Gold and silver nanoparticles in Alzheimer's and Parkinson's diagnostics and treatments.

Neurodegenerative diseases (NDs) impose substantial medical and public health burdens on people worldwide and represent one of the major threats to human health. The prevalence of these age-dependent disorders is dramatically increasing over time, a process intrinsically related to a constantly rising percentage of the elderly population in recent years. Among all the NDs, Alzheimer's and Parkinson's are considered the most debilitating as they cause memory and cognitive loss, as well as severely affecting basic physiological conditions such as the ability to move, speak, and breathe. There is an extreme need for new and more effective therapies to counteract these devastating diseases, as the available treatments are only able to slow down the pathogenic process without really stopping or resolving it. This review aims to elucidate the current nanotechnology-based tools representing a future hope for NDs treatment. Noble metal nano-systems, that is, gold and silver nanoparticles (NPs), have indeed unique physicochemical characteristics enabling them to deliver any pharmacological treatment in a more effective way within the central nervous system. This can potentially make NPs a new hope for reversing the actual therapeutic strategy based on slowing down an irreversible process into a more effective and permanent treatment.

Shape-Driven Response of Gold Nanoparticles to X-rays.

Radiotherapy (RT) involves delivering X-ray beams to the tumor site to trigger DNA damage. In this approach, it is fundamental to preserve healthy cells and to confine the X-ray beam only to the malignant cells. The integration of gold nanoparticles (AuNPs) in the X-ray methodology could be considered a powerful tool to improve the efficacy of RT. Indeed, AuNPs have proven to be excellent allies in contrasting tumor pathology upon RT due to their high photoelectric absorption coefficient and unique physiochemical properties. However, an analysis of their physical and morphological reaction to X-ray exposure is necessary to fully understand the AuNPs' behavior upon irradiation before treating the cells, since there are currently no studies on the evaluation of potential NP morphological changes upon specific irradiations. In this work, we synthesized two differently shaped AuNPs adopting two different techniques to achieve either spherical or star-shaped AuNPs. The spherical AuNPs were obtained with the Turkevich-Frens method, while the star-shaped AuNPs (AuNSs) involved a seed-mediated approach. We then characterized all AuNPs with Transmission Electron Microscopy (TEM), Uv-Vis spectroscopy, Dynamic Light Scattering (DLS), zeta potential and Fourier Transform Infrared (FTIR) spectroscopy. The next step involved the treatment of AuNPs with two different doses of X-radiation commonly used in RT, namely 1.8 Gy and 2 Gy, respectively. Following the X-rays' exposure, the AuNPs were further characterized to investigate their possible physicochemical and morphological alterations induced with the X-rays. We found that AuNPs do not undergo any alteration, concluding that they can be safely used in RT treatments. Lastly, the actin rearrangements of THP-1 monocytes treated with AuNPs were also assessed in terms of coherency. This is a key proof to evaluate the possible activation of an immune response, which still represents a big limitation for the clinical translation of NPs.

Investigation of the Effects of Pulse-Atomic Force Nanolithography Parameters on 2.5D Nanostructures' Morphology.

In recent years, Atomic Force Microscope (AFM)-based nanolithography techniques have emerged as a very powerful approach for the machining of countless types of nanostructures. However, the conventional AFM-based nanolithography methods suffer from low efficiency, low rate of patterning, and high complexity of execution. In this frame, we first developed an easy and effective nanopatterning technique, termed Pulse-Atomic Force Lithography (P-AFL), with which we were able to pattern 2.5D nanogrooves on a thin polymer layer. Indeed, for the first time, we patterned nanogrooves with either constant or varying depth profiles, with sub-nanometre resolution, high accuracy, and reproducibility. In this paper, we present the results on the investigation of the effects of P-AFL parameters on 2.5D nanostructures' morphology. We considered three main P-AFL parameters, i.e., the pulse's amplitude (setpoint), the pulses' width, and the distance between the following indentations (step), and we patterned arrays of grooves after a precise and well-established variation of the aforementioned parameters. Optimizing the nanolithography process, in terms of patterning time and nanostructures quality, we realized unconventional shape nanostructures with high accuracy and fidelity. Finally, a scanning electron microscope was used to confirm that P-AFL does not induce any damage on AFM tips used to pattern the nanostructures.

