## 4 Discussion  
The uppermost skin layer is the stratum corneum (SC). The role of this skin barrier is to prevent excessive moisture loss and to impede the penetration of potentially unsafe substances into the body. At the same time, the role of SC is crucial for the absorption process of most skin formulations by passive diffusion. SC is composed of approximately 15-25 layers of corneocytes suspended in an extracellular hydrophobic matrix of lipid bilayers [33]. The lipid bilayer space consists of a hydrophilic region in between two hydrophobic ones. Due to its hydrophobic nature, lipid-soluble substances can penetrate SC mainly through the intercellular tissue compartment. To circumvent the limited permeation properties of SC a number of methods have been proposed such as tape-stripping, laser abrasion, electroporation, sonophoresis and iontophoresis [34]. In addition, it has been noted that the SC swells considerably when hydrated and increases its permeability [35]. Penetration enhancers, such as OCAs, can effectively lower the permeation barrier of SC without damaging the viable cells. Due to their hyperosmotic properties when applied on the skin tissue surface, OCAs induce water flux from the interstitial space into the superficial tissue layers, increasing the osmolality of ISF. The process leads to the desired RI matching effect through the swelling of SC and the decrease of \(\mu_{\text{OCT}}\). In our experiments, all OCAs significantly increase the thickness of the epidermis (Figure 2b). PG induced  
Figure 2: Normalized changes in the signal attenuation coefficient \(\mu_{\text{OCT}}\) (**a**) and in epidermal thickness (**b**) of non-treated skin after the application of four different OCAs at two different times t1 = 5 min and t2 = 90 min (x-axis, no name suffix and suffix “90”, respectively). The number of asterisks denotes the decreasing \(p\)-value whereas \(p\leq\) 0.05, \(p\leq\) 0.01, \(p\leq\) 0.001 and \(p\leq\) 0.0001 are symbolized by 1–4 asterisks. Raw mean values are presented in Tables S1 and S2 (Supplementary Information). the highest increase in epidermal thickness at t2 (+33%). PG is a hydrophilic molecule and can be attached to the polar head group of the lipid bilayers increasing the interfacial area per lipid without changes in the short or long lamellar spacing of the SC membrane but with some possible alterations of the intracellular \(\alpha\)- or \(\beta\)-keratin probably due to the displacement of water [36; 37]. This results in the swelling of the intracellular hydrophilic space of the SC and in the increase of epidermal thickness. On the other hand, OA induces SC lipid fluidization and the formation of a new disordered lipid phase [38]. OA consists of a hydrophobic tail and a hydrophilic head. The hydrophobic part lowers the barrier of SC by disrupting the lipid layers, increasing the permeability of the hydrophilic PG [39]. V and LV also increase the water content in SC causing a swelling effect. However, these hydrophobic molecules (OA, V and LV) prevent the high flux of water to the upper SC. In contrast, PG enhances this phenomenon due to its hydrophilic properties, resulting in the observed maximum epidermal thickness. Still, it is known that PG is hygroscopic and adsorbs water until saturation, inhibiting complete dehydration of the tissue [40]. PGOA achieved the lowest \(\mu_{\text{OCT}}\) at t2 (Figure 2b). Previous studies have shown that when PG and OA are combined, skin permeation is increased as a result of two different synergistic mechanisms: PG solvates the \(\alpha\)-keratin of corneocytes while OA acts on the lipid disruption [29; 41]. Hence, the presence of OA enhances the multidirectional diffusion of PG into the skin and the OCA uptake saturates in 2-3 h (+t2). Within the time frame of our experiments, we observed the results of the combined PGOA OCA towards the significant decrease of \(\mu_{\text{OCT}}\) which was the highest among all the tested agents. Significant, albeit lower, reduction was also achieved by PG at t2.