Efficient mucosal delivery of optical contrast agents using imidazole-modified chitosan

Abstract

The clinical applicability of antibodies and plasmonic nanosensors as topically applied, molecule-specific optical diagnostic agents for noninvasive early detection of cancer and precancer is severely limited by our inability to efficiently deliver macromolecules and nanoparticles through mucosal tissues. We have developed an imidazole-functionalized conjugate of the polysaccharide chitosan (chitosan-IAA) to enhance topical delivery of contrast agents, ranging from small molecules and antibodies to gold nanoparticles up to 44 nm in average diameter. Contrast agent uptake and localization in freshly resected mucosal tissues was monitored using confocal microscopy. Chitosan-IAA was found to reversibly enhance mucosal permeability in a rapid, reproducible manner, facilitating transepithelial delivery of optical contrast agents. Permeation enhancement occurred through an active process, resulting in the delivery of contrast agents via a paracellular or a combined paracellular/transcellular route depending on size. Coadministration of epidermal growth factor receptor-targeted antibodies with chitosan-IAA facilitated specific labeling and discrimination between paired normal and malignant human oral biopsies. Together, these data suggest that chitosan-IAA is a promising topical permeation enhancer for mucosal delivery of optical contrast agents.

Figure 1

Chemical schematic for the synthesis of imidazole-modified chitosan (chitosan-IAA).

Figure 2

Stromal accumulation of fluorescent macromolecules following topical permeation treatment. Freshly excised bladder tissue was treated for 15 min with 0.01% w/v chitosan-IAA, chitosan, or media and then probed with a 1:1:1 mixture of 3 kDa rhodamine-dextran, 40 kDa fluorescein-dextran, and Alexa 647-IgG. The yellow/white lines indicate the boundary between the epithelium and stroma. In reflectance images, the epithelium was distinguished from the stroma by its darker appearance. Following permeation treatment, the fluorescent macromolecules accumulated in the stroma. Stromal accumulation was brighter following treatment with imidazole-modified chitosan than with nonmodified chitosan. In media-treated controls, macromolecule penetration was limited to a few cells in the superficial epithelium. The scale bar represents 100 μm. The epithelium (E) and stroma (S) are labeled in the first image. (Color online only.)

Figure 3

Stromal accumulation of gold nanoparticles following topical permeation treatment. Freshly excised bladder tissue was treated for 15 min with 0.01% w/v chitosan-IAA, chitosan, or media, and then fluorescein-PEG-gold spheres of 44- and 33-nm diameter were applied topically. Transepithelial delivery of both sizes of nanoparticles was observed by monitoring for the localization of nanoparticle-associated fluorescence. The stromal reflectance was visibly enhanced in samples showing accumulation of nanoparticles. No significant change in stromal fluorescence or reflectance was observed in media-treated controls. The scale bar represents 100 μm.

Figure 4

Fluorescence intensity as a function of time and temperature. Tissues pretreated with chitosan-IAA were washed and allowed to recover at 37 °C (left) or 4 °C (right). The recovery of barrier function was probed at regular time intervals using fluorescent macromolecules and nanoparticles. Tissues held at 37 °C rapidly recovered barrier function, while tissues held at 4 °C displayed continued permeability as evidenced by stromal fluorescence. The ability of the recovering epithelium to block contrast agents was size dependent, with larger molecules excluded more rapidly than smaller molecules.

Figure 5

Representative confocal images of antiEGFR-647 labeling in fresh human oral biopsies co-treated with chitosan-IAA, collected at the same gain. Biopsies were topically treated with antiEGFR-647 diluted in 0.01% w/v chitosan-IAA for 1 h at 37 °C, washed three times, sliced transversely, and imaged. In normal tissues (top), EGFR labeling is limited to the basal layer of epithelial cells, appearing as a faint region of labeling at the base of the epithelium. Nonspecific labeling is observed at the apical surface of keratinized tissues such as the retromolar trigone tissue shown below. EGFR labeling in squamous cell carcinoma (bottom) is characterized by bright, ring-like extracellular labeling extending downward from the tissue surface. The scale bar represents 100 μm.

Figure 6

Measured mean fluorescence intensity of paired oral biopsies collected from patients with cancer of the buccal (1), retromolar trigon (2), maxilla (3, 4), and tongue (5) regions. Biopsies were collected from clinically abnormal and contralateral normal regions. The biopsy pairs were topically labeled for EGFR in the presence of chitosan-IAA and imaged transversely. Statistically significant differences (P⩽0.01) were observed in mean fluorescence between biopsy pairs.

Video 1

Optical sectioning of bladder epithelium and stroma following the topical application of 3 kDa rhodamine-dextran. The mucosal surface was pretreated with chitosan-IAA30 for 15 min at 37 °C. Confocal fluorescence images were acquired parallel to the tissue surface in 2-μm steps with constant laser power and gain. Rhodamine-dextran permeation through the epithelium of the tissue appears to follows a paracellular route, appearing as bright rings around cells and minimal labeling within the cells. As the imaging progresses deeper, shown in the video, the stroma appears, clearly defined by a selective accumulation of contrast agents. Representative images, taken at a depth of 10 μm, are shown here. The scale bar represents 100 μm (QuickTime, 3.4 MG)..

Video 2

Optical sectioning of bladder epithelium and stroma following the topical application of 44 nm fluorescein-PEG-gold. The mucosal surface was pretreated with chitosan-IAA30 for 15 min at 37 °C. Confocal fluorescence images were acquired parallel to the tissue surface in 2-μm steps with constant laser power and gain. Representative images, taken at a depth of 10 μm, are shown here. Nanoparticle transport in the epithelium appears to follow both paracellular and transcellular routes, appearing as bright rings around cells and diffuse labeling within cells. Individual nuclei appear as a black spot within each cell. Deeper imaging within the tissue samples, seen in the video, demonstrates clear nanoparticle uptake throughout the stromal regions. The scale bar represents 100 μm (QuickTime, 3.3 MG). .