Cell culture and regents
Human prostate cancer LNCaP and human keratinocyte HaCaT cells were purchased from the American Type Culture Collection (ATCC), and human follicle dermal papilla cells (HFDPCs) were purchased from PromoCell (C-12071). Human peripheral blood mononuclear cells (PBMCs) were obtained from Cellular Technology Limited (CTL-UP1). LNCaP cells were cultured in RPMI medium (HyClone) supplemented with 1% penicillin–streptomycin (HyClone) and 10% fetal bovine serum (FBS, HyClone). HaCaT cells were cultured in DMEM (HyClone) supplemented with 1% penicillin–streptomycin and 10% FBS. HFDPCs were maintained in follicle dermal papilla cell growth medium (PromoCell). Individual human hairs were pulled out with forceps in the occipital area of the scalp, and plucked hairs with visible bulbs were selected by microscopy. The plucked hair follicles were cultured in DMEM/F12 (Gibco) supplemented with 1% penicillin–streptomycin, 10% FBS, 10 µg/mL insulin-transferrin-selenium-X supplement (Gibco), 2.5 µg/mL amphotericin B (Gibco), 1% GlutaMAX (Gibco), 20 ng/mL fibroblast growth factor (FGF, PeproTech), 20 ng/mL epidermal growth factor (EGF, Sigma), and 10 ng/mL hydrocortisol (Tokyo Chemical Industry). Plucked human hair follicles were treated with 10 μM SAMiRNA-AR68 for 48 h followed by qPCR and immunofluorescence analysis. The synthesis and quality control of the SAMiRNA nanoparticles were previously described35,37.
Measurement of SAMiRNA nanoparticle size
For determination of the long-term stability of SAMiRNA, the nanoparticle size was monitored by qNano Gold (Izon Science) according to standard operating procedures. Briefly, 35 μl of SAMiRNA-AR68 was analyzed with qNano Gold equipment using an NP80 Nanopore (Izon Science) and applying a 47 mm stretch, a current of 140 nA, and 10 mBar parametric conditions. The calibration particles (CPC100, Izon Science) were assayed before the experimental samples under identical conditions. Particle counts (≥ 50 events each) were finally determined using the qNano software provided by Izon Science (Izon Control Suite Version 3.3).
Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed according to the MIQE guidelines48. Total RNA from cells was extracted using an AccuPrep® Universal RNA Extraction Kit (K-3140, Bioneer) according to the manufacturer’s instructions. For total RNA extraction from hair follicles, SAMiRNA-AR68-treated plucked hair follicles were washed with PBS, resuspended in TRIzol® reagent (Invitrogen), homogenized with a Biomasher II® Disposable Micro Tissue Homogenizer (Polyscience), and purified with an AccuPrep® Universal RNA Extraction Kit (K-3140, Bioneer) according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed using Accupower® RocketScript™ Cycle RT PreMix (dT20) in a 20 μl reaction (K-2201, Bioneer) according to the manufacturer’s instructions. For qPCR analysis, 10 μl of tenfold diluted cDNA was amplified using AccuPower® 2X GreenStar™ qPCR MasterMix (K-6253, Bioneer). The following primer sets were used: AR, forward 5′-TTGTACACGTGGTCAAGTGG-3′ and reverse 5′-TGGAGTTGACATTGGTGAAGG-3′; RPLPO, forward 5′-TGCCATTGCCCCATGTGAAG-3′ and reverse 5′-AGCTGCACATCACTCAGGATT-3′; IL-1B, forward 5′-CTGAGCTCGCCAGTGAAAT-3′ and reverse 5′-CTGTAGTGGTGGTCGGAGA-3′; IL-6, forward 5′-AGATGCAATAACCACCCCTG-3′ and reverse 5′-TGCGCAGAATGAGATGAGTT-3′; TNF, forward 5′-CTGTAGCCCATGTTGTAGCA-3′ and reverse 5′-GGTTATCTCTCAGCTCCACG-3′; IFNG, forward 5′-GAATGTCCAACGCAAAGCAA-3′ and reverse 5′-ACCTCGAAACAGCATCTGAC-3′; RPL13A, forward 5′-TGCCATTGCCCCATGTGAAG-3′ and reverse 5′-AGCTGCACATCACTCAGGATT-3′; IL-1B, forward 5′-GTGTTTGACGGCATCCCACC-3′ and reverse 5′-TAGGCTTCAGACGCACGACC-3′. PCR amplification was performed in a 50 μl reaction as follows: one cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 5 s, 58 °C for 25 s, and 72 °C for 30 s, with one final extension step at 72 °C for 5 min. All experiments were performed in triplicate. The delta-delta Ct method was used to determine relative fold changes, and all data were normalized to the internal control gene.
