Co-reporter:H. D. Roberts-Dalton;A. Cocks;J. M. Falcon-Perez;E. J. Sayers;J. P. Webber;P. Watson;A. Clayton;A. T. Jones
Nanoscale (2009-Present) 2017 vol. 9(Issue 36) pp:13693-13706
Publication Date(Web):2017/09/21
DOI:10.1039/C7NR04128D
Extracellular vesicles, including exosomes, are naturally derived nanovesicles generated in and released by numerous cell types. As extracellular entities they have the capacity to interact with neighbouring cells and distant tissues and affect physiological processes as well as being implicated in numerous diseases including tumorigenesis and neurodegeneration. They are also under intense investigation as delivery vectors for biotherapeutics. The ways in which EVs interact with recipient cells to influence cell physiology and deliver a macromolecular payload are at the early stages of exploration. A significant challenge within these studies is the ability to label EVs directly or indirectly with fluorescent probes to allow visualization without compromising functionality. Here, we present a thiol-based fluorescence labelling method allowing comprehensive analysis of the cellular uptake of prostate cancer derived EVs in live cells using confocal microscopy. Labelling of the EVs in this way did not influence their size and had no effect on their ability to induce differentiation of lung fibroblasts to myofibroblasts. For endocytosis analyses, depletion of key endocytic proteins and the use of chemical inhibitors (Dynasore, EIPA, Rottlerin and IPA-3) indicated that fluid-phase endocytosis and/or macropinocytosis was involved in EV internalisation. Over a period of six hours EVs were observed to increasingly co-localise with lysosomes, indicating a possible termination point following internalisation. Overall this method provides new opportunities for analysing the cellular dynamics of EVs as biological entities affecting cell and whole body physiology as well as investigating their potential as drug delivery vectors.
Co-reporter:E.J. Sayers, K. Cleal, N.G. Eissa, P. Watson, A.T. Jones
Journal of Controlled Release 2014 Volume 195() pp:55-62
Publication Date(Web):10 December 2014
DOI:10.1016/j.jconrel.2014.07.055
For cell penetrating peptides (CPPs) to fulfil their promise as effective delivery vectors we need a better understanding of their mechanisms of cell binding and uptake. This is especially the case when they are linked to different types of cargo. Here we describe new studies based on our previous findings suggesting that, for peptide-CPP chimeras, distal hydrophobic residues upstream of the CPP sequence can have profound effects on the way they interact with cells. We studied peptides bearing an N-terminal Glycine or Phenylalanine linked via a neutral and flexible bridging group, SGSGSGSG, to three well-studied CPPs: octaarginine, penetratin and TP10. Using a combination of flow cytometry, live-cell imaging and image analysis we examined the effects of this single amino acid change on binding and uptake of Alexa488-fluorophore, bovine serum albumin and quantum dot cargoes. The influence of the glycine–phenylalanine switch for fluorophore delivery was most dramatic in TP10, increasing cellular uptake by 4.4 and 9.9 fold in non-adherent and adherent cells, respectively. Only penetratin showed effective uptake of bovine serum albumin with the phenylalanine variant showing an increase of 1.6 fold over the glycine variant. The uptake of quantum dots was most efficiently demonstrated by octaarginine, with the glycine variant increasing uptake 4.8 fold and the phenylalanine variant increasing uptake 9.5 fold over quantum dots alone. Overall the data demonstrate that hydrophobicity distal to the CPP could be utilised to enhance their capacity to bind to the cell membrane and deliver a range of macromolecules to the insides of cells.A substitution from glycine to the hydrophobic phenylalanine at the N-terminus of CPPs R8, TP10 and penetratin can dramatically alter their ability to deliver different cargos into different cell types.Figure optionsDownload full-size imageDownload high-quality image (230 K)Download as PowerPoint slide
Co-reporter:M. Al Soraj, L. He, K. Peynshaert, J. Cousaert, D. Vercauteren, K. Braeckmans, S.C. De Smedt, A.T. Jones
Journal of Controlled Release 2012 Volume 161(Issue 1) pp:132-141
Publication Date(Web):10 July 2012
DOI:10.1016/j.jconrel.2012.03.015
Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.
