In vivo cell microenvironments are characterized by gradients of soluble molecules, particularly oxygen, whose levels depend on the distance from blood vessels and cellular metabolic activity. In pathological conditions, oxygen levels often decrease, leading to hypoxia and steeper concentration gradients. Standard in vitro cultures provide homogeneous conditions that poorly replicate the in vivo environment. This work presents the microfabrication of devices that generate cell culture microenvironments characterized by three-dimensional oxygen gradients that resemble those found in vivo. We patterned the material as a hydrogel to create oxygen gradients, thanks to immobilized proteins that regulate oxygen. We developed a digital inkjet printing procedure based on two reactive inks to generate patterns. The resulting spatial oxygen concentrations were quantified using three-dimensional electrochemical mapping. Living cells were grown and observed in the in vivo-mimetic oxygen gradients, and the growth of malignant cells was consistent with adaptation to hypoxic conditions.

Human pluripotent stem cells (hPSCs), derived from embryos or reprogrammed from somatic cells, represent a key resource for preclinical research and personalized medicine due to their capacity for indefinite self-renewal and differentiation into all three germ layer derivatives. However, incomplete maturation of hPSC-derived cells limits their translational potential across multiple fields, including cardiovascular medicine. In particular, cardiomyocytes differentiated from hPSCs (hPSC-CMs), although valuable models to recapitulate physio-pathological traits of heart development and disease, retain fetal-like properties that constrain their utility in late-onset disease modelling, cell therapy and drug testing. While several strategies have been proposed to enhance maturation, all present some technical or biological limitations. Recently, optically active semiconductors, both organic and inorganic, have emerged as gene-free tools to modulate cellular function with high spatial and temporal precision. Here, we investigate the effects of long-term photostimulation using the far-red absorbing conjugated polymer (PCPDTBT) on hPSC-CMs. We show that 6-hour pulsed photostimulation significantly enhances maturation across transcriptional, functional, structural, and metabolic levels without inducing phototoxicity. Overall, our findings establish a simple and effective approach to improve hPSC-CM maturation, thereby expanding their applicability in disease modelling and therapeutic development.

Ovarian cancer (OC) is the most lethal gynecologic malignancy, often diagnosed at advanced stages due to clinically silent peritoneal carcinomatosis. Although intraperitoneal (IP) chemotherapy enhances drug exposure, its effectiveness is hindered by rapid clearance, toxicity, and uneven distribution. To address these challenges, we developed a novel drug delivery system integrating paclitaxel (PTX)-loaded poly(lactic-co-glycolic acid) microparticles (PLGA-MPs) within calcium-alginate microbeads (Alg-MBs). This system aims to provide sustained drug release while minimizing adverse effects. PTX-loaded PLGA-MPs were prepared via solvent evaporation and encapsulated in Alg-MBs using a coaxial air jet generator. In vitro studies showed an initial burst release over five days, followed by sustained release until day 21, confirming the role of Alg-MBs in modulating drug diffusion. Cytotoxicity tests in 2D SKOV-3 OC cultures revealed dose-dependent effects, with increased PTX concentrations reducing cell viability. A 3D bioprinted tumor model was used to better replicate in vivo conditions and evaluate long-term efficacy. Sustained PTX release resulted in progressive tumor cell death over 21 days, with delayed but potent cytotoxicity at higher doses. These findings support hierarchical PTX microencapsulation for prolonged IP chemotherapy, while the 3D bioprinted model provided a more physiologically relevant platform for evaluating long-term therapeutic efficacy in OC treatment.

Separators are critical components in batteries. In closed systems, like lithium-ion batteries, they should ensure a good ion transport between the two electrodes. In open systems, such as flow batteries, separators should also avoid the crossover of the redox species in the flowing electrolytes. We focused our studies on separators for redox flow batteries (RFBs), which are widely applied as electrochemical energy storage systems, specifically in combination with renewable energy systems like photovoltaic or wind turbines. We pioneered the use of scanning electrochemical microscopy to characterize the efficiency of different commercial membranes, specifically for all-copper RFB, by evaluating the cupric ion permeation through these membranes. In fact, one drawback of this system is represented by the permeation of cupric ions in the negative half-cell that leads to the dissolution of the copper deposit and results in the battery's self-discharge. Several types of membranes have been tested as separators to limit this process. Finite elements simulations were also performed to quantitatively interpret the electrochemical measurements and to correlate separator permeabilities to membrane molecular characteristics such as hydrophobicity and ion exchange capability. Estimates of copper ion diffusion coefficients and permeabilities were provided for a subset of the separators investigated.

