Transdermal Drug Delivery in the Pig Skin

Abstract

Transdermal delivery can be accomplished through various mechanisms including formulation optimization, epidermal stratum corneum barrier disruption, or directly by removing the stratum corneum layer. Microneedling, electroporation, a combination of both and also the intradermal injection known as mesotherapy have proved efficacy in epidermal-barrier disruption. Here we analyzed the effects of these methods of epidermal-barrier disruption in the structure of the skin and the absorption of four compounds with different characteristics and properties (ketoprofen, biotin, caffein, and procaine). Swine skin (Pietrain x Durox) was used as a human analogue, both having similar structure and pharmacological release. They were biopsied at different intervals, up to 2 weeks after application. High-pressure liquid chromatography and brightfield microscopy were performed, conducting a biometric analysis and measuring histological structure and vascular status. The performed experiments led to different results in the function of the studied molecules: ketoprofen and biotin had the best concentrations with intradermal injections, while delivery methods for obtaining procaine and caffein maximum concentrations changed on the basis of the lapsed time. The studied techniques did not produce significant histological alterations after their application, except for an observed increase in Langerhans cells and melanocytes after applying electroporation, and an epidermal thinning after using microneedles, with variable results regarding dermal thickness. Although all the studied barrier disruptors can accomplish transdermal delivery, the best disruptor is dependent on the particular molecule.

Keywords: electroporation; intradermal injection; mesotherapy; microneedling; morphology; skin; transdermal delivery.

Figure 1

Design of the experimental procedure in the pigs. The working diagram of (a) the right side and (b) the left side) shows the different techniques and time intervals. The diagrams were applied to each of the differently treated animals. The images show the ketoprofen-treated pig, labeled as KETO (c) and the biotin-treated animal, labeled as BIOTINA (d). Punch biopsies were performed according to the diagram (e), and then sutured (f). Legend for quadrants: microneedle (MN), intradermal injection (ID), electroporation (EP) and the combination MN + EP; subquadrants depict the time after drug administration in minutes (0, 15, 30, and 60 min).

Figure 2

Method of measurement of epidermal and dermal thickness. Skin samples were stained with hematoxylin–eosin and prepared to be measured through the analysis software (a–c). Images were processed to quantify epidermal (d–f) and dermal (g–i) thickness by reducing the area to spheres. Colors in images d–i refer to the diameter of the spheres, which is depicted in the color legend in the upper-right-hand corner of each picture, measured in µm. Scale bar 1 mm.

Figure 3

Features of pig skin. Masson trichrome (a–c) illustrates the three main layers in different colors with low magnification (epidermis in red, e; dermis in blue, d; hypodermis in white, h), specifically coloring collagen fibers in blue (a). Hair follicles (f) are not distinguishable from human, but closely there are some apocrine glands (g) that help in this distinction (b). A superficial vascular plexus is readily identifiable in red with Masson trichrome (blood vessels, bv, and dashed circles), highlighted amongst the bluish collagen background of the dermis (c). Vimentin intermediate filaments are demonstrated in brown due to the diaminobenzidine, and are typically present in epidermal dendritic cells and dermal fibroblasts and endothelial cells of the blood vessels (d). Scale bar 200 µm (a,b), 80 µm (c,d).

Figure 4

Microscopic lesions caused by puncture. Epidermal solutions of continuity, highlighted with black arrows, related to ID (a) were morphologically deeper than the MN-related ones (b,c). Some erythrocytes (highlighted with dashed circles) can be identified over the cutaneous disruption (d), at the dermo–epidermal junction (e), or even in the superficial dermis (f). Scale bar 40 µm (a–c,e,f), 20 µm (d).

Figure 5

Immunohistochemical location of epidermal dendritic cells after 7 days (a–d) and after 14 days (e,f). Epidermis thickness is highlighted with a blue bar, epidermal basal cell layer thickness is highlighted with a dark blue bar, and the dermo–epidermal junction is outlined by a clear blue line. The highest density of melanocytes was observed in the EP specimen, where S100 protein demonstrated a slightly dendritic population in the epidermal basal layers (a,d). Langerhans cells were also readily apparent above the basal cell layer in the Vimentin-immunostained EP specimen (b,c,e,f). Images c and f are details of the squared regions in images b and e. Scale bar 40 µm (a,b,d,e), 20 µm (c,f).

Figure 6

Values of measured cutaneous concentrations (vertical axis), achieved with the HPLC technique for procaine (n = 1), ketoprofen (n = 1), biotin (n = 1), and caffeine (n = 1). Concentrations were measured immediately after administration (T0), after 15 min (T15), after 30 min (T30), after 60 min (T60), after one week (D7), and after two weeks (D14). Values are expressed as µg/mL.

Figure 7

Average values of the HPLC measurements (n = 4 for each molecule and disruption technique) during the first hour after drug application (blue bubbles), including standard deviation values (light blue bubbles), for procaine, ketoprofen, biotin, and caffeine.

Figure 8

Area under the curve comparing the different molecules and procedures employed. Values are expressed as µg·min/mL.