Biomolecular Corona of Gold Nanoparticles: The Urgent Need for Strong Roots to Grow Strong Branches.

Gold nanoparticles (GNPs) are largely employed in diagnostics/biosensors and are among the most investigated nanomaterials in biology/medicine. However, few GNP-based nanoformulations have received FDA approval to date, and promising in vitro studies have failed to translate to in vivo efficacy. One key factor is that biological fluids contain high concentrations of proteins, lipids, sugars, and metabolites, which can adsorb/interact with the GNP's surface, forming a layer called biomolecular corona (BMC). The BMC can mask prepared functionalities and target moieties, creating new surface chemistry and determining GNPs' biological fate. Here, the current knowledge is summarized on GNP-BMCs, analyzing the factors driving these interactions and the biological consequences. A partial fingerprint of GNP-BMC analyzing common patterns of composition in the literature is extrapolated. However, a red flag is also risen concerning the current lack of data availability and regulated form of knowledge on BMC. Nanomedicine is still in its infancy, and relying on recently developed analytical and informatic tools offers an unprecedented opportunity to make a leap forward. However, a restart through robust shared protocols and data sharing is necessary to obtain "stronger roots". This will create a path to exploiting BMC for human benefit, promoting the clinical translation of biomedical nanotools.

Hydrogen Promotes the Growth of Platinum Pyramidal Nanocrystals by Size-Dependent Symmetry Breaking.

The growth of pyramidal platinum nanocrystals is studied by a combination of synthesis/characterization experiments and density functional theory calculations. It is shown that the growth of pyramidal shapes is due to a peculiar type of symmetry breaking, which is caused by the adsorption of hydrogen on the growing nanocrystals. Specifically, the growth of pyramidal shapes is attributed to the size-dependent adsorption energies of hydrogen atoms on {100} facets, whose growth is hindered only if they are sufficiently large. The crucial role of hydrogen adsorption is further confirmed by the absence of pyramidal nanocrystals in experiments where the reduction process does not involve hydrogen.

Catalytic Bioswitch of Platinum Nanozymes: Mechanistic Insights of Reactive Oxygen Species Scavenging in the Neurovascular Unit.

Oxidative stress is known to be the cause of several neurovascular diseases, including neurodegenerative disorders, since the increase of reactive oxygen species (ROS) levels can lead to cellular damage, blood-brain barrier leaking, and inflammatory pathways. Herein, we demonstrate the therapeutic potential of 5 nm platinum nanoparticles (PtNPs) to effectively scavenge ROS in different cellular models of the neurovascular unit. We investigated the mechanism underlying the PtNP biological activities, analyzing the influence of the evolving biological environment during particle trafficking and disclosing a key role of the protein corona, which elicited an effective switch-off of the PtNP catalytic properties, promoting their selective in situ activity. Upon cellular internalization, the lysosomal environment switches on and boosts the enzyme-like activity of the PtNPs, acting as an intracellular "catalytic microreactor" exerting strong antioxidant functionalities. Significant ROS scavenging was observed in the neurovascular cellular models, with an interesting protective mechanism of the Pt-nanozymes along lysosomal-mitochondrial axes.

Nanozymes based on octahedral platinum nanocrystals with {111} surface facets: glucose oxidase mimicking activity in electrochemical sensors.

The ability of shape-controlled octahedral Pt nanoparticles to act as nanozyme mimicking glucose oxidase enzyme is reported. Extended {111} particle surface facets coupled with a size comparable to natural enzymes and easy-to-remove citrate coating give high affinity for glucose, comparable to the enzyme as proven by the steady-state kinetics of glucose electrooxidation. The easy and thorough removal of the citrate coating, demonstrated by X-ray photoelectron spectroscopy analysis, allows a highly stable deposition of the nanozymes on the electrode. The glucose electrochemical detection (at -0.2 V vs SCE) shows a linear response between 0.36 and 17 mM with a limit of detection of 110 µM. A good reproducibility has been achieved, with an average relative standard deviation (RSD) value of 9.1% (n = 3). Similarly, a low intra-sensor variability has been observed, with a RSD of 6.6% (n = 3). Moreover, the sensor shows a long-term stability with reproducible performances for at least 2 months (RSD: 7.8%). Tests in saliva samples show the applicability of Pt nanozymes to commercial systems for non-invasive monitoring of hyperglycemia in saliva, with recoveries ranging from 92 to 98%.