Time-resolved, rapid-scan Fourier transform infrared (FT-IR) difference spectra have been recorded upon illumination on photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides under fixed hydration conditions (relative humidity = 76%). Two different illumination schemes were adopted. Whereas the use of a laser flash (duration: 7 ns) made it possible to follow the kinetics of recombination of the light-induced state P+QA– to the neutral state PQA, the use of a 20.5 s continuous light from a lamp made it possible to follow both the build-up of a steady-state P+QA– population and its decay to PQA. Comparison between P+QA–/PQA FT-IR difference spectra obtained under (or 650 ms after) continuous illumination and obtained after one laser flash show small but meaningful differences, reflecting structural changes in the light-adapted state produced by the 20.5 s period of illumination. These differences are strikingly similar to those observed when comparing FT-IR difference spectra reflecting charge separation in photosystem II in light-adapted states and non-light-adapted states (c.f. Sipka et al., “Light-Adapted Charge-Separated State of Photosystem II: Structural and Functional Dynamics of the Closed Reaction Center”. Plant Cell. 2021. 33(4): 1286–1302). Two-dimensional correlation spectroscopy analysis revealed that in all the observed series of time-resolved FT-IR difference spectra (under illumination, after illumination, and after a laser flash), marker bands at 1749, 1716, and 1668 cm–1 all evolve synchronously, demonstrating that electron transfer reactions and protein backbone response (at least the one reflected by the 1668 cm–1 band) are strongly correlated. Conversely, for spectra under and after continuous illumination, many asynchronicities are observed for (still unassigned) bands throughout the whole 1740–1200 cm–1 region, reflecting a more complicated molecular scenario in the RC upon build-up of the light-adapted state and during its relaxation to the resting neutral state.

Melanoma exhibits high intratumoral heterogeneity, characterized by a diverse population of cells undergoing dynamic transitions between cellular states. These adaptive changes enable melanoma cells to survive in the harsh tumor microenvironment, acquire drug resistance, and metastasize. One such state, quiescence, has been linked to both relapse and drug resistance, but its underlying biology and molecular mechanisms remain poorly understood. Our study challenges the conventional understanding of melanoma quiescence. Contrary to the notion of a rare, unique subpopulation, we demonstrate that quiescence is a highly dynamic state accessible to most, if not all, melanoma cells. This state is exquisitely sensitive to microenvironmental cues. We identify GPNMB as a marker of quiescence, that is expressed in both primary and metastatic tumors. GPNMB-positive cells exhibit a pro-metastatic phenotype and are enriched in metastatic sites, suggesting a potential role for quiescence in tumor dissemination. Our findings position GPNMB as a valuable marker for isolating quiescent melanoma cells and as a potential therapeutic target to tackle metastasis.

Dehydration is known to affect the rate of electron transfer backreaction from the light-induced charge separation state P+QA− to the neutral ground state PQA in photosynthetic bacterial Reaction Centers. On the other hand, a 20 s continuous illumination period has been demonstrated to induce (at 297 K) formation of one or more light-adapted states at different levels of dehydration; these light-adapted states are believed to be related to peculiar response(s) from the protein. In this work, we applied time-resolved rapid-scan FTIR difference spectroscopy to investigate the protein response under dehydrated conditions (RH = 11%) at 281 K both after a flash and under prolonged continuous illumination. Time-resolved FTIR difference spectra recorded after a laser flash show a protein recovery almost synchronous to the electron transfer backreaction P+QA− → PQA. Time-resolved FTIR difference spectra recorded after 20.5 s of continuous illumination (RH = 11%, T = 281 K) surprisingly show almost the same kinetics of electron transfer back reaction compared to spectra recorded after a laser flash. This means that the mechanism of formation of a light-adapted stabilized state is less effective compared to the same hydration level at 297 K and to the RH = 76% hydration level (both at 281 K and 297 K). Time-resolved FTIR difference spectra after continuous illumination also suggest that the 1666 cm−1 protein backbone band decays faster than marker bands for the electron transfer back reaction P+QA− → PQA. Finally, FTIR double-difference spectra (FTIR difference spectrum recorded after 18.4 s illumination minus flash-induced FTIR difference spectrum) suggest that at RH = 11%, a light-adapted state different from the one observed at RH = 76% is formed. A possible interpretation is that at RH = 11%, the protein response is modified by the fact that only protons can move easily, differently from water molecules, as instead observed for RH = 76%. This probably makes the formation of a real light-adapted P+QA− stabilized state at RH = 11% unfeasible.

