Cellular Dynamics of EGF Receptor–Targeted Synthetic Viruses

Abstract

The epidermal growth factor receptor (EGFR) is overexpressed on a high percentage of human carcinomas. EGFR is an attractive therapeutic target for tissue-specific targeting by non-viral vectors in cancer gene therapy. In this study we analyzed and compared the effects of EGFR-targeted and untargeted polyplexes in respect to internalization into EGFR overexpressing HuH7 cells. Uptake kinetics and internalization dynamics were evaluated by flow cytometry and single-particle tracking. Our results clearly show that EGFR targeting leads to faster and more efficient internalization compared with untargeted particles. After 5 minutes 50% of the EGFR-targeted polyplexes were internalized, whereas untargeted polyplexes reached only approximately 20% internalization even after 20 minutes. In addition, single-particle tracking revealed a three-phase dynamics of the internalization process, and this was generally observed for polyplexes independent of targeting. Phase I was characterized by slow, actin cytoskeleton-mediated movement of the particles with drift, and included the internalization process. During phase II particles displayed increased velocities with normal and confined diffusion in the cytoplasm. Phase III was characterized by fast active transport along microtubules. Targeting of polyplexes for receptor-mediated endocytosis by the EGFR resulted in shortening of phase I and strongly accelerated internalization.

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

“…When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. As expected, the speed observed in the directed diffusion matches well with the speed of the typical molecular motor-mediated transport (0.5-2 mm/s) (59)(60)(61)(62). When compared with the stimulated EGFRs, the untreated EGFRs clearly show increased mobility on membrane.…”

“…4 C). Since the 2D-SPT techniques can only probe the diffusion process on membrane (phase I) (49,50,58), very few results have been published about the endocytosis and intracellular transport dynamics of membrane receptors (34,60). In this study we report the physical parameters associated with these processes (Table 1) and show the statistics of our 3D trajectory classifications (Fig.…”

“…4, C-III) within a short period of time (10 s). Phase III was believed to be the active transportation of endosome on microtubules (i.e., directed diffusion) (34,60). Both the average instantaneous velocity of 1.20 5 1.06 mm/s (V i ) and the active transport speed of 1.39 5 0.99 mm/s (V, from Eq.…”

Epidermal Growth Factor–PEG Functionalized PAMAM-Pentaethylenehexamine Dendron for Targeted Gene Delivery Produced by Click Chemistry

“…31 The situation might even be more complex for targeted gene delivery in vivo. The polyplexes with suitable particle size had to first accumulate in tumor site, usually passively via enhanced penetration and retention (EPR) effect.…”

“…55 The EGF-EGFR interaction would be expected to locally improve the internalization of polyplexes. 31 In sum, further modifications have to be made for overcoming subsequent gene delivery steps after receptor-mediated targeted cell binding. Bioreversible PEGylation might be one possible solution.…”