Transdermal delivery of Minoxidil using HA-PLGA nanoparticles for the treatment in alopecia
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Alopecia has become a very common disease that many people around the world are suffered. Minoxidil (MXD) is the most well-known commercialized drug in its treatment. However, in the case of MXD administration, there are some problems with low efficiency of transdermal delivery and additional side effects.
MXD and Rhodamine B (Rho B) are encapsulated in poly(Lactide-co-Glycolide) grafted hyaluronate nanoparticles (HA-PLGA/MXD NPs, HA-PLGA/Rho B NPs) which is prepared with W/O/W solvent evaporation method. After then, the investigation is carried out to confirm the feasibility of NPs in alopecia treatment.
Both of HA-PLGA/MXD NPs and HA-PLGA/Rho B NPs are successfully prepared. In addition, it is confirmed that HA-PLGA NPs sufficiently delivered to cells without any significant cytotoxicity by cell viability, cellular uptake and skin permeation test.
Taken together, HA-PLGA NPs as a transdermal delivery carrier to hair follicle cells can be exploited to develop the efficient and effective platform of transdermal drug delivery for the treatment of various diseases.
KeywordsHyaluronate Poly(Lactide-co-Glycolide) Alopecia Minoxidil Transdermal drug delivery
Alopecia is a symptom that hairs were abnormally lost because of various causes including genetic disorder, healthy state, stress, and aging [1, 2]. Alopecia has become a common disease that large number of people around the world have suffered . Many kinds of medical trial have been performed to treat alopecia by activating hair regeneration at follicle cells. Among them, medication is the most common method to treat alopecia.
Minoxidil (MXD) is the most widely used drug in alopecia treatment, especially male-pattern hair loss [1, 2, 3, 4, 5, 6, 7]. It is known to have a vasodilatory effect and expedite secretion of growth factors for hair regeneration. Due to the characteristics of alopecia, it works effectively when applied to the topical skin area through transdermal delivery of MXD. However, typical MXD delivery has the disadvantages of low efficiency of transdermal delivery and various side effects, such as tolerance, inflammation and low blood pressure . Therefore, many studies have been investigated to improve the transdermal delivery efficiency of MXD applied to the skin and the therapeutic efficacy in alopecia, through the transdermal routes such as hair follicles, sweat glands or microneedle arrays [2, 4, 7, 8, 9, 10].
To resolve the above issues, we have developed the transdermal drug delivery platform for the treatment of alopecia using MXD encapsulated Hyaluronate – Poly(Lactide-co-Glycolide) nanoparticles (HA-PLGA/MXD NPs). Hyaluronic acid (HA) is a well-known natural polysaccharide for various biomedical applications [11, 12, 13, 14, 15, 16, 17]. The excellent characteristic of HA for the transdermal delivery has been reported in our previous works . Poly(Lactide-co-Glycolide) (PLGA) is also well-known biocompatible polymers and widely investigated for the various drug delivery application [11, 13, 18, 19]. Because of the hydrophobicity of PLGA, it is known to be permeable to the skin, and transdermal delivery using PLGA has been reported in some previous studies [15, 16]. Therefore, through the respective transdermal characteristics, HA-PLGA is expected to exhibit the synergistic effect in transdermal delivery of MXD. In this work, to encapsulate hydrophilic MXD in hydrophobic HA-PLGA conjugates we prepared NPs using the W/O/W emulsion method and characterized HA-PLGA/MXD NPs. Then we investigated cell viability, cellular uptakes and transdermal delivery of those NPs to confirm the enhanced transdermal delivery of MXD and the feasibility for treatment of various diseases on skin.
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.
