Quantifying Tight Junction Disruption Caused by Biomimetic pH-Sensitive Hydrogel Drug Carriers

Abstract

Facilitation of protein transport across biomimetic polymers and carriers used in drug delivery is a subject of major importance in the field of oral delivery. Quantitative immunofluorescence of epithelial tight junctions can be a valuable tool in the evaluation of paracellular permeation enhancement and macromolecular drug absorption. The tight junctional space is composed of transmembrane protein networks that provide both mechanical support and a transport barrier. Both of these may be affected by drug delivery agents that enhance paracytosis. Imaging is the only tool that can tease apart these processes. A confocal microscopy imaging method was developed to determine the effect of microparticulate poly(methacrylic acid) grafted poly(ethylene glycol) (P(MAA-g-EG)) hydrogel drug carriers on the integrity of claudin-1 and E-cadherin networks in Caco-2 monolayers. Z-stack projection images showed the lateral disruption of tight junctions in the presence of drug carriers. Tight junction image fraction measurements showed more significant differences between membranes exposed to microparticles and a control group. Mechanical disruption was much more pronounced in the presence of P(MAA-g-EG) microparticles as compared to the effect of EDTA.

Figure 1

Scanning electron micrograph of P(MAA-g-EG) microparticles crushed and passed through a 150 μm sieve.

Figure 2

TEER change over time in Caco-2 monolayers exposed to 10 mg/ml P(MAA-g-EG) microparticles in the apical chamber (n=6) (▲), 2 μM EDTA in the apical chamber(n=3) (●) and a control group (n=3) (■) . Monolayers were grown on 4.71 cm² membranes with 3 μm pore sizes. All membranes contained HBSS without calcium or magnesium in both the apical and basolateral chambers. Each data point represents ±SD. *p < 0.05, significantly different from control group at all time points.

Figure 3

Maximum projection z-stack images of polarized Caco-2 monolayers double immunolabeled for claudin-1 (a-f) and E-cadherin (g-l) and imaged with 63× objective power at 1024×1024 pixel resolutions. The left column (a, d, g, j) shows a control monolayer, the middle column (b, e, h, k) shows a monolayer exposed to 10 mg/ml P(MAA-g-EG) microparticles and the right (c, f, i, l) shows a monolayer exposed to 2 μm EDTA, both in the apical chamber. The images that result from binary conversion at a 50% intensity threshold (d-f, j-l) are show just below the original.

Figure 4

Image fraction of immunolabeled E-cadherin and claudin-1 in Caco-2 monolayers at lowest resistance value. Z-stack images were at 20x objective power and projections were converted to binary images at a 30% intensity threshold. *, ¥ donate statistically significant difference from control for p<0.00001 and p<0.0001 respectively. Data points are mean ±1 SD. The average E-cadherin image fraction increases 1.71 times the control for microparticle treated group and 1.48 times for EDTA. The average claudin-1 image fraction increased 3.34 and 4.57 times for the microparticle and EDTA treated groups, respectively, versus the control.

Figure 5

Normalized image intensity of immunolabeled E-cadherin and claudin-1 in Caco-2 monolayers at lowest resistance value. Z-stack images were at 20× objective power. *, ¥ donate statistically significant difference from control for p<0.0008 and p<0.005 respectively. Data points are mean ±1 SD.