Synthesis of the Opioid Agonist
Biphalin was synthesized by Adriano Mollica at his laboratory in Università degli Studi G. d'Annunzio Chieti e Pescara, Department of Pharmacy, Chieti, Italy. Chemical properties of the peptide were in full agreement with those already reported in the literature (Fig. 1). 
Corneal Epithelial Cell Culture
Human immortalized corneal epithelial cells (HCECs) were a generous gift from Dr. James Jester (Irvine, CA, USA). HCECs were cultured in keratinocyte serum-free medium (KSFM; Gibco, NY, USA) supplemented with bovine pituitary extract (BPE; 25 μg/mL), epidermal growth factor (EGF; 50 ng/mL), penicillin (100 IU/mL), and streptomycin (100 μg/mL). The cells were maintained in 75 cm2 flasks until experimentation.
HCECs were treated with different concentrations of biphalin (from 1 pM to 100 µM) in 96-well culture dishes (Corning, NY, USA) for 24 hours. The cytotoxicity of exposure was measured with MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay (Thermo Fisher Scientific, MA, USA). Color of MTT tetrazole salt was measured with a spectrophotometer at the wavelength of 570 nm.
In Vitro Scratch Assay
The HCECs were grown to confluence on 12-well culture dishes (Corning, NY, USA). On reaching confluence, cells were rinsed with a phosphate-buffered saline solution (PBS) and exposed to a differentiation medium consisting of Dulbecco modified eagle medium (DMEM; Gibco, NY, USA) with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL) for one day. Two perpendicular linear scratches were made using a sterile 200 µL pipette tip and the wells were washed with PBS. Immediately after the scratch, all groups incubated in KSFM. Biphalin (in two different concentrations; 1 µM [10-6 M] and 10 µM [10-5 M]) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS) was added to the cultures. The scratch area was captured hourly for 24 hours using a live cell microscopy (DMi 8; Leica, Wetzlar, Germany). The relative wound area (RWA) was measured using ImageJ software (National Institutes of Health [NIH], MD, USA) (Fig. 2).
Transwell Migration Assay
Immediately after the wounding process has been completed as described above, HCECs were trypsinized, washed, and plated (2.5×105 cells per insert) in 8.0 µm pore size transwell inserts (Corning, NY, USA) in KSFM. The lower compartment was filled with DMEM with 10% FBS, or DMEM with 10% FBS plus either biphalin (in two different concentrations; 1 µM and 10 µM) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS). After 24 hours; the cells on the upper side of the insert were removed by scraping and the cells that had migrated through were fixed on the lower side of the membrane with 4% paraformaldehyde, then stained with hematoxylin-eosin and quantified by counting the number of cells in 10 separate fields. The data were expressed as the number of migrated cells per micrograph field for each sample well.
Ki67 Proliferation Assay
The effect of biphalin on in vitro proliferation was assessed by immunofluorescence staining for Ki67. The Ki67 protein is present during G1, S, G2 and M phases of the cell cycle and is strictly associated with cell proliferation. 2.5 x 105 HCECs were plated in equal numbers in 24-well culture dishes (Corning, NY, USA). After reaching confluence, cells were rinsed twice with PBS and exposed to a stratification medium consisting of DMEM with 10% FBS. Two perpendicular linear scratches were made using a sterile 200 µL pipette tip and the wells were washed three times with PBS and incubated with KSFM without EGF and BPE. Immediately after the scratch, biphalin (in two different concentrations; 1 µM and 10 µM) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS) were added to the cell culture medium. The cells were incubated for 6 hours at 37°C. After the treatment, cells grown on 24-well culture dishes were fixed in 4% paraformaldehyde for 20 min. After three washes with PBS, the cells were incubated with 0.1% TritonX-100 in PBS for 8 min. The cells were incubated with Superblock (Thermo Fisher Scientific, MA, USA) for 10 min at room temperature and then overnight at 4°C with the rabbit anti-Ki67 primary antibody (Abcam, MA, USA) at optimal dilutions in blocking solution. After three washes with PBS, cells were incubated with the FITC-conjugated secondary antibody (Abcam, MA, USA) for 90 min at 37°C, then washed, counterstained with 406-diamidino-2-phenylindole (DAPI), and mounted. Negative controls were stained in a similar fashion (DMi 8; Leica, Wetzlar, Germany). Ki67 proliferation index calculated as proportion of Ki67 stained cell nuclei to DAPI stained cell nuclei from 20 different micrograph areas.
Gene Expression Analysis of Opioid Receptors with Quantitative Reverse Transcription
Presence and expression levels of MOR, DOR and KOR in HCECs were measured quantitatively using real-time polymerase chain reaction (qRT-PCR). The presence of OPRM1, OPRD1, and OPRK1 mRNAs, which are related with mu, delta and kappa opioid receptor proteins, respectively, was studied in differentiated and undifferentiated HCECs, and in SH-SY5Y cell lines. SH-SY5Y cell lines are human neuronal cancer cell lines, which have showed expressions of these three opioid receptors in previous literature. RNA isolation was performed with Quick-RNA MicroPrep Kit (Zymo Research, Irvine, CA, USA) by following manufacturer’s instructions. RNA was quantified with spectrophotometric read at 260 nm by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and 1000 ng cDNA was prepared by using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time expressions of mRNAs were detected and compared by using Light Cycler 480 SYBR Green I Master (Roche, Basel, Switzerland). The primers of genes used in the study are shown in Table 1.
Each experiment was performed at least two times. For blind analysis, collection of images was made by E.Y., and each of the images was assigned a number. Then, images were analyzed anonymously by K.K. Values were displayed as mean ± standard deviation. Statistical analysis was performed using two-way ANOVA for in vitro scratch assay results and one-way ANOVA with a Tukey's Honest Significant Difference test for other results to determine the degree of significance (R; R-Project, Vienna, Austria). Results were considered statistically significant when p-value was less than 0.05.