Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

Abstract

Cell-penetrating peptides (CPPs) comprise a fast growing class of short length peptides that differ in sequence, size and charge but share a common characteristic ability to translocate across the plasma membrane. It has been demonstrated that CPPs can act efficiently as nonviral delivery vehicles for macromolecules that are much larger in size than their own and lack the self-potential to enter living cells due to the The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat 2 , mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cellpenetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active b-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat 2 -mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene ⁄ ethanol (1 : 40, v ⁄ v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat 2 -mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat 2 -GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat 2 -plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat 2 -plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent LipofectamineÔ 2000 to the Tat 2 -plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.Abbreviations AID, arginine-rich intracellular delivery; CPP, cell-penetrating peptide; EIPA, 5-(N-ethyl N-isopropyl) amirolide; b-GUS, b-glucuronidase; M-Tat, mutated-Tat; pVEC, peptide vascular endothelial-cadherin.

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

“…The second step involves macropinocytosis for uptake of CPP-cargo complex, and the final step is dependent on parameters such as pH causing disruption of micropinosome resulting in release of the CPP-cargo complex into the cytosol. Our studies on Tat-cargo complex in plant cells do not rule out possibility of simultaneous involvement of various mechanisms for cellular internalization (3,62). This observation is further corroborated by the studies carried by research groups working with mammalian cell lines (64,95).…”

“…In plant system, cytotoxic effects were not observed with CPPs alone or in complex with their cargoes, at the concentration investigated in somatic embryos or microspores (3,62). Viability of the microspores immediately after treatment with CPPs or their cargoes was also similar to the control microspores (65-70%).…”

“…Additionally, CPPs alone and their cargoes have low levels of cytotoxicity at the concentration at which CPPs are used for the delivery of macromolecules (2). We have also observed that internalization of cargoes of CPPs, such as Tat and its dimmer, is nondeleterious to plant cells (3).…”

“…Cellular uptake was increased remarkably at 4 8C, as observed in both mammalian and plant cells (3,6,44). This may be due to low temperature-induced change in the physicochemical properties of the cell membrane, stabilizing inverted phospholipids micelles thereby favoring a higher cellular uptake of the peptides studied (45)(46)(47).…”

“…The cell wall remains a major obstacle for transduction of larger proteins in plant tissues, such as immature scutellum and cotyledon, a model system for genetic transformation studies owing to their amenability toward tissue culture procedures and high efficacy for plant regeneration. Permeabilization of cell wall is a prerequisite to achieve efficient translocation of CPPs and their macromolecular cargoes (3). Following such pretreatment, nonlabeled Tat monomer (Tat) and dimmer (Tat 2 ) are able to deliver the large protein b-glucuronidase (GUS, MW 270 kDa) efficiently.…”