Increasing Permeability of the Intestinal Epithelium to Enable Oral Delivery of Protein Drugs

Abstract

Oral delivery of macromolecular drugs, especially bioactive proteins, is one of the greatest unmet needs in modern biomedicine. Although engineering solutions have been developed to overcome low pH and enzymatic degradation in the stomach, poor absorption across the intestinal epithelial barrier and into the bloodstream continues to hinder clinical viability of oral protein formulations. One common solution is to employ chemical permeation enhancers of the epithelium. Unfortunately, most efficacious enhancers have been thwarted by toxicity, and the mechanisms that contribute to this behavior are poorly understood. Thus, there is an ongoing need to develop and characterize nontoxic permeation enhancers. First, this work seeks to expedite cell culture screening for permeation enhancer candidates by reducing the cost- and time inputs required for Caco-2 cell monolayers, the most common model of the intestinal epithelium. A new, 3-day system deemed “thrifty, rapid intestinal monolayers” (TRIM) is developed, comparing favorably with two current monolayer systems. Next, the work explores the mechanism by which piperazines, a family of known permeation enhancers, interact with cells. Interestingly, the pH of the piperazine solutions presents as the controlling parameter, even when accounting for effects from pH change alone. The piperazines nonetheless suffer from narrow therapeutic windows, underscoring the need for a new generation of enhancers that do not permanently damage the intestines. The next portion of this work screens an extensive, food-based library for nontoxic but effective chemicals to improve intestinal protein absorption. Of 106 crude food extracts, the vast majority are not cytotoxic, and only a small fraction increase epithelial permeability. An iterative separation-activity screening method is used to isolate a single, active compound: the polyphenolic molecule pelargonidin from strawberries. Pelargonidin is demonstrated as a reversible, efficacious permeation enhancer, enabling uptake of model drugs and insulin through the intestinal lining. Finally, this work presents the surprising ability of anionic nanoparticles to act as physicochemical permeation enhancers. Orally administered silica particles induce dose-dependent permeabilization of the intestinal epithelium, allowing for oral delivery of insulin and exenatide. Histology of treated mouse intestines shows no evidence of particle-induced necrosis, inflammation, or changes in tissue architecture. These conclusive results underscore the ability of silica nanoparticles to safely increase epithelial permeability, enabling the oral delivery of macromolecular drugs without encapsulation or conjugation.