Exploration of the Graphene Quantum Dots-Blue Light Combination: A Promising Treatment against Bacterial Infection.

Graphene Quantum Dots (GQDs) have shown the potential for antimicrobial photodynamic treatment, due to their particular physicochemical properties. Here, we investigated the activity of three differently functionalized GQDs-Blue Luminescent GQDs (L-GQDs), Aminated GQDs (NH2-GQDs), and Carboxylated GQDs (COOH-GQDs)-against E. coli. GQDs were administrated to bacterial suspensions that were treated with blue light. Antibacterial activity was evaluated by measuring colony forming units (CFUs) and metabolic activities, as well as reactive oxygen species stimulation (ROS). GQD cytotoxicity was then assessed on human colorectal adenocarcinoma cells (Caco-2), before setting in an in vitro infection model. Each GQD exhibits antibacterial activity inducing ROS and impairing bacterial metabolism without significantly affecting cell morphology. GQD activity was dependent on time of exposure to blue light. Finally, GQDs were able to reduce E. coli burden in infected Caco-2 cells, acting not only in the extracellular milieu but perturbating the eukaryotic cell membrane, enhancing antibiotic internalization. Our findings demonstrate that GQDs combined with blue light stimulation, due to photodynamic properties, have a promising antibacterial activity against E. coli. Nevertheless, we explored their action mechanism and toxicity on epithelial cells, fixing and standardizing these infection models.

Nanotechnology in the COVID-19 era: Carbon-based nanomaterials as a promising solution.

The Coronavirus Disease 2019 (COVID-19) pandemic has led to collaboration between nanotechnology scientists, industry stakeholders, and clinicians to develop solutions for diagnostics, prevention, and treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections. Nanomaterials, including carbon-based materials (CBM) such as graphene and carbon nanotubes, have been studied for their potential in viral research. CBM unique effects on microorganisms, immune interaction, and sensitivity in diagnostics have made them a promising subject of SARS-CoV-2 research. This review discusses the interaction of CBM with SARS-CoV-2 and their applicability, including CBM physical and chemical properties, the known interactions between CBM and viral components, and the proposed prevention, treatment, and diagnostics uses.

3D-printed graphene polylactic acid devices resistant to SARS-CoV-2: Sunlight-mediated sterilization of additive manufactured objects.

Additive manufacturing has played a crucial role in the COVID-19 global emergency allowing for rapid production of medical devices, indispensable tools for hospitals, or personal protection equipment. However, medical devices, especially in nosocomial environments, represent high touch surfaces prone to viral infection and currently used filaments for 3D printing can't inhibit transmission of virus [1]. Graphene-family materials are capable of reinforcing mechanical, optical and thermal properties of 3D printed constructs. In particular, graphene can adsorb near-infrared light with high efficiency. Here we demonstrate that the addition of graphene nanoplatelets to PLA filaments (PLA-G) allows the creation of 3D-printed devices that can be sterilized by near-infrared light exposure at power density analog to sunlight. This method has been used to kill SARS-CoV-2 viral particles on the surface of 3D printed PLA-G by 3 min of exposure. 3D-printed PLA-G is highly biocompatible and can represent the ideal material for the production of sterilizable personal protective equipment and daily life objects intended for multiple users.

Antenna-enhanced mid-infrared detection of extracellular vesicles derived from human cancer cell cultures.

BACKGROUND: Extracellular Vesicles (EVs) are sub-micrometer lipid-bound particles released by most cell types. They are considered a promising source of cancer biomarkers for liquid biopsy and personalized medicine due to their specific molecular cargo, which provides biochemical information on the state of parent cells. Despite this potential, EVs translation process in the diagnostic practice is still at its birth, and the development of novel medical devices for their detection and characterization is highly required. RESULTS: In this study, we demonstrate mid-infrared plasmonic nanoantenna arrays designed to detect, in the liquid and dry phase, the specific vibrational absorption signal of EVs simultaneously with the unspecific refractive index sensing signal. For this purpose, EVs are immobilized on the gold nanoantenna surface by immunocapture, allowing us to select specific EV sub-populations and get rid of contaminants. A wet sample-handling technique relying on hydrophobicity contrast enables effortless reflectance measurements with a Fourier-transform infrared (FTIR) spectro-microscope in the wavelength range between 10 and 3 µm. In a proof-of-principle experiment carried out on EVs released from human colorectal adenocarcinoma (CRC) cells, the protein absorption bands (amide-I and amide-II between 5.9 and 6.4 µm) increase sharply within minutes when the EV solution is introduced in the fluidic chamber, indicating sensitivity to the EV proteins. A refractive index sensing curve is simultaneously provided by our sensor in the form of the redshift of a sharp spectral edge at wavelengths around 5 µm, where no vibrational absorption of organic molecules takes place: this permits to extract of the dynamics of EV capture by antibodies from the overall molecular layer deposition dynamics, which is typically measured by commercial surface plasmon resonance sensors. Additionally, the described metasurface is exploited to compare the spectral response of EVs derived from cancer cells with increasing invasiveness and metastatic potential, suggesting that the average secondary structure content in EVs can be correlated with cell malignancy. CONCLUSIONS: Thanks to the high protein sensitivity and the possibility to work with small sample volumes-two key features for ultrasensitive detection of extracellular vesicles- our lab-on-chip can positively impact the development of novel laboratory medicine methods for the molecular characterization of EVs.

