(T1130-11-76) Loading of Minoxidil to Extracellular Vesicles to Enhance the Transdermal Delivery to Hair Follicle

Purpose: Exosomes are increasingly important in cell biology because of their role in cell-to-cell communication and transfer of cellular materials, such as miRNA, lncRNA, mRNA, proteins, and DNA. ^1,2 In the last few years, extracellular vesicles (EVs) have shown great promise as effective carriers for various therapeutics. This is primarily due to their natural biocompatibility, capacity to traverse physiological barriers, and minimal potential for triggering immune responses.³ Minoxidil, the most commonly utilized medication for treating alopecia, particularly male-pattern hair loss, demonstrates its effectiveness through transdermal delivery when applied topically to the skin area. Nevertheless, conventional minoxidil delivery suffers from drawbacks such as low efficiency in transdermal administration and various side effects, including tolerance, inflammation, and low blood pressure.⁴ In order to address the aforementioned concerns, we have created a transdermal drug delivery system for alopecia treatment. This platform involves loading minoxidil into the milk exosomes through various loading methods including incubation, sonication, acidic pH, and surfactant treatment.

Methods: The dispersion of milk exosomes with a concentration of 2.96×10¹⁰ vesicles/mL was prepared and 5% w/v minoxidil was made in exosomes dispersion. The mixture was aliquoted to equal parts and each part was treated with a particular loading method as follows: incubation at 37 °C for 6 h and 24 h, sonication in a bath sonicator three 15 seconds pulses with 30 seconds pauses, pH 3 treatment for three minutes, and 0.01% Sodium dodecyl sulfate (SDS) treatment for seven minutes. The loaded exosomes were purified from the other ingredients using spin filtration (10000 g three times). The concentrated exosomes and filtrates containing unloaded minoxidil were preserved at -80 °C and -20 °C respectively and were analyzed using HPLC and LC-MS/MS. The biological tests to investigate the penetration through the skin and delivery the drug to the site of action were performed on the pig ear. Briefly, RNA Select dye was incubated with exosomes at a 1:10 dilution for 30 minutes at 37 °C. Exosomes washed 3x with PBS to remove excess dye. 100 ul of stained exosomes in PBS were applied to a pig ear in a 1cm² section and incubated for 6 hours. The 1cm² section was removed from the rest of the ear. Sections were placed in OCT Compound for approximately 10 minutes. Tissues were frozen in acetone chilled with dry ice, for quick freezing. Tissue was sectioned at 5μm thickness using a cryostat and fixed using acetone. Sections were washed 3x for 10 minutes in PBS to remove excess OCT. Sections were preserved with vector shield (with DAPI) for counter stain and reducing photo bleaching. Sections were imaged with a Keyence BZ-X810 microscope.

Results: The chromatography results showed efficient loading of minoxidil into the exosomes via all loading approaches. To find out the most efficient method, more investigation is needed. To measure the delivered minoxidil to the tissue, a histological sample has been sent to LC-MS/MS analysis and the sample is under investigation.

Conclusion: This study clearly showed the impact of delivery by EVs compared to simple formulations. Chromatography results provided a strong evidence of loading the minoxidil into the EVs. Consequently, this method of transdermal drug delivery provided an efficient transdermal drug delivery approach.

References: 1. Midekessa, G. et al. Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability. ACS Omega 5, 16701–16710 (2020).
2. Haraszti, R. A. et al. High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources. J. Extracell. Vesicles 5, 1–14 (2016).
3. You, Y. et al. Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy. Nat. Biomed. Eng. (2023) doi:10.1038/s41551-022-00989-w.
4. Jeong, W. Y. et al. Transdermal delivery of Minoxidil using HA-PLGA nanoparticles for the treatment in alopecia. Biomater. Res. 23, 1–10 (2019).

Chromatogram of the minoxidil in lysed exosomes

Milk 20x Topside: Some exosomes made their way to the lower sections of the tissue sample, overall exosomes are sparse.

Milk 20x Underside: Bottom of the sample showing some autofluorescence but little else.