Supramolecular Assembly in Live Cells Mapped by Real-Time Phasor-Fluorescence Lifetime Imaging.

The complex dynamics and transience of assembly pathways in living systems complicate the understanding of these molecular to nanoscale processes. Current technologies are unable to track the molecular events leading to the onset of assembly, where real-time information is imperative to correlate their rich biology. Using a chemically designed pro-assembling molecule, we map its transformation into nanofibers and their fusion with endosomes to form hollow fiber clusters. Tracked by phasor-fluorescence lifetime imaging (phasor-FLIM) in epithelial cells (L929, A549, MDA-MB 231) and correlative light-electron microscopy and tomography (CLEM), spatiotemporal splicing of the assembly events shows time-correlated metabolic dysfunction. The biological impact begins with assembly-induced endosomal disruption that reduces glucose transport into the cells, which, in turn, stymies mitochondrial respiration.

Nanoscale Control of the Surface Functionality of Polymeric 2D Materials.

Typically, 2D nanosheets have a homogeneous surface, making them a major challenge to structure. This study proposes a novel concept of 2D organic nanosheets with a heterogeneously functionalized surface. This work achieves this by consecutively crystallizing two precisely synthesized polymers with different functional groups in the polymer backbone in a two-step process. First, the core platelet is formed and then the second polymer is crystallized around it. As a result, the central area of the platelets has a different surface functionality than the periphery. This concept offers two advantages: the resulting polymeric 2D platelets are stable in dispersion, which simplifies further processing and makes both crystal surfaces accessible for subsequent functionalization. Additionally, a wide variety of polymers can be used, making the process and the choice of surface functionalization very flexible.

Endosomal sorting results in a selective separation of the protein corona from nanoparticles.

The formation of the protein corona is a well-known effect when nanoparticles (NP) are exposed to biological environments. The protein corona is the most important factor, which determines the rate and route of endocytosis, and decisively impacts cellular processes and even the release of the active pharmaceutical ingredient from the nanoparticles. While many studies concentrate on the effect of the protein corona formation extracellularly or the uptake consequences, little is known about the fate of the protein corona inside of cells. Here, we reconstruct for the first time the separation of the protein corona from the NPs by the cell and their further fate. Ultimately, the NPs and protein corona are separated from each other and end up in morphologically different cellular compartments. The cell directs the NPs towards recycling endosomes, whereas the protein corona gathers in multivesicular bodies. From this, we conclude that the NPs are prepared for subsequent exocytosis, while the protein corona remains in the cell and is finally metabolized there.

In Situ Assembly of Platinum(II)-Metallopeptide Nanostructures Disrupts Energy Homeostasis and Cellular Metabolism.

Nanostructure-based functions are omnipresent in nature and essential for the diversity of life. Unlike small molecules, which are often inhibitors of enzymes or biomimetics with established methods of elucidation, we show that functions of nanoscale structures in cells are complex and can implicate system-level effects such as the regulation of energy and redox homeostasis. Herein, we design a platinum(II)-containing tripeptide that assembles into intracellular fibrillar nanostructures upon molecular rearrangement in the presence of endogenous H2O2. The formed nanostructures blocked metabolic functions, including aerobic glycolysis and oxidative phosphorylation, thereby shutting down ATP production. As a consequence, ATP-dependent actin formation and glucose metabolite-dependent histone deacetylase activity are downregulated. We demonstrate that assembly-driven nanomaterials offer a rich avenue to achieve broad-spectrum bioactivities that could provide new opportunities in drug discovery.

Nanocarriers Made of Proteins: Intracellular Visualization of a Smart Biodegradable Drug Delivery System.

This work analyzes the intracellular fate of protein-based nanocarriers along their endolysosomal pathway by means of correlative light and electron microscopy methods. To unambiguously identify the nanocarriers and their degradation remnants in the cellular environment, they are labeled with fluorescent, inorganic nanoplatelets. This allows tracking the nanocarriers on their intracellular pathway by means of electron microscopy imaging. From the present data, it is possible to identify different cell compartments in which the nanocarriers are processed. Finally, three different terminal routes for the intracellular destiny of the nanocarriers are presented. These findings are important to reveal the degradation process of protein nanocapsules and contribute to the understanding of the therapeutic success of an encapsulated drug.

Surface Properties of Colloidal Particles Affect Colloidal Self-Assembly in Evaporating Self-Lubricating Ternary Droplets.

