Intestinal Transcytosis of a Protein Cargo and Nanoparticles Mediated by a Non-Toxic Form of Pseudomonas aeruginosa Exotoxin A.

The low permeability of nanoparticles (NPs) across the intestinal epithelium remains a major challenge for their application of delivering macromolecular therapeutic agents via the oral route. Previous studies have demonstrated the epithelial transcytosis capacity of a non-toxic version of Pseudomonas aeruginosa exotoxin A (ntPE). Here, we show that ntPE can be used to deliver the protein cargo green fluorescent protein (GFP) or human growth hormone (hGH), as genetic fusions, across intact rat jejunum in a model where the material is administered by direct intra-luminal injection (ILI) in vivo in a transcytosis process that required less than 15 min. Next, ntPE chemically coupled onto biodegradable alginate/chitosan condensate nanoparticles (AC NPs-ntPE) were shown to transport similarly to ntPE-GFP and ntPE-hGH across rat jejunum. Finally, AC NPs-ntPE loaded with GFP as a model cargo were demonstrated to undergo a similar transcytosis process that resulted in GFP being colocalized with CD11c+ cells in the lamina propria after 30 min. Control NP preparations, not decorated with ntPE, were not observed within polarized epithelial cells or within the cells of the lamina propria. These studies demonstrate the capacity of ntPE to facilitate the transcytosis of a covalently associated protein cargo as well as a biodegradable NP that can undergo transcytosis across the intestinal epithelium to deliver a noncovalently associated protein cargo. In sum, these studies support the potential applications of ntPE to facilitate the oral delivery of macromolecular therapeutics under conditions of covalent or non-covalent association.

Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis.

Cholix (Chx) is expressed by the intestinal pathogen Vibrio cholerae as a single chain of 634 amino acids (~70.7 kDa protein) that folds into three distinct domains, with elements of the second and third domains being involved in accessing the cytoplasm of nonpolarized cells and inciting cell death via ADP-ribosylation of elongation factor 2, respectively. In order to reach nonpolarized cells within the intestinal lamina propria, however, Chx must cross the polarized epithelial barrier in an intact form. Here, we provide invitro and invivo demonstrations that a nontoxic Chx transports across intestinal epithelium via a vesicular trafficking pathway that rapidly achieves vesicular apical to basal (A→B) transcytosis and avoids routing to lysosomes. Specifically, Chx traffics in apical endocytic Rab7+ vesicles and in basal exocytic Rab11+ vesicles with a transition between these domains occurring in the ER-Golgi intermediate compartment (ERGIC) through interactions with the lectin mannose-binding protein 1 (LMAN1) protein that undergoes an intracellular re-distribution that coincides with the re-organization of COPI+ and COPII+ vesicular structures. Truncation studies demonstrated that domain I of Chx alone was sufficient to efficiently complete A→B transcytosis and capable of ferrying genetically conjoined human growth hormone (hGH). These studies provide evidence for a pathophysiological strategy where native Chx exotoxin secreted in the intestinal lumen by nonpandemic V. cholerae can reach nonpolarized cells within the lamina propria in an intact form by using a nondestructive pathway to cross in the intestinal epithelial that appears useful for oral delivery of biopharmaceuticals.One-Sentence Summary: Elements within the first domain of the Cholix exotoxin protein are essential and sufficient for the apical to basal transcytosis of this Vibrio cholerae-derived virulence factor across polarized intestinal epithelial cells.

Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation.

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein-protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4kDa fluorescent dextran but not 70kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3-4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.