Intracellular trafficking and unpacking of siRNA/quantum dot-PEI complexes modified with and without cell penetrating peptide: confocal and flow cytometric FRET analysis.
Cationic quantum dots (QDs) were utilized to complex small interfering RNA (siRNA) for studying intracellular trafficking, unpacking, and gene silencing. Positively charged polyethylenimine (PEI) was covalently conjugated on the surface of QDs to complex with cyanine dye labeled vascular endothelial growth factor siRNA (cy5-VEGF siRNA) for the formation of nanosized polyelectrolyte complexes (PEC). Fluorescence resonance energy transfer (FRET) was achieved between cy5-VEGF siRNA and PEI conjugated QDs (QD625) in the complex. From confocal microscopic analysis, intracellular uptake and release of siRNA from the PEC were visualized as a function of incubation time. The extent of cy5-siRNA release from the PEC was quantitatively evaluated by flow cytometric analysis. In addition, PEI conjugated QDs were further modified with a protein transduction domain (PTD) from human transcriptional factor, Hph-1. The two siRNA/QD-PEI complexes with and without Hph-1 have shown markedly different intracellular uptake behaviors and unpacking kinetics of cy5-siRNA. However, they exhibited similar extent of VEGF gene knockout regardless of Hph-1, but showed much higher gene silencing efficiency than siRNA/PEI complexes. The present study demonstrates that PEI conjugated QDs can be utilized as a useful siRNA carrier to analyze intracellular trafficking and unpacking pathway as well as to effectively silence a target gene.
Lee
Hyukjin
H
Department of Biological Sciences and Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
Kim
In-Kyoung
IK
Park
Tae Gwan
TG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D045663:Quantum Dots; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000172:deficiency / Q000235:genetics
D004337:Drug Carriers; D010455:Peptides; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D011094:Polyethyleneimine