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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

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.
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pone.0133154.g007: 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.

Mentions: Finally, a western blot analysis determined the effect of the 100 nM siRNA dose of 306O13 siGAPDH LNPs on GAPDH protein expression. For this experiment, protein lysate was collected every day for five days post-transfection. As seen in Fig 7, the amount of GAPDH protein began to decrease around 1 or 2 days post transfection. Alpha-tubulin was used as a protein loading control for comparison of the GAPDH protein expression over time. Band quantification using ImageJ suggested that the siRNA-loaded LNPs could reduce the protein expression of GAPDH by approximately 85% five days post-transfection.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133154.g007: 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.
Mentions: Finally, a western blot analysis determined the effect of the 100 nM siRNA dose of 306O13 siGAPDH LNPs on GAPDH protein expression. For this experiment, protein lysate was collected every day for five days post-transfection. As seen in Fig 7, the amount of GAPDH protein began to decrease around 1 or 2 days post transfection. Alpha-tubulin was used as a protein loading control for comparison of the GAPDH protein expression over time. Band quantification using ImageJ suggested that the siRNA-loaded LNPs could reduce the protein expression of GAPDH by approximately 85% five days post-transfection.

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