Comparative study on transduction and toxicity of protein transduction domains

Abstract

Background and purpose: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs. Experimental approach: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines. Key results: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev4Antp4 Tat4VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-b-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tatfused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat. Conclusions and implications: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.

Cell-penetrating and cell-targeting peptides in drug delivery

“…For example, Toro et al confirmed that the short Tat peptide was not toxic for lymphocytes at dose up to 300 M [81]. Using different cationic peptides (namely Tat, Antp, Rev and VP22), no toxicity was observed in Hela or Jurkat cells with up to 20-30 M concentrations [82].…”

Direct cell entry of gold/iron-oxide magnetic nanoparticles in adenovirus mediated gene delivery

“…This result is also useful because the new cell-transduction technology to avoid the endosome trapping is needed [20]. Even the existing magnetic nanoparticle, MagnetofectionÔ, could not escape from the endosome as previously described [21,22].…”

Transduction of Human Recombinant Proteins into Mitochondria as a Protein Therapeutic Approach for Mitochondrial Disorders

“…Although the toxicity studies carried out with PTDs are quite limited, variable response to various PTDs used has been recorded. These differences may be attributed to the nature and structural characteristics (cationic, amphipathic) of each PTD used, the nature of its linkage with the cargo, the molecular mass, and/or the pI of the attached cargo (162)(163)(164). In addition, internalization process of PTDs may influence the homeostasis of targeted cells.…”