Acidification of eukaryotic cell compartments is accomplished by vacuolar H⁺-ATPases (V-ATPases), large multisubunit complexes able to pump protons into the lumen of organelles or in the extracellular medium. V-ATPases are involved in a number of physiological cellular processes, and thus regulation of V-ATPase activity is of crucial importance for the cell. Indeed, dysfunction of V-ATPase or alterations of acidification have been recently recognized as key factors in a variety of human diseases. In this study, we applied capsule-based pH sensors and a real-time tracking method for investigating the role of the V<inf>1</inf>G<inf>1</inf> subunit of V-ATPases in regulating the activity of the proton pump. We first constructed stable cell lines overexpressing or silencing the subunit V<inf>1</inf>G<inf>1</inf>. Second, we used fluorescent capsule-based pH sensors to monitor acidification before and during internalization by modified and control living cells. By using a simple real-time method for tracking capsule internalization, we were able to identify different capsule acidification levels with respect to each analyzed cell and to establish the kinetics for each. The intracellular pH measurements indicate a delay in acidification in either V<inf>1</inf>G<inf>1</inf>-overexpressing or V<inf>1</inf>G<inf>1</inf>-silenced cells compared to controls. Finally, in an independent set of experiments, we applied transmission electron microscopy and confocal fluorescence microscopy to further investigate the internalization of the capsules. Both analyses confirm that capsules are engulfed in acidic vesicular structures in modified and control cell lines. The use of capsule-based pH sensors allowed demonstration of the importance of the V<inf>1</inf>G<inf>1</inf> subunit in V-ATPase activity concerning intravesicular acidification. We believe that the combined use of these pH-sensor system and such a real-time method for tracking their internalization path would contribute to systematically measure the proton concentration changes inside the endocytic compartments in various cell systems. This approach would provide fundamental information regarding molecular mechanisms and factors that regulate intracellular acidification, vesicular trafficking, and cytoskeletal reorganizations.

Polyelectrolyte multilayer (PEM) capsules engineered with active elements for targeting, labeling, sensing and delivery hold great promise for the controlled delivery of drugs and the development of new sensing platforms. PEM capsules composed of biodegradable polyelectrolytes are fabricated for intracellular delivery of encapsulated cargo (for example peptides, enzymes, DNA, and drugs) through gradual biodegradation of the shell components. PEM capsules with shells responsive to environmental or physical stimuli are exploited to control drug release. In the presence of appropriate triggers (e.g., pH variation or light irradiation) the pores of the multilayer shell are unlocked, leading to the controlled release of encapsulated cargos. By loading sensing elements in the capsules interior, PEM capsules sensitive to biological analytes, such as ions and metabolites, are assembled and used to detect analyte concentration changes in the surrounding environment. This Review aims to evaluate the current state of PEM capsules for drug delivery and sensing applications.

Multicompartment, spherical microcontainers were engineered through a layer-by-layer polyelectrolyte deposition around a fluorescent core while integrating a ruthenium polyoxometalate (Ru4POM), as molecular motor, vis-à-vis its oxygenic, propeller effect, fuelled upon H2O2 decomposition. The resulting chemomechanical system, with average speeds of up to 25 ?m s-1, is amenable for integration into a microfluidic set-up for mixing and displacement of liquids, whereby the propulsion force and the resulting velocity regime can be modulated upon H2O2-controlled addition.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that par- ticipates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic forcemicroscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of -cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Here we report for the first time the design and expression of highly charged, unfolded protein polymers based on elastin-like peptides (ELPs). Positively and negatively charged variants were achieved by introducing lysine and glutamic acid residues, respectively, within the repetitive pentapeptide units. Subsequently it was demonstrated that the monodisperse protein polyelectrolytes with precisely defined amino acid compositions, sequences, and stereochemistries can be transferred into superstructures exploiting their electrostatic interactions. Hollow capsules were assembled from oppositely charged protein chains by using the layer-by-layer technique. The structures of the capsules were analyzed by various microscopy techniques revealing the fabrication of multilayer containers. Due to their low toxicity in comparison to other polyelectrolytes, supercharged ELPs are appealing candidates for the construction of electrostatically induced scaffolds in biomedicine.

In this review we provide an overview of the recent progress in designing composite polymer capsules based on the Layer-by-Layer (LbL) technology demonstrated so far in material science, focusing on their potential applications in medicine, drug delivery and catalysis. The benefits and limits of current systems are discussed and the perspectives on emerging strategies for designing novel classes of therapeutic vehicles are highlighted. © 2010 The Royal Society of Chemistry.

