Hyaluronic acid (HA, 100 kDa) was purchased from Lifecore Co. (Chaska, MN). Poly(Lactide-co-Glycolide) (PLGA, 75:25, MW 10 kDa) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Hexamethylenediamine (HMDA), N-Hydroxysuccinimide (NHS), Rhodamine B (Rho B), Poly(vinyl alcohol) (PVA), Dichloromethane (DCM), Tween 80, Formaldehyde were purchased from Sigma-Aldrich (St. Louis, MO). N,N′-Dicyclohexylcarbodiimide (DCC), Dimethylsulfoxide (DMSO), Ethanol, and Methanol were obtained from Alfa Aesar (Haverhill, MA). 1-Ethyl3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride, Minoxidil (MXD) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). EZ-CYTOX was purchased from Daeil lab Service Co. (Cheongwon, Korea), Optimal cutting temperature (OCT) compound was obtained from Sakura Finetek (Zoeterwoude, Netherlands). Phosphate buffered saline (PBS) were purchased from Invitrogen Co. (Carlsbad, CA). Glycerin was purchased from DaeJung Chem Co. (Siheung, Korea). DMEM, Fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Waltham, MA), EFO gro™ HDP was purchased from DYNE Bio Co. (Seongnam, Korea), Fibroblast cell (NIH/3 T3) was obtained from American Type Culture Collection (Manassas, VA), Hair follicle dermal papillary cell (HFDP) was purchased from Cell Engineering For Origin (Seoul, Korea).
Synthesis and characterization of HA-PLGA conjugates
The synthesis of HA-PLGA was described in elsewhere . In brief, hexamethylenediamine substituted HA (HA-HMDA) were prepared and dissolved in dimethylsulfoxide (DMSO, 30 mL) with a concentration of 1 mg/mL. PLGA (14 μmol) was dissolved in 6 mL of DMSO containing N,N′-Dicyclohexylcarbodiimide (DCC, 21 μmol) and N-Hydroxysuccinimide (NHS, 21 μmol). Then, the solution of PLGA was mixed with HA-HMDA solution, and stirred overnight. After then, the mixture was dialyzed against DI water for 3 days. The HA-PLGA was lyophilized for 3 days. And then, HA-PLGA was characterized by 1H nuclear magnetic resonance spectroscopy (NMR, AVANCE NEO 500, Bruker, Germany).
Preparation and characterization of NPs encapsulating MXD or Rho B
MXD encapsulated HA-PLGA NPs were prepared by using W/O/W solvent evaporation with sonication . First, 7.5 mg of MXD was dissolved in 0.5 mL of 2% acetic acid aqueous solution. The solution was added to the 1 wt% solution of HA-PLGA in of 6.25 mL of dichloromethane (DCM) containing 11.3 μL of Tween 80. And then, the mixture was emulsified by using a probe sonicator (VC 750, Sonics Inc., CT, USA) at 200 W of energy output for 20 min in an ice bath (pulse on 2.0 s, pulse off 1.0 s). Then, the prepared water-in-oil (W/O) emulsion was added to 33 mL of 1.5 wt% Poly(vinyl alcohol) (PVA) aqueous solution and the mixture was emulsified using a probe sonicator at 200 W of energy output for 20 min in an ice bath again to obtain HA-PLGA/MXD NPs. To evaporate DCM, prepared W/O/W emulsion was stirred overnight. The NPs were collected by ultracentrifugation at 12,000 rpm for 20 min. After centrifugation, the precipitated NPs were dispersed in 2 mL of DI water. Rhodamine B (Rho B) encapsulated HA-PLGA NPs were also prepared in the same manner as described above. The hydrodynamic diameter, zeta potential, and morphology of NPs were characterized by dynamic light scattering (Zetasizer Nano ZS90, Malvern Instruments, UK) and transmission electron microscope (TEM, H-7600, HITACHI, Japan). In addition, the loading capacity of the NPs is defined as the mass of the loaded drug in NPs divided by the mass of NPs and the encapsulation efficiency is defined as the loaded drug amount in the NPs over drug quantity in solutions . To evaluate loading capacity and encapsulation efficiency, amount of MXD and Rho B in the supernatant of NPs solution were calculated through measurement of the absorbance at 289 nm for MXD and 555 nm for Rho B with UV-Vis spectrometer (Optizen 2120UV, Mecasys, Korea), respectively. Furthermore, to confirm the stability of MXD loaded HA-PLGA NPs, we measured the HA-PLGA NP’s size and amount of MXD in NPs after storing for 7 days in DI water.
