Autonomous Non-Equilibrium Self-Assembly and Molecular Movements Powered by Electrical Energy.

The ability to exploit energy autonomously is one of the hallmarks of life. Mastering such processes in artificial nanosystems can open technological opportunities. In the last decades, light- and chemically driven autonomous systems have been developed in relation to conformational motion and self-assembly, mostly in relation to molecular motors. In contrast, despite electrical energy being an attractive energy source to power nanosystems, its autonomous harnessing has received little attention. Herein we consider an operation mode that allows the autonomous exploitation of electrical energy by a self-assembling system. Threading and dethreading motions of a pseudorotaxane take place autonomously in solution, powered by the current flowing between the electrodes of a scanning electrochemical microscope. The underlying autonomous energy ratchet mechanism drives the self-assembly steps away from equilibrium with a higher energy efficiency compared to other autonomous systems. The strategy is general and might be extended to other redox-driven systems.

Stable and Biocompatible Monodispersion of C60 in Water by Peptides.

The lack of solubility in water and the formation of aggregates hamper many opportunities for technological exploitation of C60. Here, different peptides were designed and synthesized with the aim of monomolecular dispersion of C60 in water. Phenylalanines were used as recognizing moieties, able to interact with C60 through π-π stacking, while a varying number of glycines were used as spacers, to connect the two terminal phenylalanines. The best performance in the dispersion of C60 was obtained with the FGGGF peptidic nanotweezer at a pH of 12. A full characterization of this adduct was carried out. The peptides disperse C60 in water with high efficiency, and the solutions are stable for months both in pure water and in physiological environments. NMR measurements demonstrated the ability of the peptides to interact with C60. AFM measurements showed that C60 is monodispersed. Electrospray ionization mass spectrometry determined a stoichiometry of C60@(FGGGF)4. Molecular dynamics simulations showed that the peptides assemble around the C60 cage, like a candy in its paper wrapper, creating a supramolecular host able to accept C60 in the cavity. The peptide-wrapped C60 is fully biocompatible and the C60 "dark toxicity" is eliminated. C60@(FGGGF)4 shows visible light-induced reactive oxygen species (ROS) generation at physiological saline concentrations and reduction of the HeLa cell viability in response to visible light irradiation.

Electrochemical monitoring of reactive oxygen/nitrogen species and redox balance in living cells.

Levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in cells and cell redox balance are of great interest in live cells as they are correlated to several pathological and physiological conditions of living cells. ROS and RNS detection is limited due to their spatially restricted abundance: they are usually located in sub-cellular areas (e.g., in specific organelles) at low concentration. In this work, we will review and highlight the electrochemical approach to this bio-analytical issue. Combining electrochemical methods and miniaturization strategies, specific, highly sensitive, time, and spatially resolved measurements of cellular oxidative stress and redox balance analysis are possible. Graphical abstract In this work, we highlight and review the use of electrochemistry for the highly spatial and temporal resolved detection of ROS/RNS levels and of redox balance in living cells. These levels are central in several pathological and physiological conditions and the electrochemical approach is a vibrant bio-analytical trend in this field.

Surface-Confined Electrochemiluminescence Microscopy of Cell Membranes.

Herein is reported a surface-confined microscopy based on electrochemiluminescence (ECL) that allows to image the plasma membrane of single cells at the interface with an electrode. By analyzing photoluminescence (PL), ECL and AFM images of mammalian CHO cells, we demonstrate that, in contrast to the wide-field fluorescence, ECL emission is confined to the immediate vicinity of the electrode surface and only the basal membrane of the cell becomes luminescent. The resulting ECL microscopy reveals details that are not resolved by classic fluorescence microscopy, without any light irradiation and specific setup. The thickness of the ECL-emitting regions is ∼500 nm due to the unique ECL mechanism that involves short-lifetime electrogenerated radicals. In addition, the reported ECL microscopy is a dynamic technique that reflects the transport properties through the cell membranes and not only the specific labeling of the membranes. Finally, disposable transparent carbon nanotube (CNT)-based electrodes inkjet-printed on classic microscope glass coverslips were used to image cells in both reflection and transmission configurations. Therefore, our approach opens new avenues for ECL as a surface-confined microscopy to develop single cell assays and to image the dynamics of biological entities in cells or in membranes.

Single Cell Electrochemiluminescence Imaging: From the Proof-of-Concept to Disposable Device-Based Analysis.