Translating Material Science into Bone Regenerative Medicine Applications: State-of-The Art Methods and Protocols.

In the last 20 years, bone regenerative research has experienced exponential growth thanks to the discovery of new nanomaterials and improved manufacturing technologies that have emerged in the biomedical field. This revolution demands standardization of methods employed for biomaterials characterization in order to achieve comparable, interoperable, and reproducible results. The exploited methods for characterization span from biophysics and biochemical techniques, including microscopy and spectroscopy, functional assays for biological properties, and molecular profiling. This review aims to provide scholars with a rapid handbook collecting multidisciplinary methods for bone substitute R&D and validation, getting sources from an up-to-date and comprehensive examination of the scientific landscape.

INSIDIA 2.0 High-Throughput Analysis of 3D Cancer Models: Multiparametric Quantification of Graphene Quantum Dots Photothermal Therapy for Glioblastoma and Pancreatic Cancer.

Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids' death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.

Graphene-Based Materials: Biological and Biomedical Applications.

This editorial aims to summarize the eleven scientific papers published in the Special Issue "Graphene-Based Materials: Biological and Biomedical Applications" [...].

Unravelling the Potential of Graphene Quantum Dots in Biomedicine and Neuroscience.

Quantum dots (QDs) are semiconducting nanoparticles that have been gaining ground in various applications, including the biomedical field, thanks to their unique optical properties. Recently, graphene quantum dots (GQDs) have earned attention in biomedicine and nanomedicine, thanks to their higher biocompatibility and low cytotoxicity compared to other QDs. GQDs share the optical properties of QD and have proven ability to cross the blood-brain barrier (BBB). For this reason, GQDs are now being employed to deepen our knowledge in neuroscience diagnostics and therapeutics. Their size and surface chemistry that ease the loading of chemotherapeutic drugs, makes them ideal drug delivery systems through the bloodstream, across the BBB, up to the brain. GQDs-based neuroimaging techniques and theranostic applications, such as photothermal and photodynamic therapy alone or in combination with chemotherapy, have been designed. In this review, optical properties and biocompatibility of GQDs will be described. Then, the ability of GQDs to overtake the BBB and reach the brain will be discussed. At last, applications of GQDs in bioimaging, photophysical therapies and drug delivery to the central nervous system will be considered, unraveling their potential in the neuroscientific field.

Graphene Quantum Dots' Surface Chemistry Modulates the Sensitivity of Glioblastoma Cells to Chemotherapeutics.

Recent evidence has shown that graphene quantum dots (GQDs) are capable of crossing the blood-brain barrier, the barrier that reduces cancer therapy efficacy. Here, we tested three alternative GQDs' surface chemistries on two neural lineages (glioblastoma cells and mouse cortical neurons). We showed that surface chemistry modulates GQDs' biocompatibility. When used in combination with the chemotherapeutic drug doxorubicin, GDQs exerted a synergistic effect on tumor cells, but not on neurons. This appears to be mediated by the modification of membrane permeability induced by the surface of GQDs. Our findings highlight that GQDs can be adopted as a suitable delivery and therapeutic strategy for the treatment of glioblastoma, by both directly destabilizing the cell membrane and indirectly increasing the efficacy of chemotherapeutic drugs.

PE_PGRS3 of Mycobacterium tuberculosis is specifically expressed at low phosphate concentration, and its arginine-rich C-terminal domain mediates adhesion and persistence in host tissues when expressed in Mycobacterium smegmatis.

