Polyoxyethylene and Polypropylene Emulsification Enhances Retinol Palmitate Efficacy in Corneal Wound Healing

Polyoxyethylene and Polypropylene Emulsification Enhances Retinol Palmitate Efficacy in Corneal Wound Healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Polyoxyethylene and Polypropylene Emulsification Enhances Retinol Palmitate Efficacy in Corneal Wound Healing Haruki Horiuchi, Keisuke Watanabe, Hiroshi Iijima, Yoshiyuki Obayashi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6223209/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 May, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Retinol palmitate (VApal), an active ingredient in ophthalmic solution, has been reported to repair corneal injuries. Additionally, it has been suggested that the efficacy of VApal is enhanced by a specific surfactant, polyoxyethylene-polypropylene [EO 100 PO 70 EO 100 (EOPO)]. We aimed to determine the efficacy of VApal in corneal wound healing in comparison to that of hyaluronic acid (HA), carboxymethyl cellulose (CMC) and hydroxypropyl methylcellulose (HPMC) used in over-the-counter ophthalmic solutions and whether the efficacy of VApal could be enhanced by EOPO compared to the widely used surfactant, polyoxyethylene hydrogenated castor oil 60 (HCO60). To evaluate the efficacy of VApal or VApal emulsified with a surfactant, we performed a wound healing assay using corneal epithelial cells in monolayer (n = 4) or 3D culture (n = 6). Wound closure rates were calculated each time, and the efficacy was compared using the time to reach a 50% wound closure rate (ET50). The ET50 values of VApal, HA, CMC and HPMC were approximately 17.31 h, 26.99 h, 28.98 h and 26.01 h respectively. The ET50 values of VApal emulsified with EOPO or HCO60 were 34.49 h and 43.31 h, respectively. In conclusion, VApal is more beneficial than other ingredients for corneal wound healing. Additionally, the efficacy of VApal can be enhanced using EOPO instead of HCO60. Biological sciences/Cell biology/Cell migration Biological sciences/Drug discovery/Pharmaceutics wound healing cornea retinol palmitate vitamin A surfactant Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The cornea, which functions in visual information intake, tear fluid retention, and biological defense [1] , comprises the epithelium, Bowman’s membrane, stroma, Descemet’s membrane, and endothelium. The corneal epithelium is particularly vulnerable to external stimuli and can easily be injured [2] . Corneal epithelial wounds are commonly caused by contact lens wear and increased blink friction due to tear film destabilization [3] . These wounds are typically repaired through a self-healing process consisting of three phases: cell extension and migration (phase 1), division and proliferation (phase 2), and differentiation (phase 3) [4] . Retinol palmitate (VApal), a lipophilic derivative of vitamin A, is crucial for maintaining homeostasis in the eye, skin, and mucous membranes [5, 6] . VApal is used to treat conditions such as night blindness and keratoderma and is formulated as an active ingredient in over-the-counter medicines in several countries, including ophthalmic solutions for eye fatigue, blurred vision and external preparations for the skin. The biologically active form of VApal, retinoic acid, regulates various functions, such as cell growth, differentiation, and organogenesis [7] , by binding to retinoic acid receptors, which are nuclear receptors. In the corneal epithelium, VApal increases hyaluronic acid (HA) production [8] . Moreover, retinoic acid has been reported to increase mucin expression, which is involved in tear fluid retention [9] , and to upregulate lysyl oxidase-like 4, which promotes cell migration [10] . Additionally, Toshida et al. [11] demonstrated the topical application of VApal to repair corneal wounds in a mouse model. In addition to VApal, several other ingredients used in ophthalmic solutions have been reported to promote corneal wound healing. For example, HA was found to promote the migration of corneal epithelial cells by binding to CD44 [12] and accelerating corneal wound healing in a rabbit corneal alkali burn model [13] . Similarly, carboxymethyl cellulose (CMC) promotes epithelial cell migration by interacting with matrix proteins and has demonstrated efficacy in accelerating wound healing in human corneal epithelial cells [14] . Hydroxypropyl methylcellulose (HPMC), which is often used as a thickening agent, was found to accelerate corneal wound healing in a rat corneal detachment model [15] . Cyclosporine suppresses inflammation and improves ocular surface conditions. In a clinical phase II study involving patients with moderate-to-severe dry eye, cyclosporine significantly reduced the degree of corneal fluorescein staining compared to the vehicle [16] . Despite the availability of ingredients that accelerate corneal wound healing, previous studies comparing the efficacy of VApal and other ingredients are limited. In a study that compared the efficacy of VApal and cyclosporine in corneal wound healing, Reimondez-Troitiño et al. [17] reported that the efficacy of retinoic acid in corneal wound healing was equal to that of cyclosporine in a mouse injured by photorefractive keratectomy surgery. Their study compared the efficacy of ingredients with different mechanisms of action, including VApal (cell migration and water retention) and cyclosporine (anti-inflammatory). We did not find any previous studies that compared the efficacy of VApal in wound healing with ingredients that have mechanisms of action similar to those of VApal (e.g., HA). Therefore, when compared to HA, CMC, and HPMC, which have similar mechanisms of action as VApal, the relative value of VApal in corneal wound healing remains unclear. The formulation of VApal in water-based ophthalmic solutions requires surfactants. Polyoxyethylene-hydrogenated castor oil 60 (HCO60) is typically used to emulsify VApal. Previously, Miyake et al. [18] investigated a method for the formulation of VApal using polyoxyethylene-polypropylene (EO 100 PO 70 EO 100 [EOPO]) [18] . They investigated the behavior of fluorescence-labeled VApal emulsified with EOPO in relation to cell-membrane interactions, focusing on surface chemistry [18] . Their study demonstrated that VApal emulsified with EOPO exhibited greater localization around the cell nucleus than did HCO60. Similarly, Toshida et al. [19] reported the effectiveness of VApal emulsified with EOPO in patients with dry eyes. In their study, both the objective signs and subjective symptoms of dry eye significantly improved with VApal emulsified with EOPO compared with placebo. However, regarding the efficacy of VApal in corneal wound healing, it remains unclear whether EOPO is a better surfactant than commonly used surfactants (e.g., HCO60). In this study, we aimed to determine 1) the efficacy of VApal in corneal wound healing compared to ingredients with similar mechanisms of action (i.e., HA, CMC, and HPMC), and 2) whether the efficacy of VApal emulsified with EOPO was higher than that of HCO60. Results Efficacy of VApal in human corneal wound healing compared to other ingredients To investigate the efficacy of VApal in corneal wound healing, we conducted a wound healing assay using SV40-immortalized human corneal epithelial cells (HCE-T cells). We compared the efficacy of VApal with that of HA, CMC, and HPMC, which are commonly used as active ingredients in ophthalmic solutions for corneal wounds. Figure 1 a shows representative images of the wound healing process in the control, VApal, HA, CMC, and HPMC groups after 24 h. The wound area was noticeably smaller in the VApal group than in the other groups. Figure 1 b shows the wound closure rates in each group from 0 to 60 h. The VApal group had higher wound closure rates than the HA and CMC groups at all time points. Compared to the HPMC group, the VApal group had significantly higher wound closure rates at all time points, except at 36 h (Table 1 ). To quantify the efficacy of each treatment, we defined the time required to reach 50% of the total wound healing area (ET50). The ET50 of the VApal group was significantly shorter than that of the HA, CMC, and HPMC groups (p = 0.0002, p < 0.0001, and p = 0.0005, respectively) (Table 2 ). Table 1 Statistical analysis of the wound closure rates at each time. Dunnett's test (vs. VApal) P-value 12 h 24 h 36 h 48 h 60 h Control 0.0002 < 0.0001 < 0.0001 < 0.0001 < 0.0001 HA 0.0009 0.0050 0.0109 0.0033 0.0053 CMC 0.0006 < 0.0001 0.0029 0.0040 0.0099 HPMC < 0.0001 0.0084 0.1836 0.0178 0.0418 VApal, retinol palmitate; HA, hyaluronic acid; CMC, carboxymethyl cellulose; HPMC, hydroxypropyl methylcellulose Table 2 ET50 of the control, VApal, HA, CMC, and HPMC groups ET50 (h) Dunnett's test (vs. VApal) P-value Control 32.16 ± 1.50 < 0.0001 VApal 17.31 ± 1.27 - HA 26.99 ± 1.82 0.0002 CMC 28.98 ± 2.06 < 0.0001 HPMC 26.01 ± 2.19 