Fusion-mediated microinjection of lysozyme into HepG2 cells through hemagglutinin neuraminidase-depleted Sendai virus envelopes.
The potential of reconstituted Sendai viral envelopes containing only the fusion protein (F-virosomes) was evaluated for a targeted cytosolic delivery of lysozyme to human hepatoblastoma cells (HepG2) in culture. 125I-Lysozyme loaded into F-virosomes was used to monitor its fusion-mediated transfer to the HepG2 cells. Using fusion assay based on the transfer of water soluble probe, we have demonstrated the existence of aqueous connection between F-virosomes and target cells. Target specificity of the F-virosomes was ensured by the strong interaction between terminal beta-galactose moiety of F protein and the asialoglycoprotein receptor on the membrane of HepG2 cells. Incubation of the loaded F-virosomes with cells resulted in fusion-mediated injection, as inferred from the ability of cells to internalize lysozyme in the presence of azide (an inhibitor of the endocytotic process). Binding as well as fusion of the F-virosomes to HepG2 cells was solely mediated by the F protein. Introduction of 125I-lysozyme into the HepG2 cells was confirmed by selective accumulation of acid and antibody-precipitable radioactivity in the cytosolic compartment. The structural integrity of the internalized lysozyme was also assessed. The potential usefulness of F-virosomes with defined specificities as biological carrier for both in vitro and in vivo cytosolic delivery of macromolecules and drugs has been established.
Bagai
S
S
Department of Biochemistry, University of Delhi South Campus, New Delhi, India.
Sarkar
D P
DP
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D005690:Galactose; D018197:Hepatoblastoma; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008561:Membrane Fusion; D008845:Microinjections; D009113:Muramidase / Q000008:administration & dosage* / Q000378:metabolism*; D009439:Neuraminidase; D010222:Parainfluenza Virus 1, Human; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014760:Viral Fusion Proteins
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D011956:Receptors, Cell Surface; D014760:Viral Fusion Proteins; D009113:Muramidase; D009439:Neuraminidase; D005690:Galactose