105 M-Cell Targeting by Neuraminidase Functionalized Microparticles for Future Application in Oral Immunotherapy

BACKGROUND: Recently, we demonstrated in an experimental mouse study that mucosal M-cell targeting with Aleuria aurantia lectin (AAL) coated Poly(D,L-lactide-co-glycolide) (PLGA) microspheres represents a promising oral treatment approach in IgE mediated allergy. Due to its structural similarities with AAL we aimed to assess Neuraminidase (NA) from Vibrio cholerae as a novel M-cell specific targeters and compared its properties to AAL and wheat germ agglutinin (WGA) representing 2 plant lectins, which target either M-cells or epithelial cells, respectively. METHODS: The resistance against gastric digestion of NA, AAL and WGA was analyzed in simulated gastric fluid (SGF) experiments. Intestinal epithelial binding was determined using the colon carcinoma cell line Caco2, which represents a well established model for the human intestinal epithelium. Binding specificity was evaluated by inhibition experiments by incubating Caco2 cells with Biotin-labeled NA, AAL or WGA, after preincubation with a-L fucose, monoganglioside (GM1) or N,N′,N″-triacetyl-chitotriose (TCT). The stimulatory effects of the targeting substances on the intestinal microenvironment were investigated by cytokine read-out experiments in real-time PCR. Further, the transeptihelial uptake of NA-, AAL- or WGA-functionalized fluospheres was evaluated in a human M-cell co-culture model. RESULTS: All 3 targeters were stable up to 180 minutes in SGF, indicating their suitability for oral application. The binding partners were a-L fucose for AAL and TCT for WGA, whereas NA interacts with intestinal epithelial cells via a-L fucose and additionally GM1. NA skewed the cytokine production by inducing a 2-fold increase of the Th1 cytokine IFNg after 60 minutes, whereas AAL decreased the overall cytokine expression. In a human M-cell co-culture model, a higher transepithelial transport rate of fluospheres coated with NA and AAL was observed as compared to WGA and plain particles. CONCLUSIONS: NA specifically targets M-cells via a-L fucose and additionally GM1 and, thus, increases the transepithelial transport of NA coated particles. Due to the immunomodulatory capacity on intestinal epithelial cells, NA functionalized microspheres may represent a promising M-cell specific targeting approach for oral immunotherapy.