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

Ball RL, Knapp CM, Whitehead KA - PLoS ONE (2015)

Bottom Line: Recently, a class of lipid-like materials termed "lipidoids" have been shown to potently deliver siRNA to the liver and immune cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0133154.g003: 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).

Mentions: LNPs were then evaluated for their capacity to induce gene silencing in intestinal epithelial cells. In this study, we chose to work with Caco-2 cells, which are human colorectal adenocarcinoma cells commonly used to model the intestinal epithelium [31,32]. Upon differentiation, Caco-2 cells polarize into monolayers expressing microvilli and tight junctions that serve as a barrier to absorption [31–33]. For this proof-of-concept analysis, we sought to silence the model “housekeeping” protein, GAPDH, which is typically expressed at high levels and can be knocked down without causing adverse cellular events [34,35]. LNPs were delivered at an siGAPDH concentration of 100 nM to undifferentiated Caco-2 cells, and GAPDH mRNA expression was measured by qPCR 24 hours post-transfection. At this dose, 306O13 and 303O13 LNPs mediated high levels of GAPDH silencing of around 90% (Fig 3A). In order to identify the best lipidoid for Caco-2 work between the two, dose response experiments were conducted (Fig 3B). Results illustrate that 306O13 (blue curve with triangles) facilitated the most potent dose dependent response, with 75% GAPDH silencing achieved at a low dose of 10 nM. Based on this gene silencing data, the lipidoid 306O13 was used in all further gene silencing experiments.


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

Ball RL, Knapp CM, Whitehead KA - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4508104&req=5

pone.0133154.g003: 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).
Mentions: LNPs were then evaluated for their capacity to induce gene silencing in intestinal epithelial cells. In this study, we chose to work with Caco-2 cells, which are human colorectal adenocarcinoma cells commonly used to model the intestinal epithelium [31,32]. Upon differentiation, Caco-2 cells polarize into monolayers expressing microvilli and tight junctions that serve as a barrier to absorption [31–33]. For this proof-of-concept analysis, we sought to silence the model “housekeeping” protein, GAPDH, which is typically expressed at high levels and can be knocked down without causing adverse cellular events [34,35]. LNPs were delivered at an siGAPDH concentration of 100 nM to undifferentiated Caco-2 cells, and GAPDH mRNA expression was measured by qPCR 24 hours post-transfection. At this dose, 306O13 and 303O13 LNPs mediated high levels of GAPDH silencing of around 90% (Fig 3A). In order to identify the best lipidoid for Caco-2 work between the two, dose response experiments were conducted (Fig 3B). Results illustrate that 306O13 (blue curve with triangles) facilitated the most potent dose dependent response, with 75% GAPDH silencing achieved at a low dose of 10 nM. Based on this gene silencing data, the lipidoid 306O13 was used in all further gene silencing experiments.

Bottom Line: Recently, a class of lipid-like materials termed "lipidoids" have been shown to potently deliver siRNA to the liver and immune cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus