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

Abstract

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 4 kDa fluorescent dextran but not 70 kDa 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 70 kDa 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.

Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos

“…However, the importance of the presence of the CPP for exocytosis or direct translocation across the basolateral membrane subsequent to cellular internalization via the apical side of the plasma membrane is not known. In addition, paracellular delivery across the epithelium or endothelium may contribute to the net delivery of the cargo by directly affecting the opening and closure of the TJs via local high CPP concentrations [ 89 ] or indirectly through the binding to intracellular targets regulating the TJ dynamics [ 14 ].…”

“…In addition to their application as inert vectors for delivery of cargo molecules, an emerging concept is the dual-acting CPPs, which are both membrane permeating and bioactive. Within this context, studies have demonstrated that, in addition to being cell-penetrating, selected CPPs are able to safely modulate the intestinal paracellular barrier [ 14 , 15 ], to act as neuroprotectants [ 16 ], to or induce apoptosis in cancer cells [ 17 ].…”

Shortcut Approaches to Substance Delivery into the Brain Based on Intranasal Administration Using Nanodelivery Strategies for Insulin

“…Insulin co-administered with PIP peptide 250 or 640 reduced blood glucose levels by 50%, respectively, after intraluminal intestinal injection in an in vivo assay using male Wistar rats. Cy3-labeled insulin was detected predominantly in the paracellular route after intraluminal intestinal injection with PIP peptide 640 [36]. Structurally, both PIP peptides 250 and 640 belong to CPPs due to their arginine-rich cationic charge.…”

Paradigm shift in bacteriophage-mediated delivery of anticancer drugs: from targeted ‘magic bullets’ to self-navigated ‘magic missiles’

“…However, no targeting ligands have been identified to increase paracellular transport in vivo . Recently, peptides were designed to reversibly open tight junctions of polarized intestinal epithelial cells [35, 37]. It is though that targeting with these peptides may increase paracellular transport activity.…”