We report here the development of coreactant-based electrogenerated chemiluminescence (ECL) as a surface-confined microscopy to image single cells and their membrane proteins. Labeling the entire cell membrane allows one to demonstrate that, by contrast with fluorescence, ECL emission is only detected from fluorophores located in the immediate vicinity of the electrode surface (i.e., 1-2 µm). Then, to present the potential diagnostic applications of our approach, we selected carbon nanotubes (CNT)-based inkjet-printed disposable electrodes for the direct ECL imaging of a labeled plasma receptor overexpressed on tumor cells. The ECL fluorophore was linked to an antibody and enabled to localize the ECL generation on the cancer cell membrane in close proximity to the electrode surface. Such a result is intrinsically associated with the unique ECL mechanism and is rationalized by considering the limited lifetimes of the electrogenerated coreactant radicals. The electrochemical stimulus used for luminescence generation does not suffer from background signals, such as the typical autofluorescence of biological samples. The presented surface-confined ECL microscopy should find promising applications in ultrasensitive single cell imaging assays.

Complex Media and Enzymatic Kinetics.

Enzymatic reactions in complex environments often take place with concentrations of enzyme comparable to that of substrate molecules. Two such cases occur when an enzyme is used to detect low concentrations of substrate/analyte or inside a living cell. Such concentrations do not agree with standard in vitro conditions, aimed at satisfying one of the founding hypotheses of the Michaelis-Menten reaction scheme, MM. It would be desirable to generalize the classical approach and show its applicability to complex systems. A permeable micrometrically structured hydrogel matrix was fabricated by protein cross-linking. Glucose oxidase enzyme (GOx) was embedded in the matrix and used as a prototypical system. The concentration of H2O2 was monitored in time and fitted by an accurate solution of the enzymatic kinetic scheme, which is expressed in terms of simple functions. The approach can also find applications in digital microfluidics and in systems biology where the kinetics response in the linear regimes often employed must be replaced.

"Active" drops as phantom models for living cells: a mesoscopic particle-based approach.

Drops and biological cells share some morphological features and visco-elastic properties. The modelling of drops by mesoscopic non-atomistic models has been carried out to a high degree of success in recent years. We extend such treatment and discuss a simple, drop-like model to describe the interactions of the outer layer of cells with the surfaces of materials. Cells are treated as active mechanical objects that are able to generate adhesion forces. They appear with their true size and are made of "parcels of fluids" or beads. The beads are described by (very) few quantities/parameters related to fundamental chemical forces such as hydrophilicity and lipophilicity that represent an average of the properties of a patch of material or an area of the cell(s) surface. The investigation of adhesion dynamics, motion of individual cells, and the collective behavior of clusters of cells on materials is possible. In the simulations, the drops become active soft matter objects and different from regular droplets they do not fuse when in contact, their trajectories are not Brownian, and they can be forced "to secrete" molecules, to name some of the properties targeted by the modeling. The behavior that emerges from the simulations allows ascribing some cell properties to their mechanics, which are related to their biological features.

A novel neuroferritinopathy mouse model (FTL 498InsTC) shows progressive brain iron dysregulation, morphological signs of early neurodegeneration and motor coordination deficits.

Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498-499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains. Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron-ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498-499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy.

Nongenetic Optical Modulation of Pluripotent Stem Cells Derived Cardiomyocytes Function in the Red Spectral Range.

Optical stimulation in the red/near infrared range recently gained increasing interest, as a not-invasive tool to control cardiac cell activity and repair in disease conditions. Translation of this approach to therapy is hampered by scarce efficacy and selectivity. The use of smart biocompatible materials, capable to act as local, NIR-sensitive interfaces with cardiac cells, may represent a valuable solution, capable to overcome these limitations. In this work, a far red-responsive conjugated polymer, namely poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl]] (PCPDTBT) is proposed for the realization of photoactive interfaces with cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Optical excitation of the polymer turns into effective ionic and electrical modulation of hPSC-CMs, in particular by fastening Ca2+ dynamics, inducing action potential shortening, accelerating the spontaneous beating frequency. The involvement in the phototransduction pathway of Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) and Na+ /Ca2+ exchanger (NCX) is proven by pharmacological assays and is correlated with physical/chemical processes occurring at the polymer surface upon photoexcitation. Very interestingly, an antiarrhythmogenic effect, unequivocally triggered by polymer photoexcitation, is also observed. Overall, red-light excitation of conjugated polymers may represent an unprecedented opportunity for fine control of hPSC-CMs functionality and can be considered as a perspective, noninvasive approach to treat arrhythmias.

