Lecithin-Based Dermal Drug Delivery for Anti-Pigmentation Maize Ceramide

Abstract

Ceramides have several well-known biological properties, including anti-pigmentation and anti-melanogenesis, which make them applicable for use in skincare products in cosmetics. However, the efficacy of ceramides is still limited. Dermal or transdermal drug delivery systems can enhance the anti-pigmentation properties of ceramides, although there is currently no systemic evaluation method for the efficacy of these systems. Here we prepared several types of lecithin-based emulsion of maize-derived glucosylceramide, determining PC70-ceramide (phosphatidylcholine-base) to be the safest and most effective anti-pigmentation agent using zebrafish larvae. We also demonstrated the efficacy of PC70 as a drug delivery system by showing that PC70-Nile Red (red fluorescence) promoted Nile Red accumulation in the larval bodies. In addition, PC70-ceramide suppressed melanin in mouse B16 melanoma cells compared to ceramide alone. In conclusion, we developed a lecithin-based dermal delivery method for ceramide using zebrafish larvae with implications for human clinical use.

Keywords: cone ceramide; in vivo imaging; zebrafish screening.

Figure 1

PC70-ceramide suppresses pigmentation in zebrafish. (a) Structures of lecithin used in this study and electron microscope image of PC70-ceramide (10,000× magnification; yellow arrows). (b) Survival rate for zebrafish larvae treated with lecithin-ceramides. The fish were treated with each compound from 24 h post-fertilization (hpf) for 5 days. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control. n = 3, error bars indicate SD. ns indicates no significant difference. For the survival rate for lecithin alone, please see Table 1. (c) Representative images of zebrafish larvae treated with 0.1% PC70-ceramide (containing 10 µg/mL ceramide), 0.1% PC70, or 10 µg/mL ceramide. The fish were treated with each compound from 24 hpf for 48 h. (d) Quantification of pigmentation in zebrafish larvae treated with PC70-ceramide, PC70, or ceramide. * p < 0.05 vs. control. n = 12, error bars indicate SD.

Figure 1

PC70-ceramide suppresses pigmentation in zebrafish. (a) Structures of lecithin used in this study and electron microscope image of PC70-ceramide (10,000× magnification; yellow arrows). (b) Survival rate for zebrafish larvae treated with lecithin-ceramides. The fish were treated with each compound from 24 h post-fertilization (hpf) for 5 days. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control. n = 3, error bars indicate SD. ns indicates no significant difference. For the survival rate for lecithin alone, please see Table 1. (c) Representative images of zebrafish larvae treated with 0.1% PC70-ceramide (containing 10 µg/mL ceramide), 0.1% PC70, or 10 µg/mL ceramide. The fish were treated with each compound from 24 hpf for 48 h. (d) Quantification of pigmentation in zebrafish larvae treated with PC70-ceramide, PC70, or ceramide. * p < 0.05 vs. control. n = 12, error bars indicate SD.

Figure 2

Visualization of the PC70 drug delivery system using PC70-Nile Red (PC70-NR) treatment in zebrafish larvae. (a) Representative images of PC70-NR- or NR-treated zebrafish. Seventy-two hpf larvae were exposed to 1% PC70-NR (containing 200 μg/mL PC70 with 1 ng/mL NR) or 1 ng/mL NR for 2 and 20 h, respectively. Following this, the fish were washed in breeding water for 18 h. Red colouration indicated NR fluorescence. (b) Quantification of NR fluorescence in zebrafish treated with PC70-NR or NR. * p < 0.05, ** p < 0.01. Each experimental group contained 12 fish; error bars indicate SD.

Figure 3

PC70-ceramide suppresses melanin accumulation in mouse B16 melanoma cells. (a) Representative images of B16 cells treated with PC70-ceramide or ceramide. B16 4A5 cells were treated with 0.5% PC70-ceramide (containing 50 µg/mL ceramide) or 50 μg/mL ceramide for 48 h. (b) Melanin levels of B16 cells treated with PC70-ceramide, ceramide, or arbutin. Arbutin was used as a positive control, following the method used by a previous study [8]. * p < 0.05, # p < 0.1 vs. control. n = 3, error bars indicate SD. (c) Cell viability of B16 cells treated with PC70-ceramide or ceramide. “ns” indicates no significance. n = 3, error bars indicate SD.