title: Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.
abstract: Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Etienne
authors.initials: E
authors.lastname: van Bracht
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Luuk R M
authors.initials: LR
authors.lastname: Versteegden
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Sarah
authors.initials: S
authors.lastname: Stolle
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Wouter P R
authors.initials: WP
authors.lastname: Verdurmen
authors.affiliation: Department of Laboratory Medicine, Laboratory of Hematology, Radboud university medical centre, Geert Grooteplein 8, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Rob
authors.initials: R
authors.lastname: Woestenenk
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: René
authors.initials: R
authors.lastname: Raavé
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: Hafmans
authors.affiliation: Department of Urology, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Egbert
authors.initials: E
authors.lastname: Oosterwijk
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Toin H
authors.initials: TH
authors.lastname: van Kuppevelt
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Willeke F
authors.initials: WF
authors.lastname: Daamen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018938:Caco-2 Cells; D002417:Cattle; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism; D004705:Endocytosis; D005434:Flow Cytometry; D005612:Freeze Drying; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D010316:Particle Size; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D057846:Cell-Penetrating Peptides; D012710:Serum Albumin, Bovine