Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

Stereoisomer-Dependent Membrane Association and Capacity for Insulin Delivery Facilitated by Penetratin.

Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers.

Influence of Folate-Targeted Gold Nanoparticles on Subcellular Localization and Distribution into Lysosomes.

The cell interaction, mechanism of cell entry and intracellular fate of surface decorated nanoparticles are known to be affected by the surface density of targeting agents. However, the correlation between nanoparticles multivalency and kinetics of the cell uptake process and disposition of intracellular compartments is complicated and dependent on a number of physicochemical and biological parameters, including the ligand, nanoparticle composition and colloidal properties, features of targeted cells, etc. Here, we have carried out an in-depth investigation on the impact of increasing folic acid density on the kinetic uptake process and endocytic route of folate (FA)-targeted fluorescently labelled gold nanoparticles (AuNPs). A set of AuNPs (15 nm mean size) produced by the Turkevich method was decorated with 0-100 FA-PEG3.5kDa-SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG2kDa-SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KBFR-high) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG3.5kDa-SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG3.5kDa-SH molecules/particle) induces more efficient particle internalization and trafficking to lysosomes, reaching the maximum concentration in lysosomes at 2 h, than the lower FA density of 10 FA-PEG3.5kDa-SH molecules/particle. Pharmacological inhibition of endocytic pathways and TEM analysis showed that particles with high folate density are internalized predominantly by a clathrin-independent process.

Highly-fluorescent BODIPY-functionalised metallacages as drug delivery systems: synthesis, characterisation and cellular accumulation studies.

With the aim of designing new metallosupramolecular architectures for drug delivery, research has focused on porous 3-dimensional (3D)-metallacages able to encapsulate cytotoxic agents protecting them from metabolism while targeting them to cancer sites. Here, two self-assembled [Pd2L4]4+ cages (CG1 and CG2) featuring 3,5-bis(3-ethynylpyridine)phenyl ligands (L) exo-functionalised with dipyrromethene (BODIPY) groups have been synthesised and characterised by different methods, including NMR spectroscopy and mass spectrometry. 1H NMR spectroscopy studies shows that the cages are able to encapsulate the anticancer drug cisplatin in their hydrophobic cavity, as evidenced by electrostatic potential (ESP) analysis based on XRD studies. The stability of the cages in an aqueous environment, and in the presence of the intracellular reducing agent glutathione, has been confirmed by UV-visible absorption spectroscopy. The luminescence properties of the cages enabled the investigation of their cellular uptake and intracellular localisation in human cancer cells by confocal laser scanning microscopy. In melanoma A375 cells, cage CG1 is taken up via active transport and endocytic trafficking studies show little evidence of transport through the early endosome while the cages accumulated in melanosomes rather than lysosomes. The antiproliferative activity of the lead cage was investigated in A375 together with two breast cancer cell lines, SK-BR-3 and MCF7. While the cage per se is non-cytotoxic, very different antiproliferative effects with respect to free cisplatin were evidenced for the [(cisplatin)2⊂CG1·BF4] complex in the various cell lines, which correlate with its different intracellular localisation profiles. The obtained preliminary results provide a new hypothesis on how the subcellular localisation of the cage affects the cisplatin intracellular release.

Fluid-Phase Endocytosis and Lysosomal Degradation of Bovine Lactoferrin in Lung Cells.

The iron-binding protein lactoferrin and the cell-penetrating peptides derived from its sequence utilise endocytosis to enter different cell types. The full-length protein has been extensively investigated as a potential therapeutic against a range of pathogenic bacteria, fungi, and viruses, including SARS-CoV-2. As a respiratory antiviral agent, several activity mechanisms have been demonstrated for lactoferrin, at the extracellular and plasma membrane levels, but as a protein that enters cells it may also have intracellular antiviral activity. Characterisation of lactoferrin's binding, endocytic traffic to lysosomes, or recycling endosomes for exocytosis is lacking, especially in lung cell models. Here, we use confocal microscopy, flow cytometry, and degradation assays to evaluate binding, internalisation, endocytic trafficking, and the intracellular fate of bovine lactoferrin in human lung A549 cells. In comparative studies with endocytic probes transferrin and dextran, we show that lactoferrin binds to negative charges on the cell surface and actively enters cells via fluid-phase endocytosis, in a receptor-independent manner. Once inside the cell, we show that it is trafficked to lysosomes where it undergoes degradation within two hours. These findings provide opportunities for investigating both lactoferrin and derived cell-penetrating peptides activities of targeting intracellular pathogens.