0.0005 ET50, time required to reach 50% of the total wound healing area; VApal, retinol palmitate; HA, hyaluronic acid; CMC, carboxymethyl cellulose; HPMC, hydroxypropyl methylcellulose Efficacy of VApal emulsified with EOPO in human corneal wound healing We investigated the efficacy of VApal emulsified with surfactants in corneal wound healing. Prior to this, we assessed the toxicity of EOPO on corneal epithelial cells using a cell survival assay (Fig. 2 a). The exposure of corneal epithelial cells to EOPO for up to 72 h did not affect cell viability, indicating that EOPO was not toxic to corneal epithelial cells. Next, we examined whether EOPO could increase the efficacy of VApal and compared the efficacy of human corneal wound healing between the VApal and EOPO + VApal groups. Figure 2 b shows representative images of the wound healing process in the VApal and EOPO + VApal groups at 24 h. The wound area in the EOPO + VApal group was smaller than that in the VApal group. Figure 2 c shows the wound closure rates in each group from 0 to 60 h. At 24, 36, and 48 h, the wound closure rates in the EOPO + VApal group were significantly higher than those in the VApal group (p = 0.0002, p = 0.0186, and p = 0.0183, respectively). Moreover, the ET50 of the EOPO + VApal group was significantly shorter than that of the VApal group (p = 0.0013) (Table 3 ). Table 3 ET50 of the VApal and EOPO + VApal groups ET50 (h) Student’s t-test P-value VApal 20.92 ± 1.59 - EOPO + VApal 15.57 ± 0.35 0.0013 ET50, time required to reach 50% of the total wound healing area; VApal, retinol palmitate; EOPO, polyoxyethylene-polypropylene [EO 100 PO 70 EO 100 ] Finally, we compared the efficacy of VApal emulsified with EOPO to that of VApal emulsified with HCO60 in promoting corneal wound healing. HCO60 is a surfactant commonly used as an emulsifier for VApal in ophthalmic over-the-counter solutions. Before conducting the wound healing assay, we assessed the toxicity of HCO60 in corneal epithelial cells. The cell viability rates in the HCO60 group were significantly lower than those in the control group at 48 and 72 h (Fig. 2 d) (p = 0.0004 and p = 0.0004, respectively). Therefore, we concluded that a monolayer culture of corneal epithelial cells may not be suitable for investigating the efficacy of VApal emulsified with HCO60. Cytotoxicity of HCO60 in a reconstructed human corneal epithelium model Owing to the observed toxicity of HCO60 in the monolayer cultures of corneal epithelial cells, we were unable to demonstrate a difference in efficacy between VApal emulsified with EOPO and VApal emulsified with HCO60. Consequently, we opted to use a reconstructed human corneal epithelium model (3D corneal epithelial cells) that is more similar to the human corneal epithelial cells [20] . To evaluate the cytotoxicity of HCO60 in the 3D corneal epithelial cells, a cell survival assay was performed. The exposure of corneal epithelial cells to HCO60 for up to 72 h did not affect cell viability (Fig. 3 ). Therefore, HCO60 was less toxic to 3D corneal epithelial cells than to monolayer cultures of corneal epithelial cells. Comparison of the efficacy of VApal emulsified with EOPO vs. VApal emulsified with HCO60 in corneal wound healing in 3D corneal epithelial cells To investigate the difference in efficacy between VApal emulsified with EOPO and VApal emulsified with HCO60 for corneal wound healing, we performed a wound healing assay using 3D corneal epithelial cells. Figure 4 a shows representative images of the wound healing process in the EOPO + VApal and HCO60 + VApal groups at 36 h. The wound area was smaller in the EOPO + VApal group than that in the HCO60 + VApal group. Figure 4 b shows the wound closure rates in each group from 0 to 60 h. After 36 h, the wound closure rates in the EOPO + VApal group were significantly higher than those in the HCO60 + VApal group (p = 0.0007, p = 0.0004, and p = 0.0004, respectively). Comparing the EOPO + VApal and HCO60 + VApal groups, the ET50 of the EOPO + VApal group was significantly shorter than that of the HCO60 + VApal group (p = 0.0005) (Table 4 ). Table 4 ET50 of the HCO60 + VApal and EOPO + VApal groups ET50 (h) Student’s t-test P-value HCO60 + VApal 43.31 ± 4.56 - EOPO + VApal 34.49 ± 1.08 0.0005 ET50, time required to reach 50% of the total wound healing area; VApal, retinol palmitate; EOPO, polyoxyethylene-polypropylene (EO 100 PO 70 EO 100 ); HCO60, polyoxyethylene-hydrogenated castor oil 60 Discussion In this study, we assessed the efficacy of VApal in corneal wound healing compared to other ingredients with a mechanism of action similar to that of VApal. The results indicated that the efficacy of VApal in corneal wound healing was higher than that of other ingredients, such as HA. These results may be attributed to the differences in the mechanisms of action of VApal and the other ingredients. Previous reports have shown that HA and CMC promote cell migration through their binding to extracellular substrates [12, 14] . HPMC is used in ophthalmic solutions because of its water retention properties. In contrast, VApal is metabolized into retinoic acid within the corneal epithelial cells, which regulates the expression of target genes mediated by retinoic acid receptors. VApal has also been reported to enhance HA production by increasing the expression of hyaluronic acid synthetase 3 [21] . Retinoic acid has also been reported to increase the expression of lysyl oxidase-like protein 4, which is involved in cell migration [10] . In a study using a mouse dry eye model lacking aquaporin 5 in the corneal epithelium, retinoic acid enhanced cell proliferation and reduced apoptosis by inducing the expression of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and Bcl-2 [22] . As described above, ingredients such as HA promote corneal wound healing by enhancing cell migration, whereas VApal promotes cell proliferation and inhibits apoptosis. Considering these differences in the mechanisms of action, it is likely that the efficacy of VApal in corneal wound healing is higher than that of the other ingredients. However, the wound healing assay utilized in this study was an evaluation model in which there was no extracellular matrix. Therefore, it is possible that the efficacies of HA and CMC were lower because extracellular substrates affected their efficiency. In addition, because the cells are always exposed to the medium, it may be difficult to determine the efficacy of wound healing mediated by water retention. In the future, it will be necessary to evaluate the efficacy in in vivo studies to accurately understand the drug effects. Toshida et al. [19] demonstrated the effectiveness of VApal emulsified with EOPO in a clinical study; however, it remains unclear whether the effectiveness of VApal emulsified with EOPO is higher than that of VApal emulsified with HCO60. We showed that the ET50 of VApal emulsified with EOPO was shorter than that of VApal alone. This suggests that EOPO can enhance the efficacy of VApal in corneal wound healing. We hypothesize that this enhancement may be attributed to differences in the transition mechanism of VApal at the cell membrane. Generally, VApal is transported into cells via transporters in the plasma membrane. Kawaguchi et al. [23] reported that VApal binds to retinol-binding proteins (RBPs) in the extracellular space, and VApal-RBP complexes are taken up into the cell by STRA6, a plasma membrane receptor that specifically recognizes RBPs. Miyake et al. [18] reported that VApal emulsified with EOPO may be transported into cells by endocytosis. They examined the interactions between VApal emulsified with EOPO and plasma membrane-mimicking giant unilamellar vesicles (GUVs). They demonstrated that VApal emulsified with EOPO induced morphological changes in GUVs and was taken up by GUVs in a manner similar to endocytosis. This implies that VApal emulsified with EOPO is transported not only by the usual transport pathway mediated by STRA6 but also by endocytosis. Furthermore, we assessed whether the efficacy of VApal emulsified with EOPO was higher than that of VApal emulsified with HCO60. We experimentally confirmed that the efficacy of VApal could be enhanced using EOPO instead of HCO60. Miyake et al. [18] suggested that EOPO emulsions increase the efficacy of VApal; however, it has not been confirmed biochemically whether the efficacy is higher than that of widely used surfactants, such as HCO60. To the best of our knowledge, this is the first biochemical study to investigate the enhancement of VApal’s efficacy by EOPO. In this study, we constructed an experimental system using 3D corneal epithelial cells to compare the efficacy of VApal emulsified with EOPO to that of VApal emulsified with HCO60. Wound healing assays using 3D cells have been reported; Fallacara et al. [24] evaluated the efficacy of cross-linked HA through hematoxylin and eosin (HE) staining in