Cellular dynamics of EGF receptor-targeted synthetic viruses.
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.
de Bruin
Karla
K
Department Chemie und Biochemie, Ludwig-Maximilians Universität, München, Germany.
Ruthardt
Nadia
N
von Gersdorff
Katharina
K
Bausinger
Ralf
R
Wagner
Ernst
E
Ogris
Manfred
M
Bräuchle
Christoph
C
D000199:Actins / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007700:Kinetics; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D021381:Protein Transport; D013997:Time Factors
D000199:Actins; D004279:DNA, Viral; D066246:ErbB Receptors