Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA. Recently, CPPs were introduced that showed greatly enhanced delivery ...efficiency. However, the molecular basis of this increased activity is poorly understood. Here, we performed a detailed analysis of the molecular and physicochemical properties of seven different siRNA–CPP nanoparticles. In addition, we determined which complexes are internalized most efficiently into the leukemia cell-line SKNO-1, and subsequently inhibited the expression of a luciferase reporter gene. We demonstrated effective complexation of siRNA for all tested CPPs, and optimal encapsulation of the siRNA was achieved at very similar molar ratios independent of peptide charge. However, CPPs with an extreme high or low overall charge proved to be exceptions, suggesting an optimal range of charge for CPP–siRNA nanoparticle formation based on opposite charge. The most active CPP (PepFect6) displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules, indicating the necessity for partial decomplexation for efficient uptake. Surprisingly, CPP–siRNA complexes acquired a negative ζ-potential in the presence of serum. These novel insights shed light on the observation that cell association is necessary but not sufficient for activity and motivate new research into the nature of the nanoparticle–cell interaction. Overall, our results provide a comprehensive molecular basis for the further development of peptide-based oligonucleotide transfection agents.
Macromolecular drug candidates and nanoparticles are typically tested in 2D cancer cell culture models, which are often directly followed by in vivo animal studies. The majority of these drug ...candidates, however, fail in vivo. In contrast to classical small-molecule drugs, multiple barriers exist for these larger molecules that two-dimensional approaches do not recapitulate. In order to provide better mechanistic insights into the parameters controlling success and failure and due to changing ethical perspectives on animal studies, there is a growing need for in vitro models with higher physiological relevance. This need is reflected by an increased interest in 3D tumor models, which during the past decade have evolved from relatively simple tumor cell aggregates to more complex models that incorporate additional tumor characteristics as well as patient-derived material. This review will address tissue culture models that implement critical features of the physiological tumor context such as 3D structure, extracellular matrix, interstitial flow, vascular extravasation, and the use of patient material. We will focus on specific examples, relating to peptide-and protein-conjugated drugs and other nanoparticles, and discuss the added value and limitations of the respective approaches.
Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. ...Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.
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Hypoxia switches the metabolism of tumor cells and induces drug resistance. Currently, no therapeutic exists that effectively and specifically targets hypoxic cells in tumors. Development of such ...therapeutics critically depends on the availability of in vitro models that accurately recapitulate hypoxia as found in the tumor microenvironment. Here, we report on the design and validation of an easy-to-fabricate tumor-on-a-chip microfluidic platform that robustly emulates the hypoxic tumor microenvironment. The tumor-on-a-chip model consists of a central chamber for 3D tumor cell culture and two side channels for medium perfusion. The microfluidic device is fabricated from polydimethylsiloxane (PDMS), and oxygen diffusion in the device is blocked by an embedded sheet of polymethyl methacrylate (PMMA). Hypoxia was confirmed using oxygen-sensitive probes and the effect on the 3D tumor cell culture investigated by a pH-sensitive dual-labeled fluorescent dextran and a fluorescently labeled glucose analogue. In contrast to control devices without PMMA, PMMA-containing devices gave rise to decreases in oxygen and pH levels as well as an increased consumption of glucose after two days of culture, indicating a rapid metabolic switch of the tumor cells under hypoxic conditions towards increased glycolysis. This platform will open new avenues for testing anti-cancer therapies targeting hypoxic areas.
To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target ...flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved.
Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol.
We believe that this reengineered system is an important step forward to addressing efficient cell-specific delivery of proteins to the cytosol.
Over many years, a variety of delivery systems have been investigated that have the capacity to shuttle macromolecular cargoes, especially proteins, into the cytosol. Due to the lack of an objective ...way to quantify cytosolic delivery, relative delivery efficiencies of the various transport systems have remained unclear. Here, we demonstrate the use of the biotin ligase assay for a quantitative comparison of protein transport to the cytosol via cell-penetrating peptides, supercharged proteins and bacterial toxins in four different cell lines. The data illustrate large differences in both the total cellular internalization, which denotes any intracellular location including endosomes, and in the cytosolic uptake of the transport systems, with little correlation between the two. Also, we found significant differences between the cell lines. In general, protein transport systems based on cell-penetrating peptides show a modest total uptake, and mostly do not deliver cargo to the cytosol. Systems based on bacterial toxins show a modest receptor-mediated internalization but an efficient delivery to the cytosol. Supercharged proteins, on the contrary, are not receptor-specific and lead to massive total internalization into endosomes, but only low amounts end up in the cytosol.