surgically wounded 3D corneal epithelial cell, whereas Smith et al. [25] assessed wound healing by HE staining of 3D gingival and skin keratinocytes. However, both reports involved only qualitative examinations. Therefore, we developed an in vitro experimental system similar to the in vivo alkali burn model based on the report by Kato et.al [27] . In this system, we created a circular wound using 1 N NaOH treatment and evaluated the wound healing. Using this experimental system, we demonstrated that the efficacy of VApal emulsified with EOPO was higher than that of VApal emulsified with HCO60 in corneal wound healing. This experimental system may enable more extrapolated in vitro wound healing assays. The problem with this evaluation system is that it is difficult to consider the depth of the wound and the corneal tissue structure. Moreover, the wound healing time may differ depending on the depth of the wound. To improve the extrapolation of these evaluation systems, it may be necessary to measure and equalize the depth of wounds using confocal laser microscopy or tissue staining. In conclusion, our findings suggest that VApal may be more useful than other ingredients used in ophthalmic solutions for corneal wound healing. For the treatment of dry eye disease, anti-inflammatory medications such as cyclosporine and artificial tears are often used [26] . Elvan et.al [27] reported on a comparison between the combined use of cyclosporine A with artificial tears and the use of artificial tears alone in patients with dysfunctional tear syndrome. In this report, they demonstrated that the combination of an anti-inflammatory and artificial tears (0.05% cyclosporine and 0.3% hydroxypropyl methylcellulose) showed statistically significantly better break up time, corneal fluorescein staining than the use of artificial tears alone. Because VApal has been shown to have significant efficacy for corneal wound healing in comparison with artificial tear ingredients, VApal may be a more effective ingredient to use in combination with cyclosporine for patients with dry eye disease. Additionally, EOPO was found to enhance the efficacy of VApal for corneal wound healing. By utilizing EOPO, the cellular permeability and effectiveness of other lipophilic ingredients, such as vitamin E, may be enhanced. However, it should be noted that this research is limited to in vitro results. Generally, the retention of drugs on the ocular surface is critical for the efficacy of ophthalmic solutions. The wound healing assay employed in this study was conducted under conditions where cells and tissues were continuously exposed to the drug. These conditions may not accurately reflect the actual retention time of the drug in the eye. Therefore, the constant exposure of cells and tissues to the drug without accounting for retention warrant caution in interpreting the efficacy results obtained from this study. For future studies, it will be necessary to develop models that accurately replicate the actual conditions of ophthalmic solution usage as well as experimental designs that can appropriately assess ocular drug retention. For instance, using in vivo animal models or developing in vitro systems that replicate human tear fluid dynamics are promising strategies. By combining these experimental techniques, more extrapolatable and reliable data should be able to be obtained. Methods Materials VApal was purchased from DSM Nutrition Japan (Tokyo, Japan). HA was purchased from Bloomage Biotechnology (Tokyo, Japan). CMC and HPMC were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). EOPO and HCO60 were purchased from BASF Japan (Tokyo, Japan) and Nikko Chemicals (Tokyo, Japan), respectively. Preparation of solutions for the wound healing assay VApal (913,000 IU/ml), dissolved in dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), was diluted 500-fold with a low-serum medium consisting of Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F-12; Thermo Fisher Scientific, MA, USA) supplemented with 1% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Thermo Fisher Scientific, MA, USA). HA (3 mg/ml), CMC (5 mg/ml), and HPMC (3 mg/ml) were dissolved in water. These concentrations were matched with the following over-the-counter ophthalmic solutions: HA (Hyalein® ophthalmic Solution 0.3%; Santen, Osaka, Japan), CMC (Refresh plus®, Allergan, Marlow, UK), and HPMC (Genteal® Eye Drops, Novartis, Basel, Switzerland). Each solution was diluted 25-fold with a low-serum medium. VApal (50,000 IU/ml) emulsified with EOPO or HCO60 (0.2%) was diluted 25-fold with a low-serum medium. The concentration of EOPO or HCO60 was determined to be the minimum concentration necessary to emulsify VApal. Cell culture and wound healing assay SV40-immortalized human corneal epithelial cells (HCE-T: RCB2280, RIKEN BRC, Tokyo, Japan) were cultured in a growth medium consisting of DMEM/F-12 supplemented with 5% FBS, 5 mg/mL insulin (Thermo Fisher Scientific, MA, USA), 10 ng/mL epidermal growth factor (Thermo Fisher Scientific, MA, USA), 0.5% dimethyl sulfoxide, 100 units/mL penicillin, and 100 mg/mL streptomycin. The HCE-T cells were maintained at 37 ℃ in 5% CO 2 , and the growth medium was replaced every other day. The HCE-T cells were seeded into 24-well plates (Sumitomo Bakelite, Tokyo, Japan) at a density of 2×10 5 cells/well. After 24 h, the growth medium was removed, and the cells were washed with phosphate-buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). After washing, the cells were treated with solutions containing each ingredient (as indicated in the figure legends) in a low-serum medium. After 24 h, wounds were induced on the confluent cell layer using a 200 µL pipette tip. Thereafter, the cells were treated with the same solution. The wound healing process from 0 to 60 h was photographed using a confocal microscope (ORCA-Flash 4.0 V3, Nikon, Tokyo, Japan) with an attached device to maintain 37°C in 5% CO 2 (STX CO 2 O 2, TOKAI HIT, Fujinomiya, Japan) or a monitoring system (Provi CM20, OLYMPUS, Tokyo, Japan). The wound areas at 0, 12, 24, 36, 48, and 60 h were calculated using ImageJ software, and the wound closure rates were calculated using the following formula: Wound closure rates (%) = 100 × (1 - (wound area at each time/wound area at 0 h)). The ET50 was defined as the time required to reach 50% of the total wound healing area. It was calculated using a quadratic curve approximation derived from a plot of the wound closure rates. Use of 3D corneal epithelial cells Before inducing the wounds, 3D corneal epithelial cells (Japan Tissue Engineering, Gamagori, Japan) were pre-incubated according to the manufacturer’s protocol. The wound healing model using 3D corneas was based on method by Kimiko [28] . To create wounds, we first prepared a filter paper (No.5C, Toyo Roshi, Tokyo, Japan), which was punched into 3 mm diameter discs using a biopsy trepan (BP-30F, KAI MEDICAL, Tokyo, Japan). Next, we placed a circular filter paper soaked in 1 N NaOH (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) on the 3D corneal epithelial cells for 1 min. The filter paper was then removed, and the cells were washed with PBS. The 3D corneal epithelial cells were then treated with solutions (as indicated in the figure legends) in a low-serum medium for 60 h. Wound healing was observed at 0, 12, 24, 36, 48, and 60 h using a digital microscope (VHX-7000; Keyence, Tokyo, Japan). The wound area was calculated using ImageJ software, and the wound closure rates were calculated as described above. Cell viability assay Cell viability assays were performed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Tokyo, Japan) according to the manufacturer’s protocol. HCE-T cells were seeded into 96-well plates (Sumitomo Bakelite, Tokyo, Japan) at 2×10 4 cells/well, or 3D corneal epithelial cells were treated with solutions containing the ingredients indicated in the figure legends for 24, 48, and 72 h in a serum-free medium (DMEM/F-12 supplemented with 100 units/mL penicillin and 100 mg/mL streptomycin). Each solution was removed, and the cells were incubated with the CCK-8 reagent for 2 h. The absorbance was measured using a microplate reader (SYNERGY H1; Agilent Technologies, CA, USA). Statistical analysis The wound closure rates of the VApal group (n = 4) and other ingredients commonly used in over-the-counter ophthalmic solutions (n = 4) at each time point were compared using Dunnett’s test. The ET50 values of VApal and other ingredients were compared using Dunnett’s test. The wound closure rates in the VApal (n = 4) and EOPO + VApal (n = 4) groups at each time point and the ET50 of these groups were compared using Student’s t-test. The cell viabilities of the EOPO or HCO60 group (n = 3 or 4) and control (n = 3 or 4) groups at each time point were compared using Student’s t-test. The wound closure rates in the EOPO + VApal group (n = 6) and HCO60 + VApal (n = 6) at each time point and the ET50 of these groups were compared using Student’s t-test. Every experimental result is presented as mean ± standard deviation. The level of statistical significance was set at p < 0.05. All statistical analyses were performed using JMP ® 17.0.0 (JMP Statistical Discovery LLC, Tokyo, Japan). Declarations Acknowledgements We thank Dr. Hiroshi Toshida for the useful discussions. No funding was received for this research. Author contributions All authors contributed to the conception and design of the study. Data availability statements The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests All authors are current employees of Lion Corporation. 