Gum Arabic Enhances Hair Follicle-Targeting Drug Delivery of Minoxidil Nanocrystal Dispersions

Abstract

In this study, we attempted to enhance the delivery of minoxidil (MXD) nanocrystals into hair follicles for efficacious hair growth treatment. We applied a bead milling method and designed an MXD nanocrystal dispersion containing methylcellulose (MC) and gum arabic (GA), termed MG-MXD@NP, with a particle size of 110 nm. In vivo studies in C57BL/6 mice showed that MG-MXD@NP improved MXD delivery to the skin tissue, hair bulges, and hair bulbs, resulting in earlier and superior hair growth compared with a commercially available MXD lotion (Riup 5%, CA-MXD). These findings were consistent with the increased MXD contents observed in the hair bulge and hair bulb regions in the MG-MXD@NP-treated mice, and suggested a correlation between the efficiency of MXD delivery and efficacy of promotion of hair growth by products containing MXD. Furthermore, delivery of MXD using MG-MXD@NP was associated with elevated expression levels of CD34 and CD200, markers of hair follicle epithelial stem cells, which are crucial for promoting hair growth. Thus, it is possible that the upregulation of CD34 and CD200 in the MG-MXD@NP-treated mice reflects activation of the papilla cells and hair follicle stem cells known to be closely associated with hair growth enhancement. In conclusion, MG-MXD@NP, containing MXD nanocrystals in combination with MC and GA, exhibited a superior hair growth effect as compared with conventional MXD formulations. These findings suggest that this novel delivery method for MXD could represent a promising treatment approach for hair loss.

INTRODUCTION

Androgenetic Alopecia (AGA), otherwise known as male- or female-pattern baldness, refers to thinning of hair commonly seen in adult men. The major causes of AGA include heredity, stress, and the effects of male hormones, and an estimated 12.6 million men nationwide suffer from thinning of their hair.¹⁾ Both activation of follicular epithelial stem cells in the hair bulge and activation of the papilla cells in the bulb region are believed to be closely involved in hair growth and hair regrowth.²⁾ Drugs with proven hair growth and hair regrowth effects include minoxidil (MXD), carpronium chloride hydrate (topical solution 5%), finasteride, and dutasteride. Among these, MXD improves blood flow to the hair tissue, activates the papilla cells, promotes the production of cell growth factors, and inhibits apoptosis of hair matrix cells. MXD has been used not only to treat AGA in men, but also in women.³⁾ On the other hand, excessive MXD levels in the blood can results in adverse reactions such as palpitations, dizziness, and shortness of breath due to decreased blood pressure, so that subjects using MXD should be warned of the potential adverse effects and exercise caution.

Delivery of the drug to the hair bulb region is important for MXD to exert its therapeutic effects. Two routes of transdermal absorption of ingredients with hair growth effects are known: the accessory organ route through the hair follicles, sweat glands, and other accessory organs, and the route through the stratum corneum of the skin.⁴⁾ In regard to drug absorption via the accessory organ pathway, drugs entering the skin through the pores reach the hair bulb via the bulge region, and drugs that reach the hair root migrate into the skin tissue and blood by diffusion.⁴⁾ On the other hand, in drug absorption through the parenchymal horny layer, drugs infiltrating into the skin are transferred to the bulge area, hair bulb, and bloodstream by diffusion.⁴⁾ Formulations containing drug particles are superior to formulations containing drugs in the liquid form for allowing drug infiltration into the skin appendages, and in particular, nanosized particles are believed to show superior drug infiltration through the hair follicle and allow drugs to reach deep inside the hair follicles.^5–9) We previously prepared MXD nanoparticles (MXD-NPs) using a wet bead milling method and demonstrated excellent direct drug penetration of MXD-NPs into the hair follicles via the skin pores, with 10-fold higher drug retention in the bulge region and hair bulb as compared with MXD dispersion without bead mill processing (MXD-MPs).^10,11) In addition, the nanoparticle MXD formulation also showed a higher therapeutic efficacy than MXD micro-suspensions not subjected to fragmentation treatment and other commercially available MXD formulations, suggesting that targeting, or sustained release of the drug to the site of action can be achieved.¹⁰⁾ Furthermore, the exact local efficacy and safety have not yet been reported, although it has been reported that in in vitro experimental systems established using keratinocytes and corneal cells, no cell death was observed in culture treated with the formulation at 103 µg/mL for 7 d, and MXD was effective^12,13); furthermore, even among commercial products, MXD was shown to be more effective than 1% commercial MXD when used at 5 µg/mL for 7 d.^12,13) Thus, at the currently used MXD concentrations (1–5%), an increase in the local concentration has relatively few disadvantages in terms of the systemic side effects, whereas it is thought to lead to improved efficacy.