Cationic cell-penetrating peptides (CPP) are receiving increasing attention as molecular transporters of membrane-impermeable molecules. Import of cationic CPP occurs both via endocytosis and — at ...higher peptide concentrations — in an endocytosis-independent manner via localized regions of the plasma membrane. At present, this endocytosis-independent import of cationic CPP is not well understood, but has been shown to be sensitive to various pharmacological inhibitors, suggesting a role of an unidentified enzymatic activity. Here, we demonstrate that the direct translocation of cationic CPP depends on a CPP-induced translocation of acid sphingomyelinase (ASMase) to the outer leaflet of the plasma membrane and ceramide formation. The involvement of ASMase in uptake was confirmed by a pharmacological inhibition of ASMase by imipramine and a subsequent rescue of uptake through external addition of sphingomyelinase, and by using ASMase-deficient cells. We also found that the threshold for direct CPP translocation can be lowered through addition of sphingomyelinase and that sphingomyelinase enhances the translocation of R9 coupled to low-molecular weight cargos, but not high-molecular weight cargos. In conclusion, we show that a previously poorly understood mechanism of cationic CPP import depends on the ASMase-dependent formation of ceramide on the outer leaflet of the plasma membrane. To our knowledge, this is the first illustration that a class of delivery vectors operates through the induction of an enzymatic activity that changes the lipid composition of the plasma membrane.
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Variations in oxygen levels play key roles in numerous physiological and pathological processes, but are often not properly controlled in
models, introducing a significant bias in experimental ...outcomes. Recent developments in microfluidic technology have introduced a paradigm shift by providing new opportunities to better mimic physiological and pathological conditions, which is achieved by both regulating and monitoring oxygen levels at the micrometre scale in miniaturized devices. In this review, we first introduce the nature and relevance of oxygen-dependent pathways in both physiological and pathological contexts. Subsequently, we discuss strategies to control oxygen in microfluidic devices, distinguishing between engineering approaches that operate at the device level during its fabrication and chemical approaches that involve the active perfusion of fluids oxygenated at a precise level or supplemented with oxygen-producing or oxygen-scavenging materials. In addition, we discuss readout approaches for monitoring oxygen levels at the cellular and tissue levels, focusing on electrochemical and optical detection schemes for high-resolution measurements directly on-chip. An overview of different applications in which microfluidic devices have been utilized to answer biological research questions is then provided. In the final section, we provide our vision for further technological refinements of oxygen-controlling devices and discuss how these devices can be employed to generate new fundamental insights regarding key scientific problems that call for emulating oxygen levels as encountered
. We conclude by making the case that ultimately emulating physiological or pathological oxygen levels should become a standard feature in all
cell, tissue, and organ models.
Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived ...N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19)F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-1.1.1-1-ylglycine (CF3-Bpg) as a reporter group for (19)F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼ 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.
In drug development, major resources are invested into the development of cellular delivery systems to increase the effectiveness of a large array of potential therapeutics, such as proteins and ...oligonucleotides. These carriers comprise cell-penetrating peptides (CPPs), cationic lipids and cationic polymers. In recent years, evidence has been accumulating that these carriers not only act as mere pharmacokinetic modifiers but also interfere with cellular processes in various ways. In this review, we present an overview of the biological side effects associated with carrier systems. The focus will be on CPPs, which have been explored for a diverse set of cargos. Reported activities range from an induction of receptor internalization to the generation of reactive oxygen species. Ultimately, cell-penetrating molecules with such biological side effects might evolve into new bioactive agents that combine delivery capacity and pharmacophore in a single molecular entity. First examples for such molecules will be presented.
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