November 2023
Volume 12, Issue 11
Open Access
Cornea & External Disease  |   November 2023
A Novel Riboflavin Formulation for Corneal Delivery Without Damaging Epithelial Cells
Author Affiliations & Notes
  • Yutaka Yamagata
    Analytical Research Laboratory, MEDRx Co. Ltd., Kagawa, Japan
  • Takeshi Ide
    Tokyo Vision Eye Clinic Asagaya, Tokyo, Japan
  • Correspondence: Yutaka Yamagata, Analytical Research Laboratory, MEDRx Co. Ltd., 431-7 Nishiyama, Higashikagawa-city, Kagawa, Japan. e-mail: y-yamagata@medrx.co.jp 
Translational Vision Science & Technology November 2023, Vol.12, 10. doi:https://doi.org/10.1167/tvst.12.11.10
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      Yutaka Yamagata, Takeshi Ide; A Novel Riboflavin Formulation for Corneal Delivery Without Damaging Epithelial Cells. Trans. Vis. Sci. Tech. 2023;12(11):10. https://doi.org/10.1167/tvst.12.11.10.

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Abstract

Purpose: This study aimed to evaluate the trans-epithelial permeability enhancement and cell damage caused by a novel riboflavin composition for corneal delivery.

Methods: We developed a trans-epithelial formulation of riboflavin for corneal delivery using 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) and isostearic acid (ISA). The permeation enhancement was evaluated using an in vitro corneal epithelial cell culture system by measuring the amount of transferred riboflavin with high-performance liquid chromatography. Riboflavin permeation of MedioCROSS TE, a commercially available riboflavin formulation containing benzalkonium chloride, was also evaluated and compared to that of the DODAP/ISA formulation by changing the riboflavin concentration. The trans-epithelial electrical resistance (TEER) was measured after exposure to the samples in an in vitro corneal epithelial cell culture system to assess cytotoxicity.

Results: The DODAP/ISA formulation demonstrated greater permeation when used together than when each component was used individually. The permeation enhancement effect of the DODAP/ISA formulation was almost the same as that of MedioCROSS TE. However, when a 10-fold higher riboflavin concentration was used in the DODAP/ISA formulation, the permeation enhancement effect surpassed that of MedioCROSS TE. After 24 hours of exposure, the TEER of the DODAP/ISA formulation was higher than that of MedioCROSS TE, indicating that the DODAP/ISA formulation was less cytotoxic than MedioCROSS TE.

Conclusions: This study indicated that the DODAP/ISA formulation could serve as a less cytotoxic alternative to MedioCROSS TE. Further studies are required to determine the clinical efficacy and safety of the DODAP/ISA formulation in vivo.

Translational Relevance: This study may provide alternative procedures for corneal collagen crosslinking with less of a cytotoxic effect on corneal epithelial cells.