In this work, we unravel the role of surface properties of colloidal particles on the formation of supraparticles (clusters of colloidal particles) in a colloidal Ouzo droplet. Self-lubricating colloidal Ouzo droplets are an efficient and simple approach to form supraparticles, overcoming the challenge of the coffee stain effect in situ. Supraparticles are an efficient route to high-performance materials in various fields, from catalysis to carriers for therapeutics. Yet, the role of the surface of colloidal particles in the formation of supraparticles using Ouzo droplets remains unknown. Therefore, we used silica particles as a model system and compared sterically stabilized versus electrostatically stabilized silica particles─positively and negatively charged. Additionally, we studied the effect of hydration. Hydrated negatively charged silica particles and sterically stabilized silica particles form supraparticles. Conversely, dehydrated negatively charged silica particles and positively charged amine-coated particles form flat film-like deposits. Notably, the assembly process is different for all the four types of particles. The surface modifications alter (a) the contact line motion of the Ouzo droplet and (b) the particle-oil and particle-substrate interactions. These alterations modify the particle accumulation at the various interfaces, which ultimately determines the shape of the final deposit. Thus, by modulating the surface properties of the colloidal particles, we can tune the shape of the final deposit, from a spheroidal supraparticle to a flat deposit. In the future, this approach can be used to tailor the supraparticles for applications such as optics and catalysis, where the shape affects the functionality.

Super liquid repellent surfaces for anti-foaming and froth management.

Wet and dry foams are prevalent in many industries, ranging from the food processing and commercial cosmetic sectors to industries such as chemical and oil-refining. Uncontrolled foaming results in product losses, equipment downtime or damage and cleanup costs. To speed up defoaming or enable anti-foaming, liquid oil or hydrophobic particles are usually added. However, such additives may need to be later separated and removed for environmental reasons and product quality. Here, we show that passive defoaming or active anti-foaming is possible simply by the interaction of foam with chemically or morphologically modified surfaces, of which the superamphiphobic variant exhibits superior performance. They significantly improve retraction of highly stable wet foams and prevention of growing dry foams, as quantified for beer and aqueous soap solution as model systems. Microscopic imaging reveals that amphiphobic nano-protrusions directly destabilize contacting foam bubbles, which can favorably vent through air gaps warranted by a Cassie wetting state. This mode of interfacial destabilization offers untapped potential for developing efficient, low-power and sustainable foam and froth management.

How a water drop removes a particle from a hydrophobic surface.

To understand the removal of particles from surfaces by water drops, we used an inverted laser scanning confocal microscope to image the collision between a water drop and a particle on a flat polydimethylsiloxane (PDMS) surface. The dynamic drop-particle contact line was monitored by fixing the drop directly above the objective lens while moving the sample stage at well-defined speeds (10-500 µm s-1). The lateral force acting on the drop during the collision was measured as a function of speed, using a force sensor mounted on the microscope. Depending on the collision speed, the particle either stays attached at the rear of the drop or detaches from it. We propose a criterion to determine whether the particle remains attached to the drop based on the capillary and resistive forces acting on the particle during the collision. The forces measured when the particle crosses the air-water interface are compared to existing models. We adapted these to account for rolling of the particle. By comparing our experimental measurements with an analytical model for the capillary torque acting on a particle rolling at an interface, we provide detailed insights on the origins of the resistive force acting on the particle when it is pushed or pulled by the drop. A low friction force between the surface and the particle increases the likelihood of particle removal.

Adaptive Wetting of Polydimethylsiloxane.

To better understand the wetting of cross-linked polydimethylsiloxane (PDMS), we measured advancing and receding contact angles of sessile water drops on cross-linked PDMS as a function of contact line velocity (up to 100 µm/s). Three types of samples were investigated: pristine PDMS, PDMS where oligomers were removed by toluene treatment, and PDMS with an enriched concentration of oligomers. Depending on the velocity of advancing contact lines and the contact time with water, different modes of wetting were observed: one with a relatively low contact angle hysteresis (Δθ ≈ 10°) and one with a larger hysteresis. We attribute the low hysteresis state, called the lubricated state, to the enrichment of free oligomers at the water-PDMS interface. The enrichment of oligomers is induced by drop contact. The kinetics of the transition to the lubricated state can be described by adaptation theory. PDMS adapts to the presence of water by an enrichment of free oligomers at the interface and a correlated reduction in interfacial tension.

Submicrometer-Sized Roughness Suppresses Bacteria Adhesion.

Biofilm formation is most commonly combatted with antibiotics or biocides. However, proven toxicity and increasing resistance of bacteria increase the need for alternative strategies to prevent adhesion of bacteria to surfaces. Chemical modification of the surfaces by tethering of functional polymer brushes or films provides a route toward antifouling coatings. Furthermore, nanorough or superhydrophobic surfaces can delay biofilm formation. Here we show that submicrometer-sized roughness can outweigh surface chemistry by testing the adhesion of E. coli to surfaces of different topography and wettability over long exposure times (>7 days). Gram-negative and positive bacterial strains are tested for comparison. We show that an irregular three-dimensional layer of silicone nanofilaments suppresses bacterial adhesion, both in the presence and absence of an air cushion. We hypothesize that a 3D topography can delay biofilm formation (i) if bacteria do not fit into the pores of the coating or (ii) if bending of the bacteria is required to adhere. Thus, such a 3D topography offers an underestimated possibility to design antibacterial surfaces that do not require biocides or antibiotics.