Co-reporter:Hélène Bruyère, Andrew D. Westwell, Arwyn T. Jones
Bioorganic & Medicinal Chemistry Letters 2010 Volume 20(Issue 7) pp:2200-2203
Publication Date(Web):1 April 2010
DOI:10.1016/j.bmcl.2010.02.035
Orthoesters are acid-sensitive moieties that allow substantial structural diversity for biological applications including drug delivery. Here, the pH-sensitivity of a range of novel orthoester based compounds was compared in the range 7.5–4.5 that is characteristic of the increased acidification during endocytosis. We find that simple modifications close to the orthoester had major effects on both the rate and extent of hydrolysis, suggesting this could be exploited for activating drug delivery systems on endocytic pathways.Small chemical modifications influence aqueous pH-sensitivity of orthoester conjugates.
Co-reporter:Arwyn Tomos Jones
Journal of Cellular and Molecular Medicine 2007 Volume 11(Issue 4) pp:670-684
Publication Date(Web):14 JUN 2007
DOI:10.1111/j.1582-4934.2007.00062.x
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Introduction
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Membrane ruffling and the formation of the macropinosome
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Rabs and macropinocytosis
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The fate of the macropinosome on the endocytic pathway
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A role for nexins in macropinosome sorting
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Cell penetrating peptides as vectors for drug delivery
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The case for and against macropinocytosis
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Conclusions
Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short-lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides.
Co-reporter:Marjan Fretz, Jing Jin, Robin Conibere, Neal A. Penning, Saly Al-Taei, Gert Storm, Shiroh Futaki, Toshihide Takeuchi, Ikuhiko Nakase, Arwyn T. Jones
Journal of Controlled Release 2006 Volume 116(Issue 2) pp:247-254
Publication Date(Web):28 November 2006
DOI:10.1016/j.jconrel.2006.07.009
Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34+ leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV–TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV–TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells.
Co-reporter:Paul R Moody, Edward J Sayers, Johannes P Magnusson, Cameron Alexander, ... Arwyn T Jones
Molecular Therapy (December 2015) Volume 23(Issue 12) pp:1888-1898
Publication Date(Web):1 December 2015
DOI:10.1038/mt.2015.178
A major unmet clinical need is a universal method for subcellular targeting of bioactive molecules to lysosomes. Delivery to this organelle enables either degradation of oncogenic receptors that are overexpressed in cancers, or release of prodrugs from antibody–drug conjugates. Here, we describe a general method that uses receptor crosslinking to trigger endocytosis and subsequently redirect trafficking of receptor:cargo complexes from their expected route, to lysosomes. By incubation of plasma membrane receptors with biotinylated cargo and subsequent addition of streptavidin to crosslink receptor:cargo–biotin complexes, we achieved rapid and selective lysosomal targeting of transferrin, an anti-MHC class I antibody, and the clinically approved anti-Her2 antibody trastuzumab. These three protein ligands each target a receptor with a distinct cellular function and intracellular trafficking profile. Importantly, we confirmed that crosslinking of trastuzumab increased lysosomal degradation of its cognate oncogenic receptor Her2 in breast cancer cell lines SKBR3 and BT474. These data suggest that crosslinking could be exploited for a wide range of target receptors, for navigating therapeutics through the endolysosomal pathway, for significant therapeutic benefit.
Co-reporter:Mark Gumbleton, Arwyn T. Jones
Drug Discovery Today (December 2010) Volume 15(Issues 23–24) pp:1079-1080
Publication Date(Web):1 December 2010
DOI:10.1016/j.drudis.2010.10.010
Co-reporter:Helen L. Wiggins, Jennifer M. Wymant, Francesca Solfa, Stephen E. Hiscox, Kathryn M. Taylor, Andrew D. Westwell, Arwyn T. Jones
Biochemical Pharmacology (1 February 2015) Volume 93(Issue 3) pp:332-342
Publication Date(Web):1 February 2015
DOI:10.1016/j.bcp.2014.12.014