Pile-Ups Formation in AFM-Based Nanolithography: Morpho-Mechanical Characterization and Removal Strategies.

In recent decades, great efforts have been made to develop innovative, effective, and accurate nanofabrication techniques stimulated by the growing demand for nanostructures. Nowadays, mechanical tip-based emerged as the most promising nanolithography technique, allowing the pattern of nanostructures with a sub-nanometer resolution, high reproducibility, and accuracy. Unfortunately, these nanostructures result in contoured pile-ups that could limit their use and future integration into high-tech devices. The removal of pile-ups is still an open challenge. In this perspective, two different AFM-based approaches, i.e., Force Modulation Mode imaging and force-distance curve analysis, were used to characterize the structure of pile-ups at the edges of nanogrooves patterned on PMMA substrate by means of Pulse-Atomic Force Lithography. Our experimental results showed that the material in pile-ups was less stiff than the pristine polymer. Based on this evidence, we have developed an effective strategy to easily remove pile-ups, preserving the shape and the morphology of nanostructures.

Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells.

The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial-mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells' infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment.

Inorganic Nanomaterials versus Polymer-Based Nanoparticles for Overcoming Neurodegeneration.

Neurodegenerative disorders (NDs) affect a great number of people worldwide and also have a significant socio-economic impact on the aging population. In this context, nanomedicine applied to neurological disorders provides several biotechnological strategies and nanoformulations that improve life expectancy and the quality of life of patients affected by brain disorders. However, available treatments are limited by the presence of the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (B-CSFB). In this regard, nanotechnological approaches could overcome these obstacles by updating various aspects (e.g., enhanced drug-delivery efficiency and bioavailability, BBB permeation and targeting the brain parenchyma, minimizing side effects). The aim of this review is to carefully explore the key elements of different neurological disorders and summarize the available nanomaterials applied for neurodegeneration therapy looking at several types of nanocarriers. Moreover, nutraceutical-loaded nanoparticles (NPs) and synthesized NPs using green approaches are also discussed underling the need to adopt eco-friendly procedures with a low environmental impact. The proven antioxidant properties related to several natural products provide an interesting starting point for developing efficient and green nanotools useful for neuroprotection.

Impact of Nanomaterials in Biological Systems and Applications in Nanomedicine Field.

The increasingly widespread use of engineered nanomaterials in many applications increases the need to understand the mechanisms behind their toxicity [...].

AFM Characterization of Halloysite Clay Nanocomposites' Superficial Properties: Current State-of-the-Art and Perspectives.

Natural halloysite clay nanotubes (HNTs) are versatile inorganic reinforcing materials for creating hybrid composites. Upon doping HNTs with polymers, coating, or loading them with bioactive molecules, the production of novel nanocomposites is possible, having specific features for several applications. To investigate HNTs composites nanostructures, AFM is a very powerful tool since it allows for performing nano-topographic and morpho-mechanical measurements in any environment (air or liquid) without treatment of samples, like electron microscopes require. In this review, we aimed to provide an overview of recent AFM investigations of HNTs and HNT nanocomposites for unveiling hidden characteristics inside them envisaging future perspectives for AFM as a smart device in nanomaterials characterization.

Pulse-Atomic Force Lithography: A Powerful Nanofabrication Technique to Fabricate Constant and Varying-Depth Nanostructures.