Carbon nanomaterials such as single-walled carbon nanotubes (SWCNTs) can improve algal photosynthetic efficiency and increase the production of valuable compounds without decreasing biomass productivity. This work investigates the effects of SWCNTs dispersed by different proteins or functionalized with carboxylic groups (SWCNT-COOH) on several microalgae and a cyanobacterium, aiming to enhance photosynthetic performance and produce valuable compounds. The best SWCNTs' dispersion in water was achieved with lysozyme (LSZ@SWCNT), histone (HST@SWCNT) and SWCNT-COOH. In most cases, no toxicity was observed when the phototrophs were exposed for 72 h to these SWCNTs. The cyanobacterium Arthrospira platensis and the marine diatom Thalassiosira sp. were selected for evaluating the effects of exposure to LSZ@SWCNT for up to 14 days. Increased production of valuable algal compounds (i.e., phycocyanin + 38 % in A. platensis after 14 days, docosahexaenoic acid + 92 % and eicosapentaenoic acid + 63 % in Thalassiosira sp. after 7 days) was observed without any impairment of the photosynthetic efficiency (+19 % for Thalassiosira sp.). The effects observed on both the cell surface and intracellular structures (i.e., cell wall modifications, increased mucus, and vacuolization) suggested that the interaction with LSZ@SWCNT was responsible for the changes in biochemical composition and photosynthetic performance. Therefore, the proposed nanobiotechnological approach, which couples cyanobacteria and microalgae with SWCNTs, may tune the photosynthetic pathways towards the production of high-value compounds exploitable in cosmetics and nutraceuticals, ultimately improving the light-to-chemicals conversion processes without negatively impacting growth.

Print-Light-Synthesis (PLS) combines the inkjet printing of a ruthenium precursor ink with the simultaneous photo-induced generation of ruthenium oxide films. During PLS, inkjet-printing generates on conductive as well as insulating substrates micrometer-thin reaction volumes that contain with high precision defined precursor loadings. Upon direct UV light irradiation, the Ru precursor converts to RuO2 while all other ink components escape in the gas phase. No post PLS processes are required, and the as-obtained RuO2 films can be immediately used as electrochemical devices. Two-dimensional RuO2 patterns with micrometric resolution and highly-controlled ruthenium loadings (few µg/cm2) are realized. Thin RuO2 films are generated on insulating substrates, such as polyimide, as well as individual RuO2 particles on conductive substrates, such as graphene layers. The RuO2 films are characterized by electron microscopy and spectroscopic techniques. The sensoristic applicability of the PLS-RuO2 electrodes is demonstrated by potentiometric pH sensing in cell cultures and amperometric detection of L-cysteine. For pH sensing the RuO2 film electrodes show Nernstian sensitivity. L-cysteine detection of RuO2-modified graphene electrodes showed an electrocatalytical effect and resulted in the possibility of selectively detecting L-Cysteine also in presence of the interfering compound uric acid.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