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A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315 , 820–825 (2007). Fallacara, A. et al. Novel Artificial Tears containing cross-linked hyaluronic acid: an in vitro re-epithelialization study. Molecules 22 , 2104 (2017). Smith, C. J. et al . Investigating wound healing characteristics of gingival and skin keratinocytes in organotypic cultures. J. Dent. 125 , 104251 (2022). Cedric. J. et al . A novel serine protease inhibitor as potential treatment for dry eye syndrome and ocular inflammation. Sci. Rep. 14 , 10 (1) (2020). Demiryay E. et al . Effects of topical cyclosporine a plus artificial tears versus artificial tears treatment on conjunctival goblet cell density in dysfunctional tear syndrome. Eye Contact Lens. 5 , 312-5 (2011). Kimiko, K. Evaluation of corneal wound healing by sodium hyaluronate ophthalmic solution in the 3D culture human corneal epithelial model [Japanese]. Poster presented at: The Pharmaceutical Society of Japan; May 26, 2018; Kanazawa. Additional Declarations Competing interest reported. All authors are current employees of Lion Corporation. All experiments were performed at the Well-Being Research Laboratories. Cite Share Download PDF Status: Published Journal Publication published 22 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Apr, 2025 Reviews received at journal 10 Apr, 2025 Reviews received at journal 07 Apr, 2025 Reviewers agreed at journal 06 Apr, 2025 Reviewers agreed at journal 06 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers invited by journal 17 Mar, 2025 Editor assigned by journal 17 Mar, 2025 Editor invited by journal 17 Mar, 2025 Submission checks completed at journal 14 Mar, 2025 First submitted to journal 13 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6223209","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":430577226,"identity":"bbc9d927-59e1-418f-9e2b-1c18ac277a32","order_by":0,"name":"Haruki Horiuchi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACAyBmbDCQqO9nZ2yAih0gSosF48xm0rQwVDBuOEysw8zZzz6TnFEgwWx8mLnxcQGDnRwD41n81lj2pJtJbjCQYDM7zNhsPIMh2ZiB4VwCfocdSGOTfGAgwQPU0ibNw3AgsYHhjAF+LeefgbVIGDcTreUG0BagwwwMmInX8ozZcoaBRIIEyC88BsnGbAT9cj6N8WbPn7oE/vb2h495Kuzk+CUIhBi6CQwMbBJnSNEBBvw9JGsZBaNgFIyC4Q0A1s8/A9lagLIAAAAASUVORK5CYII=","orcid":"","institution":"Lion Corporation","correspondingAuthor":true,"prefix":"","firstName":"Haruki","middleName":"","lastName":"Horiuchi","suffix":""},{"id":430577227,"identity":"3b8fcd31-fd06-4d4b-bec4-2beb7104fac8","order_by":1,"name":"Keisuke Watanabe","email":"","orcid":"","institution":"Lion Corporation","correspondingAuthor":false,"prefix":"","firstName":"Keisuke","middleName":"","lastName":"Watanabe","suffix":""},{"id":430577228,"identity":"831505b5-a0f4-423d-a1f6-e251c516d56b","order_by":2,"name":"Hiroshi Iijima","email":"","orcid":"","institution":"Lion Corporation","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Iijima","suffix":""},{"id":430577229,"identity":"b5b743de-46c2-4237-92d3-3905038d9ff3","order_by":3,"name":"Yoshiyuki Obayashi","email":"","orcid":"","institution":"Lion Corporation","correspondingAuthor":false,"prefix":"","firstName":"Yoshiyuki","middleName":"","lastName":"Obayashi","suffix":""}],"badges":[],"createdAt":"2025-03-14 02:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6223209/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6223209/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-03021-8","type":"published","date":"2025-05-22T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78850303,"identity":"db12f5ed-657b-4b10-9a6e-64029938e20d","added_by":"auto","created_at":"2025-03-19 18:58:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24576,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of VApal, HA, CMC, and HPMC in the wound healing assay using HCE-T cells. (a) Representative images of the wound healing process in each group. The wound area is delimited by yellow lines. (b) Wound closure rates of each group from 0 to 60 h. The wound area was monitored every 12 h. HCE-T cells were treated with VApal (2000 IU/ml), HA (0.12 mg/ml), CMC (0.2 mg/ml), and HPMC (0.12 mg/ml). The data represent mean + standard deviation (n = 4).\u003c/p\u003e\n\u003cp\u003eVApal, retinol palmitate; HA, hyaluronic acid; CMC, carboxymethyl cellulose; HPMC, hydroxypropyl methylcellulose; HCE-T, human corneal epithelial cells\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6223209/v1/5d86249373902b05ce8f5c14.jpg"},{"id":78850304,"identity":"f08de0a7-dc53-4d10-8c5a-3af9e12704c0","added_by":"auto","created_at":"2025-03-19 18:58:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37525,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of VApal emulsified with EOPO in the wound healing assay and the cytotoxicity of EOPO and HCO60 in HCE-T cells. (a, d) Cytotoxicity of EOPO and HCO60 in HCE-T cells. HCE-T cells were exposed to EOPO (0.008%) (a) or HCO60 (0.008%) (d) for 24, 48, and 72 h. The cell viability was assessed using CCK-8, and these data were normalized to control (PBS-treated) values (100%). Each bar shows mean ± SD. Student’s t-test; **p \u0026lt; 0.01. (b) Representative images of the wound healing process in the VApal and EOPO+VApal groups. The wound area is delimited by yellow lines. (c) Wound closure rates of each group from 0 to 60 h. The wound area was monitored every 12 h. HCE-T cells were treated with VApal (2000 IU/ml) and EOPO+VApal (2000 IU/ml). The wound closure rates in the VApal and EOPO+VApal groups at each point in time were compared using Student’s t-test (*p \u0026lt; 0.05, **p \u0026lt; 0.01). All the data are expressed as the mean ± SD (n = 4).\u003c/p\u003e\n\u003cp\u003eVApal, retinol palmitate; EOPO, polyoxyethylene-polypropylene [EO\u003csub\u003e100\u003c/sub\u003ePO\u003csub\u003e70\u003c/sub\u003eEO\u003csub\u003e100\u003c/sub\u003e]; HCO60, polyoxyethylene-hydrogenated castor oil 60; HCE-T, human corneal epithelial cells; SD, standard deviation; PBS, phosphate-buffered saline; CCK-8, Cell Counting Kit-8\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6223209/v1/5216a78e36262e17491de4b8.jpg"},{"id":78850406,"identity":"a330918f-3894-4920-9858-431ac3e9d4a9","added_by":"auto","created_at":"2025-03-19 19:06:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13005,"visible":true,"origin":"","legend":"\u003cp\u003eToxicity of HCO60 on the 3D corneal epithelial cells. The 3D corneal epithelial cells were exposed to HCO60 (0.008%) for 24, 48, and 72 h. Cell viability was assessed using CCK-8, and the data were normalized to control (PBS-treated) values (100%). All the data are expressed as the mean + standard deviation (n = 3).\u003c/p\u003e\n\u003cp\u003eHCO60, polyoxyethylene-hydrogenated castor oil 60; PBS, phosphate-buffered saline; CCK-8, Cell Counting Kit-8\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6223209/v1/57745dda9ac476a9158bee20.jpg"},{"id":78850305,"identity":"42006bdb-bfe8-46e1-85b2-67d2cbc59b53","added_by":"auto","created_at":"2025-03-19 18:58:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25289,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of VApal emulsified with EOPO and VApal emulsified with HCO60 in the 3D corneal epithelial cells. (a) Representative images of the wound healing process in each group. The wound area is delimited by yellow lines. (b) Wound closure rates of each group from 0 to 60 h. The wound area was monitored every 12 h. 3D epithelial cells were treated with EOPO+VApal (2000 IU/ml) and HCO60+VApal (2000 IU/ml). The wound closure rates of the EOPO+VApal and HCO60+VApal groups at each point in time were compared using the Student’s t-test (**p \u0026lt; 0.01). The data represent mean + standard deviation (n = 6).