Gum arabic (GA), with a molecular weight of approximately 250 kDa, is an anionic polysaccharide consisting of a backbone comprising D-galactose units, with side chains comprised of D-glucuronic acid, L-rhamnose, and L-arabinose moieties. GA has the noteworthy ability to form chemical bonds with hydrophobic peptides, conferring aqueous solutions with robust emulsion stability. Consequently, GA finds multifarious applications across diverse domains, serving as a foundational ingredient in powder formulations, as a suspending agent in cosmetics and beverages, as a binder in tablet production, an anti-crystallization agent, as a constituent in microcapsule fabrication, as an emulsion stabilizer, as a glazing agent, and as a coating material. In this study, we investigated whether the addition of GA, with its emulsifying properties, could enhance hair follicle-targeting drug delivery of MXD nanocrystals. In addition, we demonstrated the efficacy of dispersions incorporating both MXD nanocrystals and GA in accelerating the hair growth rate in a C57BL/6 mouse model of AGA.

MATERIALS AND METHODS

Animals

Seven-week-old male C57BL/6 mice were obtained from CLEA Japan, Inc. (Tokyo, Japan) and the hair on their back was removed prior to their use in the experiments performed to evaluate the hair follicle (hair bulge and hair bulb) delivery and hair growth effects of MXD. All the C57BL/6 mice were housed under normal animal housing conditions (room temperature 25 °C; 12 h/12 h light/dark cycle] and had free access to water and a CE-2 diet (CLEA Japan, Inc.). The test MXD formulation (30 µL) was administered to the back (4 cm²) of the C57BL/6 mice once a day (at 10:30 a.m.). The animal experiments were performed in accordance with the Pharmacology Committee Guidelines for the Care and Use of Laboratory Animals at Kindai University (Approval No. KAPS-2021-002, April 1, 2021). The mice were euthanized by injection of pentobarbital (200 mg/kg, intraperitoneally) in accordance with the AVMA Guidelines 2020.

Chemicals

A commercially available MXD formulation (Riup 5%, CA-MXD) and MXD powder were procured from Taisho Pharmaceutical Co., Ltd. (Tokyo, Japan). TRIzol reagent was purchased from Life Technologies Inc. (Rockville, MD, U.S.A.), and GA, methyl and propyl paraoxibenzoate were purchased from Wako Pure Chemical Corporation (Osaka, Japan). Methylcellulose (MC) was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). The Mouse CD34 (Sandwich enzyme-linked immunosorbent assay (ELISA)) ELISA Kit and Mouse CD200 (Sandwich ELISA) ELISA Kit were obtained from LifeSpan BioSciences, Inc. (Shirley, MA, U.S.A.). The RNA PCR Kit (AMV Ver. 3.0) was sourced from Roche Diagnostics Applied Science (Mannheim, Germany), and The Bio-Rad Protein Assay Kit from Bio-Rad Laboratories (Hercules, CA, U.S.A.). All the other chemicals used were of the highest purity available commercially.

Preparation of Dispersions Based on the MC/GA/MXD-NP Complex (MG-MXD@NP)

The MXD was subjected to bead milling using a Micro Smash MS-100R (TOMY DIGITAL BIOLOGY Co., Ltd., Tokyo, Japan), ShakeMaster^® NEO (Bio Medical Science, Tokyo, Japan), and zirconia beads (diameter: 0.1 mm), in accordance with the protocol established by Nagai et al.^10,14) In brief, MXD powder, MC, and GA were amalgamated and subjected to milling in an agate mortar under refrigerated conditions (4 °C) for 1.5 h. The resultant mixture (MXD, 5 g, MC, 1 g, and GA, 0.1 g) was subsequently dispersed in 100 mL of purified water containing methyl p-hydroxybenzoate (0.026%) and propyl p-hydroxybenzoate (0.014%) as preservatives. This dispersion was agitated in 2-mL tubes with zirconia beads (diameter, 0.1 mm). Thereafter, the mixture was subjected to milling via a hybrid approach integrating ultrasonic treatment for 5 min (W-113MK-II, Honda Electronics Co., Ltd., Aichi, Japan) and Micro Smash MS-100R treatment at 5500 rpm for 30 s. This sequential ultrasonic and bead milling regimen was repeated 30 times at 4 °C. Subsequently, the milled dispersion was comminuted using a ShakeMaster^® NEO with zirconia beads (diameter, 0.1 mm) for 120 min at 1500 rpm, which yielded the MC/GA/MXD-NP complex (MG-MXD@NP). In addition, a dispersion incorporating MXD powder, MC, GA, methyl p-hydroxybenzoate, and propyl p-hydroxybenzoate was also formulated and, denoted as the MC/GA/MXD-MP complex (MG-MXD@MP, micronized drug). The compositions of the two MXD formulations (MG-MXD@MP and MG-MXD@NP) were as follows: MXD 5%, MC 1%, GA 0.1%, methyl p-hydroxybenzoate 0.026%, and propyl p-hydroxybenzoate 0.014%. The roles of the additives are described in the relevant reference articles. Furthermore, the pH of both MXD formulations was adjusted to 6.8 using a NaOH aqueous solution.

