No abstract

Protein therapy holds great promise for treating a variety of diseases. To act on intracellular targets, therapeutic proteins must cross the plasma membrane. This has previously been achieved by covalent attachment to a variety of cell-penetrating peptides (CPPs). However, there is limited information on the relative performance of CPPs in delivering proteins to cells, specifically the cytosol and other intracellular locations. Here we use green fluorescent protein (GFP) as a model cargo to compare delivery capacity of five CPP sequences (Penetratin, R8, TAT, Transportan, Xentry) and cyclic derivatives in different human cell lines (HeLa, HEK, 10T1/2, HepG2) representing different tissues. Confocal microscopy analysis indicates that most fusion proteins when incubated with cells at 10 µM localise to endosomes. Quantification of cellular uptake by flow cytometry reveals that uptake depends on both cell type (10T1/2 > HepG2 > HeLa > HEK), and CPP sequence (Transportan > R8 > Penetratin≈TAT > Xentry). CPP sequence cyclisation or addition of a HA-sequence increased cellular uptake, but fluorescence was still contained in vesicles with no evidence of endosomal escape. Our results provide a guide to select CPP for endosomal/lysosomal delivery and a basis for developing more efficient CPPs in the future.

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.

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.

No abstract

Conjugation of small molecules to a simple cyanine dye can lead to organelle-specific delivery.

The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors.

Recently, 3-dimensional supramolecular coordination complexes of the metallacage type have been shown to hold promise as drug delivery systems for different cytotoxic agents, including the anticancer drug cisplatin. However, so far only limited information is available on their uptake and sub-cellular localisation in cancer cells. With the aim of understanding the fate of metallacages in cells by fluorescence microscopy, three fluorescent Pd2L4 metallacages were designed and synthesised by self-assembly of two types of bispyridyl ligands (L), exo-functionalised with boron dipyrromethene (BODIPY) moieties, with Pd(II) ions. The cages show high quantum yields and are moderately stable in the presence of physiologically relevant concentration of glutathione. Furthermore, the cages are able to encapsulate the anticancer drug cisplatin, as demonstrated by NMR spectroscopy. Preliminary cytotoxicity studies in a small panel of human cancer cells showed that the metallacages are scarcely toxic in vitro. The marked fluorescence due to BODIPY allowed us to visualise the 2 cages' uptake and sub-cellular localisation inside melanoma cells using fluorescence microscopy, highlighting uptake via active transport mechanisms and accumulation in cytoplasmic vesicles.