterone solution was added to the model group, and the other drug groups were added with drug solution and 1 μM dihydrotestosterone solution, respectively, and incubated for 24 hours. The drug groups were finasteride, nanoparticles, and finasteride nanoparticles, respectively. The dosage of finasteride nanoparticles was 0.15 μg / mL, and the dosage of finasteride and the corresponding drug in the nanoparticles was consistent. The cells were treated according to the instructions of the PCR kit, and fluorescence quantification was performed using a real-time fluorescence quantitative PCR instrument.

[0121] The experimental results of the relative mRNA expression of CTNNBIP1, DKK1, SRD5A2 and VEGF in human dermal papilla cells are shown as follows: Figure 11-14 As shown in the figure, after DHT modeling, the expression of CTNNBIP1 and VEGF was downregulated, and the expression of SRD5A2 and DKK1 was upregulated. The FIN, Blank NPs and FIN-NPs groups could upregulate CTNNBIP1 and VEGF, downregulate SRD5A2 and DKK1, and the effect of finasteride nanoparticles in reversing the abnormal effects of DHT was better than that of other groups.

[0122] 2. Antioxidant test

[0123] Hair loss is usually associated with the local oxidative stress microenvironment around the hair follicles. Oxidative stress is mainly caused by excessive reactive oxygen species (ROS), which leads to apoptosis of hair papilla cells and hinders the transition of hair follicles from the resting phase to the growth phase. Therefore, this study examined the antioxidant capacity of nanoparticles through antioxidant damage experiments and ROS scavenging experiments.

[0124] (1) Antioxidant damage experiment

[0125] Use H 2 O 2 The cell oxidative damage model was created to simulate the state of human dermal papilla cells under oxidative stress. HDPC in the logarithmic growth phase were digested and inoculated in a 96-well plate at a density of 8,000 cells / well. The plate was then placed in an incubator for overnight culture. After the cells adhered to the wall, the original culture medium was removed and different drugs were used for treatment. The control group was only treated with culture medium without any drugs, and the model group was treated with 700 μM H 2O 2 , the drug group 2 O 2 Finasteride, nanoparticles and finasteride nanoparticles were added on the basis of the above. The dosage of finasteride nanoparticles was 0.15 μg / mL based on finasteride, and the dosage of finasteride and the corresponding drugs in the nanoparticles were consistent. After 24 hours of drug incubation, the cell survival rate was detected by MTT method to understand the protective ability of different drugs against cell oxidative damage.

[0126] The experimental results are as follows Fig.15 As shown. 2 O 2 After modeling, the survival rate of HDPC cells decreased and cell viability was damaged. Both nanoparticles and finasteride nanoparticles can reduce H 2 O 2 Oxidative damage caused by modeling.

[0127] (2) ROS scavenging experiment

[0128] HDPC cells in the logarithmic growth phase were digested and seeded in 12-well plates at a seeding density of 1×10 5 The cells were placed in an incubator overnight, and the original culture medium was removed after the cells adhered to the wall. Different drugs were then applied. The control group was treated with culture medium only without any drugs, and the model group was treated with 700 μM H 2 O 2 The drug group 2 O 2 Finasteride, nanoparticles and finasteride nanoparticles were added on the basis of the above. The dosage of finasteride nanoparticles was 0.15 μg / mL based on finasteride, and the dosage of finasteride and the corresponding drugs in the nanoparticles were consistent. After incubation for 24 hours, the original culture medium was removed, the cells were washed with PBS three times, and the diluted ROS fluorescent probe DCFH-DA was incubated with the cells for 1 hour. Then, the cells were washed with PBS three times to remove the fluorescent probes that did not enter the cells. Finally, a fluorescence microscope was used to take pictures to evaluate the drug's ability to scavenge ROS.

[0129] The experimental results are as follows Fig.16 As shown in the results, nanoparticles and finasteride nanoparticles can reduce the production of intracellular ROS. Consistent with the results of cell activity experiments, nanoparticles and finasteride nanoparticles can reduce H 2 O 2 Oxidative damage caused by modeling.

[0130] 3. Cell migration assay

[0131] The formation of microvessels around hair follicles can provide rich nutrients, immune cells and biological factors for hair follicle growth, thereby promoting the transformation of hair follicle cycle and repairing damaged hair follicles. Human umbilical vein endothelial cells (HUVEC) are a commonly used model cell for studying angiogenesis. Therefore, the effect of nanoparticles on angiogenesis was evaluated by examining the proliferation effect of nanoparticles on HUVEC cells and improving their migration ability.

[0132] HUVEC cells were plated at 5 × 10 5 The cells were inoculated at a density of 100 μg / cm2 in a 6-well plate and cultured overnight in an incubator. A 200 μL sterilized pipette tip was used to draw a line perpendicular to the bottom edge of the 6-well plate, and the culture medium was removed. The cells were rinsed 2-3 times with sterile PBS to remove the scratched cells. Finasteride, nanoparticles, and finasteride nanoparticles were diluted to the corresponding concentrations with incomplete DMEM culture medium and incubated with the cells. Only fresh culture medium was added to the control group. Cell migration was observed under a microscope at 0 h, 24 h, and 48 h after drug incubation, and the scratch area at different times was counted using Image J software. The scratch area at 0 h was defined as the initial scratch area, and the cell migration rate was calculated according to formula (4).

