Lipidoid Nanoparticles for siRNA Delivery to the Intestinal Epithelium: In Vitro Investigations in a Caco-2 Model

PLOS ONE, Dec 2019

Short interfering ribonucleic acid (siRNA) therapeutics show promise for the treatment of intestinal diseases by specifically suppressing the expression of disease relevant proteins. Recently, a class of lipid-like materials termed “lipidoids” have been shown to potently deliver siRNA to the liver and immune cells. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of siRNA delivery to the intestinal epithelium. Initial studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal epithelial cells, with a single 10 nM dose depressing GAPDH mRNA expression for one week. Transfection with siRNA-loaded LNPs did not induce significant cytotoxicity in Caco-2 cells or alter intestinal barrier function. Protein silencing was confirmed by Western blotting, with the lowest levels of GAPDH protein expression observed five days post-transfection. Together, these data underscore the potential of LNPs for the treatment of intestinal disorders.

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Lipidoid Nanoparticles for siRNA Delivery to the Intestinal Epithelium: In Vitro Investigations in a Caco-2 Model

July Lipidoid Nanoparticles for siRNA Delivery to the Intestinal Epithelium: In Vitro Investigations in a Caco-2 Model Rebecca L. Ball 0 2 3 Christopher M. Knapp 0 2 3 Kathryn A. Whitehead 0 2 3 0 1 Department of Chemical Engineering, Carnegie Mellon University , Pittsburgh , Pennsylvania, United States of America, 2 Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania , United States of America 1 was provided. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Funding: This work was funded by the Center for Nucleic Acids Science and Technology ( 3 Editor: Michael Koval, Emory University School of Medicine, UNITED STATES Short interfering ribonucleic acid (siRNA) therapeutics show promise for the treatment of intestinal diseases by specifically suppressing the expression of disease relevant proteins. Recently, a class of lipid-like materials termed “lipidoids” have been shown to potently deliver siRNA to the liver and immune cells. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of siRNA delivery to the intestinal epithelium. Initial studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal epithelial cells, with a single 10 nM dose depressing GAPDH mRNA expression for one week. Transfection with siRNA-loaded LNPs did not induce significant cytotoxicity in Caco-2 cells or alter intestinal barrier function. Protein silencing was confirmed by Western blotting, with the lowest levels of GAPDH protein expression observed five days post-transfection. Together, these data underscore the potential of LNPs for the treatment of intestinal disorders. - Competing Interests: KAW has a pending patent application related to the materials contained in this manuscript. The application title is “Amine-Containing Lipidoids and Uses Thereof”, U.S. Patent Application Intestinal diseases such as gastrointestinal cancer and inflammatory bowel disease (IBD) adversely affect the health and quality of life of millions of people worldwide[1,2]. Although numerous factors such as genetics, microbiota composition, environment and the immune system have been shown to play a specific role in development of these diseases, the underlying causal biology is complex and not fully understood[1,3–7]. However, there are some instances in which diseases of the gut have been associated with an over-expression of one or more proteins[6,8–10]. Accordingly, such intestinal maladies could potentially be treated with RNA interference (RNAi) theraputics, which rely on short interfering RNA (siRNA) to transiently knockdown the expression of problematic genes. As with the rest of the RNAi space, one of the key challenges in developing a clinically viable therapy for the intestines is the identification of a stable siRNA delivery vehicle that achieves potent gene silencing without inducing toxicity or immune stimulation[11,12]. To date, there has been limited work done in this area. Some classes of nanoparticles, including polymers and lipids, have been shown to deliver various drugs, including aminosalicylates, corticosteriods, and immunosuppresives, to intestinal epithelial cells in vitro and in vivo[12–14]. Other studies have shown that several types of nanoparticles and microspheres can be used to deliver siRNA to the macrophages within the gastrointestinal (GI) tract [15–17]. The most common gene target studied has been the inflammatory cytokine, TNF-α, which is produced by macrophages and has been implicated in the progression of inflammatory bowel disease[4,18]. Although anti-TNF-α therapies can be effective in alieviating symptoms of IBD in some patients, serious side effects associated with continual immunosuppressive therapy often limits their use in the clinic [19,20]. Little focus has been placed on gene silencing within the epithelial cells that provide barrier function within the small and large intestines. In one study, amphiphilic polyallylamine polymeric micelles were shown to facilitate moderate levels of siRNA delivery to intestinal cells in vitro[21]. We believe delivery to epithelial cells warrants greater attention, as this approach may have utility in the treatment of several intestinal maladies. For example, intestinal cells prone to polyp formation have been shown to upregulate beta-catenin [22,23]. Another condition that may benefit from gene silencing therapy is salmonella infection, as salmonella bacteria mediate an upregulation in the tight junction protein claudin-2, leading to a leaky intestinal barrier[24]. Our approach seeks to use degradable lipidoid nanoparticles (LNPs) for siRNA delivery to the intestinal epithelium. The active delivery conponents of LNPs are lipid-like molecules, termed lipidoids, that are synthesized by the Michael addition of alkyl-amines to alkyl-acrylates[25]. Degradable (...truncated)


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Rebecca L. Ball, Christopher M. Knapp, Kathryn A. Whitehead. Lipidoid Nanoparticles for siRNA Delivery to the Intestinal Epithelium: In Vitro Investigations in a Caco-2 Model, PLOS ONE, 2015, Volume 10, Issue 7, DOI: 10.1371/journal.pone.0133154