Nanotopography facilitates in vivo transdermal delivery of high molecular weight therapeutics through an integrin-dependent mechanism

Abstract

Transdermal delivery of therapeutics is restricted by narrow limitations on size and hydrophobicity. Nanotopography has been shown to significantly enhance high molecular weight paracellular transport in vitro. Herein, we demonstrate for the first time that nanotopography applied to microneedles significantly enhances transdermal delivery of etanercept, a 150 kD therapeutic, in both rats and rabbits. We further show that this effect is mediated by remodeling of the tight junction proteins initiated via integrin binding to the nanotopography, followed by phosphorylation of myosin light chain (MLC) and activation of the actomyosin complex, which in turn increase paracellular permeability.

Keywords: Nanotopography; drug delivery; etanercept; integrin; transdermal.

Figures

Figure 1

Nanotopography significantly enhances transdermal delivery of etanercept in vivo. (a) Schematic representation of the transdermal delivery devices used in this study. Progressing from top to bottom: each device is made of an impermeable backing (tan), drug reservoir (green), rate-controlling membrane (yellow), and silicon microneedle array (MNA) (gray). Each 290 μm long, 100 μm wide microneedle has longitudinal perforations along the side, through which drug flows out. Drug flow from the reservoir down the grooves of the microneedles is indicated by a green dashed arrow. Perforations are denoted with a white arrowhead. “Smooth” microneedles were not coated with a film, while “nanostructured” microneedles were coated with a nanostructured film (scale bar represents 300 μm). Inset on furthest right depicts an SEM image of the nanostructures coated onto each microneedle (scale bar = 3 μm). (b) Nanostructured MNAs deliver significantly more etanercept transdermally than either drug reservoir patch alone or unstructured, smooth MNA controls in rats. After 72 h, the nanostructured MNA cumulatively delivered 10.6 times more etanercept (*p < 0.01) and achieved a maximal serum concentration (Cmax) 13.9 times higher (*p < 0.01) than the smooth microneedles (n = 4 animals for each category). (c) Nanostructured MNAs deliver significantly more etanercept transdermally than unstructured, smooth MNA controls in rabbits. After 72 h, the nanostructured MNA devices cumulatively delivered 35 times more etanercept (*p < 0.01) and achieved a Cmax 10.2 times higher than smooth MNA controls (*p < 0.01) (n = 4 animals for each category).

Figure 2

Nanotopography leads to reversible downregulation of claudin-1 and -4 expression in cultured human keratinocytes. (a,b) Day 8 primary human keratinocytes showed marked diminishment in claudin-1 and -4 expression after 24 h incubation with nanotopography, compared to controls that were exposed to no device or to unstructured, flat films. Claudin-1 was decreased by 58% with polyether ether ketone (PEEK)-based nanostructured films (NS PEEK) and by 36% with polypropylene-based nanostructured films (NS PP), relative to no device controls (**p < 0.01). Claudin-4 was decreased by 49% with PEEK nanostructured films and by 39% with polypropylene nanostructured films, relative to no device controls (**p < 0.01). Exposure to unstructured, flat films (Flat PP) had no effect on claudin-1 or -4 expression relative to no device controls (scale bar = 10 μm). Fluorescence intensity was normalized by area. (c,d) Twenty four hours following removal of nanotopography, the decrease in claudin-1 and -4 expression was reversed, and there was no statistically significant difference between nanotopography-treated keratinocytes (NS PP and NS PEEK) versus no device or unstructured, flat controls (scale bar = 10 μm).

