Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy
Abstract
Quantitatively understanding how nonviral gene delivery vectors (polyplexes) are transported inside cells is essential before they can be optimized for gene therapy and medical applications. In this study, we used spatio-temporal image correlation spectroscopy (ICS) to follow polymer-nucleic acid particles (polyplexes) of various sizes and analyze their diffusive-like and flow behaviors intracellularly to elucidate the mechanisms responsible for their transport. ICS is a quantitative imaging technique that allows the assessment of particle motion in complex systems, although it has not been widely used to date. We find that the internalized polyplexes are able to use microtubule motors for intracellular trafficking and exhibit different transport behaviors for short (<10 s) versus long (≈60 s) correlation times. This motion can be explained by a memory effect of the microtubule motors. These results reveal that, although microtubule motor biases may be present for short periods of time, resulting in a net directional velocity, the overall long-term motion of the polyplexes is best described as a random walk-like process. These studies suggest that spatio-temporal ICS is a powerful technique for assessing the nature of intracellular motion and provides a quantitative tool to compare the transport of different objects within a living cell.
Additional Information
© 2005 by The National Academy of Sciences of the USA. Communicated by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, March 9, 2005 (received for review September 15, 2004). Published ahead of print May 16, 2005. We thank Jeremy Heidel and Swaroop Mishra for assistance in making the polyplexes, Tania Demyanenko for assistance with tissue culture, and Mag Bak, Nils Petersen, Paul Wiggins, Paul Grayson, Michael Liebling, Helen McBride, Mary Dickinson, and Elaine Bearer for helpful discussion and comments on the manuscript. This work was supported by a National Institute of Child Health and Human Development grant (to S.E.F.) and a National Defense Science and Engineering Graduate Fellowship (to R.P.K.). Author contributions: R.P.K., M.E.D., and S.E.F. designed research; R.P.K. performed research; R.P.K. and D.D.W. contributed new reagents/analytic tools; R.P.K., S.E.F., and M.E.D. analyzed data; and R.P.K., M.E.D., and S.E.F. wrote the paper.Attached Files
Published - PNAS-2005-Kulkarni-7523-8.pdf
Supplemental Material - 01950Fig5.pdf
Supplemental Material - 01950Fig6.pdf
Supplemental Material - 01950Fig7.pdf
Supplemental Material - 01950Fig8.pdf
Supplemental Material - 01950Fig9.pdf
Supplemental Material - 01950Movie1.mov
Supplemental Material - 01950SuppMethods.pdf
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Additional details
- PMCID
- PMC1140437
- Eprint ID
- 52035
- Resolver ID
- CaltechAUTHORS:20141121-093134521
- National Institute of Child Health and Human Development
- National Defense Science and Engineering Graduate (NDSEG) Fellowship
- Created
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2014-11-21Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field