\u003c/p\u003e\n\u003cp\u003eVApal, retinol palmitate; EOPO, polyoxyethylene-polypropylene [EO\u003csub\u003e100\u003c/sub\u003ePO\u003csub\u003e70\u003c/sub\u003eEO\u003csub\u003e100\u003c/sub\u003e]; HCO60, polyoxyethylene-hydrogenated castor oil 60\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6223209/v1/84c753b0e3b24bd0796afde8.jpg"},{"id":83460678,"identity":"89f36274-0d81-42cb-b05c-84efab5a9aea","added_by":"auto","created_at":"2025-05-26 16:13:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":904934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6223209/v1/d4fdd882-80d5-404a-8c65-a27f6f7947f0.pdf"}],"financialInterests":"Competing interest reported. All authors are current employees of Lion Corporation. All experiments were performed at the Well-Being Research Laboratories.","formattedTitle":"Polyoxyethylene and Polypropylene Emulsification Enhances Retinol Palmitate Efficacy in Corneal Wound Healing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cornea, which functions in visual information intake, tear fluid retention, and biological defense\u003csup\u003e[1]\u003c/sup\u003e, comprises the epithelium, Bowman\u0026rsquo;s membrane, stroma, Descemet\u0026rsquo;s membrane, and endothelium. The corneal epithelium is particularly vulnerable to external stimuli and can easily be injured\u003csup\u003e[2]\u003c/sup\u003e. Corneal epithelial wounds are commonly caused by contact lens wear and increased blink friction due to tear film destabilization\u003csup\u003e[3]\u003c/sup\u003e. These wounds are typically repaired through a self-healing process consisting of three phases: cell extension and migration (phase 1), division and proliferation (phase 2), and differentiation (phase 3)\u003csup\u003e[4]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRetinol palmitate (VApal), a lipophilic derivative of vitamin A, is crucial for maintaining homeostasis in the eye, skin, and mucous membranes\u003csup\u003e[5, 6]\u003c/sup\u003e. VApal is used to treat conditions such as night blindness and keratoderma and is formulated as an active ingredient in over-the-counter medicines in several countries, including ophthalmic solutions for eye fatigue, blurred vision and external preparations for the skin.\u003c/p\u003e \u003cp\u003eThe biologically active form of VApal, retinoic acid, regulates various functions, such as cell growth, differentiation, and organogenesis\u003csup\u003e[7]\u003c/sup\u003e, by binding to retinoic acid receptors, which are nuclear receptors. In the corneal epithelium, VApal increases hyaluronic acid (HA) production\u003csup\u003e[8]\u003c/sup\u003e. Moreover, retinoic acid has been reported to increase mucin expression, which is involved in tear fluid retention\u003csup\u003e[9]\u003c/sup\u003e, and to upregulate lysyl oxidase-like 4, which promotes cell migration\u003csup\u003e[10]\u003c/sup\u003e. Additionally, Toshida et al.\u003csup\u003e[11]\u003c/sup\u003e demonstrated the topical application of VApal to repair corneal wounds in a mouse model.\u003c/p\u003e \u003cp\u003eIn addition to VApal, several other ingredients used in ophthalmic solutions have been reported to promote corneal wound healing. For example, HA was found to promote the migration of corneal epithelial cells by binding to CD44\u003csup\u003e[12]\u003c/sup\u003e and accelerating corneal wound healing in a rabbit corneal alkali burn model\u003csup\u003e[13]\u003c/sup\u003e. Similarly, carboxymethyl cellulose (CMC) promotes epithelial cell migration by interacting with matrix proteins and has demonstrated efficacy in accelerating wound healing in human corneal epithelial cells\u003csup\u003e[14]\u003c/sup\u003e. Hydroxypropyl methylcellulose (HPMC), which is often used as a thickening agent, was found to accelerate corneal wound healing in a rat corneal detachment model\u003csup\u003e[15]\u003c/sup\u003e. Cyclosporine suppresses inflammation and improves ocular surface conditions. In a clinical phase II study involving patients with moderate-to-severe dry eye, cyclosporine significantly reduced the degree of corneal fluorescein staining compared to the vehicle\u003csup\u003e[16]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the availability of ingredients that accelerate corneal wound healing, previous studies comparing the efficacy of VApal and other ingredients are limited. In a study that compared the efficacy of VApal and cyclosporine in corneal wound healing, Reimondez-Troiti\u0026ntilde;o et al.\u003csup\u003e[17]\u003c/sup\u003e reported that the efficacy of retinoic acid in corneal wound healing was equal to that of cyclosporine in a mouse injured by photorefractive keratectomy surgery. Their study compared the efficacy of ingredients with different mechanisms of action, including VApal (cell migration and water retention) and cyclosporine (anti-inflammatory). We did not find any previous studies that compared the efficacy of VApal in wound healing with ingredients that have mechanisms of action similar to those of VApal (e.g., HA). Therefore, when compared to HA, CMC, and HPMC, which have similar mechanisms of action as VApal, the relative value of VApal in corneal wound healing remains unclear.\u003c/p\u003e \u003cp\u003eThe formulation of VApal in water-based ophthalmic solutions requires surfactants. Polyoxyethylene-hydrogenated castor oil 60 (HCO60) is typically used to emulsify VApal. Previously, Miyake et al.\u003csup\u003e[18]\u003c/sup\u003e investigated a method for the formulation of VApal using polyoxyethylene-polypropylene (EO\u003csub\u003e100\u003c/sub\u003ePO\u003csub\u003e70\u003c/sub\u003eEO\u003csub\u003e100\u003c/sub\u003e [EOPO]) \u003csup\u003e[18]\u003c/sup\u003e. They investigated the behavior of fluorescence-labeled VApal emulsified with EOPO in relation to cell-membrane interactions, focusing on surface chemistry\u003csup\u003e[18]\u003c/sup\u003e. Their study demonstrated that VApal emulsified with EOPO exhibited greater localization around the cell nucleus than did HCO60. Similarly, Toshida et al.\u003csup\u003e[19]\u003c/sup\u003e reported the effectiveness of VApal emulsified with EOPO in patients with dry eyes. In their study, both the objective signs and subjective symptoms of dry eye significantly improved with VApal emulsified with EOPO compared with placebo. However, regarding the efficacy of VApal in corneal wound healing, it remains unclear whether EOPO is a better surfactant than commonly used surfactants (e.g., HCO60).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to determine 1) the efficacy of VApal in corneal wound healing compared to ingredients with similar mechanisms of action (i.e., HA, CMC, and HPMC), and 2) whether the efficacy of VApal emulsified with EOPO was higher than that of HCO60.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of VApal in human corneal wound healing compared to other ingredients\u003c/h2\u003e \u003cp\u003eTo investigate the efficacy of VApal in corneal wound healing, we conducted a wound healing assay using SV40-immortalized human corneal epithelial cells (HCE-T cells). We compared the efficacy of VApal with that of HA, CMC, and HPMC, which are commonly used as active ingredients in ophthalmic solutions for corneal wounds. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea shows representative images of the wound healing process in the control, VApal, HA, CMC, and HPMC groups after 24 h. The wound area was noticeably smaller in the VApal group than in the other groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb shows the wound closure rates in each group from 0 to 60 h. The VApal group had higher wound closure rates than the HA and CMC groups at all time points. Compared to the HPMC group, the VApal group had significantly higher wound closure rates at all time points, except at 36 h (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To quantify the efficacy of each treatment, we defined the time required to reach 50% of the total wound healing area (ET50). The ET50 of the VApal group was significantly shorter than that of the HA, CMC, and HPMC groups (p\u0026thinsp;=\u0026thinsp;0.0002, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, and p\u0026thinsp;=\u0026thinsp;0.0005, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical analysis of the wound closure rates at each time.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eDunnett's test (vs. VApal) P-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60 h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0053\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0418\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eVApal, retinol palmitate; HA, hyaluronic acid; CMC, carboxymethyl cellulose; HPMC, hydroxypropyl methylcellulose\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eET50 of the control, VApal, HA, CMC, and HPMC groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eET50 (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDunnett's test (vs. VApal) P-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e32.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVApal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e17.