The revolutionary discovery by Abe & Kimura that H2S exerts a beneficial role in human body has renewed interest in this small molecule, long known for its toxicity. Understanding the (bio)reactivity of H2S with biological and bioinorganic targets is therefore of increasing importance, yet studies on its interaction with nonheme metalloproteins remain limited. Here, we investigate the reactivity of HS- with two natural multicopper proteins, SLAC and NiR. We demonstrate that SLAC, a two-domain blue-copper oxidase, can function as a multiwavelength, multireadout fluorescent sensor for H2S in complex environments. Comparative studies on NiR support the proposed mechanism of H2S recognition via selective reduction of copper centers. Finally, we benchmark the performance of these multicopper proteins against Cu-azurin, previously reported as a H2S recognition element, highlighting the advantages of multicopper architectures in terms of sensitivity, selectivity, and reversibility. Our findings establish multicopper proteins as versatile platforms for H2S sensing with potential applications in biomedical and environmental monitoring.

In this contribution, the synthesis and the reactivity with HS- of a family of half-salen Zn complexes are reported. We provide evidence that HS- binds the zinc center of all the complexes under investigation. DFT and CCSD(T) calculations were performed to model the reactivity of these complexes with HS-. We successfully applied a homoleptic zinc complex bearing a Schiff-based ligand with pyridine pendant arms as a probe for the monitoring of exogenous and endogenous H2S levels in live cells.

In the current contribution, the synthesis and characterization of new diaminomaleonitrile-based salen cobalt complexes L1Co and L2Co (L1-H = 2,3-bis[[(2,4-dihydroxy-phenyl)(methylene)]amino]-2-butenedinitrile, L2-H = 2,3-bis[[(2-hydroxy-4-(diethylamino)phenyl)(methylene)]amino]-2-butenedinitrile) are reported. Structural analysis and electronic properties of the complexes were assessed, providing a basis for their application as H2S sensors. Spectroscopic investigations revealed an enhancement in the fluorescence intensities upon H2S addition. A reversible fluorescence response was observed when the systems were shifted from H2S-rich to oxygenated environments, demonstrating their reusability and adaptability. To further establish the mechanism of interaction of the HS- with the cobalt complexes, electrochemical analysis was conducted, providing evidence of HS- coordination to the cobalt centers. Moreover, biological assessment of the sensors was performed in HepG2 cells, revealing their low cytotoxicity and efficient cell permeability. Fluorescence imaging experiments provided evidence of intracellular detection of exogenous H2S. The reversibility of the sensing mechanism was confirmed in living cells: subsequent addition of HS- and oxygen-saturated buffer to the cells tunes their fluorescence accordingly, in a cyclable manner, as in the experiments performed in vitro. This study provides evidence that cobalt-based DAMN complexes work as robust, reversible, and selective detection systems for H2S, demonstrating their suitability for applications in both biological and environmental contexts.

The study of nano‐biointeractions, at the forefront of interdisciplinary research, unveils intricate interplays between nanomaterials (NMs) and intracellular organelles, which are pivotal hubs orchestrating diverse cellular processes. Thanks also to the formation of dynamic contacts among their membranes, organelles regulate lipid exchange, calcium signaling, and metabolic pathways. Recently, the potential role of NMs in cellular homeostasis through the regulation of organelle membrane contact sites (MCSs) is emerging, and a complete overview of this issue is still lacking. This perspective aims at elucidating the synergy between functional NMs and organelle contact site research, underscoring the pivotal role of NMs in advancing the comprehension of cell biology mechanisms and fostering therapeutic breakthroughs. This subject represents a crucial aspect of nano‐biointeractions, as it can reveal new molecular targets for NMs and potentially revolutionize therapeutic strategies. Nanotechnology may offer unprecedented tools to decipher and manipulate dynamic organelle interfaces with remarkable precision. Engineered nanomaterials may serve as versatile probes and effectors, enabling targeted modulation of organelle contact sites and unraveling the molecular intricacies governing organelle dynamics. Furthermore, nano‐biointeraction‐driven insights hold promise for therapeutic innovations, offering novel avenues in diseases linked to dysregulated organelle contacts.