In vitro release of HA-PLGA/MXD NPs
To investigate in vitro release of MXD, HA-PLGA/MXD NPs were dispersed in 2 mL of phosphate buffered saline (PBS, pH 7.4) with 50 μg/mL concentration of MXD. Then, the suspension was transferred in dialysis membrane (MWCO 10,000 Da) and placed in 38 mL of PBS. The sample tube was shaken at 60 rpm in 37 °C conditions. At a predetermined time interval, 1 mL of PBS was collected and replaced by equivalent volume of fresh PBS. Then, each sample was analyzed with UV-Vis spectroscopy to quantify the amount of MXD released from HA-PLGA/MXD NPs. In addition, in vitro release test of MXD was also carried out using MXD aqueous solution and PLGA/MXD NPs in the same method as control experiment .
Cell viability test
For cell viability test, fibroblasts were cultured in DMEM containing 10% FBS and 1% antibiotics and 5% CO2 condition. The cytotoxicity of HA-PLGA/MXD NPs against fibroblast was evaluated by cell proliferation (WST) assay using EZ-CYTOX kit (water soluble tetrazolium salt). First, fibroblasts (2.5 × 103 cells/well) were seeded in a 96-well plate for 24 h. Then, various concentrations (2, 4, 10, 20, 40, 100, and 200 μg/mL) of HA-PLGA/MXD NPs were added to each well and incubated for 12 and 24 h, respectively. At the end of incubation, 10 μL of WST solution (EZ-CYTOX) was added to the cell and incubated for 2 h. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader (AMR-100, ALLSHENG, China).
Cellular uptake test
For cellular uptake test, four dishes of hair follicle dermal papilla cells (2.5 × 106 cells/dish) were cultured with EFO gro™ HDP containing 10% FBS and 1% antibiotics in 37 °C and 5% CO2 condition for 24 h. Then, the growth medium in all of the dishes was replaced to HA-PLGA/Rho B NPs or PLGA/Rho B NPs contained serum-free medium with 10 μM concentration of Rho B and incubated for 2 h. After that, the cells of each dish were washed with fresh PBS and 500 μL of 4% formaldehyde solution was added to each dish for cell fixation. The fixed cells were washed twice with fresh PBS and mounted with 1 mL of glycerin. And then, the cells were visualized using a fluorescence microscope (Eclipse TS100, Intensilight C-HGFI, Nikon, Japan).
Skin permeation test
PLGA/MXD and HA-PLGA/MXD NPs with 300 μg of MXD was applied on the 3 surfaces of rat skin, respectively. The rat skin was placed between the receptor and donor chambers at 37 °C. The receptor chamber was filled with 22 mL of PBS buffer (pH 7.4). Then, at predetermined time points (4, 6, 8, 12, and 24 h), 1 mL of the sample solution was collected from a sidearm of receptor chamber and replaced by the same volume of fresh PBS. Then, each sample was analyzed with High Performance Liquid Chromatography (HPLC, e2695, Waters, USA) and UV-Vis spectrometer to measure the permeated MXD amount through the skin. The HPLC analysis was performed with a SunFire C18 column (100 Å, 5 μm, 4.6 × 250 mm, Waters, USA). The mobile phase was methanol/DI water/acetic acid (75:25:1, HPLC grade, v/v/v) and the flow rate was 1.0 mL/min. The detection wavelength was 285 nm.
Ten microliters of diluted solution of HA-PLGA/Rho B NPs and PLGA/Rho B NPs with 30 μg/mL concentration of Rho B were applied on rat skin in the same area. Then, at the predetermined time of 4, 6, 8, and 12 h, the skin tissues were harvested. The retrieved skin tissues were embedded into optimal cutting temperature (OCT) compound at − 30 °C, and cut into 10 μm thick sections using cryotome (CM1860, Leica Biosystem, Germany). The sections were fixed with the 1:1 (v/v) mixture of ethanol and acetone and washed with distilled water to remove residual OCT resins on the slide. And then, the histological tissue sections were imaged by using a fluorescence microscope. The image analysis was performed using Image J (NIH).
Data are expressed as means ± standard deviation in a few separate experiments. Statistical analysis was carried out with the t-test using Prism 8 (GraphPad Software, San Diego, CA), P values less than 0.05 were considered statistically significant.