SKIN PENETRATION BY COPPER-PEPTIDE COMPLEXES 63 ml/ min -1, and the eluate was monitored at 2 00 nm using the UV /Vis detector. The concentration of peptide was determined by measuring the peak area. Electrospray MS was applied to identify copper complexes present in the acceptor cell. ESI-MS spectra were acquired in the range of 150-1500 µ using 20 ms dwell time and 0.1 µ of step size. The ion spray voltage of 4000 V was applied for positive and negative ion acquisition. The orifice potential was established at 80 V, as the one offering the best signal intensity and causing partial fragmentation of the molecular ion at the peptide bounds (34). DATA ANALYSIS To calculate the permeability coefficient, the cumulative amount of copper ions was plotted against the flux CT) of a compound across the membrane, determined at steady state (35). The permeability coefficient of the Cu2+ ion in the lipid membrane K P (cm·s-1) was calculated by Fick's first law of diffusion. Figure 1 shows exemplary permeation profiles of ligands and the amount of copper vs time. RESULTS AND DISCUSSION The aim of our research was to determine the influence of ligands (peptides) on the permeation process of copper ions. First, our studies confirmed the ability of copper ions to penetrate the model membrane without the determination of a compound form (copper ions or copper complexes). Second, we investigated the concentration of peptides that permeated the membrane. Finally, from the obtained data and ESI-MS results we were able to establish the form in which copper and peptides permeate. PERMEABILITY COEFFICIENT STUDY The results introduced in Figure 2 reveal a high influence of complexing agent (GHK or GSH) on the permeability coefficient of copper ions. In all cases, the permeation rates of copper ions were lower than those obtained for complexed copper. For this reason, it may be concluded that the complexing agents (GHK and GSH) accelerate the migration of copper ions through the model membranes. As shown in Figure 2, the influence of peptide complexes on the permeation of copper ions has different levels the influence of GHK on copper ion penetration was confirmed to be twice as strong as that of GSH. Research determined the permeation coefficient of peptides from the copper complexes. The concentration of G HK and GSH in the acceptor cell was determined by reversed­ phase liquid chromatography (RPLC) with UV-VIS detection. In Figure 3 the compari­ son of the permeation coefficients of GHK and GSH peptides from the copper complexes is presented. The figure proves that GHK and GHK-Cu have very similar values. What is more, on the basis of Figure 3 the conclusion that tripeptide complexation of copper does not change the K P value of GHK may be drawn. The GSH values were different: the K P for GSH was higher than that for the GSH copper complex. Similar properties of the penetration abilities of the GHK peptides confirmed the thesis that the structure and high affinity to the lipid structures of the membrane strongly influence the per­ meation process. The permeation coefficients of the peptides are significantly lower than those of copper ions.

64 JOURNAL OF COSMETIC SCIENCE a) 6 a ::1. I � 4- + "C I!! + C • G) a. 0 2 c :::J E a, ♦ 0 I I I 7 0 8 16 24 32 40 48 56 64 72 80 time [h] b) 4 a :i � 3- en (!) ..... I!! 2 • C • 0 • c :::J 1 0 E (U ♦ 0 I I 0 8 16 24 32 40 48 56 64 72 time [h] Figure 1. The permeation profiles of peptide GSH from (a) copper complex GSH-Cu and (b) peptide. ESI-MS STUDY The other significant factor playing a key role in the migration processes is the equi­ librium of the complexes in the acceptor solution. Finally, from the obtained data and ESI-MS results, we were able to establish the form in which copper and the peptides permeate. As shown in Figure 2, we could find a lot of molecules in the acceptor cell by ESI-MS study. During the study of the penetration ability of peptides we could find species in acceptor cells (GHK and GSH).