Nanostructure-mediated transport of biologics across epithelial tissue: enhancing permeability via nanotopography

Abstract

Herein, we demonstrate that nanotopographical cues can be utilized to enable biologics >66 kDa to be transported across epithelial monolayers. When placed in contact with epithelial monolayers, nanostructured thin films loosen the epithelial barrier and allow for significantly increased transport of FITC-albumin, FITC-IgG, and a model therapeutic, etanercept. Our work highlights the potential to use drug delivery systems which incorporate nanotopography to increase the transport of biologics across epithelial tissue.

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1

Nanostructured thin film fabrication. Molds for NIL were fabricated using (a) electron beam lithography followed by anisotropic reactive ion etching to generate small features on the nanometer length scale. (b) Next, nanoimprint lithography was employed to imprint the nanofeatures from the nanofeatured mold into a polypropylene thin film through a stamping process. (c) A scanning electron microscopy image of the low AR nanostructured film, P(1.5), shows nanopillar features with an average height of 300 nm and an average diameter of 200 nm. The scale bar is 400 nm. The high AR nanostructured film, P(20), has features with an average height of 16 μm and an average diameter of 800 nm. The scale bar is 3 μm. The schematic demonstrates how P(1.5) with the lower surface roughness is capable of more focal contact points with the cell (yellow dots) compared to the fewer contact points between the cell and P(20).

Figure 2

In vitro transport studies. Transport studies show that P(1.5) significantly enhances the transport of high MW species across the Caco-2 cell monolayers over 2 h. Data are displayed as the mean mass in micrograms (± standard deviation). Parts (a), (b), and (c) are the transport of FITC-BSA, FITC-IgG, and etanercept, respectively. Part (d) is a schematic of the transport study setup. The nanostructured thin film is placed directly in contact with the Caco-2 monolayer. The drug solution is placed in the apical chamber and is sampled (with PBS replacement) periodically from the basal camber. (e) It appears that the IgG-FITC (green) is located around the Caco-2 cells (blue Hoechst) directly in the paracellular space. The scale bars represent 20 μm.

Figure 3

Active transport processes. (a) Etanercept transport studies performed at 4 and 37°C show significantly higher drug concentration in the basal chamber at physiological temperature. The transport is retarded at 4°C. These results indicate that the enhanced transport is due to active transport instead of passive mechanisms. (b) Dynasore was used to inhibit dynamin-mediated endocytosis and shows no significant effect on the transport of etanercept across the epithelial monolayer. (c) Similarly, genistein was used to inhibit caveolae-mediated endocytosis and also does not affect the transport of the etanercept.

Figure 4

Tight junction morphological changes. Immunofluorescence staining of the tight junction protein, zonula occluden (ZO-1), was performed. (a) Staining of the untreated caco-2 monolayer shows a normal cobblestone morphology. (b) When the flat unimprinted polypropylene control film is placed on the monolayer, minimal disruptions in ZO-1 are observed as indicated by the discontinuous lines (pointed out by the white arrows). (c) When the flat film is removed, the relatively low staining intensity of the ZO-1 remains the same after 24 h. (d) However, it is apparent that the nanostructured thin film induces a dramatic ruffled morphology after 2 h (see arrows), indicating tight junction remodeling and a loosening of the epithelial barrier to allow for paracellular transport. (e) After the nanostructured thin film was removed from the monolayer and incubated for 24 h, the ZO-1 morphology reverted back to the normal cobblestone architecture, indicating a reversible rearrangement. (f) TEER measurements before and after the nanostructured surface is placed in contact with the cells. TEER measurements decreased in the presence of both the nanostructured and the flat films. However, the monolayer that had been in contact with the nanostructured film eventually increased after 24 h which demonstrates the reversible and nondeleterious effects of the nanostructures on the cells. In contrast, the monolayer that had been in contact with the flat film does not recover to a higher TEER value after 24 h. Scale bars for the three top images and three bottom images are 10 and 20 μm, respectively.

Figure 5

Quantitative PCR studies. The gene expression levels of the signaling molecules, myosin light chain kinase (MLCK) and focal adhesion kinase (FAK), are displayed in (a) and (b). The gene expression levels of tight junction proteins zonula occludin-1 (ZO-1) and occludin (ocln) are also displayed in (c) and (d). Data are normalized by expression levels of each gene by the controls (Untreated) and presented as an average ± standard deviation. *p < 0.05, **p < 0.005, ***p < 0.002, and ****P < 0.001, n = 3.