Nano-Electrochemical Characterization of a 3D Bioprinted Cervical Tumor Model.

Current cancer research is limited by the availability of reliable in vivo and in vitro models that are able to reproduce the fundamental hallmarks of cancer. Animal experimentation is of paramount importance in the progress of research, but it is becoming more evident that it has several limitations due to the numerous differences between animal tissues and real, in vivo human tissues. 3D bioprinting techniques have become an attractive tool for many basic and applied research fields. Concerning cancer, this technology has enabled the development of three-dimensional in vitro tumor models that recreate the characteristics of real tissues and look extremely promising for studying cancer cell biology. As 3D bioprinting is a relatively recently developed technique, there is still a lack of characterization of the chemical cellular microenvironment of 3D bioprinted constructs. In this work, we fabricated a cervical tumor model obtained by 3D bioprinting of HeLa cells in an alginate-based matrix. Characterization of the spheroid population obtained as a function of culturing time was performed by phase-contrast and confocal fluorescence microscopies. Scanning electrochemical microscopy and platinum nanoelectrodes were employed to characterize oxygen concentrations-a fundamental characteristic of the cellular microenvironment-with a high spatial resolution within the 3D bioprinted cervical tumor model; we also demonstrated that the diffusion of a molecular model of drugs in the 3D bioprinted construct, in which the spheroids were embedded, could be measured quantitatively over time using scanning electrochemical microscopy.

Glucose micro-biosensor for scanning electrochemical microscopy characterization of cellular metabolism in hypoxic microenvironments.

Mapping of the metabolic activity of tumor tissues represents a fundamental approach to better identify the tumor type, elucidate metastatic mechanisms and support the development of targeted cancer therapies. The spatially resolved quantification of Warburg effect key metabolites, such as glucose and lactate, is essential. Miniaturized electrochemical biosensors scanned over cancer cells and tumor tissue to visualize the metabolic characteristics of a tumor is attractive but very challenging due to the limited oxygen availability in the hypoxic environments of tumors that impedes the reliable applicability of glucose oxidase-based glucose micro-biosensors. Herein, the development and application of a new glucose micro-biosensor is presented that can be reliably operated under hypoxic conditions. The micro-biosensor is fabricated in a one-step synthesis by entrapping during the electrochemically driven growth of a polymeric matrix on a platinum microelectrode glucose oxidase and a catalytically active Prussian blue type aggregate and mediator. The as-obtained functionalization improves significantly the sensitivity of the developed micro-biosensor for glucose detection under hypoxic conditions compared to normoxic conditions. By using the micro-biosensor as non-invasive sensing probe in Scanning Electrochemical Microscopy (SECM), the glucose uptake by a breast metastatic adenocarcinoma cell line, with an epithelial morphology, is measured.

Nature-inspired functional porous materials for low-concentration biomarker detection.

Nanostructuration is a promising tool for enhancing the performance of sensors based on electrochemical transduction. Nanostructured materials allow for increasing the surface area of the electrode and improving the limit of detection (LOD). In this regard, inverse opals possess ideal features to be used as substrates for developing sensors, thanks to their homogeneous, interconnected pore structure and the possibility to functionalize their surface. However, overcoming the insulating nature of conventional silica inverse opals fabricated via sol-gel processes is a key challenge for their application as electrode materials. In this work, colloidal assembly, atomic layer deposition and selective surface functionalization are combined to design conductive inverse opals as an electrode material for novel glucose sensing platforms. An insulating inverse opal scaffold is coated with uniform layers of conducting aluminum zinc oxide and platinum, and subsequently functionalized with glucose oxidase embedded in a polypyrrole layer. The final device can sense glucose at concentrations in the nanomolar range and is not affected by the presence of common interferents gluconolactone and pyruvate. This method may also be applied to different conductive materials and enzymes to generate a new class of highly efficient biosensors.

Semiconducting Polymer Nanoporous Thin Films as a Tool to Regulate Intracellular ROS Balance in Endothelial Cells.