[0133]

[0134] The experimental results are as follows Figure 17-19 As shown in the figure, it can be seen that at a concentration of 0.15 μg / mL, blank nanoparticles and drug-loaded nanoparticles have the effect of promoting HUVEC cell proliferation. The HUVEC scratch area decreases with time, and at 48 hours, the relative migration area of ​​the finasteride nanoparticle group is significantly higher than that of the finasteride group and the nanoparticle group. This shows that finasteride nanoparticles can promote the proliferation and migration of HUVEC cells and promote angiogenesis.

[0135] Experimental Example 5 Study on the Hair Follicle Enrichment of Nanoparticle Microneedles

[0136] In order to observe whether the nanoparticles can transport drugs to the hair follicles, coumarin 6 (green fluorescence) was used to simulate the lipophilic drug finasteride, and an in vivo hair follicle enrichment visualization experiment was performed. The distribution of coumarin 6 in the skin was observed using a laser confocal microscope.

[0137] Take SD rats weighing about 180g, anesthetize them with 20% (w / v) urethane, shave the abdominal hair with an electric shaver, and then use depilatory cream to remove hair. Let the skin recover for 24h. Anesthetize the rats with 20% (w / v) urethane, fix them on the mouse board, and glue the supply pool of the diffusion cell (the drug administration area is 3.14cm 2) was pasted on the abdominal skin of the rat, and the microneedle was pressed on the stratum corneum of the skin with a fixed force of 30N. After maintaining for 3 minutes, the microneedle was fixed on the skin with medical 3M tape. Example 6, Comparative Example 1 and Comparative Example 2 were selected as controls and added to the supply pool, and the dosage per unit area was kept consistent with that of the microneedle group, and it was evenly distributed in the supply pool. The upper end of the supply pool was sealed with sealing film and tin foil to prevent water evaporation, and the rats were placed in a dark place for 24 hours of in vivo transdermal experiment. Three rats were taken out at 1, 4, 8, 12, and 24 hours respectively, and the rats were killed by cervical dislocation to remove residual drugs. The skin of the administration site was wiped 3 times each with cotton balls soaked in water, methanol, and water, and the skin was quickly removed. The skin at the administration site was cut into small pieces of about 2 mm × 5 mm using surgical scissors, embedded in OCT frozen section embedding medium, and then longitudinally sectioned on a frozen section machine with a thickness of 20 μm. The obtained skin section samples were adhered to a glass slide. The frozen skin section samples were placed under a laser confocal microscope to observe the distribution of coumarin 6 in the skin.

[0138] Fig. 20 The results of visualization of coumarin 6 enrichment in hair follicles in vivo; the green fluorescence of free coumarin 6 is almost exclusively distributed in the stratum corneum. The fluorescence of the nanoparticle and microneedle groups penetrates deep into the skin over time and is mainly concentrated in the hair follicle area, indicating that nanoparticles and microneedles can deliver drugs to the hair follicle area.

[0139] Experimental Example 6 In vivo pharmacodynamic evaluation of microneedles

[0140] Seven-week-old C57BL / 6 male mice were used to establish a hair loss animal model. The control group of mice did not receive any treatment after the back hair was removed, and the remaining mice were modeled with a testosterone solution containing 50% ethanol as a solvent, and 0.1 mL of a 0.5% testosterone solution was applied topically every day for 2 consecutive weeks. Except for the control group, the remaining mice were randomly divided into 7 groups (n=6), and the administration methods of each group were as follows: (1) Control group: 0.1 mL of normal saline was applied topically every day; (2) Model group: 0.1 mL of 0.5% testosterone solution was applied topically every day; (3) Blank nanoparticle group: 0.1 mL of Example 2 (1) was applied topically every 2 days; (4) Finasteride nanoparticle group: 0.1 mL of Example 2 (2) was applied topically every 2 days; (5) Nanoparticle microneedle group: blank nanoparticle microneedle was applied once every 2 days; (6) Finasteride nanoparticle microneedle group: Example 6 was applied once every 2 days; (7) 5% minoxidil group: 0.1 g of 5% minoxidil commercial tincture was applied topically every day, as in Comparative Example 3; (8) 0.25% finasteride group: 0.1 mL of 0.25% finasteride solution was applied topically every day, as in Comparative Example 4. In the above groups, except for the control group, the other groups applied 0.1 mL of 0.5% testosterone solution topically every day on the basis of drug administration. The total dosage of the finasteride nanoparticle group and the finasteride microneedle group during the drug administration period was 0.9 mg, and the total dosage of the 0.25% finasteride solution group was 3 mg. During the drug administration period, photos were taken on days 0, 3, 6, 9, 12, 14, and 16 to record the hair regeneration of the mice, and the hair coverage of the back skin of the mice was calculated using Image J software. After the experiment, the mice were killed by cervical dislocation, their back hair and skin were removed, and the length of the regenerated hair of the mice was measured using a vernier caliper.