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eET50, time required to reach 50% of the total wound healing area; VApal, retinol palmitate; HA, hyaluronic acid; CMC, carboxymethyl cellulose; HPMC, hydroxypropyl methylcellulose\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEfficacy of VApal emulsified with EOPO in human corneal wound healing\u003c/h3\u003e\n\u003cp\u003eWe investigated the efficacy of VApal emulsified with surfactants in corneal wound healing. Prior to this, we assessed the toxicity of EOPO on corneal epithelial cells using a cell survival assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The exposure of corneal epithelial cells to EOPO for up to 72 h did not affect cell viability, indicating that EOPO was not toxic to corneal epithelial cells. Next, we examined whether EOPO could increase the efficacy of VApal and compared the efficacy of human corneal wound healing between the VApal and EOPO\u0026thinsp;+\u0026thinsp;VApal groups. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb shows representative images of the wound healing process in the VApal and EOPO\u0026thinsp;+\u0026thinsp;VApal groups at 24 h. The wound area in the EOPO\u0026thinsp;+\u0026thinsp;VApal group was smaller than that in the VApal group. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec shows the wound closure rates in each group from 0 to 60 h. At 24, 36, and 48 h, the wound closure rates in the EOPO\u0026thinsp;+\u0026thinsp;VApal group were significantly higher than those in the VApal group (p\u0026thinsp;=\u0026thinsp;0.0002, p\u0026thinsp;=\u0026thinsp;0.0186, and p\u0026thinsp;=\u0026thinsp;0.0183, respectively). Moreover, the ET50 of the EOPO\u0026thinsp;+\u0026thinsp;VApal group was significantly shorter than that of the VApal group (p\u0026thinsp;=\u0026thinsp;0.0013) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eET50 of the VApal and EOPO\u0026thinsp;+\u0026thinsp;VApal groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eET50 (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStudent\u0026rsquo;s t-test P-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVApal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEOPO\u0026thinsp;+\u0026thinsp;VApal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eET50, time required to reach 50% of the total wound healing area; VApal, retinol palmitate; EOPO, polyoxyethylene-polypropylene [EO\u003csub\u003e100\u003c/sub\u003ePO\u003csub\u003e70\u003c/sub\u003eEO\u003csub\u003e100\u003c/sub\u003e]\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFinally, we compared the efficacy of VApal emulsified with EOPO to that of VApal emulsified with HCO60 in promoting corneal wound healing. HCO60 is a surfactant commonly used as an emulsifier for VApal in ophthalmic over-the-counter solutions. Before conducting the wound healing assay, we assessed the toxicity of HCO60 in corneal epithelial cells. The cell viability rates in the HCO60 group were significantly lower than those in the control group at 48 and 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) (p\u0026thinsp;=\u0026thinsp;0.0004 and p\u0026thinsp;=\u0026thinsp;0.0004, respectively). Therefore, we concluded that a monolayer culture of corneal epithelial cells may not be suitable for investigating the efficacy of VApal emulsified with HCO60.\u003c/p\u003e\n\u003ch3\u003eCytotoxicity of HCO60 in a reconstructed human corneal epithelium model\u003c/h3\u003e\n\u003cp\u003eOwing to the observed toxicity of HCO60 in the monolayer cultures of corneal epithelial cells, we were unable to demonstrate a difference in efficacy between VApal emulsified with EOPO and VApal emulsified with HCO60. Consequently, we opted to use a reconstructed human corneal epithelium model (3D corneal epithelial cells) that is more similar to the human corneal epithelial cells\u003csup\u003e[20]\u003c/sup\u003e. To evaluate the cytotoxicity of HCO60 in the 3D corneal epithelial cells, a cell survival assay was performed. The exposure of corneal epithelial cells to HCO60 for up to 72 h did not affect cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, HCO60 was less toxic to 3D corneal epithelial cells than to monolayer cultures of corneal epithelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComparison of the efficacy of VApal emulsified with EOPO vs. VApal emulsified with HCO60 in corneal wound healing in 3D corneal epithelial cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the difference in efficacy between VApal emulsified with EOPO and VApal emulsified with HCO60 for corneal wound healing, we performed a wound healing assay using 3D corneal epithelial cells. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows representative images of the wound healing process in the EOPO\u0026thinsp;+\u0026thinsp;VApal and HCO60\u0026thinsp;+\u0026thinsp;VApal groups at 36 h. The wound area was smaller in the EOPO\u0026thinsp;+\u0026thinsp;VApal group than that in the HCO60\u0026thinsp;+\u0026thinsp;VApal group. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb shows the wound closure rates in each group from 0 to 60 h. After 36 h, the wound closure rates in the EOPO\u0026thinsp;+\u0026thinsp;VApal group were significantly higher than those in the HCO60\u0026thinsp;+\u0026thinsp;VApal group (p\u0026thinsp;=\u0026thinsp;0.0007, p\u0026thinsp;=\u0026thinsp;0.0004, and p\u0026thinsp;=\u0026thinsp;0.0004, respectively). Comparing the EOPO\u0026thinsp;+\u0026thinsp;VApal and HCO60\u0026thinsp;+\u0026thinsp;VApal groups, the ET50 of the EOPO\u0026thinsp;+\u0026thinsp;VApal group was significantly shorter than that of the HCO60\u0026thinsp;+\u0026thinsp;VApal group (p\u0026thinsp;=\u0026thinsp;0.0005) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eET50 of the HCO60\u0026thinsp;+\u0026thinsp;VApal and EOPO\u0026thinsp;+\u0026thinsp;VApal groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eET50 (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStudent\u0026rsquo;s t-test P-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCO60\u0026thinsp;+\u0026thinsp;VApal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e43.31\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEOPO\u0026thinsp;+\u0026thinsp;VApal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e34.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eET50, time required to reach 50% of the total wound healing area; VApal, retinol palmitate; EOPO, polyoxyethylene-polypropylene (EO\u003csub\u003e100\u003c/sub\u003ePO\u003csub\u003e70\u003c/sub\u003eEO\u003csub\u003e100\u003c/sub\u003e); HCO60, polyoxyethylene-hydrogenated castor oil 60\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we assessed the efficacy of VApal in corneal wound healing compared to other ingredients with a mechanism of action similar to that of VApal. The results indicated that the efficacy of VApal in corneal wound healing was higher than that of other ingredients, such as HA. These results may be attributed to the differences in the mechanisms of action of VApal and the other ingredients.\u003c/p\u003e \u003cp\u003ePrevious reports have shown that HA and CMC promote cell migration through their binding to extracellular substrates\u003csup\u003e[12, 14]\u003c/sup\u003e. HPMC is used in ophthalmic solutions because of its water retention properties. In contrast, VApal is metabolized into retinoic acid within the corneal epithelial cells, which regulates the expression of target genes mediated by retinoic acid receptors. VApal has also been reported to enhance HA production by increasing the expression of hyaluronic acid synthetase 3\u003csup\u003e[21]\u003c/sup\u003e. Retinoic acid has also been reported to increase the expression of lysyl oxidase-like protein 4, which is involved in cell migration\u003csup\u003e[10]\u003c/sup\u003e. In a study using a mouse dry eye model lacking aquaporin 5 in the corneal epithelium, retinoic acid enhanced cell proliferation and reduced apoptosis by inducing the expression of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and Bcl-2\u003csup\u003e[22]\u003c/sup\u003e. As described above, ingredients such as HA promote corneal wound healing by enhancing cell migration, whereas VApal promotes cell proliferation and inhibits apoptosis. Considering these differences in the mechanisms of action, it is likely that the efficacy of VApal in corneal wound healing is higher than that of the other ingredients. However, the wound healing assay utilized in this study was an evaluation model in which there was no extracellular matrix. Therefore, it is possible that the efficacies of HA and CMC were lower because extracellular substrates affected their efficiency. In addition, because the cells are always exposed to the medium, it may be difficult to determine the efficacy of wound healing mediated by water retention. In the future, it will be necessary to evaluate the efficacy in in vivo studies to accurately understand the drug effects.\u003c/p\u003e \u003cp\u003eToshida et al.