Immunoblot analysis
LNCaP and HFDP cells were collected and lysed using cell lysis buffer (Cell Signaling Technology) with protease inhibitor cocktail (Thermo Fisher Scientific). Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Bio-Rad). After the membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) for at least 1 h, they were incubated with the indicated antibodies overnight, washed with TBS three times, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Immunoblotting was performed with the following antibodies: anti-AR (ab133273, Abcam), anti-GAPDH (2118, Cell Signaling Technology), and horseradish peroxidase-conjugated anti-rabbit (7074, Cell Signaling Technology). Immunoblot band intensities of AR and GAPDH were quantified by ImageJ software, and the level of AR protein expression was normalized to the GAPDH values.
Enzyme-linked immunosorbent assay (ELISA)
ELISA to detect the AR protein was performed using a human AR ELISA kit (LS-F4505, LSBio) according to the manufacturer’s manual. In brief, HFDP cells (5 × 104 cells/well) were collected and lysed using cell lysis buffer (Cell Signaling Technology), and then, 100 μl of cell lysate was added to each well for 1 h at 37 °C. After incubation, a biotin-conjugated detection antibody (detection reagent A) was added that bound to the captured antigen. An avidin-horseradish peroxidase conjugate (detection reagent B) that binds to biotin was then added. The TMB substrate reacts with the HRP enzyme, resulting in color development. A sulfuric acid solution (stop solution) terminated the color development reaction, and the optical density (OD) of each well was measured at a wavelength of 450 nm. The OD of samples was computed with an OD standard curve generated using known standard AR concentrations (0.313 ~ 20 ng/ml) to determine its AR concentration. The incubation and washing steps were performed according to the supplier’s manual. Absorbance at 450 nm was monitored with a FLUOstar Omega microplate reader (BMG Labtech).
Cell viability assay
For the cell viability assay, HaCaT and HFDP cells were seeded at a density of 4 × 103 cells/well in a 96-well plate and then treated with the indicated conditions for 72 h. After incubation, 10 μl of WST (water soluble tetrazolium salt) reagent (EZ-Cytox, DoGen) was added to each well for 30 min at 37 °C. Absorbance at 450 nm was monitored with a FLUOstar Omega microplate reader (BMG Labtech).
Immunofluorescence (IF)
Plucked hairs from healthy donors were obtained from the occipital area of the scalp. After incubation, hair follicles were washed three times with 0.05% Tween-20 (Sigma) in PBS for 5 min and permeabilized in 0.1% Triton X-100 (Sigma) in PBS for 1 min. Hair follicles were fixed in 4% formaldehyde (Sigma) in PBS for 20 min, placed on a cryotray and covered with OCT compound (Tissue-Tek). The mold was slowly placed in liquid nitrogen until the entire tissue block was completely frozen. The frozen tissue blocks were sectioned at 10 µm using a cryotome (Leica Biosystems). Hair follicle sections were incubated overnight at 4 °C with an anti-AR antibody (ab133273, Abcam). The sections were subsequently incubated with anti-rabbit Fluor 568 (A11011, Invitrogen) secondary antibody for 1 h at room temperature. After at least three washes, the sections were counterstained with DAPI (D9542, Sigma-Aldrich) and mounted in Immu-Mount solution (Thermo Fisher Scientific). All incubations were conducted in dark and humid chambers. The fluorescence signal was visualized using a confocal microscope (LSM880, Carl Zeiss) at excitation wavelengths of 568 nm (Alexa Fluor 568) and 405 nm (DAPI). At least three fields per section were analyzed. ZEN software (Zeiss) was used to analyze images of the desired area to measure the intensity on a pixel-by-pixel basis and calculate the mean intensity automatically.
Statistical analysis
All data are presented as the mean ± standard deviation (SD), and the number of samples is indicated in each figure legend. The statistical significance of differences was assessed using the two-sided Student’s t test. The results shown are representative of at least three independent experiments. Significance was denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.
Clinical study I: low-dose SAMiRNA-AR68 (0.5 mg/ml) treatment three times per week
Clinical study I was a double-blind, randomized, placebo-controlled study in accordance with the Korea Ministry of Food and Drugs Safety (MFDS) Guidelines on the Efficacy Study of Cosmetics for Hair Loss Relief (July 2018). The study was approved by the Institutional Review Board of the local committee, Seowon Skin Research Center, and was conducted on 10/09/2020 (IRB No. 1040820–202,009-HR004–02) and all experimental protocols were carried out in accordance with relevant guidelines and regulations. All subjects provided written informed consent for publication of identifying information/images in an online open-access publication prior to study participation. Summary information of the study is listed and available at CRIS Registration No. KCT0005501: https://cris.nih.go.kr/cris/search/detailSearch.do/20301. Subjects were selected on the basis of inclusion and exclusion criteria and consisted of 48 Korean males and females ranging from 22 to 53 years of age. Male subjects were diagnosed with more than the M1, C1 and U1 range as the basic type and more than the V1 and F1 range as the specific type according to the basic and specific (BASP) classification49. Female subjects were diagnosed with more than the F1 and L1 ranges according to the BASP and Ludwig classification, respectively. The exclusion criteria were participation in a previous study within 6 months, prior surgical correction of scalp hair loss, use of topical minoxidil or finasteride within 6 months, and other skin diseases of the scalp, including severe seborrheic dermatitis, psoriasis, lichenoid eruption or other scalp infections. Alterations in hair styling and dyeing of the hair were not allowed during the study.
