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Quantifying the uptake and distribution of arginine rich peptides at therapeutic concentrations using Fluorescence Correlation Spectroscopy and Image Correlation Spectroscopy techniques

Ben Staley, Egor Zindy and Alain Pluen

In: 3rd Cellular Delivery of Therapeutic Macromolecules meeting; 26 Jun 2010-29 Jun 2010; University of Cardiff. 2010.

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Abstract

Due an apparent ability to enter cells in an energy independent manner, cell-penetrating peptides (CPPs) are increasingly being used as vectors for the delivery of macromolecules into cells. But 20 years on, their mechanism of entry is still disputed1. Additionally cellular uptake and delivery have been studied at relatively high concentrations (micromolar), while therapeutic doses more likely to be in the nanomolar range. Thus, we hypothesised that taking advantage of Fluorescence Correlation Spectroscopy (FCS) and Image Correlation Spectroscopy (ICS) should help to understand the delivery mechanisms of arginine rich peptides Tat and nona-arginine (R9) at therapeutic doses. The peptides (at bulk concentrations lower than 500nM) were incubated for one hour with both Caco-2 and HeLa cells. Subcellular distribution was analysed with confocal microscopy revealing three localised areas -discrete, punctuate and cytoplasmic regions- sampled initially with FCS. To improve our understanding at the cytoplasmic level, ICS2,3 was implemented to parallel and correlate FCS. R9 and Tat-TAMRA peptide presence, molecular concentrations and diffusion times were determined in the different areas of Caco-2 and HeLa cells using ICS and FCS. Our results indicate that arginine rich peptides are observed at both 250 and 500nM concentrations in all three areas sampled. The amount in cytoplasm appears to be linearly dependant with concentration. On the other hand, in the punctuate and discrete areas, the amount of peptide did not appear to be concentration dependent. To conclude, FCS and ICS have shown they can provide valuable information on the delivery of peptides at therapeutic levels.References:1. Lee, H. L.; Dubikovskaya, E. A.; Hwang, H.; Semyonov, A. N.; Wang, H.; Jones, L. R.; Twieg, R. J.; Moerner, W. E.; Wender, P. A. Journal of the American Chemical Society 2008, 130, 9364-9370.2. Kolin, D. L.; Wiseman, P. W. Cell Biochemistry and Biophysics 2007, 49, 141-164.3. Hebert, B.; Costantino, S.; Wiseman, P. W. Biophysical Journal 2005, 88, 3601-3614.

Bibliographic metadata

Type of resource:
Content type:
Publication date:
Conference title:
3rd Cellular Delivery of Therapeutic Macromolecules meeting
Conference venue:
University of Cardiff
Conference start date:
2010-06-26
Conference end date:
2010-06-29
Abstract:
Due an apparent ability to enter cells in an energy independent manner, cell-penetrating peptides (CPPs) are increasingly being used as vectors for the delivery of macromolecules into cells. But 20 years on, their mechanism of entry is still disputed1. Additionally cellular uptake and delivery have been studied at relatively high concentrations (micromolar), while therapeutic doses more likely to be in the nanomolar range. Thus, we hypothesised that taking advantage of Fluorescence Correlation Spectroscopy (FCS) and Image Correlation Spectroscopy (ICS) should help to understand the delivery mechanisms of arginine rich peptides Tat and nona-arginine (R9) at therapeutic doses. The peptides (at bulk concentrations lower than 500nM) were incubated for one hour with both Caco-2 and HeLa cells. Subcellular distribution was analysed with confocal microscopy revealing three localised areas -discrete, punctuate and cytoplasmic regions- sampled initially with FCS. To improve our understanding at the cytoplasmic level, ICS2,3 was implemented to parallel and correlate FCS. R9 and Tat-TAMRA peptide presence, molecular concentrations and diffusion times were determined in the different areas of Caco-2 and HeLa cells using ICS and FCS. Our results indicate that arginine rich peptides are observed at both 250 and 500nM concentrations in all three areas sampled. The amount in cytoplasm appears to be linearly dependant with concentration. On the other hand, in the punctuate and discrete areas, the amount of peptide did not appear to be concentration dependent. To conclude, FCS and ICS have shown they can provide valuable information on the delivery of peptides at therapeutic levels.References:1. Lee, H. L.; Dubikovskaya, E. A.; Hwang, H.; Semyonov, A. N.; Wang, H.; Jones, L. R.; Twieg, R. J.; Moerner, W. E.; Wender, P. A. Journal of the American Chemical Society 2008, 130, 9364-9370.2. Kolin, D. L.; Wiseman, P. W. Cell Biochemistry and Biophysics 2007, 49, 141-164.3. Hebert, B.; Costantino, S.; Wiseman, P. W. Biophysical Journal 2005, 88, 3601-3614.

Institutional metadata

University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:93663
Created by:
Pluen, Alain
Created:
2nd November, 2010, 15:51:30
Last modified:
2nd November, 2010, 15:51:30

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