Particle Distribution of the MXD Formulations

The dimensional characteristics of both MXD powder particles and nanoparticles spanning the range of 0.01–50 µm were evaluated using a particle size analyzer SALD-7100 (Shimadzu Corp., Kyoto, Japan). Within the SALD-7100, the refractive index was maintained at 1.60–0.010 i. Furthermore, the dimensions and population density of the nanoparticles constituting MXD within the 0–600 nm spectrum were determined using NANOSIGHT LM10 (QuantumDesign Japan, Tokyo, Japan). The experimental parameters for the examination using NANOSIGHT LM10 included a viscosity of 1.45 mPa·s, a wavelength of 405 nm, and a measurement duration of 60 s. Furthermore, nanoparticle quantification was conducted utilizing the same NANOSIGHT LM10 instrument. Visual examination of the MXD-NPs was performed through a scanning probe microscope (SPM)-9700 (Shimadzu Corp.), and Scanning Electron Microscopy (SEM) imaging was performed with NeoScope™ JCM-7000 (JEOL Ltd., Tokyo, Japan). Atomic Force Microscopy (AFM) images were constructed by integrating the phase and height images.

Characterization of the MXD Formulations

Separation of the soluble and insoluble fractions of MXD within the MXD formulations was achieved through centrifugation at 100000 × g employing a Beckman Optima™ MAX-XP Ultracentrifuge (Beckman Coulter, Osaka, Japan). Subsequently, the MXD levels in each fraction were quantified using a previously described HPLC method.^10,11) Dispersibility evaluation was performed incubating the MXD formulations at 22 °C for 28 d, with periodic sampling conducted from 5 mm beneath the surface. Analysis of MXD concentrations in the sampled solutions was conducted by an HPLC method. Zeta potential measurements were performed using a Zeta Potential Meter Model 502 (Nihon Rufuto Co., Ltd., Tokyo, Japan). Viscosity measurements of the MXD formulations were performed across temperatures ranging from 10–40 °C using a Brookfield digital viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA, U.S.A.). Crystallographic investigations were conducted using the Mini Flex II (Rigaku Corporation, Tokyo, Japan) X-ray diffractometer under conditions of 15 mA and 30 kV, scanning from 5° to 90° diffraction angles at a rate of 10°/min. The preparation of the X-ray diffraction (XRD) samples involved lyophilization.

Measurement of the MXD Levels in the Formulations by an HPLC Method

Quantification of MXD levels in the formulations was performed in accordance with established protocols described in previously published literature, using an LC-20AT system (HPLC, Shimadzu Corp.) equipped with an Inertsil^® ODS-3 column (GL Sciences Inc., Tokyo, Japan).^10,11) Samples of 50 µL were mixed with 100 µL of methanol containing ethyl p-hydroxybenzoate (1 µg/mL), and 10 µL of the resulting solution was injected. In this study, ethyl p-hydroxybenzoate was used as the designated internal standard. The mobile phase consisted of methanol/purified water containing 3 mM docusate sodium (1/1, v/v) operating at a flow rate of 0.2 mL/min, with measurements conducted at 35 °C. Detection of MXD was achieved at a wavelength of 254 nm.

Redispersibility of the MXD Formulations

After a 28-d incubation period at 22 °C, the MXD formulations were subjected to gentle agitation 10 times to ensure homogeneous mixing. Subsequently, the particle distribution of the redispersed formulations was evaluated using NANOSIGHT LM10. Furthermore, the redispersed formulations were allowed to equilibrate for 10 min before samples were collected from a depth of 5 mm beneath the surface. The MXD levels in these samples, which are indicative of the redispersibility of the MXD formulations, were quantified using a previously described HPLC method.