Introduction
Corneal collagen crosslinking (CXL) therapy, proposed by Wollensak et al.,1 is becoming a useful option to treat corneal ectatic diseases such as keratoconus. CXL is commonly conducted by using riboflavin 5′-phosphate sodium and ultraviolet-A irradiation.2 Initially, CXL was conducted under epithelium-off (epi-off) conditions, otherwise referred to as the Dresden protocol, which includes such risks as pain, infection, persistent epithelial defect, and corneal melt.3,4 To address these adverse events, the epi-on CXL treatment was developed which does not require epithelial cell removal. However, the hydrophilic nature of riboflavin 5′-phosphate presents a challenge due to its permeation through corneal epithelial cells. To address this problem, several studies utilized calcium-sequestering compounds,5 cyclodextrin derivates,6 nanostructured lipid carriers,7 and a channel-forming peptide (NC-1059)8 and reported enhancement of the permeability of riboflavin 5′-phosphate through the corneal epithelium. The most utilized technique in clinical applications is a formulation containing benzalkonium chloride (BAC), and several commercial products with this formulation are used clinically.9 Though BAC is mostly used in clinical products, damage to corneal epithelial cells10 is still observed due to loosening of the tight junction.1113 In recent years, ionic liquids (ILs) have attracted attention for peptide delivery through the gastrointestinal root by enhancing transmucosal permeation.14,15 Several ILs have been used to dissolve water-insoluble compounds in combination with bases and acids. However, ILs electrostatically separate into cation and anion pairs when diluted in the gastrointestinal tract, where the target drugs are absorbed. In addition, some lipid nanoparticles composed of 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) have recently been utilized for gene delivery into cells.16 In the present study, we investigated the enhanced effect of corneal epithelial cell permeation by a DODAP/isostearic acid (ISA) combination formulation. The concentration of riboflavin 5′-phosphate in commercially available medical products is set between 0.1% and 0.25%,9 although the solubility of sodium riboflavin 5′-phosphate in water is 37 mg/mL.17 Because material transport obeys Fick's first law of diffusion,18 the amount of riboflavin transported would increase if the riboflavin 5′-phosphate concentration is increased. Therefore, we investigated the permeation enhancement effect of a higher concentration of riboflavin 5′-phosphate in the DODAP/ISA formulation. 
Methods
Materials
Riboflavin 5′-phosphate sodium, arginine (Arg), and lactic acid (LA) were purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan) (Table). DODAP and ISA were purchased from MedChemExpress (Wembley, UK) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). MedioCROSS TE was purchased from Avedro, Inc. (Waltham, MA). Human corneal epithelial cells were obtained from the Japan Tissue Engineering Co., Ltd. (Aichi, Japan) as LabCyte CORNEA-MODEL. Other substances purchased were of research reagent grade. 
Table.
 
Components Used for Riboflavin Permeation Enhancement
Table.
 
Components Used for Riboflavin Permeation Enhancement
Preparation of Riboflavin 5′-Phosphate Test Samples
To prepare aqueous suspensions of riboflavin 5′-phosphate sodium containing the DODAP/ISA combination, DODAP alone, and ISA alone, a 10-mM DODAP/ISA, DODAP, or ISA suspension was initially prepared. Riboflavin 5′-phosphate sodium was then dissolved to 0.25% and 2.5%. To prepare an aqueous solution of Arg/LA, a 10-mM Arg/LA aqueous solution was prepared, and riboflavin 5′-phosphate sodium was dissolved in it to reach 0.25%. 
Riboflavin Permeation Through Corneal Epithelial Cells
Human corneal epithelial culture kits (LabCyte CORNEA-MODEL) were used after overnight incubation, as instructed. Each test suspension or solution was added to culture caps (n = 3), and 0.2 mL culture media in the bottom wells was periodically withdrawn and replaced with fresh media. Riboflavin concentration in the sampled media was measured using high-performance liquid chromatography (HPLC). After 24 hours of incubation, trans-epithelial electrical resistance (TEER) was measured using a Millicell ERS-2 (Merck Millipore, Burlington, MA). 
HPLC Assay
Riboflavin concentration in the withdrawn culture media was measured by using an Shimazu HPLC system (LC2050; Shimadzu, Kyoto, Japan) with an isocratic elution mode (mobile phase A/mobile phase B) on a Waters XTerra MS C18 column (4.6 mm × 150 mm, 5 µm; Waters Corporation, Milford, MA). The HPLC system (LC-2010C HT; Shimadzu, Kyoto, Japan) consisted of a quaternary pump and ultraviolet/visible light detector. Mobile phase A consisted of 893 mL of 8.0-mM sodium-1-hexanesulfonate, glacial acetic acid (7.5 mL), and methanol (100 mL), and the pH of the solution was adjusted to 3.0 with the ion-pairing reagent diethylamine to enhance peak shape. Mobile phase B consisted of HPLC-grade methanol. The instrumental setup included a flow rate of 1.0 mL/min, and the temperature of the column oven was set at 30°C. The injection volume was set to 100 mL with a run time of 12 minutes. The effluent was detected at 268 nm using a photodiode array detector. 
Statistical Analysis
Student's t-test was used to compare each riboflavin concentration in the bottom wells with the test samples and TEER values after 24 hours of incubation of each sample. Data from riboflavin concentrations and TEER measurements (n = 3), expressed as average ± standard deviation (SD), were considered statistically significant for P < 0.05. 
Results
Corneal Cell Permeability Enhancement by DODAP/ISA Ion Pairs
Riboflavin permeation was enhanced when the DODAP/ISA combination was applied to corneal epithelial cells (Fig. 1). The DODAP/ISA combination accelerated riboflavin permeation more effectively than either DODAP or ISA alone. 
Figure 1.
 