Immunotherapy is considered a powerful clinical strategy aiming to boost the immune system to fight cancer. In this context, nanomaterials (NMs) are uniquely suited to improve the development and the broad implementation of cancer immunotherapies by overcoming several challenges. In fact, NMs can be rationally designed to navigate complex physical barriers, respond to tumor microenvironments, and enhance/modulate immune system activation. Here, we present a method to prepare stimuli-responsive biocompatible nanoparticles (NPs) able to target the tumor microenvironment. Moreover, we describe protocols to characterize the physical-chemical properties of NPs as well as to evaluate their biocompatibility and therapeutic potential in vitro on three-dimensional (3D) tumor spheroids.

The catalytic and antioxidant properties of platinum nanoparticles (PtNPs) make them promising candidates for several applications in nanomedicine. However, an open issue, still shared among most nanomaterials, is the understanding on how internalized PtNPs, which are confined within endo-lysosomal compartments, can exert their activities. To address this problem, here we study the protective effect of 5 nm PtNPs on a human hepatic (HepG2) cell line exposed to dichlorodiphenylethylene (DDE) as a model of oxidative stress. Our results indicate that PtNPs are very efficient to reduce DDE-induced damage in HepG2 cells, in an extent that depends on DDE dose. PtNPs can contrast the unbalance of mitochondrial dynamics induced by DDE and increase the expression of the SOD2 mitochondrial enzyme that recovers cells from oxidative stress. Interestingly, in cells treated with PtNPs—alone or in combination with DDE—mitochondria form contact sites with a rough endoplasmic reticulum and endo-lysosomes containing nanoparticles. These findings indicate that the protective capability of PtNPs, through their intrinsic antioxidant properties and modulating mitochondrial functionality, is mediated by an inter-organelle crosstalk. This study sheds new light about the protective action mechanisms of PtNPs and discloses a novel nano-biointeraction mechanism at the intracellular level, modulated by inter-organelle communication and signaling.

Platinum nanoparticles (PtNPs) are considered promising nanomaterials for biomedical applications thanks to their formidable catalytic and antioxidant properties. To improve their therapeutic potential and to boost their applicability in nanomedicine, it is fundamental to understand the biological mechanisms that control PtNP interaction with cells and tissues. In this context, an important issue, still open and shared among other nanomaterials, is how internalized PtNPs, that are confined within endo-lysosomal compartments, can exert their activities within the cells. In this framework, here we overviewed the antioxidant and catalytic properties of PtNPs, that make them useful tools for theragnostic applications, and their interactions with cells. In particular, we focused the attention on the endocytic pathways of NPs and their intracellular localization and discussed the formation of inter-organelle contact sites as new and possible mechanisms of communications between cells and NPs, based on recent findings and never reported before. This brief review sheds light on possible new players in the process of nano-biointeractions at the intracellular level (i.e., the interorganelle contact sites), offering new insights about the mechanisms of action of PtNPs compared to previous works. In this context, we contemplated the opportunity to exploit this new mechanism of nano-biointeraction, e.g., by modulating the formation of inter-organelle contact sites and by targeting them to increase thepotential applications of PtNPs in biomedicine and theranostics.

In this contribution we report the synthesis and full characterization, via a combination of different spectroscopies (e.g., 1H NMR, UV-vis, fluorescence, MALDI), of a new family of fluorescent zinc complexes with extended π-conjugated systems, with the final aim of setting up higher performance H2S sensing devices. Immobilization of the systems into a polymeric matrix for use in a solid-state portable device was also explored. The results provided proof-of-principle that the title complexes could be successfully implemented in a fast, simple and cost-effective H2S sensing device.

To improve the efficacy of nanoparticles (NPs) and boost their theragnostic potential for brain diseases, it is key to understand the mechanisms controlling blood-brain barrier (BBB) crossing. Here, the capability of 100 nm carboxylated polystyrene NPs, used as a nanoprobe model, to cross the human brain endothelial hCMEC/D3 cell layer, as well as to be consequently internalized by human brain tumor U87 cells, is investigated as a function of NPs' different intracellular localization. We compared NPs confined in the endo-lysosomal compartment, delivered to the cells through endocytosis, with free NPs in the cytoplasm, delivered by the gene gun method. The results indicate that the intracellular behavior of NPs changed as a function of their entrance mechanism. Moreover, by bypassing endo-lysosomal accumulation, free NPs were released from cells more efficiently than endocytosed NPs. Most importantly, once excreted by the endothelial cells, free NPs were released in the cell culture medium as aggregates smaller than endocytosed NPs and, consequently, they entered the human glioblastoma U87 cells more efficiently. These findings prove that intracellular localization influences NPs' long-term fate, improving their cellular release and consequent cellular uptake once in the brain parenchyma. This study represents a step forward in designing nanomaterials that are able to reach the brain effectively.