The widespread use of nanotechnology in different application fields, resulting in the integration of nanostructures in a plethora of devices, has addressed the research toward novel and easy-to-setup nanofabrication techniques to realize nanostructures with high spatial resolution and reproducibility. Owing to countless applications in molecular electronics, data storage, nanoelectromechanical, and systems for the Internet of Things, in recent decades, the scientific community has focused on developing methods suitable for nanopattern polymers. To this purpose, Atomic Force Microscopy-based nanolithographic techniques are effective methods that are relatively less complex and inexpensive than equally resolute and accurate techniques, such as Electron Beam lithography and Focused Ion Beam lithography. In this work, we propose an evolution of nanoindentation, named Pulse-Atomic Force Microscopy, to obtain continuous structures with a controlled depth profile, either constant or variable, on a polymer layer. Due to the modulation of the characteristics of voltage pulses fed to the AFM piezo-scanner and distance between nanoindentations, it was possible to indent sample surface with high spatial control and fabricate highly resolved 2.5D nanogrooves. That is the real strength of the proposed technique, as no other technique can achieve similar results in tailor-made graded nanogrooves without the need for additional manufacturing steps.

High Doses of Silica Nanoparticles Obtained by Microemulsion and Green Routes Compromise Human Alveolar Cells Morphology and Stiffness Differently.

Among all the inorganic nanomaterials used in commercial products, industry, and medicine, the amorphous silica nanoparticles (SiO2 NPs) appeared to be often tolerated in living organisms. However, despite several toxicity studies, some concerns about the exposure to high doses of SiO2 NPs with different sizes were raised. Then, we used the microemulsion method to obtain stable SiO2 NPs having different sizes (110 nm, 50 nm, and 25 nm). In addition, a new one-pot green synthetic route using leaves extract of Laurus nobilis was performed, obtaining monodispersed ultrasmall SiO2 NPs without the use of dangerous chemicals. The NPs achieved by microemulsion were further functionalized with amino groups making the NPs surface positively charged. Then, high doses of SiO2 NPs (1 mg/mL and 3 mg/mL) achieved from the two routes, having different sizes and surface charges, were used to assess their impact on human alveolar cells (A549), being the best cell model mimicking the inhalation route. Cell viability and caspase-3 induction were analyzed as well as the cellular uptake, obtaining that the smallest (25 nm) and positive-charged NPs were more able to induce cytotoxicity, reaching values of about 60% of cell death. Surprisingly, cells incubated with green SiO2 NPs did not show strong toxicity, and 70% of them remained vital. This result was unusual for ultrasmall nanoobjects, generally highly toxic. The actin reorganization, nuclear morphology alteration, and cell membrane elasticity analyses confirmed the trend achieved from the biological assays. The obtained data demonstrate that the increase in cellular softness, i.e., the decrease in Young's modulus, could be associated with the smaller and positive NPs, recording values of about 3 kPa. On the contrary, green NPs triggered a slight decrease of stiffness values (c.a. 6 kPa) compared to the untreated cells (c.a. 8 kPa). As the softer cells were implicated in cancer progression and metastasization, this evidence strongly supported the idea of a link between the cell elasticity and physicochemical properties of NPs that, in turn, influenced the interaction with the cell membrane. Thus, the green SiO2 NPs compromised cells to a lesser extent than the other SiO2 NPs types. In this scenario, the elasticity evaluation could be an interesting tool to understand the toxicity of NPs with the aim of predicting some pathological phenomena associated with their exposure.

Green Silver Nanoparticles Promote Inflammation Shutdown in Human Leukemic Monocytes.

The use of silver nanoparticles (Ag NPs) in the biomedical field deserves a mindful analysis of the possible inflammatory response which could limit their use in the clinic. Despite the anti-cancer properties of Ag NPs having been widely demonstrated, there are still few studies concerning their involvement in the activation of specific inflammatory pathways. The inflammatory outcome depends on the synthetic route used in the NPs production, in which toxic reagents are employed. In this work, we compared two types of Ag NPs, obtained by two different chemical routes: conventional synthesis using sodium citrate and a green protocol based on leaf extracts as a source of reduction and capping agents. A careful physicochemical characterization was carried out showing spherical and stable Ag NPs with an average size between 20 nm and 35 nm for conventional and green Ag NPs respectively. Then, we evaluated their ability to induce the activation of inflammation in Human Leukemic Monocytes (THP-1) differentiated into M0 macrophages using 1 µM and 2 µM NPs concentrations (corresponded to 0.1 µg/mL and 0.2 µg/mL respectively) and two-time points (24 h and 48 h). Our results showed a clear difference in Nuclear Factor κB (NF-κb) activation, Interleukins 6-8 (IL-6, IL-8) secretion, Tumor Necrosis Factor-α (TNF-α) and Cyclooxygenase-2 (COX-2) expression exerted by the two kinds of Ag NPs. Green Ag NPs were definitely tolerated by macrophages compared to conventional Ag NPs which induced the activation of all the factors mentioned above. Subsequently, the exposure of breast cancer cell line (MCF-7) to the green Ag NPs showed that they exhibited antitumor activity like the conventional ones, but surprisingly, using the MCF-10A line (not tumoral breast cells) the green Ag NPs did not cause a significant decrease in cell viability.

