Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways. J Control Relase

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.
Journal of Controlled Release (Impact Factor: 7.71). 08/2011; 155(2):134-43. DOI: 10.1016/j.jconrel.2011.08.030
Source: PubMed


Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands.

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    • "Recent studies in which the cell-penetrating properties of cyclotides were identified (Cascales et al., 2011; Contreras et al., 2011; Greenwood et al., 2007) revealed that in addition to being stable scaffolds, cyclotides have the potential to be used to deliver bioactive peptides into cells, and thus target intracellular proteins. This was recently confirmed in a study wherein the cyclotide Momordica cochinchinensis trypsin inhibitor I (MCoTI-I), engineered to incorporate a peptide epitope that mimics the N-terminal fragment of the intracellular protein p53, displayed significant resistance to degradation in vivo (Ji et al., 2013). "
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    ABSTRACT: Cyclotides combine the stability of disulfide-rich peptides with the intracellular accessibility of cell-penetrating peptides, giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein-protein interactions. To realize and optimize the application of cyclotides as a drug framework and delivery system, we studied the ability of the prototypic cyclotide, kalata B1, to enter mammalian cells. We show that kalata B1 can enter cells via both endocytosis and direct membrane translocation. Both pathways are initiated by targeting phosphatidylethanolamine phospholipids at the cell surface and inducing membrane curvature. This unusual approach to initiate internalization might be harnessed to deliver drugs into cells and, in particular, cancer cells, which present a higher proportion of surface-exposed phosphatidylethanolamine phospholipids. Our findings highlight the potential of these peptides as drug leads for the modulation of traditionally "undruggable" targets, such as intracellular protein-protein interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & biology 08/2015; 22(8). DOI:10.1016/j.chembiol.2015.07.012 · 6.65 Impact Factor
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    • "Both MCoTI-I, which has high sequence similarity to MCoTI-II, and MCoTI-II are internalized by an energy-dependent process (e.g. endocytosis) and are unable to cross the cell by direct membrane permeation [9] [18] [19]. Specifically, internalization of MCoTI-II into MCF-7 (breast cancer) cells was found to occur through macropinocytosis (~40%) and other endocytic pathways. "
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    ABSTRACT: Peptides are emerging as a new class of therapeutics due to their high potency and specificity for a range of targets, including the inhibition of protein-protein interactions. Disulfide-rich cyclic peptides, in particular, have attracted much attention in drug design due to their ultra-stable structure. Moreover, some of them have been shown to internalize into cells, which makes them potential scaffolds to deliver pharmaceutically bioactive sequences to intracellular targets. Here we examined the effects of structural modifications on the cell-penetrating properties of two disulfide-rich cyclic cell-penetrating peptides, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1). We found that the cellular uptake of MCoTI-II can be improved by increasing the overall positive charge of the native sequence. On the other hand, mutations to SFTI-1 did not significantly influence its cellular uptake, suggesting a non-specific endocytosis-dependent mechanism of cellular uptake. This study provides an understanding of the structural features affecting the internalization of MCoTI-II and SFTI-1, and hence provides a guide for the development of these disulfide-rich cyclic scaffolds into potential drug leads.
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