Parkinson’s disease (PD) represents one of the most common neurodegenerative disorders, characterized by a dopamine (DA) deficiency in striatal synapses and misfolded toxic α-synuclein aggregates with concomitant cytotoxicity. In this regard, the misfolded proteins accumulation in neurodegenerative disorders induces a remarkable perturbations of endoplasmic reticulum (ER) homeostasis leading to persistent ER stress, which in turn, effects protein synthesis, modification, and folding quality control. A large body of evidence suggests that natural products target the ER stress signaling pathway, exerting a potential action in cancers, diabetes, cardiovascular and neurodegenerative diseases. This study aims to assess the neuroprotective effect of cocoa extract and its purified fractions against a cellular model of Parkinson’s disease represented by 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y human neuroblastoma. Our findings demonstrate, for the first time, the ability of cocoa to specifically targets PERK sensor, with significant antioxidant and antiapoptotic activities as both crude and fractioning extracts. In addition, cocoa also showed antiapoptotic properties in 3D cell model and a notable ability to inhibit the accumulation of α-synuclein in 6-OHDA-induced cells. Overall, these results indicate that cocoa exerts neuroprotective effects suggesting a novel possible strategy to prevent or, at least, mitigate neurodegenerative disorders, such as PD.

In this work, the authors explored the interaction of a suite of fluorescent zinc complexes with H2S. The authors provide evidence that HS− binds the zinc center of all the complexes under investigation, allowing them to possibly function as sensors by a ‘coordinative-based’ approach. Naked-eye color changes occur when treating the systems with HS−, so the fluorescence responses are modulated by the presence of HS−, which has been related to a change in the energy level and coupling of excited states through a computational study. The results show the potential of the systems to function as HS−/H2S colorimetric and fluorescent sensors. Paper-strip-based sensing experiments foresee the potential of using this family of complexes as chemosensors of HS− in more complex biological fluids.

The presence of micro- and nanoplastics in the marine environment is raising strong concerns since they can possibly have a negative impact on human health. In particular, the lack of appropriate methodologies to collect the nanoplastics from water systems imposes the use of engineered model nanoparticles to explore their interactions with biological systems, with results not easily correlated with the real case conditions. In this work, we propose a reliable top-down approach based on laser ablation of polymers to form polyethylene terephthalate (PET) nanoplastics, which mimic real environmental nanopollutants, unlike synthetic samples obtained by colloidal chemistry. PET nanoparticles were carefully characterized in terms of chemical/physical properties and stability in different media. The nanoplastics have a ca. 100 nm average dimension, with significant size and shape heterogeneity, and they present weak acid groups on their surface, similarly to photodegraded PET plastics. Despite no toxic effects emerging by in vitro studies on human Caco-2 intestinal epithelial cells, the formed nanoplastics were largely internalized in endolysosomes, showing intracellular biopersistence and long-term stability in a simulated lysosomal environment. Interestingly, when tested on a model of intestinal epithelium, nano-PET showed high propensity to cross the gut barrier, with unpredictable long-term effects on health and potential transport of dispersed chemicals mediated by the nanopollutants.

In this work we explore the interaction of HS- with a family of fluorescent zinc complexes. In particular we selected a family of complexes with N,O-bidentate ligands aiming at assessing whether the zinc-chelating ligand plays a role in influencing the reactivity of HS- with the complexes under investigation. Different experiments, performed by diverse spectroscopic techniques, provide evidence that HS- binds the zinc center of all the complexes included in this study. The results highlight the potential of the devised systems to be used as HS-/H2S fluorescent sensors via a coordinative-based approach. To shed light on the species formed in solution when HS-/H2S interacts with the title complexes and aiming to rationalize the photophysical properties of the sensing constructs, we performed a computational analysis based on the time dependent density functional theory (TD-DFT). Preliminary bio-imaging experiments were also performed and the results indicate the potential of this class of compounds as probes for the detection of H2S in living cells.

