Purpose: We designed formulations based on minoxidil (MXD) nanoparticles (N-MXD) and examined whether N-MXD can increase drug delivery into the follicles. In addition, we investigated the effect of N-MXD on hair growth in C57BL/6 mice. Methods: N-MXD (1%) was prepared as follows: methylcellulose, p-hydroxyalkylbenzoates, mannitol, and MXD were dispersed in purified water and milled using zirconia beads under refrigeration (5500 rpm, 30 s×15 times, intermittent milling). C57BL/6 mice were used to evaluate hair-growth effects. The expression levels of mRNA and protein for vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) were determined by real-time PCR and ELISA methods, respectively. Results: The ratio of solid-MXD was approximately 60% in N-MXD, and the MXD nanoparticles (90–300 nm) were oblong in shape. For the design of nanomedicines, usability is important. Therefore, we measured the stability and toxicity after N-MXD treatment. No agglutination of MXD nanoparticles was detected for 2 weeks, and no redness or MXD powder residue was observed in the skin after repetitive applications of N-MXD. Next, we evaluated hair-growth effects by N-MXD treatment. MXD contents in the skin tissue from N-MXD were lower than for commercially available MXD formulations (CA-MXD). Conversely, MXD contents in the hair bulbs were higher for N-MXD than for CA-MXD, and the drug efficacy of N-MXD was also higher than that of CA-MXD. In addition, the mRNA and protein levels of IGF-1 and VEGF were enhanced by the repetitive application of N-MXD and CA-MXD, and the enhanced IGF-1 and VEGF levels were significantly higher for N-MXD than for CA-MXD. Conclusion: We designed a novel nanomedicine based on MXD nanoparticles and showed that N-MXD can deliver MXD into hair bulbs via hair follicles and that the therapeutic efficiency for hair growth is higher than for CA-MXD (solution type).

We previously found that 1% minoxidil (MXD) nanoparticles prepared using a bead mill method led to an increase I n hair follicle delivery and hair growth in C57BL/6 mice. In the present study, we designed a nanoparticle formulation containing 5% MXD (MXD-NPs) using the bead mill method and investigated the hair-growth effect of MXD-NPs and a commercially available MXD solution (CA-MXD). Hair growth and in vivo permeation studies were conducted using C57BL/6 mice. Moreover, we examined the MXD contents in the upper (hair bulge) and the lower hair follicle (hair bulb) and observed the hair follicle epithelial stem cells (HFSC) by immunohistochemical staining using the CD200 antibody. The mean particle size of the MXD in the MXD-NPs was 139.8 nm ± 8.9 nm. The hair-growth effect of the MXD-NPs was higher than that of CA-MXD, and the MXD content in the hair bulge of mice treated with MXD-NPs was 7.4-fold that of the mice treated with CA-MXD. In addition, the activation of HFSC was observed around the bulge in the MXD-NPs-treated mice. We showed that MXD-NPs enable the accumulation of MXD in the upper hair follicles more efficiently than CA-MXD, leading the activation of HFSC and the hair growth.

Background/Objectives: Transdermal formulations are widely utilized in the pharmaceutical and cosmetic fields because they enable non-invasive administration and sustained local drug delivery. Conventional ex vivo skin permeation experiments using Franz diffusion cells have limitations in capturing the spatial and temporal dynamics of skin penetration. This study aimed to develop a confocal laser scanning microscopy (CLSM)-based approach to visualize and semi-quantitatively assess the penetration behavior of fluorescent dyes with differing lipophilicities. Methods: Four fluorescent dyes with different Log P values—Rhodamine B (Rho-B), Rhodamine 123 (Rho-123), Fluorescein Sodium (Flu-Na), and Nile Red (NR)—were formulated into lotion-based vehicles and applied to excised human abdominal skin. CLSM imaging was performed from 10 min to 240 min post-application. Fluorescence intensities were extracted from depth-resolved regions (R1–R4, 30-μm intervals) to examine penetration kinetics and distribution. Results: CLSM imaging demonstrated that Rho-B penetrated through stratum corneum and entered deep into the skin via the hair follicles. Rho-123 and Flu-Na exhibited intercellular and follicular penetration; however, Flu-Na showed only a slight increase in intensity over time; NR showed negligible penetration into the deeper layers. The results of our analysis indicated that moderately lipophilic substances such as Rho-B and Rho-123 diffused deeply into the skin via both transdermal and follicular routes, whereas highly hydrophobic or lipophilic substances remained in the superficial layers. Conclusions: The CLSM-based approach enabled spatially and temporally resolved, semi-quantitative evaluation of transdermal penetration in a single, non-destructive experiment. Although restricted to fluorescent probes, this approach provides a practical early-stage screening tool for comparing route-specific and time-dependent penetration behaviors of compounds with different lipophilicities.