Measurement of MXD Levels in the Hair Bulge, Bulb, Skin Tissue, and Blood

One day prior to the experiment, the hair on the dorsal region of the C57BL/6 mice was trimmed to a length of approximately 2 mm using an electric razor and clippers. On the following day, the skin surface was cleansed with saline, followed by the topical application of MXD. The mice were gently restrained for approximately 3 min post-application to minimize agitation, and the application area was promptly occluded with an adhesive tape. A 30 µL volume of the MXD formulation was applied to the dorsal area (2 × 2 cm, 4 cm²) of the C57BL/6 mice and left undisturbed for 4 h. Subsequently, the C57BL/6 mice were euthanized by intraperitoneal injection of pentobarbital (200 mg/kg) under isoflurane anesthesia, followed by the collection of hair follicles, skin tissue, and blood from the animals. Hair was extracted using tweezers, and the hair follicle was isolated into the hair bulge and bulb region. The hair bulbs and skin tissues were homogenized in ethanol, and the resultant homogenates, along with the blood samples, were subjected to centrifugation at 20400 × g for 20 min at 4 °C. The MXD levels in the supernatants were determined using the previously described HPLC method. In this study, the MXD concentrations in the hair bulges and bulbs and skin tissue were expressed as mg of MXD per mg of protein, with the total protein quantification conducted using a Bio-Rad Protein Assay Kit.

Measurement of the Effect of the Drug on Hair Growth in the Mice

Two days prior to the start of the experiment, the dorsal fur of the C57BL/6 mice was depilated using an electric razor and clippers. Then, a 30 µL volume of the MXD formulation was topically applied daily to the dorsal area (2 × 2 cm, 4 cm²) of the C57BL/6 mice at 11:00 am. Hair growth progress was monitored at 10:00 a.m. daily using a digital camera, and the changes in the area with visible hair over time were quantified using the Image J software.

Quantitative Real-Time RT-PCR

Two days prior to the start of the experiment, the dorsal fur of C57BL/6 mice was depilated using an electric razor and clippers. Then, a 30 µL volume of the MXD formulation was applied daily at 11:00 am to the dorsal area (2 × 2 cm, 4 cm²) of the C57BL/6 mice for 12 d. Subsequently, the C57BL/6 mice were euthanized by intraperitoneal injection of pentobarbital (200 mg/kg) under isoflurane anesthesia, and the hair follicles within the treated area were excised at 3:00 p.m. Total RNA was extracted from the hair bulbs using the acid guanidinium thiocyanate-phenol-chloroform extraction method with Trizol reagent. RT and PCR reactions were conducted utilizing an RNA PCR Kit and LightCycler FastStart DNA Master SYBR Green I, as per the manufacturer’s instructions. Briefly, 1 µg of total RNA was combined with a buffer (pH 8.3) containing Tris (10 mM), MgCl₂ (5 mM), and KCl (50 mM), along with oligo dT-adaptor primer, ribonuclease inhibitor, reverse transcriptase, and deoxynucleotide triphosphate. The mixture was incubated at 42 °C for 15 min, followed by 5 min at 95 °C to synthesize cDNA (total volume 10 µL). Subsequently, 2 µL of the cDNA was mixed with 2 µL of LightCycler FastStart DNA Master SYBR Green I Reaction Mix and specific primers (10 pmol) for CD34 (FOR 5′-CTGAGGCTGATGCTGGTGCTA-3′, REV 5′-GTTTGCTGGGAAGTTCTGTGCTA-3′), CD200 (for: 5′-ACAGCTTGCCTTACCCTCTATGTA-3′, REV 5′-CAAGTGATGTTTAGGTGGTCTTCAAA-3′), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FOR 5′-TGCACCACCAACTGCTTAGC-3′, rev: 5′-GGCATGGACTGTGGTCATGAG-3′). The PCR conditions comprised an initial denaturation step at 95 °C for 10 min, followed by 50 cycles of denaturation at 95 °C for 10 s, annealing at 61 °C for 10 s, and extension at 72 °C for 5 s. Differences in the threshold cycles between GAPDH and the target genes (CD34 and CD200) were determined to ascertain the expression levels of CD34 mRNA and CD200 mRNA in the C57BL/6 mice.