Analysis of the Physico-Chemical, Mechanical and Biological Properties of Crosslinked Type-I Collagen from Horse Tendon: Towards the Development of Ideal Scaffolding Material for Urethral Regeneration.

Urethral stenosis is a pathological condition that consists in the narrowing of the urethral lumen because of the formation of scar tissue. Unfortunately, none of the current surgical approaches represent an optimal solution because of the high stricture recurrence rate. In this context, we preliminarily explored the potential of an insoluble type-I collagen from horse tendon as scaffolding material for the development of innovative devices for the regeneration of injured urethral tracts. Non-porous collagen-based substrates were produced and optimized, in terms of crosslinking density of the macromolecular structure, to either provide mechanical properties compliant with the urinary tract physiological stress and better sustain tissue regeneration. The effect of the adopted crosslinking strategy on the protein integrity and on the substrate physical-chemical, mechanical and biological properties was investigated in comparison with a decellularized matrix from porcine small intestinal submucosa (SIS patch), an extensively used xenograft licensed for clinical use in urology. The optimized production protocols allowed the preservation of the type I collagen native structure and the realization of a substrate with appealing end-use properties. The biological response, preliminarily investigated by immunofluorescence experiments on human adult renal stem/progenitor cells until 28 days, showed the formation of a stem-cell monolayer within 14 days and the onset of spheroids within 28 days. These results suggested the great potential of the collagen-based material for the development of scaffolds for urethral plate regeneration and for in vitro cellular studies.

Improvement of PMMA Dental Matrix Performance by Addition of Titanium Dioxide Nanoparticles and Clay Nanotubes.

Over the last decades, several materials have been proposed for the fabrication of dental and mandibular prosthetic implants. Today, the poly(methyl-methacrylate) (PMMA) resin is the most spread material, due to its ease of processing, low cost, aesthetic properties, low weight, biocompatibility, and biostability in the oral cavity. However, the porous surface (which favors the adhesion of microorganisms) and the weak mechanical properties (which lead to wear or fracture) are the major concerns. The inclusion of engineered nanomaterials in the acrylic matrix could improve the performances of PMMA. In this study, we added two different kind of nanomaterials, namely titanium dioxide nanoparticles (TiO2NPs) and halloysite clay nanotubes (HNTs) at two concentrations (1% and 3% w/w) in PMMA. Then, we assessed the effect of nanomaterials inclusion by the evaluation of specific physical parameters: Young's modulus, roughness, and wettability. In addition, we investigated the potential beneficial effects regarding the Candida albicans (C. albicans) colonization reduction, the most common yeast responsible of several infections in oral cavity. Our experimental results showed an improvement of PMMA performance, following the addition of TiO2NPs and HNTs, in a dose dependent manner. In particular, the presence of TiO2NPs in the methacrylate matrix induced a greater increase in PMMA stiffness respect to HNTs addition. On the other hand, HNTs reduced the rate of C. albicans colonization more significantly than TiO2NPs. The results obtained are of great interest for the improvement of PMMA physico-chemical properties, in view of its possible application in clinical dentistry.

Synergistic Effect Induced by Gold Nanoparticles with Polyphenols Shell during Thermal Therapy: Macrophage Inflammatory Response and Cancer Cell Death Assessment.

