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 (Ϸ60 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.transfection agent ͉ molecular mobility ͉ cytoplasmic crowding ͉ cyclodextrin R ecently, significant progress has been made in developing alternatives to viral-based gene therapy, motivated by safety concerns of viral infection. Understanding such nonviral gene delivery (one type of vector that involves the combination of synthetic polymers and nucleic acids to form particles called polyplexes and is used here) requires knowledge of how they behave within cells, including the mechanisms involved in polyplex trafficking such as endocytosis, cytoplasmic transport, endosomal escape, and nuclear localization (1-3). Recent reports have indicated the presence of significant bottlenecks in the delivery process, especially problems with endosomal escape (4). Measuring these dynamics, especially transport parameters, is important for understanding both how such delivery methods compare with viruses and how to improve their efficiencies.Confocal microscopy allows for good visualization of small quantities of fluorescently labeled species. However, only a few studies of cytoplasmic transport have focused on quantitative biophysical parameters such as effective diffusion constants or transport velocities (4-6). Most experiments measuring these parameters have focused on examining small injected oligonucleotides or proteins (7-11). In contrast, polyplexes that range in size from 75 to 250 nm or larger enter cells by endocytosis and become enclosed in endosomes (12). For transport of large entities (Ͼ30 nm), such as the polyplexes used for gene therapy, issues such as crowding and microtubule transport become critical (13,14).Here, we describe spatio-temporal image correlation spectroscopy (IC...

Targeted intracellular codelivery of chemotherapeutics and nucleic acid with a well-defined dendrimer-based nanoglobular carrier

“…Recent studies have also showed that RGD targeted carriers have greater accumulation outside acidic endosomes and RGD could mediate intracellular trafficking via microtubule associated motor proteins closer to the nucleus [21,22]. We further investi- This study showed that the dendrimeric nanoglobules with three-dimensional compact globular morphology were effective for loading targeting agent, anticancer drug and siRNA for targeted intracellular codelivery of chemotherapeutics and siRNA for combination therapy.…”

Advances in Image Correlation Spectroscopy: Measuring Number Densities, Aggregation States, and Dynamics of Fluorescently labeled Macromolecules in Cells

“…Kulkarni et al [42] probed the dynamics of endosomes containing fluorescently labeled polyplexes, in living HeLa cells. They used TICS in conjunction with SPT to characterize the motion of an ensemble of polyplexes, and found that their motion was Brownian the majority of the time, with brief periods of directed motion, which they attributed to transport along microtubules.…”

“…Unlike single molecule techniques, such as SPT, fluctuation techniques may not be able to detect a subpopulation made up of a small fraction of the particles [38]. However, correlation techniques are generally more straightforward, and less timeconsuming to implement (see supplemental material in [42] for a comparison of SPT and ICS). Another key advantage of fluctuation techniques is that they can by applied to both relatively low and high density samples [43], unlike SPT which requires individual particles to be well separated.…”

Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells

“…This method is distinct from previously developed image correlation spectroscopy (ICS) (Petersen et al, 1993) and spatiotemporal ICS (STICS) (Wiseman et al, 2004;Hebert et al, 2005;Kulkarni et al, 2005;Rossow et al, 2009) In a typical EGFP-b-actin-labeled cell (Fig. 5A-C), the EGFP-b-actin time-average is a weak indication of where and for how long accumulations occur (Fig.…”