Larazotide acetate regulates epithelial tight junctions in vitro and in vivo.

Tight junctions (TJs) control paracellular permeability and apical-basolateral polarity of epithelial cells, and can be regulated by exogenous and endogenous stimuli. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. Herein we studied the mechanism by which larazotide acetate, an 8-mer peptide and TJ regulator, inhibits the cellular changes elicited by gliadin fragments, AT-1002, and cytokines. Previously, we demonstrated that AT-1002, a 6-mer peptide derived from the Vibrio cholerae zonula occludens toxin ZOT, caused several biochemical changes in IEC6 and Caco-2 cells resulting in decreased transepithelial electrical resistance (TEER) and increased TJ permeability. In this study, larazotide acetate inhibited the redistribution and rearrangement of zonula occludens-1 (ZO-1) and actin caused by AT-1002 and gliadin fragments in Caco-2 and IEC6 cells. Functionally, larazotide acetate inhibited the AT-1002-induced TEER reduction and TJ opening in Caco-2 cells. Additionally, larazotide acetate inhibited the translocation of a gliadin 13-mer peptide, which has been implicated in celiac disease, across Caco-2 cell monolayers. Further, apically applied larazotide acetate inhibited the increase in TJ permeability elicited by basolaterally applied cytokines. Finally, when tested in vivo in gliadin-sensitized HLA-HCD4/DQ8 double transgenic mice, larazotide acetate inhibited gliadin-induced macrophage accumulation in the intestine and preserved normal TJ structure. Taken together, our data suggest that larazotide acetate inhibits changes elicited by AT-1002, gliadin, and cytokines in epithelial cells and preserves TJ structure and function in vitro and in vivo.

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes.

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern-Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells.

Structure-activity relationship studies of permeability modulating peptide AT-1002.

AT-1002 a 6-mer synthetic peptide belongs to an emerging novel class of compounds that reversibly increase paracellular transport of molecules across the epithelial barrier. The aim of this project was to elaborate on the structure-activity relationship of this peptide with the specific goal to replace the P2 cysteine amino acid. Herein, we report the discovery of peptides that exhibit reversible permeability enhancement properties with an increased stability profile.

Endocytosis inhibitors prevent poly(amidoamine) dendrimer internalization and permeability across Caco-2 cells.

Previous studies from our group demonstrated visual evidence that endocytosis mechanism(s) contribute to the internalization and intracellular trafficking of cationic and anionic poly(amidoamine) (PAMAM) dendrimers across Caco-2 cells. These dendrimers colocalized with established endocytosis markers, which suggested PAMAM dendrimers may be internalized by a clathrin-dependent endocytosis mechanism and are rapidly trafficked to endosomal and lysosomal compartments. In the present study, generation 4 PAMAM-NH2 (G4NH2) dendrimer was labeled with tritium to measure the rate of uptake and permeability in Caco-2 cells. The effect of endocytosis inhibitors brefeldin A, colchicine, filipin, and sucrose on G4NH2 absorption and transport was examined to give further insight into the endocytosis mechanisms that transport PAMAM dendrimers across Caco-2 cell monolayers. G4NH2 showed linear uptake at lower concentrations, and rapidly increased as a function of concentration. The rate of G4NH2 uptake significantly declined at high concentrations in the presence of the endocytosis inhibitors, and the apparent permeability similarly reduced in the presence of these inhibitors. A significant reduction in G4NH2 permeability was observed in the presence of brefeldin A and colchicine, which generally disrupt vesicular trafficking and formation during the endocytosis process. Coincubation with filipin and sucrose reduced G4NH2 permeability to a lesser extent, which suggests G4NH2 could be nonspecifically internalized in coated vesicles at the plasma membrane. The observations from this study further confirm that G4NH2 internalization and transport involves an endocytosis pathway.

Surface acetylation of polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane permeability.

Improving the oral bioavailability of therapeutic compounds remains a challenging area of research. Polyamidoamine (PAMAM) dendrimers are promising candidates for oral drug delivery due to their well-defined compact structure, versatility of surface functionalities, low polydispersity, and ability to enhance transepithelial transport. However, potential cytotoxicity has hampered the development of PAMAM dendrimers for in vivo applications. In this article, we have systematically modified the surface groups of amine-terminated PAMAM dendrimers with acetyl groups. The effect of this modification on cytotoxicity, permeability, and cellular uptake was investigated on Caco-2 cell monolayers. Cytotoxicity was reduced by more than 10-fold as the number of surface acetyl groups increased while maintaining permeability across the cell monolayers. Furthermore, a decrease in nonspecific binding was evident for surface-modified dendrimers compared to their unmodified counterparts. These studies point to novel strategies for minimizing PAMAM dendrimer toxicity while maximizing their transepithelial permeability.

Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells.

PURPOSE: To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. MATERIALS AND METHODS: PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. RESULTS: Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. CONCLUSION: These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.

Transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers: Influence of size, charge and fluorescent labeling.

PURPOSE: To investigate the transport of poly(amidoamine) (PAMAM) dendrimers with positive, neutral and negatively charged surface groups across Caco-2 cell monolayers. METHODS: Cationic PAMAM-NH2 (G2 and G4), neutral PAMAM-OH (G2), and anionic PAMAM-COOH (G1.5-G3.5) dendrimers were conjugated to fluorescein isothiocyanate (FITC). The permeability of fluorescently labeled PAMAM dendrimers was measured in the apical-to-basolateral direction. 14C-Mannitol permeability was measured in the presence of unlabeled and FITC labeled PAMAM dendrimers. Caco-2 cells were incubated with the dendrimers followed by mouse anti-occludin or rhodamine phalloidin, and visualized using confocal laser scanning microscopy to examine tight junction integrity. RESULTS: The overall rank order of PAMAM permeability was G3.5COOH > G2NH2 > G2.5COOH > G1.5COOH > G2OH. 14C-Mannitol permeability significantly increased in the presence of cationic and anionic PAMAM dendrimers with significantly greater permeability in the presence of labeled dendrimers compared to unlabeled. PAMAM dendrimers had a significant influence on tight junction proteins occludin and actin, which was microscopically evidenced by disruption in the occludin and rhodamine phalloidin staining patterns. CONCLUSIONS: These studies demonstrate that enhanced PAMAM permeability is in part due to opening of tight junctions, and that by appropriate engineering of PAMAM surface chemistry it is possible to increase polymer transepithelial transport for oral drug delivery applications.

Transepithelial and endothelial transport of poly (amidoamine) dendrimers.

This article summarizes our efforts to evaluate the potential of poly (amidoamine) (PAMAM) dendrimers as carriers for oral drug delivery. Specifically, the permeability of a series of cationic PAMAM-NH2 (G0-G4) dendrimers across Caco-2 cell monolayers was evaluated as a function of dendrimer generation, concentration, and incubation time. The influence of dendrimer surface charge on the integrity, paracellular permeability, and viability of Caco-2 cell monolayers was monitored by measuring the transepithelial electrical resistance (TEER), 14C-mannitol permeability, and leakage of lactate dehydrogenase (LDH) enzyme, respectively. Microvascular extravasation of PAMAM-NH2 dendrimers in relation to their size, molecular weight, and molecular geometry is also discussed. Results of these studies show that transepithelial transport and microvascular extravasation of PAMAM dendrimers are dependent on their structural features including molecular size, molecular geometry, and surface chemistry. These results suggest that by optimizing the size and surface charge of PAMAM dendrimers, it is possible to develop oral delivery systems based on these carriers for targeted drug delivery.