BACKGROUND: In recent decades, gold nanoparticle (Au NP)-based cancer therapy has been heavily debated. The physico-chemical properties of AuNPs can be exploited in photothermal therapy, making them a powerful tool for selectively killing cancer cells. However, the synthetic side products and capping agents often induce a strong activation of the inflammatory pathways of macrophages, thus limiting their further applications in vivo. METHODS: Here, we described a green method to obtain stable polyphenol-capped AuNPs (Au NPs@polyphenols), as polyphenols are known for their anti-inflammatory and anticancer properties. These NPs were used in human macrophages to test key inflammation-related markers, such as NF-κB, TNF-α, and interleukins-6 and 8. The results were compared with similar NPs obtained by a traditional chemical route (without the polyphenol coating), proving the potential of Au NPs@polyphenols to strongly promote the shutdown of inflammation. This was useful in developing them for use as heat-synergized tools in the thermal treatment of two types of cancer cells, namely, breast cancer (MCF-7) and neuroblastoma (SH-SY5Y) cells. The cell viability, calcium release, oxidative stress, HSP-70 expression, mitochondrial, and DNA damage, as well as cytoskeleton alteration, were evaluated. RESULTS: Our results clearly demonstrate that the combined strategy markedly exerts anticancer effects against the tested cancer cell, while neither of the single treatments (only heat or only NPs) induced significant changes. CONCLUSIONS: Au NP@polyphenols may be powerful agents in cancer treatment.

Physico-Chemical Properties of Inorganic NPs Influence the Absorption Rate of Aquatic Mosses Reducing Cytotoxicity on Intestinal Epithelial Barrier Model.

Noble metals nanoparticles (NPs) and metal oxide NPs are widely used in different fields of application and commercial products, exposing living organisms to their potential adverse effects. Recent evidences suggest their presence in the aquifers water and consequently in drinking water. In this work, we have carefully synthesized four types of NPs, namely, silver and gold NPs (Ag NPs and Au NPs) and silica and titanium dioxide NPs (SiO2 NPs and TiO2 NPs) having a similar size and negatively charged surfaces. The synthesis of Ag NPs and Au NPs was carried out by colloidal route using silver nitrate (AgNO3) and tetrachloroauric (III) acid (HAuCl4) while SiO2 NPs and TiO2 NPs were achieved by ternary microemulsion and sol-gel routes, respectively. Once the characterization of NPs was carried out in order to assess their physico-chemical properties, their impact on living cells was studied. We used the human colorectal adenocarcinoma cells (Caco-2), known as the best representative intestinal epithelial barrier model to understand the effects triggered by NPs through ingestion. Then, we moved to explore how water contamination caused by NPs can be lowered by the ability of three species of aquatic moss, namely, Leptodictyum riparium, Vesicularia ferriei, and Taxiphyllum barbieri, to absorb them. The experiments were conducted using two concentrations of NPs (100 µM and 500 Μm as metal content) and two time points (24 h and 48 h), showing a capture rate dependent on the moss species and NPs type. Then, the selected moss species, able to actively capture NPs, appear as a powerful tool capable to purify water from nanostructured materials, and then, to reduce the toxicity associated to the ingestion of contaminated drinking water.

The New Frontiers in Neurodegenerative Diseases Treatment: Liposomal-Based Strategies.

In the last decade, the onset of neurodegenerative (ND) diseases is strongly widespread due to the age increase of the world population. Despite the intensive investigations boosted by the scientific community, an efficacious therapy has not been outlined yet. The drugs commonly used are only able to relieve symptom severity; following their oral or intravenous administration routes, their effectiveness is strictly limited due to their low ability to reach the Central Nervous System (CNS) overcoming the Blood Brain Barrier (BBB). Starting from these assumptions, the engineered-nanocarriers, such as lipid-nanocarriers, are suitable agents to enhance the delivery of drugs into the CNS due to their high solubility, bioavailability, and stability. Liposomal delivery systems are considered to be the ideal carriers, not only for conventional drugs but also for neuroprotective small molecules and green-extracted compounds. In the current work, the LP-based drug delivery improvements in in vivo applications against ND disorders were carefully assessed.