Figure 3

Nanotopography-induced disruption of TJ structure is conserved among different epithelia. (a,b) In Caco-2 cells, nanostructured films (NS) induce decreased expression of claudin-1 and -4 at cell borders, relative to controls exposed to no film or to unstructured, flat film (Flat). Claudin-1 was decreased 50% compared to no film control and 73% compared to the flat film control (**p < 0.01). Claudin-4 was decreased 76% and 66% compared with the no film or flat film controls, respectively (**p < 0.01). Occludin immunostaining reveals a ruffled pattern when Caco-2 cells are in contact with the nanostructured film, in contrast to the stereotypical cobblestone pattern in control cells exposed to no film or unstructured, flat film (scale bar = 20 μm). Occludin ruffling, as measured by cell perimeter, was increased with nanotopography by 87% over cells alone and 118% over flat (**p < 0.01). Fluorescence intensity was normalized by number of cells. (c) TEM imaging of the tight junction (TJ) (solid black arrow), adherens junction (AJ) (dashed black arrow), and desmosome (solid white arrow). Cells exposed to unstructured, flat films (Flat) show a partial loss of electron density in the TJ and AJ, relative to no film controls. Cells exposed to nanostructured films (NS) show near complete loss of electron density in the TJ and AJ, as well as partial loss in the underlying desmosome, relative to no film controls (scale bar = 200 nm).

Figure 4

Nanotopography leads to clustering of focal adhesion proteins and activation of myosin light chain. (a–c) In Caco-2 cells, exposure to the nanostructured film (NS) leads to increased staining of pFAK, vinculin, and pMLC, relative to controls cells exposed to either no film or to flat, unstructured film (Flat) (scale bar = 20 μm). Nuclear staining is highlighted by DAPI in blue, while protein immunostaining is highlighted in green. (d–f) pFAK was increased by 112% and 43% compared with the no film or flat film controls, respectively (**p < 0.01). Vinculin was increased by 117% and 91% compared with the no film or flat film controls, respectively (**p < 0.01). pFAK was increased by 263% and 161% compared with the no film or flat film controls, respectively (**p < 0.01).

Figure 5

Nanotopography-mediated drug delivery and down-regulation of TJs requires integrin activation and phosphorylation of myosin light chain. (a) The integrin-binding RGDS peptide is suficient to recapitulate the effect of nanotopography on Caco-2 permeability. As expected, cells exposed to nanostructured films without RGDS (open diamond) showed decreased transepithelial electrical resistance (TEER) relative to controls exposed to flat, unstructured film without RGDS (solid square). The addition of RGDS led to diminishment of TEER in both nanostructured and flat film groups, abrogating the previous difference observed in the absence of RGDS (n = 4). (b) Integrin function is necessary for nanotopography-mediated drug transport. In the absence of integrin blockade, nanostructured films (NS) significantly increase transport of FITC-BSA (left panel) (*p < 0.05). In the presence of integrin blockade, this nanotopography-induced increase in BSA transport is abolished, and there are no differences between the nanotopography and unstructured, flat film groups (n = 4). (c) Myosin light chain kinase (MLCK) function is necessary for nanotopography-mediated drug transport. In the absence of MLCK blockade, nanostructured films (NS) significantly increase transport of FITC-BSA (left panel) (*p < 0.05). In the presence of MLCK blockade, this nanotopography-induced increase in BSA transport is abolished, and there are no differences between the nanotopography and unstructured, flat film groups (n = 4). (d) Myosin light chain kinase (MLCK) function is necessary for nanotopography-mediated changes in TJ structure. ZO-1 immunostaining adopts a ruffled pattern in Caco2 cells exposed to nanostructured films (NS), indicating a disruption of the TJ complex. After addition of MLCK inhibitor, this ruffling is abolished, and cells exposed to nanotopography resume the canonical ZO-1 pattern shown in control cells exposed to either no film or flat, unstructured films.

Figure 6

Nanotopography downregulates epidermal tight junctions via integrin activation and myosin light chain phosphorylation. Proposed mechanism for nanotopography-mediated enhancement of transdermal drug delivery: Integrins bind to adsorbed proteins on the nanotopography and induce downstream clustering of focal adhesion proteins such as pFAK and vinculin. Formation of focal adhesions, in turn, leads to phosphorylation of myosin light chain and increased myosin contraction of the actin cytoskeleton. Actin dynamics ultimately remodel and downregulate proteins at the epidermal tight junction, thereby allowing increased paracellular transport within the epidermis. By contrast, we propose that the lack of topography on the flat surfaces fails to induce integrin clustering and the downstream initiation of focal adhesions and actomyosin activation (right lower image).