Platinum Nanozyme-Enabled Colorimetric Determination of Total Antioxidant Level in Saliva.

Redox imbalance and oxidative stress-related biomarkers are raising increasing consensus in the scientific community for their significant role in a wide range of human disorders. In this framework, the total antioxidant capacity (TAC), namely, the overall pattern of both enzymatic and nonenzymatic antioxidant compounds within the body, represents an important bioanalytical parameter. To date, however, antioxidant assays require costly instrumentations, laboratory setups, and reagents, and they are invasive. Yet, their accuracy typically suffers from strong sensitivity to interfering matrices and inability to detect the complete pattern of physiological antioxidant molecules, due to the use of reaction schemes and probes/substrates that are not sensitive to the diverse range of relevant target species. Here, we exploit the enzyme-mimetic properties of platinum nanoparticles combined with hydroxyl radical probes produced at the particle surface to develop an effective detection scheme that is sensitive to both single electron transfer (SET) and hydrogen atom transfer (HAT) reactions, thus covering all the physiologically relevant antioxidant species. Importantly, the nanozyme-enabled method allows fast (5 min), accurate, and noninvasive evaluation of the body TAC through saliva via simple naked-eye or smartphone-based inspection.

Delivery of biologically active miR-34a in normal and cancer mammary epithelial cells by synthetic nanoparticles.

Functional RNAs, such as microRNAs, are emerging as innovative tools in the treatment of aggressive and incurable cancers. In this study, we explore the potential of silica dioxide nanoparticles (SiO2NPs) in the delivery of biologically active miRNAs. Focusing on the tumor-suppressor miR-34a, we evaluated miRNAs delivery by SiO2NPs into the mammary gland, using in vitro as well as in vivo model systems. We showed that silica nanoparticles can efficiently deliver miR-34a into normal and cancer epithelial cells grown in culture without major signs of toxicity. Delivered miRNA retained the ability to silence artificial as well endogenous targets and can reduce the growth of mammospheres in 3D culture. Finally, miR-34a delivery through intra-tumor administration of SiO2NPs leads to a reduced mammary tumor growth. In conclusion, our studies suggest that silica nanoparticles can mediate the delivery of miR-34a directly into mammary tumors while preserving its molecular and biological activity.

Nanocatalyst/Nanoplasmon-Enabled Detection of Organic Mercury: A One-Minute Visual Test.

We present a fast and sensitive nanosensor that can detect organic mercury, exploiting the combination of the catalytic and plasmonic properties of gold nanoparticles (AuNPs). The method is one-step and completely instrument-free, and has a colorimetric readout clearly detectable by simple visual inspection. The AuNPs catalyze efficient organic mercury reduction to the metallic form (Hg0 ), allowing its nucleation and amalgam formation on particle surface, with consequent aggregation-induced plasmon shift. This leads to very rapid (1 min) and specific colorimetric detection of mercury species. The achieved limit of detection (20 ppb) is compliant with current regulatory limits in food.

Biotransformation and Biological Interaction of Graphene and Graphene Oxide during Simulated Oral Ingestion.

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.

Enhancement of Tumor Homing by Chemotherapy-Loaded Nanoparticles.

Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma.

Relevance to investigate different stages of pregnancy to highlight toxic effects of nanoparticles: The example of silica.

Amorphous silica nanoparticles (SiO2NPs) have been recognized as safe nanomaterial, hence their use in biomedical applications has been explored. Data, however, suggest potential toxicity of SiO2 NPs in pregnant individuals. However, no studies relating nanoparticle biokinetic/toxicity to the different gestational stages are currently available. In this respect, we have investigated the possible embryotoxic effects of three-size and two-surface functionalization SiO2NPs in mice. After intravenous administration of different concentrations at different stages of pregnancy, clinical and histopathological evaluations, performed close to parturition, did not show signs of maternal toxicity, nor effects on placental/fetal development, except for amino-functionalized 25 nm NPs. Biodistribution was studied by ICP-AES 24 h after administration, and demonstrates that all particles distributed to placenta and conceptuses/fetuses, although size, surface charge and gestational stage influenced biodistribution. Our data suggest the need of comprehensive toxicological studies, covering the entire gestation to reliably assess the safety of nanoparticle exposure during pregnancy.

Platinum nanoparticles in nanobiomedicine.