Measurement of CD34 and CD200 Protein in the Hair Follicles

Two days prior to the start of the experiment, the dorsal fur of the C57BL/6 mice was depilated using an electric razor and clippers. Then, 30 µL volume of the MXD formulation was applied every day at 11:00 am to the dorsal area (2 × 2 cm, 4 cm²) of the C57BL/6 mice for 12 d. Following this treatment regimen, the C57BL/6 mice were euthanized by intraperitoneal injection of pentobarbital (200 mg/kg) under isoflurane anesthesia. The hair follicles from the MXD-treated area were then collected at 3:00 p.m. The CD34 and CD200 expression levels were measured using a Mouse CD34 and CD200 (Sandwich ELISA) ELISA Kit, respectively, in accordance with the manufacturer's instructions. Briefly, the hair follicle samples were homogenized in 0.02 M phosphate-buffered saline (pH 7.0) and then centrifuged at 20400 × g for 20 min at 4 °C. The resultant supernatant (100 µL) was added to microplate wells pre-coated with specific antibodies for Mouse CD34 and CD200, respectively, and incubated for 1 h at 37 °C. Thereafter, detection reagents A and B were sequentially added and the microplates were incubated for 1 h 30 min at 37 °C. After each incubation step, the plates were washed five times with Wash Buffer. Subsequently, 90 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added, followed by incubation for 15 min at 37 °C. The reaction was then halted by adding Stop Solution, and the absorbance was measured using a microplate reader at 450 nm. The detection range of the assay spanned 0.156–10 ng/mL, with a typical sensitivity of less than 0.056 ng/mL for CD34 and less than 0.156 ng/mL for CD200.

Statistical Analysis

The data are presented as the means ± standard error of the mean. The statistical analyses were performed using the JMP software version 5.1 (SAS Institute, Tokyo, Japan). One-way repeated-measures ANOVA, followed by the Tukey–Kramer test, was employed for comparing multiple groups. An unpaired Student’s t-test was used for comparisons between two groups. p < 0.05 was considered as denoting statistical significance.

RESULTS

MXD Nanocrystal Dispersion and Evaluation of Their Physical Properties

First, we investigated whether the dispersion of the MXD nanocrystals was prepared by the bead milling treatment using the additives MC and GA. Figure 1 shows the appearances and particle sizes of the MG-MXD@MP and MG-MXD@NP formulations. MG-MXD@NP showed uniform dispersion, while precipitation was observed in MG-MXD@MP (Fig. 1A). The mean particle sizes of MG-MXD@MP and MG-MXD@NP were 5.10 ± 0.87 µm and 109.5 ± 7.9 nm, respectively (Figs. 1B, 1C), and both SEM and SPM images also confirmed that the MXD particles treated by the bead milling method were nanosized (Figs. 1E, 1F). Moreover, no changes in the positions of the crystalline peaks of MXD were observed after the bead milling treatment, indicating that the bead milling method did not alter the crystal structure of MXD (Fig. 2B). Figure 2A shows the MXD solubility in MG-MXD@MP and MG-MXD@NP. The MXD solubility in MG-MXD@NP was 1.8-fold higher than that in MG-MXD@MP. Figure 2C shows the viscosity of MG-MXD@MP and MG-MXD@NP. No difference in the viscosity was observed between MG-MXD@MP and MG-MXD@NP, and the viscosity was similar to that of CA-MXD (1.19 mPa·s at 20 °C). Figures 2D–2F show the zeta potential and dispersibility of MG-MXD@MP and MG-MXD@NP. The zeta potentials were similar between the two formulations, but the dispersibility of MG-MXD@NP was superior to that of MG-MXD@MP. MG-MXD@NP showed a mean particle size of 110.3 ± 7.1 nm immediately after preparation (Fig. 1D), with a particle size of 147.3 ± 9.6 nm following redispersed after 28 d’ storage (Fig. 3B), which indicated that the precipitated drug in MG-MXD@NP was easily redispersed by agitation, with recovery of the original nanoparticle suspension (Fig. 3).

Fig. 1. Changes in the Particle Size Frequency of MXD before and after Bead Mill Treatment

A: Digital photos of MG-MXD@MP and MG-MXD@NP. B, C: The particle size distribution data of MG-MXD@MP (B) and MG-MXD@NP (C) using SALD-7100. D: Particle size distributions of MG-MXD@NP determined with NANOSIGHT LM10. E: SEM images of MG-MXD@MP and MG-MXD@NP. F: AFM image of MG-MXD@NP.

Fig. 2. Physical Properties of MG-MXD@MP and MG-MXD@NP

A: Solubility of MXD in MG-MXD@MP and MG-MXD@NP. B: XRD patterns of MXD before and after bead mill treatment. C: Viscosity of MG-MXD@MP and MG-MXD@NP at 10C–40 °C. D: Zeta potential of MG-MXD@MP and MG-MXD@NP. E, F: Digital images (E) and dispersibility (F) of MG-MXD@MP and MG-MXD@NP. N = 6. * p < 0.05 vs. MG-MXD@MP in each comparison. ^#p < 0.05 vs. MG-MXD@MP at 10 °C for each category.