Microdroplet Contaminants: When and Why Superamphiphobic Surfaces Are Not Self-Cleaning.

Superamphiphobic surfaces are commonly associated with superior anticontamination and antifouling properties. Visually, this is justified by their ability to easily shed off drops and contaminants. However, on micropillar arrays, tiny droplets are known to remain on pillars' top faces while the drop advances. This raises the question of whether remnants remain even on nanostructured superamphiphobic surfaces. Are superamphiphobic surfaces really self-cleaning? Here we investigate the presence of microdroplet contaminants on three nanostructured superamphiphobic surfaces. After brief contact with liquids having different volatilities and surface tension (water, ethylene glycol, hexadecane, and an ionic liquid), confocal microscopy reveals a "blanket-like" layer of microdroplets remaining on the surface. It appears that the phenomenon is universal. Notably, when placing subsequent drops onto the contaminated surface, they are still able to roll off. However, adhesion forces can gradually increase by up to 3 times after repeated liquid drop contact. Therefore, we conclude that superamphiphobic surfaces do not warrant self-cleaning and anticontamination capabilities at sub-micrometric length scales.

When and how self-cleaning of superhydrophobic surfaces works.

Despite the enormous interest in superhydrophobicity for self-cleaning, a clear picture of contaminant removal is missing, in particular, on a single-particle level. Here, we monitor the removal of individual contaminant particles on the micrometer scale by confocal microscopy. We correlate this space- and time-resolved information with measurements of the friction force. The balance of capillary and adhesion force between the drop and the contamination on the substrate determines the friction force of drops during self-cleaning. These friction forces are in the range of micro-Newtons. We show that hydrophilic and hydrophobic particles hardly influence superhydrophobicity provided that the particle size exceeds the pore size or the thickness of the contamination falls below the height of the protrusions. These detailed insights into self-cleaning allow the rational design of superhydrophobic surfaces that resist contamination as demonstrated by outdoor environmental (>200 days) and industrial standardized contamination experiments.

One-Step Preparation of Fuel-Containing Anisotropic Nanocapsules with Stimuli-Regulated Propulsion.

One of the dreams of nanotechnology is to create tiny objects, nanobots, that are able to perform difficult tasks in dimensions and locations that are not directly accessible. One basic function of these nanobots is motility. Movements created by self-propelled micro- and nanovehicles are usually dependent on the production of propellants from catalytic reactions of fuels present in the environment. Developing self-powered nanovehicles with internally stored fuels that display motion regulated by external stimuli represents an intriguing and challenging alternative. Herein, a one-step preparation of fuel-containing nanovehicles that feature a motion that can be regulated by external stimuli is reported. Nanovehicles are prepared via a sol-gel process confined at the oil/water interface of miniemulsions. The nanovehicles display shapes ranging from mushroom-like to truncated cones and a core-shell structure so that the silica shell acts as a hull for the nanovehicles while the core is used to store the fuel. Azo-based initiators are loaded in the nanovehicles, which are activated to release nitrogen gas upon increase of temperature or exposure to UV light. Enhanced diffusion of nanovehicles is achieved upon decomposition of the fuel.

Flow-Induced Long-Term Stable Slippery Surfaces.

Slippery lubricant-infused surfaces allow easy removal of liquid droplets on surfaces. They consist of textured or porous substrates infiltrated with a chemically compatible lubricant. Capillary forces help to keep the lubricant in place. Slippery surfaces hold promising prospects in applications including drag reduction in pipes or food packages, anticorrosion, anti-biofouling, or anti-icing. However, a critical drawback is that shear forces induced by flow lead to depletion of the lubricant. In this work, a way to overcome the shear-induced lubricant depletion by replenishing the lubricant from the flow of emulsions is presented. The addition of small amounts of positively charged surfactant reduces the charge repulsion between the negatively charged oil droplets contained in the emulsion. Attachment and coalescence of oil droplets from the oil-in-water emulsion at the substrate surface fills the structure with the lubricant. Flow-induced lubrication of textured surfaces can be generalized to a broad range of lubricant-solid combinations using minimal amounts of oil.

How to Coat the Inside of Narrow and Long Tubes with a Super-Liquid-Repellent Layer-A Promising Candidate for Antibacterial Catheters.