The design of soft and nanometer-scale photoelectrodes able to stimulate and promote the intracellular concentration of reactive oxygen species (ROS) is searched for redox medicine applications. In this work, we show semiconducting polymer porous thin films with an enhanced photoelectrochemical generation of ROS in human umbilical vein endothelial cells (HUVECs). To achieve that aim, we synthesized graft copolymers, made of poly(3-hexylthiophene) (P3HT) and degradable poly(lactic acid) (PLA) segments, P3HT-g-PLA. In a second step, the hydrolysis of sacrificial PLA leads to nanometer-scale porous P3HT thin films. The pore sizes in the nm regime (220-1200 nm) were controlled by the copolymer composition and the structural arrangement of the copolymers during the film formation, as determined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The porous P3HT thin films showed enhanced photofaradaic behavior, generating a higher concentration of ROS in comparison to non-porous P3HT films, as determined by scanning electrochemical microscopy (SECM) measurements. The exogenous ROS production was able to modulate the intracellular ROS concentration in HUVECs at non-toxic levels, thus affecting the physiological functions of cells. Results presented in this work provide an important step forward in the development of new tools for precise, on-demand, and non-invasive modulation of intracellular ROS species and may be potentially extended to many other physiological or pathological cell models.

Highly electroconductive multiwalled carbon nanotubes as potentially useful tools for modulating calcium balancing in biological environments.

Aiming to explore the mechanisms modulating cell-carbon nanotube interactions, we investigated whether Ca(2+) ion balancing between intra- and extracellular environments could be affected by multiwalled carbon nanotubes (MWCNTs). We analyzed the effects induced by two different kinds of MWCNTs (as prepared and annealed at 2400°C) on the intracellular Ca(2+) ion levels in rat electrically sensitive cells and on the intercellular junction integrity of rat adenocarcinoma colon cells and platelet aggregation ability, which depend on the Ca(2+) concentration in the medium. MWCNTs, purified by annealing and more electroconductive as compared to nonannealed MWCNTs, affected Ca(2+) ion balancing between extra- and intracellular environments and induced changes on Ca(2+) ion-dependent cellular junctions and platelet aggregation, behaving as the calcium chelator ethylene glycol tetraacetic acid. This could be due to the sorption of cationic Ca(2+) ions on CNTs surface because of the excess of negatively charged electrons on the aromatic units formed on MWCNTs after annealing. From the ClinicAL Editor: The authors investigated whether Ca(2+) ion balance between intra- and extracellular space can be modulated by multiwalled carbon nanotubes (MWCNTs). Annealed nanotubes induced changes on Ca(2+) dependent cellular junctions and platelet aggregation, behaving similary to ethylene glycol tetraacetic acid, an established calcium chelator.

Enhanced Uptake and Phototoxicity of C60@albumin Hybrids by Folate Bioconjugation.

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C60@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C60 molecules in a physiological environment, (ii) HSA, upon C60 binding, maintains its biological identity and biocompatibility, (iii) the C60@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C60@HSA complex in HeLa cells.

Human Serum Albumin-Oligothiophene Bioconjugate: A Phototheranostic Platform for Localized Killing of Cancer Cells by Precise Light Activation.

The electronic, optical, and redox properties of thiophene-based materials have made them pivotal in nanoscience and nanotechnology. However, the exploitation of oligothiophenes in photodynamic therapy is hindered by their intrinsic hydrophobicity that lowers their biocompatibility and availability in water environments. Here, we developed human serum albumin (HSA)-oligothiophene bioconjugates that afford the use of insoluble oligothiophenes in physiological environments. UV-vis and electrophoresis proved the conjugation of the oligothiophene sensitizers to the protein. The bioconjugate is water-soluble and biocompatible, does not have any "dark toxicity", and preserves HSA in the physiological monomeric form, as confirmed by dynamic light scattering and circular dichroism measurements. In contrast, upon irradiation with ultralow light doses, the bioconjugate efficiently produces reactive oxygen species (ROS) and leads to the complete eradication of cancer cells. Real-time monitoring of the photokilling activity of the HSA-oligothiophene bioconjugate shows that living cells "explode" upon irradiation. Photodependent and dose-dependent apoptosis is one of the primary mechanisms of cell death activated by bioconjugate irradiation. The bioconjugate is a novel theranostic platform able to generate ROS intracellularly and provide imaging through the fluorescence of the oligothiophene. It is also a real-time self-reporting system able to monitor the apoptotic process. The induced phototoxicity is strongly confined to the irradiated region, showing localized killing of cancer cells by precise light activation of the bioconjugate.

Doxorubicin-Loaded Squid Pen Plaster: A Natural Drug Delivery System for Cancer Cells.