PE_PGRSs of Mycobacterium tuberculosis (Mtb) represent a family of complex and peculiar proteins whose role and function remain elusive. In this study, we investigated PE_PGRS3 and PE_PGRS4, two highly homologous PE_PGRSs encoded by two contiguous genes in the Mtb genome. Using a gene-reporter system in Mycobacterium smegmatis (Ms) and transcriptional analysis in Mtb, we show that PE_PGRS3, but not PE_PGRS4, is specifically expressed under low phosphate concentrations. Interestingly, PE_PGRS3, but not PE_PGRS4, has a unique, arginine-rich C-terminal domain of unknown function. Heterologous expression of PE_PGRS3 in Ms was used to demonstrate cellular localisation of the protein on the mycobacterial surface, where it significantly affects net surface charge. Moreover, expression of full-length PE_PGRS3 enhanced adhesion of Ms to murine macrophages and human epithelial cells and improved bacterial persistence in spleen tissue following infection in mice. Expression of the PE_PGRS3 functional deletion mutant lacking the C-terminal domain in Ms did not enhance adhesion to host cells, showing a phenotype similar to the Ms parental strain. Interestingly, enhanced persistence of Ms expressing PE_PGRS3 did not correlate with increased concentrations of inflammatory cytokines. These results point to a critical role for the ≈ 80 amino acids long, arginine-rich C-terminal domain of PE_PGRS3 in tuberculosis pathogenesis.

A novel method for post-mortem interval estimation based on tissue nano-mechanics.

Forensic estimation of post-mortem interval relies on different methods, most of which, however, have practical limitations or provide insufficient results, still lacking a gold standard method. In order to better understand the phenomenon of rigor mortis and its applicability to the post-mortem interval estimation, we decided to use atomic force microscopy, a tool often employed to measure mechanical properties of adherent cells. Thus, we surgically removed skeletal muscle samples of three forensic cases from 0 to 120 h post-mortem and quantitatively evaluate two parameters: the Young's modulus (E), which gives information about the sample stiffness, and the hysteresis (H), which estimates the contribution of viscous forces. Despite being a preliminary study, the obtained results show that the temporal behavior of E well correlates with the expected evolution of rigor mortis between 0 and 48 h post-mortem, and then monotonically decreases over time. Unfortunately, it is strongly affected by inter-individual variability. However, we found that H provides measurable data along a time-dependent curve back to the starting point, and these data measured on different subjects collapse onto a single master curve, getting rid of the inter-individual variability. Although a larger sampling should be performed to improve the result reliability, this finding is strongly suggestive that the evaluation of rigor mortis should involve the measure of the nanoscale dissipative behavior of muscular tissues.

Graphene Oxide Induced Osteogenesis Quantification by In-Situ 2D-Fluorescence Spectroscopy.

Graphene and graphene oxide can promote the adhesion, growth and differentiation of mesenchymal stem cells. Further, graphene surface coatings accelerate the differentiation of human mesenchymal stem cells acting as osteogenic inducers. Quantification of the osteogenic induction is conventionally performed with Alizarin Red S (ARS), an anthraquinone derivative used to identify calcium deposits in tissue sections and cell cultures. The ARS staining is quite versatile because the dye forms an Alizarin Red S⁻calcium complex that can be extracted from the stained monolayer of cells and readily assayed by absorbance measurements. Direct visualization of stained deposits is also feasible; however, an in-situ visualization and quantification of deposits is possible only on transparent supports and not on thick opaque materials like ceramics and graphene composites that are well-known inducers of osteogenesis. In this manuscript, the shape of the 2D-fluorescence spectra of the ARS-calcium complex is used to develop a method to detect and monitor the in-situ differentiation process occurring during the osteogenic induction mediated by opaque graphene oxide surfaces.

Nanoindentation characterisation of human colorectal cancer cells considering cell geometry, surface roughness and hyperelastic constitutive behaviour.

Characterisation of the mechanical behaviour of cancer cells is an issue of crucial importance as specific cell mechanical properties have been measured and utilized as possible biomarkers of cancer progression. Atomic force microscopy certainly occupies a prominent place in the field of the mechanical characterisation devices. We developed a hybrid approach to characterise different cell lines (SW620 and SW480) of the human colon carcinoma submitted to nanoindentation measurements. An ad hoc algorithm was written that compares the force-indentation curves experimentally retrieved with those predicted by a finite element model that simulates the nanoindentation process and reproduces the cell geometry and the surface roughness. The algorithm perturbs iteratively the values of the cell mechanical properties implemented in the finite element model until the difference between the experimental and numerical force-indentation curves reaches the minimum value. The occurrence of this indicates that the implemented material properties are very close to the real ones. Different hyperelastic constitutive models, such as Arruda-Boyce, Mooney-Rivlin and Neo-Hookean were utilized to describe the structural behaviour of indented cells. The algorithm was capable of separating, for all the cell lines investigated, the mechanical properties of cell cortex and cytoskeleton. Material properties determined via the algorithm were different with respect to those obtained with the Hertzian contact theory. This demonstrates that factors such as: the cell geometry/anatomy and the hyperelastic constitutive behaviour, which are not contemplated in the Hertz's theory hypotheses, do affect the nanoindentation measurements. The proposed approach represents a powerful tool that, only on the basis of nanoindentation measurements, is capable of characterising material at the subcellular level.

