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

Abstract

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.

Fig 1. The three lipidoids used in this study were synthesized by the Michael addition of alkyl-acrylates to alkyl-amines.

Fig 2. The three LNPs had average diameters on the order of 100 nm as determined by dynamic light scattering.

The mean light intensity is shown vs. diameter size distribution for each lipidoid nanoparticle. Error bars represent s.d. (n = 3).

Fig 3. All three LNPs facilitated GAPDH mRNA silencing in Caco-2 cells.

A) LNPs 306O₁₃ and 303O₁₃ mediated ~90% silencing at an siRNA dose of 100 nM while control LNPs containing off-targeted siRNA did not induce statistically significant changes in gene expression. ** p < 0.001 as determined by an unpaired student’s t-test to the untreated control. B) The 306O₁₃ LNPs had the most potent dose-dependent GAPDH gene silencing in Caco-2 cells. The LNP control consisted of 100 nM siGFP 306O₁₃ LNPs. Error bars represent s.d. (n = 3).

Fig 4. Neither A) Control (siGFP) nor B) siGAPDH loaded LNPs adversely affected Caco-2 cell viability at siRNA doses up to 200 nM.

All LNPs were incubated with cells for 24 hours prior to measuring viability with an MTT assay. Error bars represent s.d. (n = 7).

Fig 5. GAPDH silencing in Caco-2 cells with the LNP 306O₁₃ was potent and durable.

A single dose of 10 nM siGAPDH depressed mRNA expression for one week, with maximal silencing of 80% observed 24–60 hours post-transfection. Error bars represent s.d. (n = 3).

Fig 6. The LNP 306O₁₃ facilitated dose dependent GAPDH mRNA silencing in Caco-2 monolayers 24 hours post-transfection.

Caco-2 monolayer barrier function as measured by TEER was not affected by LNP mediated gene silencing. Dose response data (blue circles) indicate an EC₅₀ siGAPDH dose of 10 nM. TEER values (black squares) are reported 24 hours post-transfection relative to the time of transfection (t = 0), normalized to untreated cells. Error bars represent s.d. (n = 3–6).

Fig 7. A single dose of 100 nM siGAPDH 306O₁₃ LNPs gradually reduced GAPDH protein silencing in Caco-2 cells over a period of 5 days.

a) α-tubulin was used as a loading control and blot shows chemiluminescence signal from a PVDF membrane. b) Plotting the band density of GAPDH relative to α-tubulin, as quantified by ImageJ, suggests that a high level of protein silencing 85% was achieved by Day 5.