The native structure of the ß-chitin in the gladius (squid pen) of Loligo vulgaris squid can be used as a natural plaster to entrap and release a model drug, doxorubicin, in a targeted and controlled way. Local pH determines the protonation state of the doxorubicin molecules, controlling the two phenomena. Confocal microscopy shows that doxorubicin is uniformly embedded in the ß-chitin squid pen and is not simply adsorbed on its surface. Coculture with HeLa cells reveals that the ß-chitin squid pen plaster is perfectly biocompatible, while when it is loaded with doxorubicin it shows high cytotoxicity toward the cancer cells. The drug, once released, rapidly accumulates inside the cells. In conclusion, the native structure of a ß-chitin squid pen can be potentially applied as a "green" pH-responsive drug vehicle for controlled release.

Light-Triggered Electron Transfer between a Conjugated Polymer and Cytochrome C for Optical Modulation of Redox Signaling.

Protein reduction/oxidation processes trigger and finely regulate a myriad of physiological and pathological cellular functions. Many biochemical and biophysical stimuli have been recently explored to precisely and effectively modulate intracellular redox signaling, due to the considerable therapeutic potential. Here, we propose a first step toward an approach based on visible light excitation of a thiophene-based semiconducting polymer (P3HT), demonstrating the realization of a hybrid interface with the Cytochrome c protein (CytC), in an extracellular environment. By means of scanning electrochemical microscopy and spectro-electrochemistry measurements, we demonstrate that, upon optical stimulation, a functional interaction between P3HT and CytC is established. Polymer optical excitation locally triggers photoelectrochemical reactions, leading to modulation of CytC redox activity, either through an intermediate step, involving reactive oxygen species formation, or via a direct photoreduction process. Both processes are triggered by light, thus allowing excellent spatiotemporal resolution, paving the way to precise modulation of protein redox signaling.

Structural and electrochemical characterization of lawsone-dependent production of tellurium-metal nanoprecipitates by photosynthetic cells of Rhodobacter capsulatus.

Cells of the facultative photosynthetic bacterium Rhodobacter capsulatus exploit the simultaneous presence in the cultural medium of the toxic oxyanion tellurite (TeO32-) and the redox mediator lawsone (2-hydroxy-1,4-naphthoquinone) by reducing tellurite to metal Te0 nanoprecipitates (TeNPs) outside the cells. Here we have studied the mechanism by which lawsone interacts with metabolically active cells and analysed both structure and composition of the TeNPs collected from the growth medium of phototrophycally grown R. capsulatus. High Resolution Transmission Electron Microscopy (HR-TEM) images and Energy-Dispersive X-ray (EDX) microanalysis of TeNPs showed a central core of polycrystalline tellurium interspersed in an organic matrix with a predominant protein-based composition. The main proteins from Te0 nanostructures were identified by Liquid Chromatography tandem-Mass Spectrometry and were all correlated with the cell outer membrane composition. The interaction of reduced lawsone with tellurite and with the bacterial cells was probed by Cyclic Voltammetry and Scanning ElectroChemical Microscopy (SECM). We concluded that lawsone is required for the reduction of tellurite to metal Te0 in a reaction mechanism dependent on reducing equivalents deriving from the cell photosynthetic metabolism. SECM experiments demonstrate that lawsone, by diffusing inside the bacterial cells, is effectively available at the membrane site of the photosynthetic electron transport chain.

Specific, Surface-Driven, and High-Affinity Interactions of Fluorescent Hyaluronan with PEGylated Nanomaterials.

Hybrid nanomaterials are a subject of extensive research in nanomedicine, and their clinical application is reasonably envisaged in the near future. However, the fate of nanomaterials in biological environments poses serious limitations to their application; therefore, schemes to monitor them and gain control on their toxicity could be of great help for the development of the field. Here, we propose a probe for PEGylated nanosurfaces based on hyaluronic acid (HA) functionalized with rhodamine B (RB). We show that the high-affinity interaction of this fluorogenic hyaluronan (HA-RB) with nanoparticles exposing PEGylated surfaces results in their sensing, labeling for super-resolution imaging, and synergistic cellular internalization. HA-RB forms nanogels that interact with high affinity-down to the picomolar range-with silica nanoparticles, selectively when their surface is covered by a soft and amphiphilic layer. This surface-driven interaction triggers the enhancement of the luminescence intensity of the dyes, otherwise self-quenched in HA-RB nanogels. The sensitive labeling of specific nanosurfaces also allowed us to obtain their super-resolution imaging via binding-activated localization microscopy (BALM). Finally, we show how this high-affinity interaction activates a synergistic cellular uptake of silica nanoparticles and HA-RB nanogels, followed by a differential fate of the two partner nanomaterials inside cells.