Riboflavin is transferred through corneal epithelial cells. The DODAP/ISA, DODAP, or ISA formulations were applied to LabCyte CORNEA-MODEL tissue, and the transferred riboflavin concentration was measured by HPLC. Data are shown as average ± SD (n = 3).
Figure 1.
 
Riboflavin is transferred through corneal epithelial cells. The DODAP/ISA, DODAP, or ISA formulations were applied to LabCyte CORNEA-MODEL tissue, and the transferred riboflavin concentration was measured by HPLC. Data are shown as average ± SD (n = 3).
Corneal Cell Permeability Comparison to MedioCROSS
The DODAP/ISA combination enhanced riboflavin permeation when a riboflavin concentration of approximately 18 µg/mL was reached within the initial 8 hours; however, the permeation was maintained and reached approximately 20 µg/mL after 24 hours. The MedioCROSS formulation achieved riboflavin concentrations of approximately 40 µg/mL (Fig. 2A). In contrast, the Arg/LA formulation was less effective (under 25%) in enhancing riboflavin permeation than the DODAP/ISA combination. After 24 hours of incubation, the TEER of the corneal epithelium incubated with the MedioCROSS formulation was approximately 600 Ωcm2, whereas that of the DODAP/ISA and Arg/LA formulations were approximately 1500 and 2400 Ωcm2, respectively (Fig. 2B). There were no significant differences between the DODAP/ISA and Arg/LA formulations. 
Figure 2.
 
(A) Riboflavin transferred through corneal epithelial cells. The DODAP/ISA, MedioCROSS, and Arg/LA formulations were applied to LabCyte CORNEA-MODEL tissue, and the concentration of transferred riboflavin was measured by HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, MedioCROSS versus Arg//LA; **P < 0.05, DODAP/ISA versus Arg/LA; +P < 0.05, MedioCROSS versus DODAP/ISA. (B) TEER values after incubation of DODAP/ISA, MedioCROSS, and Arg/LA formulations for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, Arg//LA versus MedioCROSS; **P < 0.05, DODAP/ISA versus MedioCROSS.
Figure 2.
 
(A) Riboflavin transferred through corneal epithelial cells. The DODAP/ISA, MedioCROSS, and Arg/LA formulations were applied to LabCyte CORNEA-MODEL tissue, and the concentration of transferred riboflavin was measured by HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, MedioCROSS versus Arg//LA; **P < 0.05, DODAP/ISA versus Arg/LA; +P < 0.05, MedioCROSS versus DODAP/ISA. (B) TEER values after incubation of DODAP/ISA, MedioCROSS, and Arg/LA formulations for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, Arg//LA versus MedioCROSS; **P < 0.05, DODAP/ISA versus MedioCROSS.
Effect of Riboflavin Concentration on Corneal Permeability
The DODAP/ISA formulation containing 2.5% riboflavin 5′-phosphate accelerated riboflavin permeation through corneal epithelial cells approximately twofold higher compared to the formulation containing 0.25% riboflavin 5′-phosphate, and this enhancement was more effective than that observed with the MedioCROSS formulation (Fig. 3A). The TEER values for the DODAP/ISA formulations containing 0.25% and 2.5% riboflavin-5-phosphate after 24-hour incubations were both approximately 1000 Ωcm2, whereas that of the MedioCROSS formulation was approximately 600 Ωcm2 (Fig. 3B). 
Figure 3.
 