\u003csup\u003e[19]\u003c/sup\u003e demonstrated the effectiveness of VApal emulsified with EOPO in a clinical study; however, it remains unclear whether the effectiveness of VApal emulsified with EOPO is higher than that of VApal emulsified with HCO60. We showed that the ET50 of VApal emulsified with EOPO was shorter than that of VApal alone. This suggests that EOPO can enhance the efficacy of VApal in corneal wound healing. We hypothesize that this enhancement may be attributed to differences in the transition mechanism of VApal at the cell membrane. Generally, VApal is transported into cells via transporters in the plasma membrane. Kawaguchi et al.\u003csup\u003e[23]\u003c/sup\u003e reported that VApal binds to retinol-binding proteins (RBPs) in the extracellular space, and VApal-RBP complexes are taken up into the cell by STRA6, a plasma membrane receptor that specifically recognizes RBPs. Miyake et al.\u003csup\u003e[18]\u003c/sup\u003e reported that VApal emulsified with EOPO may be transported into cells by endocytosis. They examined the interactions between VApal emulsified with EOPO and plasma membrane-mimicking giant unilamellar vesicles (GUVs). They demonstrated that VApal emulsified with EOPO induced morphological changes in GUVs and was taken up by GUVs in a manner similar to endocytosis. This implies that VApal emulsified with EOPO is transported not only by the usual transport pathway mediated by STRA6 but also by endocytosis. Furthermore, we assessed whether the efficacy of VApal emulsified with EOPO was higher than that of VApal emulsified with HCO60. We experimentally confirmed that the efficacy of VApal could be enhanced using EOPO instead of HCO60. Miyake et al.\u003csup\u003e[18]\u003c/sup\u003e suggested that EOPO emulsions increase the efficacy of VApal; however, it has not been confirmed biochemically whether the efficacy is higher than that of widely used surfactants, such as HCO60. To the best of our knowledge, this is the first biochemical study to investigate the enhancement of VApal\u0026rsquo;s efficacy by EOPO.\u003c/p\u003e \u003cp\u003eIn this study, we constructed an experimental system using 3D corneal epithelial cells to compare the efficacy of VApal emulsified with EOPO to that of VApal emulsified with HCO60. Wound healing assays using 3D cells have been reported; Fallacara et al.\u003csup\u003e[24]\u003c/sup\u003e evaluated the efficacy of cross-linked HA through hematoxylin and eosin (HE) staining in surgically wounded 3D corneal epithelial cell, whereas Smith et al.\u003csup\u003e[25]\u003c/sup\u003e assessed wound healing by HE staining of 3D gingival and skin keratinocytes. However, both reports involved only qualitative examinations. Therefore, we developed an in vitro experimental system similar to the in vivo alkali burn model based on the report by Kato et.al \u003csup\u003e[27]\u003c/sup\u003e. In this system, we created a circular wound using 1 N NaOH treatment and evaluated the wound healing. Using this experimental system, we demonstrated that the efficacy of VApal emulsified with EOPO was higher than that of VApal emulsified with HCO60 in corneal wound healing. This experimental system may enable more extrapolated in vitro wound healing assays. The problem with this evaluation system is that it is difficult to consider the depth of the wound and the corneal tissue structure. Moreover, the wound healing time may differ depending on the depth of the wound. To improve the extrapolation of these evaluation systems, it may be necessary to measure and equalize the depth of wounds using confocal laser microscopy or tissue staining.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings suggest that VApal may be more useful than other ingredients used in ophthalmic solutions for corneal wound healing. For the treatment of dry eye disease, anti-inflammatory medications such as cyclosporine and artificial tears are often used \u003csup\u003e[26]\u003c/sup\u003e. Elvan et.al \u003csup\u003e[27]\u003c/sup\u003e reported on a comparison between the combined use of cyclosporine A with artificial tears and the use of artificial tears alone in patients with dysfunctional tear syndrome. In this report, they demonstrated that the combination of an anti-inflammatory and artificial tears (0.05% cyclosporine and 0.3% hydroxypropyl methylcellulose) showed statistically significantly better break up time, corneal fluorescein staining than the use of artificial tears alone. Because VApal has been shown to have significant efficacy for corneal wound healing in comparison with artificial tear ingredients, VApal may be a more effective ingredient to use in combination with cyclosporine for patients with dry eye disease. Additionally, EOPO was found to enhance the efficacy of VApal for corneal wound healing. By utilizing EOPO, the cellular permeability and effectiveness of other lipophilic ingredients, such as vitamin E, may be enhanced. However, it should be noted that this research is limited to in vitro results. Generally, the retention of drugs on the ocular surface is critical for the efficacy of ophthalmic solutions. The wound healing assay employed in this study was conducted under conditions where cells and tissues were continuously exposed to the drug. These conditions may not accurately reflect the actual retention time of the drug in the eye. Therefore, the constant exposure of cells and tissues to the drug without accounting for retention warrant caution in interpreting the efficacy results obtained from this study. For future studies, it will be necessary to develop models that accurately replicate the actual conditions of ophthalmic solution usage as well as experimental designs that can appropriately assess ocular drug retention. For instance, using in vivo animal models or developing in vitro systems that replicate human tear fluid dynamics are promising strategies. By combining these experimental techniques, more extrapolatable and reliable data should be able to be obtained.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eVApal was purchased from DSM Nutrition Japan (Tokyo, Japan). HA was purchased from Bloomage Biotechnology (Tokyo, Japan). CMC and HPMC were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). EOPO and HCO60 were purchased from BASF Japan (Tokyo, Japan) and Nikko Chemicals (Tokyo, Japan), respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of solutions for the wound healing assay\u003c/h3\u003e\n\u003cp\u003eVApal (913,000 IU/ml), dissolved in dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), was diluted 500-fold with a low-serum medium consisting of Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/Ham\u0026rsquo;s F12 (DMEM/F-12; Thermo Fisher Scientific, MA, USA) supplemented with 1% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Thermo Fisher Scientific, MA, USA).\u003c/p\u003e \u003cp\u003eHA (3 mg/ml), CMC (5 mg/ml), and HPMC (3 mg/ml) were dissolved in water. These concentrations were matched with the following over-the-counter ophthalmic solutions: HA (Hyalein\u0026reg; ophthalmic Solution 0.3%; Santen, Osaka, Japan), CMC (Refresh plus\u0026reg;, Allergan, Marlow, UK), and HPMC (Genteal\u0026reg; Eye Drops, Novartis, Basel, Switzerland). Each solution was diluted 25-fold with a low-serum medium.\u003c/p\u003e \u003cp\u003eVApal (50,000 IU/ml) emulsified with EOPO or HCO60 (0.2%) was diluted 25-fold with a low-serum medium. The concentration of EOPO or HCO60 was determined to be the minimum concentration necessary to emulsify VApal.\u003c/p\u003e\n\u003ch3\u003eCell culture and wound healing assay\u003c/h3\u003e\n\u003cp\u003eSV40-immortalized human corneal epithelial cells (HCE-T: RCB2280, RIKEN BRC, Tokyo, Japan) were cultured in a growth medium consisting of DMEM/F-12 supplemented with 5% FBS, 5 mg/mL insulin (Thermo Fisher Scientific, MA, USA), 10 ng/mL epidermal growth factor (Thermo Fisher Scientific, MA, USA), 0.5% dimethyl sulfoxide, 100 units/mL penicillin, and 100 mg/mL streptomycin. The HCE-T cells were maintained at 37 ℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e, and the growth medium was replaced every other day. The HCE-T cells were seeded into 24-well plates (Sumitomo Bakelite, Tokyo, Japan) at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well. After 24 h, the growth medium was removed, and the cells were washed with phosphate-buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). After washing, the cells were treated with solutions containing each ingredient (as indicated in the figure legends) in a low-serum medium. After 24 h, wounds were induced on the confluent cell layer using a 200 \u0026micro;L pipette tip. Thereafter, the cells were treated with the same solution. The wound healing process from 0 to 60 h was photographed using a confocal microscope (ORCA-Flash 4.0 V3, Nikon, Tokyo, Japan) with an attached device to maintain 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e (STX CO\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e TOKAI HIT, Fujinomiya, Japan) or a monitoring system (Provi CM20, OLYMPUS, Tokyo, Japan). The wound areas at 0, 12, 24, 36, 48, and 60 h were calculated using ImageJ software, and the wound closure rates were calculated using the following formula:\u003c/p\u003e \u003cp\u003eWound closure rates (%)\u0026thinsp;=\u0026thinsp;100 \u0026times; (1 - (wound area at each time/wound area at 0 h)).