The graphene oxide contradictory effects against human pathogens.

Standing out as the new wonder bidimensional material, graphene oxide (GO) has aroused an exceptional interest in biomedical research by holding promise for being the antibacterial of future. First, GO possesses a specific interaction with microorganisms combined with a mild toxicity for human cells. Additionally, its antibacterial action seems to be directed to multiple targets in pathogens, causing both membranes mechanical injury and oxidative stress. Lastly, compared to other carbon materials, GO has easy and low-cost processing and is environment-friendly. This remarkable specificity and multi-targeting antibacterial activity come at a time when antibiotic resistance represents the major health challenge. Unfortunately, a comprehensive framework to understand how to effectively utilize this material against microorganisms is still lacking. In the last decade, several groups tried to define the mechanisms of interaction between GO flakes and pathogens but conflicting results have been reported. This review is focused on all the contradictions of GO antimicrobial properties in solution. Flake size, incubation protocol, time of exposure and species considered are examples of factors influencing results. These parameters will be summarized and analyzed with the aim of defining the causes of contradictions, to allow fast GO clinical application.

Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin-Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements.

A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.

Time evolution of noise induced oxidation in outer hair cells: role of NAD(P)H and plasma membrane fluidity.

BACKGROUND: Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation. METHODS: Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy. RESULTS: In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6h. Peroxidation occurs after ~4h from noise insult, while ROS are produced in the first 0.2h and damage cells for a period of time after noise exposure has ended (~7.5h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment. CONCLUSIONS: Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation in hair cells. GENERAL SIGNIFICANCE: Our results could pave the way for therapeutic intervention targeting the onset of redox umbalance.

Effect of AFM probe geometry on visco-hyperelastic characterization of soft materials.

Atomic force microscopy (AFM) nanoindentation is very suited for nano- and microscale mechanical characterization of soft materials. Although the structural response of polymeric networks that form soft matter depends on viscous effects caused by the relative slippage of polymeric chains, the usual assumption made in the AFM-based characterization is that the specimen behaves as a purely elastic material and viscous forces are negligible. However, for each geometric configuration of the AFM tip, there will be a limit indentation rate above which viscous effects must be taken into account to correctly determine mechanical properties. A parametric finite element study conducted on 12 geometric configurations of a blunt cone AFM tip (overall, the study included about 200 finite element analyses) allowed us to determine the limit indentation rate for each configuration. The selected tip dimensions cover commercially available products and account for changes in tip geometry caused by serial measurements. Nanoindentation rates cover typical experimental conditions set in AFM bio-measurements on soft matter. Viscous effects appear to be more significant in the case of sharper tips. This implies that, if quantitative data on sample viscosity are not available, using a rounded indenter and carrying out experiments below the limit indentation rate will allow errors in the determination of mechanical properties to be minimized.

An integrated superhydrophobic-plasmonic biosensor for mid-infrared protein detection at the femtomole level.

In this work we present an integrated biosensor that enables FTIR (Fourier Transform-Infrared) detection of analytes contained in diluted solutions. The fabricated nanosensor allows for the detection of proteins through the identification of the fine structure of their amide I and II bands, up to the nanomolar concentration range. We exploited two distinct effects to enhance the sensitivity: (i) the concentration effect due to the presence of the superhydrophobic surface that conveys molecules dispersed in solution directly inside the focus of a FTIR spectromicroscope; (ii) the plasmonic resonance of the nanoantenna array that provides electromagnetic field enhancement in the amide I and II spectral region (1500-1700 cm(-1)). We demonstrate the detection of ferritin in the nanomolar concentration range, a blood protein that is usually available in small amounts in typical blood samples.

In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.

Janus-faced liposomes enhance antimicrobial innate immune response in Mycobacterium tuberculosis infection.

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1ß, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-ß without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1ß, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.