Fouling of thin tubes is a major problem, leading to various infections and associated morbidities, while cleaning is difficult or even impossible. Here, a generic method is introduced to activate and coat the inside of meter-long and at the same time thin (down to 1 mm) tubes with a super-liquid-repellent layer of nanofilaments, exhibiting even antibacterial properties. Activation is facilitated by pumping an oxidative Fenton solution through the tubes. Subsequent pumping of a silane solution renders the surface of the tubes super-liquid-repellent. The wide applicability of the method is demonstrated by coating stiff and flexible tubes made of polymers, inorganic/organic hybrids, metals, and ceramics. Coated medical catheters show excellent antibacterial properties. Notably, the nanofilaments retain their antibacterial properties even in the superhydrophilic state. These findings open new avenues toward the design of biocide-free, antibacterial tubings and catheters.

Pre-adsorption of antibodies enables targeting of nanocarriers despite a biomolecular corona.

To promote drug delivery to exact sites and cell types, the surface of nanocarriers is functionalized with targeting antibodies or ligands, typically coupled by covalent chemistry. Once the nanocarrier is exposed to biological fluid such as plasma, however, its surface is inevitably covered with various biomolecules forming the protein corona, which masks the targeting ability of the nanoparticle. Here, we show that we can use a pre-adsorption process to attach targeting antibodies to the surface of the nanocarrier. Pre-adsorbed antibodies remain functional and are not completely exchanged or covered by the biomolecular corona, whereas coupled antibodies are more affected by this shielding. We conclude that pre-adsorption is potentially a versatile, efficient and rapid method of attaching targeting moieties to the surface of nanocarriers.

Visible light active nanofibrous membrane for antibacterial wound dressing.

Chronic wound infections, especially due to the emergence of multidrug resistance in bacteria, require the urgent development of alternative antibacterial therapies. Here, we developed a new class of hydrogel nanofibrous membranes that show visible light-induced disinfection. The presented photocatalytic disinfection is based on the generation of reactive singlet oxygen from a conjugated microporous polymer upon visible light irradiation. Therefore, sustained protection of the wound area can be provided in the presence of visible light. Fabrication of the photoactive wound dressing consists of first synthesizing photoactive conjugated microporous polymer nanoparticles by miniemulsion polymerization and subsequently embedding the nanoparticles in polyvinyl alcohol hydrogel nanofibers by colloid-electrospinning. The fibers were then crosslinked in glutaraldehyde/HCl vapor to be water-insoluble. This nanoparticle-in-nanofiber structure allows for a flexible combination of the properties of the nanoparticles and supporting nanofibers. The disinfecting properties of the membranes were evaluated with the inactivation of Escherichia coli K-12 and Bacillus subtilis as model systems of Gram-negative and Gram-positive bacteria, as well as the inhibition of biofilm growth under irradiation of visible light. Cytotoxicity tests on fibroblast cells revealed a high cytocompatibility of the membranes. Furthermore, the good mechanical properties of the membranes allow for their facile removal after use and prevent the leakage of the embedded nanoparticles into the wound, making the photoactive hydrogel membranes a promising candidate for active wound dressing materials.

Genotoxic effects of zinc oxide nanoparticles.

The potential toxicity of nanoparticles has currently provoked public and scientific discussions, and attempts to develop generally accepted handling procedures for nanoparticles are under way. The investigation of the impact of nanoparticles on human health is overdue and reliable test systems accounting for the special properties of nanomaterials must be developed. Nanoparticular zinc oxide (ZnO) may be internalised through ambient air or the topical application of cosmetics, only to name a few, with unpredictable health effects. Therefore, we analysed the determinants of ZnO nanoparticle (NP) genotoxicity. ZnO NPs (15-18 nm in diameter) were investigated at concentrations of 0.1, 10 and 100 µg mL(-1) using the cell line A549. Internalised NPs were only infrequently detectable by TEM, but strongly increased Zn(2+) levels in the cytoplasm and even more in the nuclear fraction, as measured by atom absorption spectroscopy, indicative of an internalised zinc and nuclear accumulation. We observed a time and dosage dependent reduction of cellular viability after ZnO NP exposure. ZnCl2 exposure to cells induced similar impairments of cellular viability. Complexation of Zn(2+) with diethylene triamine pentaacetic acid (DTPA) resulted in the loss of toxicity of NPs, indicating the relevant role of Zn(2+) for ZnO NP toxicity. Foci analyses showed the induction of DNA double strand breaks (DSBs) by ZnO NPs and increased intracellular reactive oxygen species (ROS) levels. Treatment of the cells with the ROS scavenger N-acetyl-l-cysteine (NAC) resulted in strongly decreased intracellular ROS levels and reduced DNA damage. However, a slow increase of ROS after ZnO NP exposure and reduced but not quashed DSBs after NAC-treatment suggest that Zn(2+) may exert genotoxic activities without the necessity of preceding ROS-induction. Our data indicate that ZnO NP toxicity is a result of cellular Zn(2+) intake. Subsequently increased ROS-levels cause DNA damage. However, we found evidence for the assumption that DNA-DSBs could be caused by Zn(2+) without the involvement of ROS.