Quantitative Subcellular Analysis of Cyclic Cell-Penetrating Peptide EJP18 in Nonadherent Cells.

Cyclization of cell-penetrating peptides (CPPs) often results in improved capacity for intracellular delivery of a range of cargoes but quantitating the distinct subcellular localization of them, and their linear counterparts, remains a challenge. Here we describe an optimized method for recombinant generation and purification of eGFP attached to the cyclic form of the newly discovered CPP EJP18 in E. coli. We also demonstrate a novel microscopy method for quantifying its subcellular distribution in leukemia cells.

Anti-schistosomal activities of quinoxaline-containing compounds: From hit identification to lead optimisation.

Schistosomiasis is a neglected disease of poverty that is caused by infection with blood fluke species contained within the genus Schistosoma. For the last 40 years, control of schistosomiasis in endemic regions has predominantly been facilitated by administration of a single drug, praziquantel. Due to limitations in this mono-chemotherapeutic approach for sustaining schistosomiasis control into the future, alternative anti-schistosomal compounds are increasingly being sought by the drug discovery community. Herein, we describe a multi-pronged, integrated strategy that led to the identification and further exploration of the quinoxaline core as a promising anti-schistosomal scaffold. Firstly, phenotypic screening of commercially available small molecules resulted in the identification of a moderately active hit compound against Schistosoma mansoni (1, EC50 = 4.59 µM on schistosomula). Secondary exploration of the chemical space around compound 1 led to the identification of a quinoxaline-core containing, non-genotoxic lead (compound 22). Compound 22 demonstrated substantially improved activities on both intra-mammalian (EC50 = 0.44 µM, 0.20 µM and 84.7 nM, on schistosomula, juvenile and adult worms, respectively) and intra-molluscan (sporocyst) S. mansoni lifecycle stages. Further medicinal chemistry optimisation of compound 22, resulting in the generation of 20 additional analogues, improved our understanding of the structure-activity relationship and resulted in considerable improvements in both anti-schistosome potency and selectivity (e.g. compound 30; EC50 = 2.59 nM on adult worms; selectivity index compared to the HepG2 cell line = 348). Some derivatives of compound 22 (e.g. 31 and 33) also demonstrated significant activity against the two other medically important species, Schistosoma haematobium and Schistosoma japonicum. Further optimisation of this class of anti-schistosomal is ongoing and could lead to the development of an urgently needed alternative to praziquantel for assisting in schistosomiasis elimination strategies.

Strategic Trastuzumab Mediated Crosslinking Driving Concomitant HER2 and HER3 Endocytosis and Degradation in Breast Cancer.

Efficacious anticancer therapies for targeting plasma membrane receptors with antibody based therapeutics are often contingent on sufficient endocytic delivery of receptor and conjugate to lysosomes. This results in downregulation of receptor activity and, in the case of antibody-drug conjugates (ADCs), intracellular release of a drug payload. The oncogenic receptor HER2 is a priority therapeutic target in breast cancer. Known as an "endocytosis resistant" receptor, HER2 thwarts the receptor downregulating efficiency of the frontline treatment trastuzumab and reduces the potential of trastuzumab-based therapies such as trastuzumab-emtansine. We previously demonstrated that strategically inducing trastuzumab and HER2 crosslinking in breast cancer cells promoted endocytosis and lysosomal delivery of the HER2-trastuzumab complex, stimulating downregulation of the receptor. Here we reveal that HER3, but not EGFR, is also concomitantly downregulated with HER2 after crosslinking. This is accompanied by strong activation of MEK/ERK pathway that we show does not directly contribute to HER2/trastuzumab endocytosis. We show that crosslinking induced trastuzumab endocytosis occurs via clathrin-dependent and independent pathways and is an actin-dependent process. Detailed ultrastructural studies of the plasma membrane highlight crosslinking-specific remodelling of microvilli and induction of extensive ruffling. Investigations in a cell model of acquired trastuzumab resistance demonstrate, for the first time, that they are refractory to crosslinking induced HER2 endocytosis and downregulation. This implicates further arrest of HER2 internalisation in developing trastuzumab resistance. Overall our findings highlight the potential of receptor crosslinking as a therapeutic strategy for cancer while exposing the ability of cancer cells to develop resistance via endocytic mechanisms.

Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold.

Four-wave-mixing microscopy reveals non-colocalisation between gold nanoparticles and fluorophore conjugates inside cells.

Gold nanoparticles have been researched for many biomedical applications in diagnostics, theranostics, and as drug delivery systems. When conjugated to fluorophores, their interaction with biological cells can be studied in situ and real time using fluorescence microscopy. However, an important question that has remained elusive to answer is whether the fluorophore is a faithful reporter of the nanoparticle location. Here, our recently developed four-wave-mixing optical microscopy is applied to image individual gold nanoparticles and in turn investigate their co-localisation with fluorophores inside cells. Nanoparticles from 10 nm to 40 nm diameter were conjugated to fluorescently-labeled transferrin, for internalisation via clathrin-mediated endocytosis, or to non-targeting fluorescently-labelled antibodies. Human (HeLa) and murine (3T3-L1) cells were imaged at different time points after incubation with these conjugates. Our technique identified that, in most cases, fluorescence originated from unbound fluorophores rather than from fluorophores attached to nanoparticles. Fluorescence detection was also severely limited by photobleaching, quenching and autofluorescence background. Notably, correlative extinction/fluorescence microscopy of individual particles on a glass surface indicated that commercial constructs contain large amounts of unbound fluorophores. These findings highlight the potential problems of data interpretation when reliance is solely placed on the detection of fluorescence within the cell, and are of significant importance in the context of correlative light electron microscopy.

Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression.

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

Highly luminescent metallacages featuring bispyridyl ligands functionalised with BODIPY for imaging in cancer cells.

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 cages' uptake and sub-cellular localisation inside melanoma cells using fluorescence microscopy, highlighting uptake via active transport mechanisms and accumulation in cytoplasmic vesicles.

Targeted delivery and endosomal cellular uptake of DARPin-siRNA bioconjugates: Influence of linker stability on gene silencing.

Specific cell targeting and efficient intracellular delivery are major hurdles for the widespread therapeutic use of nucleic acid technologies, particularly siRNA mediated gene silencing. To enable receptor-mediated cell-specific targeting, we designed a synthesis scheme that can be generically used to engineer Designed Ankyrin Repeat Protein (DARPin)-siRNA bioconjugates. Different linkers, including labile disulfide-, and more stable thiol-maleimide- and triazole- (click chemistry) tethers were employed. Crosslinkers were first attached to a 3'-terminal aminohexyl chain on the siRNA sense strands. On the protein side thiols of a C-terminal cysteine were used as anchoring sites for disulfide- and thiol-maleimide conjugate formations, while strain-promoted azido-alkyne cycloadditions were carried out at a metabolically introduced N-terminal azidohomoalanine. After establishing efficient purification methods, highly pure products were obtained. Bioconjugates of EpCAM-targeted DARPins with siRNA directed at the luciferase gene were evaluated for cell-specific binding, uptake and gene silencing. As shown by flow cytometry and fluorescence microscopy, all constructs retained the highly specific and high-affinity antigen recognition properties of the native DARPin. As expected, internalization was observed only in EpCAM-positive cell lines, and predominantly endolysosomal localization was detected. Disulfide linked conjugates showed lower serum stability against cleavage at the linker and thus lower internalization into endosomes compared to thiol-maleimide- and triazole-linked conjugates, yet induced more pronounced gene silencing. This indicates that the siRNA payload needs to be liberated from the protein in the endosome. Our data confirm the promise of DARPin-siRNA bioconjugates for tumor targeting, but also identified endosomal retention and limited cytosolic escape of the siRNA as the rate-limiting step for more efficient gene silencing.

Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines.

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.

Switching of Macromolecular Ligand Display by Thermoresponsive Polymers Mediates Endocytosis of Multiconjugate Nanoparticles.

Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.

Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes.

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.

siRNA Inhibition of Endocytic Pathways to Characterize the Cellular Uptake Mechanisms of Folate-Functionalized Glycol Chitosan Nanogels.