Multiplexed detection of analytes is a challenge for numerous medical and biochemical applications. Many fluorescent particulate devices are being developed as ratiometric optical sensors to measure the concentration of intracellular analytes. The response of these sensors is based on changes of the emission intensity of analyte-sensitive probes, entrapped into the carrier system, which depends on the concentration of a specific analyte. However, there are a series of technical limits that prevent their use for quantitative detection of several analytes in parallel (e.g., emission crosstalk between different sensor molecules). Here we demonstrate that double-wall barcoded sensor capsules can be used for multiplexed analysis of proton, sodium, and potassium ions. The sensor detection methodology is based on porous microcapsules which carry ion-sensitive probes in their inner cavity for ion detection and a unique QD barcode in their outermost wall as tag for identification of individual sensors. The engineering of QD barcodes to capsules walls represents a promising strategy for optical multianalyte determination. © 2011 American Chemical Society.

On page 6417, L. L. del Mercato, D. Pisignano, and co-workers report a new type of 3D nanostructured pH-sensing organic fiber with embedded ratiometric fluorescent capsules. Upon proton-induced switching, the fibers undergo optical changes that are recorded by fluorescence detectors and correlated to the analyte concentration. The developed electrospinning fabrication approach is facile and versatile and enables the creation of sensitive and highly robust pH-sensing 3D scaffolds for environmental monitoring and biomedical applications, including tissue engineering and wound healing.

In this review we would like to aim at pharmaceuticals engineered on the nanoscale, i.e. pharmaceuticals where the nanomaterial plays the pivotal therapeutic role or adds additional functionality to the previous compound. Those cases would be considered as nanopharmaceuticals. The development of inorganic systems is opening the pharmaceutical nanotechnology novel horizons for diagnosis, imaging and therapy mainly because of their nanometer-size and their high surface area to volume ratios which allow for specific functions that are not possible in the micrometer-size particles. This review will focus on pharmaceutical forms that are based on inorganic nanoparticles where the nanosize of the inorganic component provides unique characteristics to the pharmaceutical form. Several examples of these systems that are either in pre-clinical investigation and under examination by the Food and Drug Administration (FDA) or that have been already approved by the FDA and are in clinical practice today like Gastromark (R), NanoTherm (R), Colloidal Gold for Lateral Flow tests, HfO-NPs, BioVant (TM) will be described and reviewed. (c) 2010 Elsevier Ltd. All rights reserved.

A fundamental issue in biomedical and environmental sciences is the development of sensitive and robust sensors able to probe the analyte of interest, under physiological and pathological conditions or in environmental samples, and with very high spatial resolution. In this work, novel hybrid organic fibers that can effectively report the analyte concentration within the local microenvironment are reported. The nanostructured and flexible wires are prepared by embedding fluorescent pH sensors based on seminaphtho-rhodafluor-1-dextran conjugate. By adjusting capsule/polymer ratio and spinning conditions, the diameter of the fibers and the alignment of the reporting capsules are both tuned. The hybrid wires display excellent stability, high sensitivity, as well as reversible response, and their operation relies on effective diffusional kinetic coupling of the sensing regions and the embedding polymer matrix. These devices are believed to be a powerful new sensing platform for clinical diagnostics, bioassays and environmental monitoring. Novel pH-sensing organic fibers are prepared by capsule-based sensors. Appropriate choice of capsule loading during electrospinning ensures alignment within the fibers, leading to pH-sensing devices with excellent stability, high sensitivity, ?m-spatial resolution as well as reversible response to proton switches. These results show a promising strategy for the development of sensitive and robust self-reporting fiber-optic sensors for bioassays and environmental monitoring.

Micrometer-sized polyelectrolyte capsules are synthesized, which have ion-sensitive fluorophores embedded in their cavities. As the membranes of the capsules are permeable to ions, the fluorescence of the capsules changed with the ion concentration. In particular, capsules sensitive to protons, sodium, potassium, and chloride ions are fabricated and their fluorescence response analyzed. In order to allow for ratiometric measurements, additional fluorophores whose emission do not depend on the ion concentration and which emit a different wavelength are co-embedded in the capsule cavities.

The Layer-by-Layer fabrication of polyelectrolyte capsules with and without Au nanoparticles embedded into their walls is reported. We have studied the behaviour of these capsules under microwave irradiation. Their properties have been investigated by transmission electron microscopy and dynamic light scattering measurements. We demonstrate that microwaves affect the structure of both capsules types by inducing remarkable damage to the multilayer wall. We also show that upon microwave exposure the walls of polyelectrolyte capsules which are modified with Au nanoparticles undergo rapid damage compared to capsules without incorporated nanoparticles. These results indicate that microwaves can be used to control the opening of cargo-loaded capsules, which could be harnessed for drug delivery purposes. © 2011 The Royal Society of Chemistry.