Oxidative stress-dependent inflammatory diseases represent a major concern for the population's health worldwide. Biocompatible nanomaterials with enzymatic properties could play a crucial role in the treatment of such pathologies. In this respect, platinum nanoparticles (PtNPs) are promising candidates, showing remarkable catalytic activity, able to reduce the intracellular reactive oxygen species (ROS) levels and impair the downstream pathways leading to inflammation. This review reports a critical overview of the growing evidence revealing the anti-inflammatory ability of PtNPs and their potential applications in nanomedicine. It provides a detailed description of the wide variety of synthetic methods recently developed, with particular attention to the aspects influencing biocompatibility. Special attention has been paid to the studies describing the toxicological profile of PtNPs with an attempt to draw critical conclusions. The emerging picture suggests that the material per se is not causing cytotoxicity, while other physicochemical features related to the synthesis and surface functionalization may play a crucial role in determining the observed impairment of cellular functions. The enzymatic activity of PtNPs is also summarized, analyzing their action against ROS produced by pathological conditions within the cells. In particular, we extensively discuss the potential of these properties in nanomedicine to down-regulate inflammatory pathways or to be employed as diagnostic tools with colorimetric readout. A brief overview of other biomedical applications of nanoplatinum is also presented.

DNA Barcoding Meets Nanotechnology: Development of a Universal Colorimetric Test for Food Authentication.

Food trade globalization and the growing demand for selected food varieties have led to the intensification of adulteration cases, especially in the form of species substitution and mixing with cheaper taxa. This phenomenon has huge economic impact and sometimes even public health implications. DNA barcoding represents a well-proven molecular approach to assess the authenticity of food items, although its use is hampered by analytical constraints and timeframes that are often prohibitive for the food market. To address such issues, we have introduced a new technology, named NanoTracer, that allows for rapid and naked-eye molecular traceability of any food and requires limited instrumentation and cost-effective reagents. Moreover, unlike sequencing, this method can be used to identify not only the substitution of a fine ingredient, but also its dilution with cheaper ones.

Antimicrobial Lemongrass Essential Oil-Copper Ferrite Cellulose Acetate Nanocapsules.

Cellulose acetate (CA) nanoparticles were combined with two antimicrobial agents, namely lemongrass (LG) essential oil and Cu-ferrite nanoparticles. The preparation method of CA nanocapsules (NCs), with the two antimicrobial agents, was based on the nanoprecipitation method using the solvent/anti-solvent technique. Several physical and chemical analyses were performed to characterize the resulting NCs and to study their formation mechanism. The size of the combined antimicrobial NCs was found to be ca. 220 nm. The presence of Cu-ferrites enhanced the attachment of LG essential oil into the CA matrix. The magnetic properties of the combined construct were weak, due to the shielding of Cu-ferrites from the polymeric matrix, making them available for drug delivery applications where spontaneous magnetization effects should be avoided. The antimicrobial properties of the NCs were significantly enhanced with respect to CA/LG only. This work opens novel routes for the development of organic/inorganic nanoparticles with exceptional antimicrobial activities.

A Universal Polymerase Chain Reaction Developer.

The versatility of PCR, the gold standard for amplification of DNA targets, is hampered by the laborious, multi-step detection based on gel electrophoresis. We propose a one-step, one-tube method for the rapid (5 min) naked-eye detection of PCR products, based on controlled aggregation of gold nanoparticles. Our method is universal, instrument-free, and ultra-sensitive, as it could detect as low as 0.01 zeptomoles of HIV template DNA in an excess of interfering human genomic DNA.

Negligible particle-specific toxicity mechanism of silver nanoparticles: the role of Ag+ ion release in the cytosol.

Toxicity of silver nanoparticles (AgNPs) is supported by many observations in literature, but no mechanism details have been proved yet. Here we confirm and quantify the toxic potential of fully characterized AgNPs in HeLa and A549 cells. Notably, through a specific fluorescent probe, we demonstrate the intracellular release of Ag(+) ions in living cells after nanoparticle internalization, showing that in-situ particle degradation is promoted by the acidic lysosomal environment. The activation of metallothioneins in response to AgNPs and the possibility to reverse the main toxic pathway by Ag(+) chelating agents demonstrate a cause/effect relationship between ions and cell death. We propose that endocytosed AgNPs are degraded in the lysosomes and the release of Ag(+) ions in the cytosol induces cell damages, while ions released in the cell culture medium play a negligible effect. These findings will be useful to develop safer-by-design nanoparticles and proper regulatory guidelines of AgNPs. From the clinical editor: The authors describe the toxic potential of silver nanoparticles (AgNP) in human cancer cell lines. Cell death following the application of AgNPs is dose-dependent, and it is mostly due to Ag+ ions. Further in vivo studies should be performed to gain a comprehensive picture of AgNP-toxicity in mammals.