Synthesis of HA-PLGA
Characterization of PLGA/MXD and HA-PLGA/MXD NPs
Characterization PLGA/MXD and HA-PLGA/MXD NPs
Mean volume diameter (nm)
Minoxidil content in nanoparticles (%)
Encapsulation efficiency (%)
159 ± 11.8
243 ± 44.5
Drug release profiles of MXD-loaded HA-PLGA NPs
In vitro cellular uptake of NPs and cytotoxicity test
Skin permeation of MXD encapsulated NPs
Transdermal drug delivery is the most attractive candidate for replacing needle-injection. However, there are some limitations, such as low delivery efficiency and bioavailability. In case of the treatment of alopecia, MXD is widely used, but using MXD also has disadvantages when it is applied on the skin. To increase the delivery efficiency of MXD, we designed novel transdermal carriers in forms of nanoparticles. To use effectively each material with good properties in the delivery of MXD, we used an emulsification method through solvent evaporation [21, 23, 24]. Emulsion method can facilitate more efficient storage, improve stability of hydrophobic drugs, and reduce the size of particles as drug delivery carrier . Therefore, we prepared the HA-PLGA NPs using W/O/W emulsion method which has been widely investigated in drug delivery system .
At first, in the HA-PLGA synthesis, we could confirm the successful synthesis of HA-PLGA from the characteristic peaks in NMR spectrum and 4.4% of substitution of PLGA for HA. Then, after characterization of HA-PLGA NPs, although PDI was increased with the conjugation of HA, the size distribution of HA-PLGA NPs was still narrow and located in appropriate size for the hair follicle permeation, which could be expected to be delivered to hair follicles. In addition, HA-PLGA NPs were shown core-shell structure composed by hydrophobic PLGA core and hydrophilic HA shell by TEM analysis.
In vitro release and cellular uptake test, the HA-PLGA NPs showed the controlled release of MXD and high efficiency of cell uptake compared to PLGA NPs. The mechanism of cellular uptake is HA receptor-mediated endocytosis by the highly expressed CD 44 which is well-known to HA receptor on dermal papillary cells . Therefore, the biocompatible HA conjugated NPs can be more utilized in the treatment of alopecia.
Through the permeation test, we could confirm that HA-PLGA NPs gradually penetrated through the hair follicles. 8 h later, the fluorescence intensity in the epidermis and follicle cells were almost constant, but the total intensity in stratum corneum was decreased continuously and the those in the dermis and follicle cells were increased. Thus, these results suggest that HA-PLGA NPs delivered to the follicle cells were dispersed to the dermis and took 8 h to approach the surrounding tissue.
Taken together, HA conjugated NPs could show a synergistic effect and enable more efficient delivery of MXD by their excellent skin permeability characteristics. Therefore, we expected HA-PLGA NPs would be a good candidate of MXD carrier for alopecia treatment.
In this work, we prepared MXD-encapsulated HA-PLGA NPs using W/O/W solvent evaporation method and confirmed the biocompatibility in cell viability test and the ability of HA-PLGA NPs to delivery to follicle cells in cellular uptake test and skin permeation test. From the characterization of HA-PLGA NPs, we could confirm higher efficiency of transdermal delivery that HA-PLGA NPs had not only suitable hydrodynamic diameter, but also more drug loading efficiency than PLGA NPs. In addition, a sufficient amount of MXD was uptaken into cells without cytotoxicity and HA-PLGA NPs were successfully delivered to hair follicle cells. With these results, HA-PLGA NPs are suitable in transdermal delivery of MXD for alopecia treatment and can be exploited to develop an efficient and effective platform for the treatment in various other diseases.
The authors gratefully thank Seonyeong An for helping in in vitro evaluation.
WYJ and SK performed all experiments, collected samples, analyzed and interpreted data, prepared the figures for the manuscript, and wrote the manuscript. KSK designed all experiment and conceived and supervised the project, interpreted data and wrote the manuscript. SYL contributed to the chemical synthesis and HL carried out skin permeation test. DWH and SYY contributed to the scientific discussion. All authors contributed to critical reading of this manuscript. All authors read and approved the final manuscript.
This work was supported by National Research Foundation of Korea (#2018R1C1B5035631, # 2019R1F1A1062216 and # 2019R1A4A1024116) and Pusan National University Research Grant, 2017.
Ethics approval and consent to participate
Consent for publication
The manuscript has been submitted with the consent of all authors for publication.
The authors declare that they have no competing interests.
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