Fig. 3. Changes in the Particle Distribution and Content of Restirred MXD Formulations at 1 Month after Preparation

A: Digital image. B: Particle size frequency. C: Concentration of MXD in restirred MG-MXD@MP and MG-MXD@NP. The formulations were stored at 20 °C for 1 month. N = 6. * p < 0.05 vs. MG-MXD@MP in each comparison.

Localization of MXD in the Skin Tissue Following Topical Application of the Formulation

Figure 4 shows the absorption behavior of MXD after the application of the formulations. The concentrations of MXD in the skin tissue, hair bulge, hair bulb, and blood of mice were significantly higher after MG-MXD@NP application than after MG-MXD@MP application. Moreover, the amounts of MXD in the skin tissue, hair bulge, and hair bulb of the mice at 4 h after application were 2.3-, 2.6- and 3.9-fold higher, respectively, following MG-MXD@NP application than following application of CA-MXD (Figs. 4A–4C). MXD transfer to the blood was lower in the mice treated with MG-MXD@NP, and the MXD level in the blood of the mice treated with MG-MXD@NP was 19.8% of that in the mice treated with CA-MXD (Fig. 4D).

Fig. 4. MXD Contents in the Skin Tissue of Mice after MXD Application

MXD contents in the skin tissue (A), hair bulge (B), hair bulb (C) and blood (D) of C57BL/6 mice treated with MG-MXD@MP, MG-MXD@NP, and CA-MXD. The MXD formulations were applied for 4 h. N = 6–7. * p < 0.05 vs. MG-MXD@MP in each comparison. ^# p < 0.05 vs. CA-MXD in each comparison.

Effect of MG-MXD@NP on the Hair Growth in the Model Mice

Figure 5 shows the changes in the hair growth rate in the C57BL/6 mice treated by repetitive applications of MXD formulations. The hair growth appearance and behavior of the mice treated with MG-MXD@MP were similar to those of the mice treated with vehicle (Figs. 5A, 5B). In contrast, the hair growth rate was accelerated in the mice treated with MG-MXD@NP and CA-MXD as compared with that in the mice treated with MG-MXD@MP or vehicle. Hair growth appeared earlier after the initial application and the hair area at each evaluation point was significantly higher in the mice treated with MG-MXD@NP as compared with the observations in the mice treated with CA-MXD. Figure 6 shows the changes in the mRNA and protein levels of CD34 and CD200 in the hair follicles of C57BL/6 mice treated by repeated applications of the MXD formulations. Expressions of CD34 and CD200 mRNA were induced and those of the CD34 and CD200 proteins tended to be higher in the mice treated with CA-MXD as compared with the observations in the mice treated with vehicle or MG-MXD@MP. Furthermore, the mRNA and protein expression levels of both CD34 and CD200 in the mice treated with MD-MXD@NP were significantly higher than those in the mice treated with CA-MXD.

Fig. 5. Effect of the Repetitive Application of MXD Formulations on the Hair Growth in C57BL/6 Mice (Once a Day)

A: Image of C57BL/6 mice skin at 12 d after the start of repetitive applications. B: Changes in the hair area in C57BL/6 mice that were treated by repetitive application of MXD formulations. N = 6–7. * p < 0.05 vs. Vehicle for each category. ^# p < 0.05 vs. MG-MXD@MP in each comparison. ^$ p < 0.05 vs. CA-MXD in each comparison.

Fig. 6. Effect of MXD Formulations on the Expression of CD34 and CD200 in the Hair Follicles of C57BL/6 Mice at 12 d after the Start of Repetitive Application (Once a Day)

A, B: Expression of CD34 (A) and CD200 (B) mRNA in C57BL/6 mice treated with MXD formulations. C, D: CD34 (C) and CD200 (D) protein expression levels in C57BL/6 mice treated with MXD formulations. Samples for examination were collected from the hair follicles (hair bulge and hair bulb). N = 8–13. * p < 0.05 vs. Vehicle in each comparison. ^# p < 0.05 vs. MG-MXD@MP in each comparison. ^$ p < 0.05 vs. CA-MXD in each comparison.