Nanosilver-based antibacterial drugs and devices: mechanisms, methodological drawbacks, and guidelines.

Despite the current advancement in drug discovery and pharmaceutical biotechnology, infection diseases induced by bacteria continue to be one of the greatest health problems worldwide, afflicting millions of people annually. Almost all microorganisms have, in fact, an intrinsic outstanding ability to flout many therapeutic interventions, thanks to their fast and easy-to-occur evolutionary genetic mechanisms. At the same time, big pharmaceutical companies are losing interest in new antibiotics development, shifting their capital investments in much more profitable research and development fields. New smart solutions are, thus, required to overcome such concerns, and should combine the feasibility of industrial production processes with cheapness and effectiveness. In this framework, nanotechnology-based solutions, and in particular silver nanoparticles (AgNPs), have recently emerged as promising candidates in the market as new antibacterial agents. AgNPs display, in fact, enhanced broad-range antibacterial/antiviral properties, and their synthesis procedures are quite cost effective. However, despite their increasing impact on the market, many relevant issues are still open. These include the molecular mechanisms governing the AgNPs-bacteria interactions, the physico-chemical parameters underlying their toxicity to prokaryotes, the lack of standardized methods and materials, and the uncertainty in the definition of general strategies to develop smart antibacterial drugs and devices based on nanosilver. In this review, we analyze the experimental data on the bactericidal effects of AgNPs, discussing the complex scenario and presenting the potential drawbacks and limitations in the techniques and methods employed. Moreover, after analyzing in depth the main mechanisms involved, we provide some general strategies/procedures to perform antibacterial tests of AgNPs, and propose some general guidelines for the design of antibacterial nanosystems and devices based on silver/nanosilver.

Absolute and direct microRNA quantification using DNA-gold nanoparticle probes.

DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.

Lab-on-a-chip-based high-throughput screening of the genotoxicity of engineered nanomaterials.

The continuous increasing of engineered nanomaterials (ENMs) in our environment, their combinatorial diversity, and the associated genotoxic risks, highlight the urgent need to better define the possible toxicological effects of ENMs. In this context, we present a new high-throughput screening (HTS) platform based on the cytokinesis-block micronucleus (CBMN) assay, lab-on-chip cell sorting, and automated image analysis. This HTS platform has been successfully applied to the evaluation of the cytotoxic and genotoxic effects of silver nanoparticles (AgNPs) and silica nanoparticles (SiO2NPs). In particular, our results demonstrate the high cyto- and genotoxicity induced by AgNPs and the biocompatibility of SiO2NPs, in primary human lymphocytes. Moreover, our data reveal that the toxic effects are also dependent on size, surface coating, and surface charge. Most importantly, our HTS platform shows that AgNP-induced genotoxicity is lymphocyte sub-type dependent and is particularly pronounced in CD2+ and CD4+ cells.

Nanotechnology-based strategies for the detection and quantification of microRNA.

MicroRNAs (miRNAs) are important regulators of gene expression, and many pathological conditions, including cancer, are characterized by altered miRNA expression levels. Therefore, accurate and sensitive quantification of miRNAs may result in correct disease diagnosis establishing these small noncoding RNA transcripts as valuable biomarkers. Aiming at overcoming some limitations of conventional quantification strategies, nanotechnology is currently providing numerous significant alternatives to miRNA sensing. In this review an up-to-date account of nanotechnology-based strategies for miRNA detection and quantification is given. The topics covered are: nanoparticle-based approaches in solution, sensing based on nanostructured surfaces, combined nanoparticle/surface sensing approaches, and single-molecule approaches.

Synthesis of fluorescent metal nanoparticles in aqueous solution by photochemical reduction.

A facile green chemistry approach for the synthesis of sub-5 nm silver and gold nanoparticles is reported. The synthesis was achieved by a photochemical method using tyrosine as the photoreducing agent. The size of the gold and silver nanoparticles was about 3 and 4 nm, respectively. The nanoparticles were characterized using x-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy and photoluminescence spectroscopy. Both silver and gold nanoparticles synthesized by this method exhibited fluorescence properties and their use for cell imaging applications has been demonstrated.