Grouping approaches of nanomaterials have the potential to facilitate high throughput and cost effective nanomaterial screening. However, an effective grouping of nanomaterials hinges on the application of suitable physicochemical descriptors to identify similarities. To address the problem, we developed an integrated testing approach coupling acellular and cellular phases, to study the full life cycle of ingested silver nanoparticles (NPs) and silver salts in the oro-gastrointestinal (OGI) tract including their impact on cellular uptake and integrity. This approach enables the derivation of exposure-dependent physical descriptors (EDPDs) upon biotransformation of undigested nanoparticles, digested nanoparticles and digested silver salts. These descriptors are identified in: size, crystallinity, chemistry of the core material, dissolution, high and low molecular weight Ag-biomolecule soluble complexes, and are compared in terms of similarities in a grouping hypothesis. Experimental results indicate that digested silver nanoparticles are neither similar to pristine nanoparticles nor completely similar to digested silver salts, due to the presence of different chemical nanoforms (silver and silver chloride nanocrystals), which were characterized in terms of their interactions with the digestive matrices. Interestingly, the cellular responses observed in the cellular phase of the integrated assay (uptake and inflammation) are also similar for the digested samples, clearly indicating a possible role of the soluble fraction of silver complexes. This study highlights the importance of quantifying exposure-related physical descriptors to advance grouping of NPs based on structural similarities.

Functionalized metal nanoparticles (NPs) hold great promise as innovative tools in nanomedicine. However, one of the main challenges is how to optimize their association with the cell membrane, which is critical for their effective delivery. Recent findings show high cellular uptake rates for NPs coated with the polycationic cell-penetrating peptide gH625-644 (gH), although the underlying internalization mechanism is poorly understood. Here, we use extended coarse-grained simulations and free energy calculations to study systems that simultaneously include metal NPs, peptides, lipids, and sterols. In particular, we investigate the first encounter between multicomponent model membranes and 2.5 nm metal NPs coated with gH (gHNPs), based on the evidence from scanning transmission electron microscopy. By comparing multiple membrane and (membranotropic) NP models, we found that gHNP internalization occurs by forming an intermediate state characterized by specific stabilizing interactions formed by peptide-coated nanoparticles with multicomponent model membranes. This association mechanism is mainly characterized by interactions of gH with the extracellular solvent and the polar membrane surface. At the same time, the NP core interacts with the transmembrane (cholesterol-rich) fatty phase.

In this work, we exploited an integrated approach combining systematic analysis of cytotoxicity, angiogenic potential, and metabolomics to shed light on the effects of graphene oxide (GO) on primary human endothelial Huvec cells. Contrary to the outcomes observed in immortalized cell lines able to internalize a similar amount of GO, significant toxicity was found in Huvec cells at high GO concentrations (25 and 50 μg/mL). In particular, we found that the steric hindrance of GO intracellular aggregates perturbed the correct assembly of cytoskeleton and distribution of mitochondria. This was found to be primarily associated with oxidative stress and impairment of cell migration, affecting the formation of capillary-like structures. In addition, preliminary metabolomics characterization demonstrated that GO affects the consumption of niacinamide, a precursor of energy carriers, and several amino acids involved in the regulation of angiogenesis. Our findings suggest that GO acts at different cellular levels, both directly and indirectly. More precisely, the combination of the physical hindrance of internalized GO aggregates, induction of oxidative stress, and alteration of some metabolic pathways leads to a significant antiangiogenic effect in primary human endothelial cells.