Clinical study II: high-dose SAMiRNA-AR68 (5 mg/ml) treatment once a week
The second clinical study was conducted as a double-blind, randomized, placebo-controlled study in accordance with the Korea MFDS Guideline on the Efficacy Study of Cosmetics for Hair Loss Relief (July 2018). A summary of the study details is available through the following link: https://cris.nih.go.kr/cris/search/detailSearch.do/20367 (CRIS Registration No. KCT0005618). Clinical study II was approved by the Institutional Review Board of the local committee, Ellead Skin Research Center, and was conducted on 07/09/2020 (IRB No. 200804T001) and all experimental protocols were carried out in accordance with relevant guidelines and regulations. All subjects provided written informed consent for publication of identifying information/images in an online open-access publication prior to study participation. The subjects were selected on the basis of inclusion and exclusion criteria and consisted of 60 Korean males and females ranging from 22 to 54 years of age. The AR68 high-dose (5 mg/ml) formulation was the same as the composition used in clinical study I, and randomly blinded products were applied once per week after shampooing. The measurement and analysis of hair density and total hair counts were conducted using the same protocol as described for clinical study I. The total hair count was measured using a Folliscope® 5.0 phototrichogram system (LeadM). Statistical analysis was also performed using the same program, statistical software SPSS statistics version 26.0 (IBM), as in clinical study I.
SAMiRNA-AR68 formulation and treatment
SAMiRNA-AR68 was formulated as a hair tonic aqueous solution with ethanol (15%, v/v), niacinamide (1% w/v), betaine (1% w/v), biotin (0.02% w/v) and buffer. The AR68 hair tonic was packaged in a piston-type bottle with a silicon adaptor for massage (Supplementary Fig. S5). The placebo was prepared in the same manner as the formula except for the addition of AR68 and packaged in the same bottle. In clinical study I, randomly selected products (AR68, 0.5 mg/ml) were used three times per week after shampooing, whereby the scalp was massaged for 5 min after application. In clinical study II, randomly selected products (AR68, 5 mg/ml) were used once a week after shampooing, whereby the scalp was massaged for 5 min after application. Product usage and compliance were monitored for each subject by reviewing subject diaries and weighing the returned test products at each scheduled visit (weeks 8, 16 and 24).
Measurement of hair density and total hair count
All subjects placed their head into a hair photograph device (Canfield Scientific), and photographs were taken at fixed distance, angle and lighting with an EOS Rebel T6i digital camera (Canon). Global evaluation was performed by comparing clinical images at baseline with those at 8, 16 and 24 weeks after treatment with the test product. The investigator qualitatively assessed clinical images on a 7-point scale (− 3, marked decrease; − 2, intermediate decrease; − 1, slight decrease; 0, no change; + 1, slight increase; + 2, intermediate increase; and + 3, marked increase).
For evaluation of total hair counts, the hair was clipped approximately 2 mm after a red spot tattoo in the evaluation area (1 cm2) of the hair loss region (forehead hairline or vertex). The total hair counts were assessed using a Folliscope® 2.8 phototrichogram system (magnification 14 times, LeadM) at baseline and at 16 weeks and 24 weeks after treatment. The total hair count (number/cm2) was calculated as the number of hairs within an area50.
Subject self-assessment questionnaires
Subject self-assessment questionnaires on efficacy and safety at 8 weeks, 16 weeks, and 24 weeks after using the product were collected.
Safety assessments
Dermatologists observed and assessed the subjects for the occurrence of objective irritation, such as edema, itching, and burning. The details, including start and end dates of occurrence, degree of severity, treatment, and causal relationship with the test product, were recorded on the form. If any adverse reaction occurred, the dermatologist’s assessment of the adverse reaction was further conducted in accordance with the standard operating procedures for adverse reactions.
Statistical analysis
Data are expressed as the mean and a changing rate between the baseline value and the value obtained at each time point. All statistical analyses were performed using the SPSS® package (IBM). For photographic assessment of hair density, statistical analysis for variables for comparison between time points or groups was conducted using the Wilcoxon signed-rank test and Mann–Whitney U test (p < 0.05). For analysis of total hair counts, the significance of a difference between time points was calculated using the paired t test, with p < 0.05 as the significance level.