Colorimetric Paper-Based Device for Hazardous Compounds Detection in Air and Water: A Proof of Concept.

In the last decades, the increase in global industrialization and the consequent technological progress have damaged the quality of the environment. As a consequence, the high levels of hazardous compounds such as metals and gases released in the atmosphere and water, have raised several concerns about the health of living organisms. Today, many analytical techniques are available with the aim to detect pollutant chemical species. However, a lot of them are not affordable due to the expensive instrumentations, time-consuming processes and high reagents volumes. Last but not least, their use is exclusive to trained operators. Contrarily, colorimetric sensing devices, including paper-based devices, are easy to use, providing results in a short time, without particular specializations to interpret the results. In addition, the colorimetric response is suitable for fast detection, especially in resource-limited environments or underdeveloped countries. Among different chemical species, transition and heavy metals such as iron Fe(II) and copper Cu(II) as well as volatile compounds, such as ammonia (NH3) and acetaldehyde (C2H4O) are widespread mainly in industrialized geographical areas. In this work, we developed a colorimetric paper-based analytical device (PAD) to detect different contaminants, including Fe2+ and Cu2+ ions in water, and NH3 and C2H4O in air at low concentrations. This study is a "proof of concept" of a new paper sensor in which the intensity of the colorimetric response is proportional to the concentration of a detected pollutant species. The sensor model could be further implemented in other technologies, such as drones, individual protection devices or wearable apparatus to monitor the exposure to toxic species in both indoor and outdoor environments.

Green Plasmonic Nanoparticles and Bio-Inspired Stimuli-Responsive Vesicles in Cancer Therapy Application.

: In the past years, there is a growing interest in the application of nanoscaled materials in cancer therapy because of their unique physico-chemical properties. However, the dark side of their usability is limited by their possible toxic behaviour and accumulation in living organisms. Starting from this assumption, the search for a green alternative to produce nanoparticles (NPs) or the discovery of green molecules, is a challenge in order to obtain safe materials. In particular, gold (Au NPs) and silver (Ag NPs) NPs are particularly suitable because of their unique physico-chemical properties, in particular plasmonic behaviour that makes them useful as active anticancer agents. These NPs can be obtained by green approaches, alternative to conventional chemical methods, owing to the use of phytochemicals, carbohydrates, and other biomolecules present in plants, fungi, and bacteria, reducing toxic effects. In addition, we analysed the use of green and stimuli-responsive polymeric bio-inspired nanovesicles, mainly used in drug delivery applications that have revolutionised the way of drugs supply. Finally, we reported the last examples on the use of metallic and Au NPs as self-propelling systems as new concept of nanorobot, which is able to respond and move towards specific physical or chemical stimuli in biological entities.

Silver Nanoparticles Addition in Poly(Methyl Methacrylate) Dental Matrix: Topographic and Antimycotic Studies.

The widespread use of nanoparticles (NPs) in medical devices has opened a new scenario in the treatment and prevention of many diseases and infections owing to unique physico-chemical properties of NPs. In this way, silver nanoparticles (AgNPs) are known to have a strong antimicrobial activity, even at low concentrations, due to their ability to selectively destroy cellular membranes. In particular, in the field of dental medicine, the use of AgNPs in different kinds of dental prosthesis matrixes could be a fundamental tool in immunodepressed patients that suffer of different oral infections. Candida albicans (C. albicans), an opportunistic pathogenic yeast with high colonization ability, is one of the causative agents of oral cavity infection. In our work, we added monodispersed citrate-capping AgNPs with a size of 20 nm at two concentrations (3 wt% and 3.5 wt%) in poly(methyl methacrylate) (PMMA), the common resin used to develop dental prostheses. After AgNPs characterization, we evaluated the topographical modification of PMMA and PMMA with the addition of AgNPs by means of atomic force microscopy (AFM), showing the reduction of surface roughness. The C. albicans colonization on PMMA surfaces was assessed by the Miles and Misra technique as well as by scanning electron microscopy (SEM) at 24 h and 48 h with encouraging results on the reduction of yeast viability after AgNPs exposure.