Subcutaneous injection of genetically engineered exosomes for androgenic alopecia treatment

Abstract

Androgenetic alopecia (AGA) is a common disorder that negatively impacts quality of life but remains challenging to treat effectively. The hair loss observed in AGA is the consequence of a gradual reduction in the duration of the anagen phase concomitant of the miniaturization of the hair follicles and subsequent atrophy. This process of miniaturization is associated with abnormalities in the Dihydrotestosterone (DHT) induced dermal papilla cells (DPCs). DHT induces DPCs senescence and promotes apoptosis of vascular endothelial cells and keratinocytes via the DPCs paracrine pathway, which ultimately leads to follicular miniaturization. In this study, we developed a multifunctional exosome-based targeted delivery platform, designated as EX104, through the engineering of HEK-293 cells to express a combination of therapeutical molecules, including WNT10B, VEGFA, and FGF7. EX104 reversed the hair follicle miniaturization phenotype in DHT-induced DPCs. Furthermore, it demonstrated significant hair growth-promoting effects in the murine model of androgenetic alopecia by remodeling the follicular microenvironment and restoring miniaturized hair follicles. Topical EX104 application demonstrated comparable hair growth promotion to first-line minoxidil, while significantly outperforming it in stimulating capillary growth and follicular proliferation. EX104 represents a promising and innovative strategy for AGA management and follicular regenerative therapy.

Keywords: EX104; alopecia; androgenetic alopecia; engineered exosomes; hair regrowth.

FIGURE 1

Characterization of EX104. Representative size distribution (A) of EX104 and murine EX104. (B) TEM images of EX104 and murine EX104 engineered exosomes. (C) Protein level of VEGFA, FGF7 and WNT10B by ELISA from Mock (293-exo), EX104 and murine EX104 (1E+12 particles/mL). Data are shown as mean ± standard deviation (SD). Statistical analysis was performed by Student’s t-test. Significance level: ****P < 0.0001.

FIGURE 2

Effects of EX104 on the viability of DPCs with or without DHT-induced. Effects of different concentrations of EX104 alone on viability of mDPCs (A) and hDPCs (B). Effects of different concentrations of DHT alone on the viability of mDPCs (C) and hDPCs (D). mDPCs (E) or hDPCs (F) were pretreated with DHT (100 μM) for 24 h and subsequently incubated with or without different concentrations of murine EX104 or EX104, respectively. Cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay. Statistical analysis was performed by one-way ANOVA. Significance level: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 3

Effects of EX104 on hair follicle miniaturization phenotype of DHT-induced DPCs. mDPCs (A–C) and hDPCs (D–F) were pretreated with DHT (100 μM) for 24 h and subsequently incubated with or without different concentrations of murine EX104 and EX104 for 24 h, respectively. Whole cells were then collected for the purpose of determining the gene expression of DKK1, AR, IL-6 and LEF1 using real-time RT-PCR. Statistical analysis was performed by one-way ANOVA. Significance level: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 4

Therapeutic efficacy of murine EX104 in DHT-induced AGA C57BL/6 mouse models. (A) Animal experimental schedule for inducing androgenetic alopecia mice model. DHT was hypodermic injection, and murine EX104 was injected subcutaneously once every 3 days, Minoxidil was topically administered once every day. (B) Hair coverage percntage (%) on the dorsal skin for each group on days 15 after depilation. (C) Each photograph documents the hair regrowth state of individual mouse on day 15 post-administration. Statistical analysis was performed by one-way ANOVA. Significance level: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 5

Effects of murine EX104 on hair follicles and inflammation in DHT-induced AGA C57BL/6 mouse models. (A) Depilated mice skin tissue was paraffin sectioned and histologic examination was performed by HE staining. Images were captured by digital slide scanner. N = 6. Scale bars are 200 μm. (B) Hair follicle number, Hair follicle lengths and Anagen/Telogen hair follicle (%) was measured in the same random area. Quantitative Analysis of β-Catenin (C) and TNF-α (D) levels in mouse skin tissues was performed by ELISA. Statistical analysis was performed by one-way ANOVA. Significance level: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 6

Effects of murine EX104 on blood vessels and proliferating hair follicles in DHT-induced AGA C57BL/6 mouse models. (A) CD31 immunohistochemical staining of dermal sections with 20 × objective lens (black arrows indicate CD31-positive micro-vessels). Scale bar = 50 μm. (B) Ki67 immunohistochemically staining of dermal sections with 20 × objective lens (black arrows indicate Ki67-positive hair follicles). Scale bar = 50 μm. (C) Quantitative analysis of CD31 staining surrounding hair follicles. (D) Quantitative analysis of Ki67 staining inside hair follicles. Statistical analysis was performed by one-way ANOVA. Significance level: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.