DISCUSSION

Our previous studies showed that suspensions containing MXD-NPs delivered MXD into the hair follicles effectively and showed a high therapeutic efficacy for hair growth.^10,11) In the previous study,¹¹⁾ we conducted the experiments using MXD nanocrystal dispersions without GA, and compared to CA-MXD, it was shown that the concentration of MXD in the hair bulge increased significantly. In this study, we designed MXD nanocrystal dispersions containing MC and GA (MG-MXD@NP), and found that the inclusion of GA in the MXD nanocrystal formulation significantly enhanced the delivery of the MXD into the skin tissue, hair bulge, and hair bulb of mice compared to CA-MXD. Therefore, it was found that the addition of GA to MXD nanocrystal dispersion can deliver MXD into the deeper parts of the hair follicle. Moreover, the enhanced MXD levels in the skin tissue, hair bulge, and hair bulb were associated with enhanced expressions of CD34 and CD200 mRNA and proteins, resulting in the acceleration of hair growth.

First, we attempted to prepare MXD nanocrystals. We used two methods for producing the nanoparticles: the breakdown method, in which smaller particles were obtained by mechanical pulverization, and the buildup method, in which the raw material was dissolved once, and physical and chemical reactions were used to stop deposition at the desired particle size. The breakdown method is considered as being advantageous, as the particle size obtained by this method ranges from micrometers to several tens of nanometers, as compared with the buildup method, which yields less than submicron-size particle and also, the nanoparticles generated by this method aggregate more easily. Furthermore, we have reported previously that good-quality indomethacin nanoparticles could be produced using MC and the bead milling method, which is one of the breakdown methods.¹⁴⁾ Based on this, we used MC and the bead-milling method to prepare MXD-NPs. In this study, we explored the effects of the additives in the formulations on the transdermal absorption behaviors of the drug nanoparticles. GA, also known as acacia gum, is a resin obtained from trees of the Acacia genus, including Acacia senegal and Acacia seyal, and is highly valued across various industries for its versatile properties and wide-ranging applications. In particular, GA has emulsifying and viscosity-enhancing properties and is used as an emulsifier and thickener in the pharmaceutical, cosmetic, and food Industries. In this study, we selected GA for preparing the MXD formulation, since we considered that the emulsifying and thickening properties of GA might have a positive effect on the drug behavior in the skin of nanoparticle formulations, and evaluated whether the inclusion of GA affected the localization of MXD in the skin tissue or its hair growth effect in model mice.

The crystal structure of MXD was maintained after the bead mill treatment, and the particle size of MXD was approximately 110 nm (Figs. 1C, 2B). Moreover, improvement of the solubility, a characteristic of drugs with particle sizes of under 100 nm, was also confirmed (Fig. 2A). In addition, MG-MXD@NP showed superior dispersibility (Figs. 2E, 2F) and good redispersibility, as it was easily redispersed by agitation with restoration of the original nanoparticle suspension (Fig. 3). Although the zeta potential and viscosity are known to be involved in the dispersibility of drug particles, no significant changes in the zeta potential or viscosity of MXD were observed after the bead milling process as compared with before the process (Figs. 2C, 2D). These findings could suggest that the improvement in the dispersibility was attributable to the changes in the particle size.

Next, we investigated the MXD contents in the skin tissue, hair bulge, hair bulb, and blood of mice treated with MXD formulations. Our previous study reported that the MXD contents in the skin tissue and blood of mice treated with MXD nanosuspensions without GA were lower than those in the mice treated with CA-MXD. Conversely, the MXD contents in the hair bulbs were higher in the mice treated with MXD nanosuspensions not containing GA than in those treated with CA-MXD, and we concluded that nanoparticles of MXD can deliver MXD directly into the hair bulbs via the hair follicles.^10,11) With the aim of delivering MXD into the deeper parts of the hair follicle, we designed the MXD nanocrystal dispersion with reduced formulation viscosity by adding GA to the MXD nanosuspension, while ensuring the grinding efficacy and dispersion stability. The viscosity of the MXD nanosuspension without GA was 30 mPa ∙ s at 30 °C,¹¹⁾ however, the viscosity of the MXD nanosuspensions with GA was approximately 1.1 mPa·s at 30 °C (Fig. 2C). In this study, in contrast to MG-MXD@NP, where the MXD reached the deep part of the hair follicle via the hair follicular route, MXD of CA-MXD reached the deep layers of the skin tissue via the stratum corneum, and the blood concentration of MXD in MG-MXD@NP was significantly lower than in CA-MXD. Moreover, in MG-MXD@NP, the drug delivery to the skin tissue, hair bulge, and hair bulb area were higher as compared with the case for CA-MXD (Fig. 4). In addition, the MXD contents in the hair bulge and hair bulb area in the MG-MXD@NP-treated mice (MXD nanosuspensions containing GA) were higher than those in the mice treated with MXD nanosuspensions not containing GA that we had prepared before.^10,11) These results show that the addition of GA allowed more effective delivery of the MXD particles into the hair bulge and hair bulb area, and the emulsifying action of GA or decrease in formulation viscosity due to the addition of GA could be involved in the higher efficiency of delivery of MXD-NPs into the hair bulb. Further studies are needed for a clearer elucidation of these issues.

