Reverse Iontophoretic Extraction of Skin Cancer-Related Biomarkers

Abstract

Non-invasive methods for early diagnosis of skin cancer are highly valued. One possible approach is to monitor relevant biomarkers such as tryptophan (Trp) and kynurenine (Kyn), on the skin surface. The primary aim of this in vitro investigation was, therefore, to examine whether reverse iontophoresis (RI) can enhance the extraction of Trp and Kyn, and to demonstrate how the Trp/Kyn ratio acquired from the skin surface reflects that in the epidermal tissue. The study also explored whether the pH of the receiver medium impacted on extraction efficiency, and assessed the suitability of a bicontinuous cubic liquid crystal as an alternative to a simple buffer solution for this purpose. RI substantially enhanced the extraction of Trp and Kyn, in particular towards the cathode. The Trp/Kyn ratio obtained on the surface matched that in the viable skin. Increasing the receiver solution pH from 4 to 9 improved extraction of both analytes, but did not significantly change the Trp/Kyn ratio. RI extraction of Trp and Kyn into the cubic liquid crystal was comparable to that achieved with simple aqueous receiver solutions. We conclude that RI offers a potential for non-invasive sampling of low-molecular weight biomarkers and further investigations in vivo are therefore warranted.

Keywords: bicontinuous cubic liquid crystals; cancer-related biomarkers; kynurenine; non-invasive extraction; tryptophan; tryptophan-to-kynurenine ratio.

Figure 1

Graphical representation of the reverse iontophoresis experiments with anodal and cathodal receptor phases comprising either (A) an aqueous buffer (n = 5–12, see Table 2), or (B) a cubic phase in contact with an aqueous buffer (n = 3–5, see Table S7 in Supplementary Materials).

Figure 2

Cumulative amounts of Trp (A, C, and E) and Kyn (B, C, and F) extracted passively and by reverse iontophoresis as a function of time at three different receptor solution pH values: pH 4.0 (A,B), pH 7.4 (C,D), and pH 9.0 (E,F). The data points show the mean ± SEM (n = 4–12, see Table 2).

Figure 2

Cumulative amounts of Trp (A, C, and E) and Kyn (B, C, and F) extracted passively and by reverse iontophoresis as a function of time at three different receptor solution pH values: pH 4.0 (A,B), pH 7.4 (C,D), and pH 9.0 (E,F). The data points show the mean ± SEM (n = 4–12, see Table 2).

Figure 3

Flux of endogenous Trp extracted by reverse iontophoresis into electrode receiver solutions at pH 7.4. The data are presented as mean ± SEM (n = 6). Corresponding cumulative amount is shown in Figure S9 in Supplementary Materials.

Figure 4

Cumulative anodal, cathodal and passive extraction of Trp and Kyn during 6 h of reverse iontophoresis (filled bars) and during a subsequent (no-current) period of 18 h (stippled bars). Data presented are mean ± SEM; n = 4–11, see Table S4 for detail.

Figure 5

Cumulative Trp and Kyn extraction in 6 h of reverse iontophoresis into the cubic matrix (Cub+aq) and compared to that when an aqueous pH 7.4 solution was used as before. The data presented are mean ± SEM (n = 3 and 5 for extraction into the cubic matrix at anode and cathode, respectively; n = 6 for extraction into aqueous solution).

Figure 6

Trp/Kyn ratios of the cumulative amounts extracted (black) and the corresponding fluxes (red) to the cathode as a function of time when the receiver medium was an aqueous pH 7.4 buffer. Data are presented as mean ± SEM (n = 1 for the first hour, n = 4 for the second hour, and n = 6 thereafter).