Glycol chitosan nanogels have been widely used in gene, drug, and contrast agent delivery in an effort to improve disease diagnosis and treatment. Herein, we evaluate the internalization mechanisms and intracellular fate of previously described glycol chitosan nanogels decorated with folate to target the folate receptor. Uptake of the folate-decorated nanogel was impaired by free folate, suggesting competitive inhibition and shared internalization mechanisms via the folate receptor. Nanogel uptake was shown to occur mainly through flotillin-1 and Cdc42-dependent endocytosis. This was determined by inhibition of uptake reduction observed upon siRNA depletion of these two proteins and the pathways that they regulate. The data also suggest the involvement of the actin cytoskeleton in nanogel uptake via macropinocytosis. After 7 h of incubation with HeLa cells, approximately half of the nanogel population was localized in endolysosomal compartments, whereas the remaining 50% of the material was in undefined regions of the cytoplasm. Glycol chitosan nanogels may thus have potential as drug delivery vectors for targeting different intracellular compartments.

Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections.

PURPOSE: To investigate the suitability of three antimicrobial peptides (AMPs) as cell-penetrating antimicrobial peptides. METHODS: Cellular uptake of three AMPs (PK-12-KKP, SA-3 and TPk) and a cell-penetrating peptide (penetratin), all 5(6)-carboxytetramethylrhodamine-labeled, were tested in HeLa WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effects of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the disrupting ability of the peptides in the presence of bilayer membranes of different composition were analyzed. RESULTS: AMP uptake relative to penetratin was ~13% (PK-12-KKP), ~66% (SA-3) and ~50% (TPk). All four peptides displayed a punctate uptake pattern in HeLa WT cells with co-localization to lysosomes and no indication that clathrin-mediated endocytosis was the predominant uptake mechanism. TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. CONCLUSION: PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development.

Cell entry of cell penetrating peptides: tales of tails wagging dogs.

Cell penetrating peptides hold considerable potential for academic and pharmaceutical remits with an interest in delivering macromolecules to the insides of cells. Hundreds of sequences now fall within the cell penetrating peptide classification and HIV-Tat, penetratin, transportan, and octaarginine represent extensively studied variants. The process by which membrane translocation is achieved has received significant interest in an aim to exploit new mechanistic knowledge to gain higher efficiency of penetration. There is evidence that many of the most well studied peptides are able to deliver themselves, relatively small cargo and possibly large macromolecular structures directly across the plasma membrane but there is also support for the involvement of an endocytic pathway or pathways. This review focuses on recent findings relating to experimental protocols and cell penetrating peptide modifications or extensions that yield significant effects on penetration capability. Relatively small changes in extracellular peptide concentrations, the inclusion or absence of serum from the incubation medium and the in vitro model exemplify variables that significantly influence the capacity of CPPs to penetrate membranes. Attachment of any type of cargo to these entities has the potential to affect their interaction with cells. There is increasing evidence to suggest that this is true for relatively small molecules such as fluorescent probes and hydrophobic adducts such as lipids and short peptide sequences designed as peptide therapeutics. Information gained from these findings will improve our knowledge of, and capacity to study the interactions of CPPs with cells, and this will accelerate their translation as efficient vectors from the in vitro setting into the clinical arena.

Co-operative membrane disruption between cell-penetrating peptide and cargo: implications for the therapeutic use of the Bcl-2 converter peptide D-NuBCP-9-r8.

Delivering apoptosis inducing peptides to cells is an emerging area in cancer and molecular therapeutics. Here, we have identified an alternative mechanism of action for the proapoptotic chimeric peptide D-NuBCP-9-r8. Integral to D-NuBCP-9-r8 is the Nur-77-derived D-isoform sequence fsrslhsll that targets Bcl-2, and the cell-penetrating peptide (CPP) octaarginine (r8) that is required for intracellular delivery. We find that the N-terminal phenylalanine of fsrslhsll acts in synergy with the cell-penetrating moiety to enhance peptide uptake at low nontoxic levels and cause rapid membrane blebbing and cell necrosis at higher (IC(50)) concentrations. These effects were not observed when a single phenylalanine-alanine mutation was introduced at the N-terminus of D-NuBCP-9-r8. Using primary samples from chronic lymphocytic leukemia (CLL) patients and cancer cell lines, we show that NuBCP-9-r8 induced toxicity, via membrane disruption, is independent of Bcl-2 expression. Overall, this study demonstrates a new mechanism of action for this peptide and cautions its use as a highly specific entity for targeting Bcl-2. For delivery of therapeutic peptides the work emphasizes that key amino acids in cargo, located several residues away from the cell-penetrating sequence, can significantly influence their cellular uptake and mode of action.