We also examined the relationships between the hair growth rate and the MXD formulation used in the C57BL/6 mice (Figs. 5, 6). When MG-MXD@NP was repeatedly applied, hair growth was observed from an earlier stage (on 8 d of administration) than when CA-MXD was repeatedly applied (Fig. 5). These results are consistent with the higher MXD content in the hair bulge and hair bulb area of the mice treated with MG-MXD@NP than in the mice treated with CA-MXD (Fig. 4). Furthermore, in previous research, the hair growth rate of mice and the local concentration of MXD were compared with CA-MXD for MXD-NPs (MXD nanocrystal dispersion that does not contain GA). We observed that the concentration of MXD after applying MXD-NPs was significantly higher than that of CA-MXD only at the hair bulge. Additionally, we observed that hair growth from an earlier stage (on the 8th day of administration) when applying MXD-NPs repeatedly than when applying CA-MXD repeatedly.¹¹⁾ These findings suggest that follicular targeting of MXD may enhance hair growth. However, there are still parts of MXD’s mechanism of action that are not fully understood. For example, it is not clear whether the effect on the hair bulge, which contains hair follicle epithelial stem cells, or the effect on the hair bulb is more likely to cause hair growth.

A high MXD content in the hair bulb is known to improve the hair tissue blood flow, activate papilla cells, promote the production of cell growth factors such as insulin-like growth factor 1 and vascular endothelial growth factor, and inhibit apoptosis of the hair matrix cells, thereby promoting hair growth. On the other hand, the bulge containing the hair follicle stem cells also plays a crucial role in regulating the hair cycle.¹⁵⁾ The cells in the bulge region are known to possess stem cell characteristics and to generate secondary germ cells that produce new hair shafts at anagen onset.²⁾ CD34 and CD200 have been reported as being useful markers of hair follicle epithelial stem cells.¹⁶⁾ CD34 is primarily considered as a marker of hematopoietic stem and progenitor cells, as well as of vascular endothelial progenitor cells and embryonic fibroblasts. In humans, CD34 is located below the bulge zone, in the suprabulbar region, as well as in the skin between the hair follicles, in the basal cells of the interfollicular epidermis. Throughout the hair cycle, CD34 is expressed during the anagen phase, but not during the catagen or telogen phases.¹⁷⁾ This is believed to indicate that the CD34 function is relevant only to epithelial cells that proliferate, and is also related to the adhesion of the root sheath cells to the surrounding stroma. CD200 is a molecule associated with reduction in the rate of graft rejection and immune system regulation.^18,19) CD200 mRNA, in addition to sebaceous gland lineage markers, was found in the bulge within reconstituted pilosebaceous structures in the dog, suggesting that the bulge stem cells might contribute to reorganization of not just hair follicles, but also of sebaceous glands.²⁰⁾ CD200 has been found in the hair follicle bulge of human skin.^16,21,22) AGA is reportedly associated with a loss of CD34 and CD200. Our results showed that the mRNA and protein expressions of both CD34 and CD200 were enhanced in the MG-MXD@NP-treated mice, and that the levels were higher in these mice than in the mice treated with CA-MXD. Taken together, these results suggest that MG-MXD@NP delivers a high MXD content into both the hair bulge and hair bulb, which may lead to the proliferation and activation of papilla cells and hair follicle epithelial stem cells and thereby, enhancement of hair growth (Figs. 5, 6). However, our present understanding of the mechanisms by which MXD fosters hair follicle growth remains limited, with scant insight having been obtained so far into the specific interface between MXD and stem cell populations. We propose to conduct further investigations to elucidate the direct impact of MXD on these stem cells.

In conclusion, we succeeded in designing a 5% MXD formulation containing MXD nanocrystals, MC, and GA (MG-MXD@NP) and showed that application of MG-MXD@NP was associated with more effective delivery of MXD to both the hair bulge and hair bulb areas than that of CA-MXD or previously prepared MXD nanodispersions not containing GA.^10,11) In addition, we found that the effect of MG-MXD@NP on hair growth was superior to that of CA-MXD. Our results suggest that the delivery to the target areas of hair follicle-targeting MXD based on nanocrystals was enhanced by the addition of GA to the formulation, and that this formulation may show satisfactory therapeutic efficacy and safety in patients with AGA.

Conflict of Interest

The authors declare no conflict of interest.

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