[0141] Fig.21 Representative photos of the back hair growth of each group of mice during the drug administration period. All groups had a therapeutic effect relative to the Model group.

[0142] The best effect group: 5% minoxidil group and finasteride nanoparticle microneedle group and control group, which are close to each other and better than blank nanoparticle microneedle group and finasteride nanoparticle group. Fig. 22 The hair coverage area of ​​the finasteride nanoparticle microneedle group and the 5% minoxidil group was comparable, which was better than that of the blank nanoparticle microneedle group, the 0.25% finasteride group, and the finasteride nanoparticle group. Fig.23 The finasteride nanoparticle microneedle group and the 5% minoxidil group showed pigmentation the earliest. Fig.24 The hair growth length in the finasteride nanoparticle microneedle group was the longest, which was statistically different from that in the 5% minoxidil group.

[0143] In summary, polyferulic acid nanoparticles have a positive effect on the treatment of hair loss. After loading finasteride and preparing into microneedles, the promotion effect is further enhanced. Compared with commercially available preparations, the dosage and frequency of administration are reduced. This can provide a new strategy for the treatment of hair loss.

[0144] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A nanoparticle microneedle, characterized in that: It comprises a backing layer and a needle body arranged on the surface of the backing layer, wherein the backing layer comprises a polymer matrix; the needle body comprises drug-loaded nanoparticles and the polymer matrix; the polymer matrix wraps the drug-loaded nanoparticles; the drug-loaded nanoparticles are a core-shell structure, wherein the core material is a 5α-reductase inhibitor and the shell material is a ferulic acid polymer.

2. The nanoparticle microneedle according to claim 1, characterized in that: The polymer matrix includes at least one of hyaluronic acid, sodium hyaluronate, chitosan, dextran, sodium carboxymethyl cellulose, sodium alginate, polyvinyl alcohol, polyvinyl pyrrolidone and polyethylene glycol.

3. The nanoparticle microneedle according to claim 1, characterized in that: The needles form a needle array on the backing layer, and the tops of the needles are 500-800 μm away from the backing layer; and / or the tops of the needles are spaced 500-800 μm apart from each other.

4. The nanoparticle microneedle according to claim 1, characterized in that: The 5α-reductase inhibitor is selected from finasteride or dutasteride.

5. The nanoparticle microneedle according to claim 1, characterized in that: The particle size of the drug-loaded nanoparticles is 30 to 120 nm; The preparation method of the drug-loaded nanoparticles comprises the following steps: adding an oil phase containing a ferulic acid polymer and a 5α-reductase inhibitor into water, and preparing the drug-loaded nanoparticles by a nanoprecipitation method.

6. The nanoparticle microneedle according to claim 5, characterized in that: The oil phase also includes the use of a stabilizer; Preferably, the stabilizer is selected from polysorbate derivatives, polyoxyethylene castor oil derivatives, and vitamin E derivatives.

7. The method for preparing the nanoparticle microneedle according to any one of claims 1 to 6, characterized in that: The steps include: S1, dispersing the drug-loaded nanoparticles and the polymer matrix into water to obtain a needle body solution; dispersing the polymer matrix into water to obtain a backing layer solution; S2, adding the needle body solution into the microneedle mold, centrifuging, so that the needle body solution fills the cavity of the microneedle mold; Then, the backing layer solution is added to the microneedle mold filled with the needle body solution, and centrifuged again to allow the backing layer solution to fill the base of the microneedle mold; The microneedle mold filled with the needle body solution and the backing layer solution is dried and demoulded to obtain the nanoparticle microneedle.

8. The method for preparing nanoparticle microneedles according to claim 7, characterized in that: In the needle solution, the mass ratio of the drug-loaded nanoparticles to the polymer matrix is ​​(0.1-0.5):1, and the dosage ratio of the drug-loaded nanoparticles to water is (0.01-0.3) g:1 mL; And / or, in the backing layer solution, the usage ratio of the polymer matrix to water is (0.1-1) g:1 mL.

9. The method for preparing nanoparticle microneedles according to claim 7, characterized in that: Step S1 also includes the following steps: dispersing the drug-loaded nanoparticles and the polymer matrix into water to obtain a needle body solution, refrigerating the needle body solution for 8-16 hours to allow it to fully swell, and centrifuging to remove bubbles before use; dispersing the polymer matrix into water to obtain a backing layer solution, refrigerating the backing layer solution for 8-16 hours to allow it to fully swell, and centrifuging to remove bubbles before use.

10. Use of the nanoparticle microneedle according to any one of claims 1 to 6 in any one of the following A) or B): A) preparing products for treating androgen-dependent diseases; B) preparing a product for treating hair loss diseases.

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