\u003c/p\u003e \u003cp\u003eThe ET50 was defined as the time required to reach 50% of the total wound healing area. It was calculated using a quadratic curve approximation derived from a plot of the wound closure rates.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eUse of 3D corneal epithelial cells\u003c/h2\u003e \u003cp\u003eBefore inducing the wounds, 3D corneal epithelial cells (Japan Tissue Engineering, Gamagori, Japan) were pre-incubated according to the manufacturer\u0026rsquo;s protocol. The wound healing model using 3D corneas was based on method by Kimiko\u003csup\u003e[28]\u003c/sup\u003e. To create wounds, we first prepared a filter paper (No.5C, Toyo Roshi, Tokyo, Japan), which was punched into 3 mm diameter discs using a biopsy trepan (BP-30F, KAI MEDICAL, Tokyo, Japan). Next, we placed a circular filter paper soaked in 1 N NaOH (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) on the 3D corneal epithelial cells for 1 min. The filter paper was then removed, and the cells were washed with PBS. The 3D corneal epithelial cells were then treated with solutions (as indicated in the figure legends) in a low-serum medium for 60 h. Wound healing was observed at 0, 12, 24, 36, 48, and 60 h using a digital microscope (VHX-7000; Keyence, Tokyo, Japan). The wound area was calculated using ImageJ software, and the wound closure rates were calculated as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eCell viability assays were performed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Tokyo, Japan) according to the manufacturer\u0026rsquo;s protocol. HCE-T cells were seeded into 96-well plates (Sumitomo Bakelite, Tokyo, Japan) at 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well, or 3D corneal epithelial cells were treated with solutions containing the ingredients indicated in the figure legends for 24, 48, and 72 h in a serum-free medium (DMEM/F-12 supplemented with 100 units/mL penicillin and 100 mg/mL streptomycin). Each solution was removed, and the cells were incubated with the CCK-8 reagent for 2 h. The absorbance was measured using a microplate reader (SYNERGY H1; Agilent Technologies, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe wound closure rates of the VApal group (n\u0026thinsp;=\u0026thinsp;4) and other ingredients commonly used in over-the-counter ophthalmic solutions (n\u0026thinsp;=\u0026thinsp;4) at each time point were compared using Dunnett\u0026rsquo;s test. The ET50 values of VApal and other ingredients were compared using Dunnett\u0026rsquo;s test. The wound closure rates in the VApal (n\u0026thinsp;=\u0026thinsp;4) and EOPO\u0026thinsp;+\u0026thinsp;VApal (n\u0026thinsp;=\u0026thinsp;4) groups at each time point and the ET50 of these groups were compared using Student\u0026rsquo;s t-test. The cell viabilities of the EOPO or HCO60 group (n\u0026thinsp;=\u0026thinsp;3 or 4) and control (n\u0026thinsp;=\u0026thinsp;3 or 4) groups at each time point were compared using Student\u0026rsquo;s t-test. The wound closure rates in the EOPO\u0026thinsp;+\u0026thinsp;VApal group (n\u0026thinsp;=\u0026thinsp;6) and HCO60\u0026thinsp;+\u0026thinsp;VApal (n\u0026thinsp;=\u0026thinsp;6) at each time point and the ET50 of these groups were compared using Student\u0026rsquo;s t-test. Every experimental result is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The level of statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were performed using JMP \u0026reg; 17.0.0 (JMP Statistical Discovery LLC, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Hiroshi Toshida for the useful discussions.\u003c/p\u003e\n\u003cp\u003eNo funding was received for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conception and design of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors are current employees of Lion Corporation. All experiments were performed at the Well-Being Research Laboratories.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKaur, S. \u003cem\u003eet al\u003c/em\u003e. Molecular nature of ocular surface barrier function, diseases that affect it, and its relevance for ocular drug delivery. \u003cem\u003eOcul. Surf.\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 3\u0026ndash;13 (2023).\u003c/li\u003e\n\u003cli\u003eLi, L. \u003cem\u003eet al\u003c/em\u003e. Trans-corneal drug delivery strategies in the treatment of ocular diseases. \u003cem\u003eAdv. Drug Deliv. Rev.\u003c/em\u003e\u003cstrong\u003e198\u003c/strong\u003e, 114868 (2023).\u003c/li\u003e\n\u003cli\u003eCraig, J. P. \u003cem\u003eet al.\u003c/em\u003e TFOS DEWS II report executive summary. \u003cem\u003eOcul. Surf.\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 802\u0026ndash;812 (2017).\u003c/li\u003e\n\u003cli\u003eLoureiro, R. R. \u0026amp; Gomes, J. \u0026Aacute;. P. 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Poster presented at: The Pharmaceutical Society of Japan; May 26, 2018; Kanazawa.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"wound healing, cornea, retinol palmitate, vitamin A, surfactant","lastPublishedDoi":"10.21203/rs.3.rs-6223209/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6223209/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRetinol palmitate (VApal), an active ingredient in ophthalmic solution, has been reported to repair corneal injuries. Additionally, it has been suggested that the efficacy of VApal is enhanced by a specific surfactant, polyoxyethylene-polypropylene [EO\u003csub\u003e100\u003c/sub\u003ePO\u003csub\u003e70\u003c/sub\u003eEO\u003csub\u003e100\u003c/sub\u003e (EOPO)]. We aimed to determine the efficacy of VApal in corneal wound healing in comparison to that of hyaluronic acid (HA), carboxymethyl cellulose (CMC) and hydroxypropyl methylcellulose (HPMC) used in over-the-counter ophthalmic solutions and whether the efficacy of VApal could be enhanced by EOPO compared to the widely used surfactant, polyoxyethylene hydrogenated castor oil 60 (HCO60). To evaluate the efficacy of VApal or VApal emulsified with a surfactant, we performed a wound healing assay using corneal epithelial cells in monolayer (n\u0026thinsp;=\u0026thinsp;4) or 3D culture (n\u0026thinsp;=\u0026thinsp;6). Wound closure rates were calculated each time, and the efficacy was compared using the time to reach a 50% wound closure rate (ET50). The ET50 values of VApal, HA, CMC and HPMC were approximately 17.31 h, 26.99 h, 28.98 h and 26.01 h respectively. The ET50 values of VApal emulsified with EOPO or HCO60 were 34.49 h and 43.31 h, respectively. In conclusion, VApal is more beneficial than other ingredients for corneal wound healing. Additionally, the efficacy of VApal can be enhanced using EOPO instead of HCO60.\u003c/p\u003e","manuscriptTitle":"Polyoxyethylene and Polypropylene Emulsification Enhances Retinol Palmitate Efficacy in Corneal Wound Healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-19 18:58:51","doi":"10.21203/rs.3.rs-6223209/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-11T05:29:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-10T20:57:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T12:21:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54028190055262921090633553031570192334","date":"2025-04-06T08:27:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317952633222491064956395701320886241270","date":"2025-04-06T07:34:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333541123043676579417902557749233584132","date":"2025-04-05T21:38:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-17T15:52:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-17T15:48:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-17T15:01:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-14T09:31:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-14T02:24:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c54da9e4-3559-4818-9e47-b91e4922d6c1","owner":[],"postedDate":"March 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45859307,"name":"Biological sciences/Cell biology/Cell migration"},{"id":45859308,"name":"Biological sciences/Drug discovery/Pharmaceutics"}],"tags":[],"updatedAt":"2025-05-26T16:10:28+00:00","versionOfRecord":{"articleIdentity":"rs-6223209","link":"https://doi.org/10.1038/s41598-025-03021-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-22 15:57:18","publishedOnDateReadable":"May 22nd, 2025"},"versionCreatedAt":"2025-03-19 18:58:51","video":"","vorDoi":"10.1038/s41598-025-03021-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-03021-8","workflowStages":[]},"version":"v1","identity":"rs-6223209","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6223209","identity":"rs-6223209","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}