(A) Riboflavin transferred through corneal epithelial cells. DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS were applied to LabCyte, and the concentration of transferred riboflavin was measured using HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5%) versus DODAP/ISA (0.25%); +P < 0.05, DODAP/ISA (2.5%) versus MedioCROSS. **P < 0.05, MedioCROSS versus DODAP/ISA (0.25%). (B) TEER values after incubation of DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5% VB2-P) versus MedioCROSS; **P < 0.05, DODAP/ISA (0.25% VB2-P) versus MedioCROSS. VB2-P, riboflavin 5′-phosphate.
Figure 3.
 
(A) Riboflavin transferred through corneal epithelial cells. DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS were applied to LabCyte, and the concentration of transferred riboflavin was measured using HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5%) versus DODAP/ISA (0.25%); +P < 0.05, DODAP/ISA (2.5%) versus MedioCROSS. **P < 0.05, MedioCROSS versus DODAP/ISA (0.25%). (B) TEER values after incubation of DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5% VB2-P) versus MedioCROSS; **P < 0.05, DODAP/ISA (0.25% VB2-P) versus MedioCROSS. VB2-P, riboflavin 5′-phosphate.
Discussion
Riboflavin formulations containing BAC are most frequently used in clinical applications for CXL therapy, and several commercial products are available for use in clinical settings.9 However, adverse effects on corneal epithelial cells,10 such as have been observed after epi-on CXL treatment with riboflavin application, are still a concern due to the loosening effect of the tight junctions.1113 Kissner et al.19 reported that corneal epithelial cells require long-term recovery from injury caused by BAC. BAC has been used as a preservative for eye drop formulations, but several studies have reported that it causes corneal epithelial cell damages both in vitro and in vivo.12,20 Such studies report that the damages involve cell-detachment and cell lysis at a BAC concentration of 0.01%.12 Therefore, we searched for alternative substances to enhance corneal epithelial permeation, focusing on ILs. Banerjee et al.14 reported that ILs, such as choline/geranate (CAGE), enhance insulin absorption through the intestinal tract. Because CAGE is considered to enhance insulin absorption by loosening tight junctions in microvilli, it is possible that it could cause damage to corneal epithelial cells, similar to the damage caused by BAC. Further research led to the discovery of DODAP, a cationic lipid that has been used recently to prepare lipid nanoparticles for the delivery of short interfering RNA (siRNA) into cells without significant damage to the cells.16 Thus, the enhancement effect of DODAP was attractive for transferring riboflavin with fewer damages to corneal epithelial cells than those observed with BAC.12,20 The DODAP/ISA combination formulation enhanced the riboflavin formulation more effectively than DODAP or ISA alone (Fig. 1). Riboflavin 5′-phosphate is taken orally and converted to riboflavin by intrinsic esterase.21 Therefore, the transferred form of riboflavin-5-phosphate was de-esterified riboflavin, measured by HPLC (data not shown). The DODAP/ISA formulation transferred riboflavin more effectively than did the Arg/LA formulation, which lacked a fatty acid chain (Fig. 2A). These data indicate that the combination of DODAP and ISA enhanced the permeation compared to the small-molecule combination of cations (Arg) and anions (LA). The DODAP/ISA formulation enhanced riboflavin permeation, similar to MedioCROSS TE, which contained 0.01% BAC (Fig. 2A); however, the MedioCROSS TEER after 24 hours of incubation was approximately 50% of that after incubation with DODAP/ISA and Arg/LA formulations. The TEER is an attractive indicator to predict in vitro barrier levels of several cell culture models.22 For a cornea epithelial cell culture system, TEER was used to measure the damage levels of corneal epithelial cells.23 Therefore, we investigated whether TEER values could be used as an indicator of corneal epithelial cell damage in vitro. The TEER results for the Arg/LA formulation are consistent with the fact that Arg and LA are generally recognized as safe (GRAS)-grade materials. The TEER of the DODAP/ISA formulation was the same as that of the Arg/LA formulation, suggesting that the DODAP/ISA formulation was less harmful to corneal epithelial cells. The TEER of the MedioCROSS formulation was approximately half that of the DODAP/ISA formulation, indicating that BAC, as one of preservatives, caused cell damage.12 An exact understanding of the DODAP/ISA formulation permeability enhancement without loss of TEER is currently lacking and is a target of further investigation. 
We hypothesized that riboflavin 5′-phosphate permeation would follow Fick's first law of diffusion.18 The 10-fold concentration of riboflavin 5′-phosphate (2.5%) in the DODAP/ISA formulation accelerated riboflavin permeation compared to the 0.25% formulation, and both formulations maintained a similar TEER (Fig. 3A). These data will contribute to formulating a more effective riboflavin eye drop formulation with fewer adverse events. 
Acknowledgments
The authors thank members of the Analytical Research Laboratory at MEDRx Co. Ltd. for their assistance in conducting this research. 
Disclosure: Y. Yamagata, None; T. Ide, None 
References
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Figure 1.
 