Cardiomyocytes differentiated from pluripotent stem cells (PSC-CMs) hold a great potential for the study and the cure of cardiovascular disease; indeed they have been largely used as platform for disease modeling and drugs testing, and represent a promising source of cells for regenerative therapies. However, their immature phenotype represents a major hurdle for their full application; indeed, PSC-CMs differ from adult ones for molecular, metabolic and morpho-functional properties. Recently, a new technology based on optical excitation of organic semiconductors (OS) has been shown to be able to modulate the behavior of many cell types by targeting different cellular processes, as proliferation, angiogenesis and calcium signalling. Here, we adopted a multidisciplinary approach based on morpho-functional, metabolic and transcriptional analyses to study the effect of OS-photoexcitation on PSC-CMs, by the use of a red-light sensitive OS polymer, namely Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b']dithiophene)-alt-4,7(2,1,3-benzothiadiazole)] (PCPDTBT), in the form of a thin film. Our results revealed a significant modulation of markers of maturation in PSC-CMs exposed to PCPDTBT and photomodulation, showing increased size, a shift of their maximal diastolic potential toward more negative values, and augmented Ca 2+ transient amplitude. Moreover, by Scanning ElectroChemical Microscopy (SECM) we found a decrease in glucose uptake and lactate release upon PCPDTBT light stimulation, potentially indicating a switch toward a more adult-like metabolism in stimulated PSC-CMs. Recently obtained RNA sequencing data are in line with these results, and will provide us with hints on the underlying molecular mechanisms. In conclusion, although not definitive, our data are in support of a potential effect of polymer-mediated optical photoexcitation in boosting PSC-CMs toward a more mature phenotype. The demonstration of a relevant effect of OS-photomodulation on PSC-CM maturation and functionality will significantly promote their full applications toward personalized medicine.This research was supported by EU Horizon 2020 FETOPEN- 2018-2020 Programme ‘LION- HEARTED’-GA #828984

Abstract 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.

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.

Photoactivation of conjugated polymers has been shown to be an attractive approach to modulate biological functions in several cell models; it is characterized by minimal invasiveness, high modulability, spatial selectivity at the level of single cells and even intracellular compartments. Reactive oxygen species produced by polymer photostimulation, in particular, were reported to trigger cell proliferation and recently proposed to finely modulate the cell cycle. Light-activated proliferation is extremely attractive for cardiac muscle cells, to overcome pathological conditions as heart failure and cardiovascular diseases with minimal invasiveness. Here, we specifically address the target to optically modulate the cell redox balance of HL-1 cells, a cardiac muscle cell model, by localized photoexcitation of a light sensitive polymer, namely poly-3-hexyl-thiophene. Scanning electrochemical microscopy is employed to quantify the changes of the cellular redox balance. Both extracellular and intracellular production of reactive oxygen species upon illumination is investigated, by employing poly-3-hexyl-thiophene respectively in the form of thin films or nanoparticles. Our results show that light induced, spatially controlled production of reactive oxygen species by poly-3-hexyl-thiophene films in the extracellular compartment determines the shift of HL-1 redox balance towards more reducing values. Thus, highly resolved spatial control of the illuminated area enables modulation of HL-1 redox balance at the single cell level. The effect on cell redox balance of cytosol internalized poly-3-hexyl-thiophene nanoparticles is also presented. Our work shows that photostimulation of conjugated polymers can be employed as a wireless, geneless technique to modulate on demand the intracellular redox balance, with high spatial resolution and minimal invasiveness, in a biologically relevant model of contractile, functioning cardiomyocytes. In perspective, this approach may be easily extended to other cell systems, wherever a fine control of the cell redox state is desirable, and open the path to innovative therapeutic tools.

ABSTRACT Melanoma is a heterogeneous tumor composed of many interacting cellular populations and highly plastic melanoma cells that pass through distinct cell states to adapt to the surrounding microenvironment. Slow cycling is a transient state that defines a minor population of cells with cancer-initiating features. These cells are enriched upon drug therapy and can trigger cancer relapse and metastasis dissemination when they acquire proliferative potential. This population is still not entirely characterized. Here we provide evidence of the existence of a slow cycling melanoma population isolated in vivo from melanoma PDXs using the H2B-GFP system. These cells display a highly invasive phenotype and are able to dynamically respond to cancer microenvironmental stimuli. Single cell transcriptomic analysis unveils a significant transcriptional heterogeneity of GFP-retaining slow cycling cells, defining a quiescent subpopulation of cells. These cells show a different phenotype in primary tumors and matched metastases, suggesting that tumor niche pressure drives a transcriptional reprogramming of quiescent cells during melanoma progression.

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.

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.