The biomedical applications of physically entangled polymeric hydrogels are generally limited due to their weak mechanical properties, rapid swelling and dissolution in physiologically relevant environment. Chemical crosslinking helps stabilizing hydrogel structure and enhancing mechanical properties, thereby allowing a higher stability in phisiological environment. In this context, it is known that the mechanical properties of the hydrogel are affected by both the molecular weight (MW) of the starting polymer and the concentration of the crosslinker. Here, our aim was to assess the influence of polymer MW and concentration in the precursor solution on the mechanical features of the final hydrogel and their influence on cells-material interaction. In detail, 3D synthetic matrices based on poly(ethylene glycol) diacrylate (PEGDA) at two molecular weights (PEG 700 and PEG 3400) and at three different concentrations (10, 20, 40 w/v %), which were photopolymerized using darocour as an initiator, were studied. Then, infrared and swelling analyses, along with a comprehensive mechanical characterization of the obtained hydrogels (i.e. oscillatory shear and confined compression tests), were performed. Finally, to evaluate the influence of the mechanical features on the biological behaviour, the hydrogels were characterized in terms of cell adhesion percentage and cell viability after functionalizing the substrates with RGD peptide at three different concentrations. Results have demonstrated that both the Young's modulus (E) in compression and the elastic modulus (G') in shear of the hydrogels increase with increasing polymer precursor concentration. E decreased as MW increased, and the differences are more relevant for more concentrated hydrogels. On the contrary, G' appears to increase with increasing PEGDA MW and in particular for the lowest polymer precursor concentration. The biological results have demonstrated that cells cultured for longer times seem to prefer PEG 3400 hydrogels with a larger mesh size structure that posses higher viscoelastic properties in shear.

In this work, we investigate the mode of interaction of a family of fluorescent zinc complexes with HS- and H2S. Different experiments, performed by diverse spectroscopic techniques, provide evidence that HS- binds the zinc center of all the complexes under investigation. Treatment with neutral H2S exhibits a markedly different reactivity which indicates selectivity for HS- over H2S of the systems under investigation. Striking color changes, visible to the naked eye, occur when treating the systems with HS- or by an H2S flow. Accordingly, also the fluorescence is modulated by the presence of HS-, with the possible formation of multiple adducts. The results highlight the potential of the devised systems to be implemented as HS-/H2S colorimetric and fluorescent sensors. Bioimaging experiments indicate the potential of using this class of compounds as probes for the detection of H2S in living cells.

Predicting the therapeutic efficacy of a nanocarrier, in a rapid and cost-effective way, is pivotal for the drug delivery to the central nervous system (CNS). In this context, in vitro testing platforms, like the transwell systems, offer numerous advantages to study the passage through the blood-brain barrier (BBB), such as overcoming ethical and methodological issues of in vivo models. However, the use of different transwell filters and nanocarriers with various physical-chemical features makes it difficult to assess the nanocarrier efficacy and achieve data reproducibility. In this work, we performed a systematic study to elucidate the role of the most widely used transwell filters in affecting the passage of nanocarriers, as a function of filter pore size and density. In particular, the transport of carboxyl- and amine-modified 100 nm polystyrene nanoparticles (NPs), chosen as model nanocarriers, was quantified and compared to the behavior of Lucifer yellow (LY), a molecular marker of paracellular transport. Results indicate that the filter type affects the growth and formation of the confluent endothelial barrier, as well as the transport of NPs. Interestingly, the in situ dispersion of NPs was found to play a key role in governing their passage through the filters, both in absence and in presence of the cellular barrier. By framing the underlying nanobiointeractions, we found that particle-specific effects modulated cellular uptake and barrier intracellular distribution, eventually governing transcytosis through their interplay with "size exclusion effects" by the porous filters. This study highlights the importance of a careful evaluation of the physical-chemical profile of the tested nanocarrier along with filter parameters for a correct methodological approach to test BBB permeability in nanomedicine.

The biotransformation and biological impact of few layer graphene (FLG) and graphene oxide (GO) are studied, following ingestion as exposure route. An in vitro digestion assay based on a standardized operating procedure (SOP) is exploited. The assay simulates the human ingestion of nanomaterials during their dynamic passage through the different environments of the gastrointestinal tract (salivary, gastric, intestinal). Physical-chemical changes of FLG and GO during digestion are assessed by Raman spectroscopy. Moreover, the effect of chronic exposure to digested nanomaterials on integrity and functionality of an in vitro model of intestinal barrier is also determined according to a second SOP. These results show a modulation of the aggregation state of FLG and GO nanoflakes after experiencing the complex environments of the different digestive compartments. In particular, chemical doping effects are observed due to FLG and GO interaction with digestive juice components. No structural changes/degradation of the nanomaterials are detected, suggesting that they are biopersistent when administered by oral route. Chronic exposure to digested graphene does not affect intestinal barrier integrity and is not associated with inflammation and cytotoxicity, though possible long-term adverse effects cannot be ruled out.