Riboflavin is transferred through corneal epithelial cells. The DODAP/ISA, DODAP, or ISA formulations were applied to LabCyte CORNEA-MODEL tissue, and the transferred riboflavin concentration was measured by HPLC. Data are shown as average ± SD (n = 3).
Figure 1.
 
Riboflavin is transferred through corneal epithelial cells. The DODAP/ISA, DODAP, or ISA formulations were applied to LabCyte CORNEA-MODEL tissue, and the transferred riboflavin concentration was measured by HPLC. Data are shown as average ± SD (n = 3).
Figure 2.
 
(A) Riboflavin transferred through corneal epithelial cells. The DODAP/ISA, MedioCROSS, and Arg/LA formulations were applied to LabCyte CORNEA-MODEL tissue, and the concentration of transferred riboflavin was measured by HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, MedioCROSS versus Arg//LA; **P < 0.05, DODAP/ISA versus Arg/LA; +P < 0.05, MedioCROSS versus DODAP/ISA. (B) TEER values after incubation of DODAP/ISA, MedioCROSS, and Arg/LA formulations for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, Arg//LA versus MedioCROSS; **P < 0.05, DODAP/ISA versus MedioCROSS.
Figure 2.
 
(A) Riboflavin transferred through corneal epithelial cells. The DODAP/ISA, MedioCROSS, and Arg/LA formulations were applied to LabCyte CORNEA-MODEL tissue, and the concentration of transferred riboflavin was measured by HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, MedioCROSS versus Arg//LA; **P < 0.05, DODAP/ISA versus Arg/LA; +P < 0.05, MedioCROSS versus DODAP/ISA. (B) TEER values after incubation of DODAP/ISA, MedioCROSS, and Arg/LA formulations for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, Arg//LA versus MedioCROSS; **P < 0.05, DODAP/ISA versus MedioCROSS.
Figure 3.
 
(A) Riboflavin transferred through corneal epithelial cells. DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS were applied to LabCyte, and the concentration of transferred riboflavin was measured using HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5%) versus DODAP/ISA (0.25%); +P < 0.05, DODAP/ISA (2.5%) versus MedioCROSS. **P < 0.05, MedioCROSS versus DODAP/ISA (0.25%). (B) TEER values after incubation of DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5% VB2-P) versus MedioCROSS; **P < 0.05, DODAP/ISA (0.25% VB2-P) versus MedioCROSS. VB2-P, riboflavin 5′-phosphate.
Figure 3.
 
(A) Riboflavin transferred through corneal epithelial cells. DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS were applied to LabCyte, and the concentration of transferred riboflavin was measured using HPLC. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5%) versus DODAP/ISA (0.25%); +P < 0.05, DODAP/ISA (2.5%) versus MedioCROSS. **P < 0.05, MedioCROSS versus DODAP/ISA (0.25%). (B) TEER values after incubation of DODAP/ISA formulations containing 0.25% or 2.5% riboflavin 5′-phosphate and MedioCROSS for 24 hours. Data are shown as average ± SD (n = 3). *P < 0.05, DODAP/ISA (2.5% VB2-P) versus MedioCROSS; **P < 0.05, DODAP/ISA (0.25% VB2-P) versus MedioCROSS. VB2-P, riboflavin 5′-phosphate.
Table.
 
Components Used for Riboflavin Permeation Enhancement
Table.
 
Components Used for Riboflavin Permeation Enhancement
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