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.
11–13 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.