Ionic Liquid-Assisted Thermal Evaporation of Bimetallic Ag-Au Nanoparticle Films as a Highly Reproducible SERS Substrate for Sensitive Nanoplastic Detection in Complex Environments.

Nanoplastic particles are emerging as an important class of environmental pollutants in the atmosphere that have adverse effects on our ecosystems and human health. While many methods have been developed to quantitatively detect nanoplastics; however, sensitive detection at low concentrations in a complex environment remains elusive. Herein, we demonstrate a greener method to fabricate a surface-enhanced Raman spectroscopy (SERS) substrate consisting of self-assembled plasmonic Ag-Au bimetallic nanoparticle (NP) films for quantitative SERS detection of nanoplastics in complex media. The self-assembly of Ag-Au bimetallic NPs was achieved through thermal evaporation onto a vapor-phase compatible ionic liquid based on deep eutectic solvent over the growth substrate. The finite-difference time-domain simulation revealed that the localized field enhancement is strong in the gaps, which generate uniform SERS "hotspots" in the obtained substrate. Benefiting from highly accessible SERS "hotspots" at the gaps, the SERS substrate exhibits excellent sensitivity for detecting crystal violet with a limit of detection (LOD) as low as 10-14 M and excellent reproducibility (RSD of 5.8%). The SERS substrate is capable of detecting PET nanoplastics with LOD as low as 1 µg/mL and about 100 µg/mL in real samples such as tap water, lake water, diluted milk, and wine. Moreover, we also validated the feasibility of the designed SERS substrate for the practical detection of PET nanoplastics collected from commercial drinking water bottles, and it showed great potential applications for sensitive detection in actual environments.

Glucose oxidase virus-based nanoreactors for smart breast cancer therapy.

BACKGROUND: Breast cancer is the most common malignant tumor disease and the leading cause of female mortality. The evolution of nanomaterials science opens the opportunity to improve traditional cancer therapies, enhancing therapy efficiency and reducing side effects. METHODS AND MAJOR RESULTS: Herein, protein cages conceived as enzymatic nanoreactors were designed and produced by using virus-like nanoparticles (VLPs) from Brome mosaic virus (BMV) and containing the catalytic activity of glucose oxidase (GOx) enzyme. The GOx enzyme was encapsulated into the BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The effect of the synthesized GOx nanoreactors on breast tumor cell lines was studied in vitro. Both nanoreactor preparations VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell cultures. Cytotoxicity for human embryonic kidney cells was also found. The monitoring of nanoreactor treatment on triple-negative breast cancer cells showed an evident production of oxygen by the catalase antioxidant enzyme induced by the high production of hydrogen peroxide from GOx activity. CONCLUSIONS AND IMPLICATIONS: The nanoreactors containing GOx activity are entirely suitable for cytotoxicity generation in tumor cells. The HSA functionalization of the VLP-GOx nanoreactors, a strategy designed for selective cancer targeting, showed no improvement in the cytotoxic effect. The GOx containing enzymatic nanoreactors seems to be an interesting alternative to improve the current cancer therapy. In vivo studies are ongoing to reinforce the effectiveness of this treatment strategy.

A Comparative Study of Cancer Cells Susceptibility to Silver Nanoparticles Produced by Electron Beam.

INTRODUCTION: Silver nanoparticles (AgNPs) have a wide range of bioactivity, which is highly dependent on particle size, shape, stabilizer, and production method. Here, we present the results of studies of AgNPs cytotoxic properties obtained by irradiation treatment of silver nitrate solution and various stabilizers by accelerating electron beam in a liquid medium. METHODS: The results of studies of morphological characteristics of silver nanoparticles were obtained by transmission electron microscopy, UV-vis spectroscopy, and dynamic light scattering measurements. MTT test, alamar blue test, flow cytometry, and fluorescence microscopy were used to study the anti-cancer properties. As biological objects for standard tests, adhesive and suspension cell cultures of normal and tumor origin, including prostate cancer, ovarian cancer, breast cancer, colon cancer, neuroblastoma, and leukemia, were studied. RESULTS: The results showed that the silver nanoparticles obtained by irradiation with polyvinylpyrrolidone and collagen hydrolysate are stable in solutions. Samples with different stabilizers were characterized by a wide average size distribution from 2 to 50 nm and low zeta potential from -7.3 to +12.4 mV. All AgNPs formulations showed a dose-dependent cytotoxic effect on tumor cells. It has been established that the particles obtained with the combination of polyvinylpyrrolidone/collagen hydrolysate have a relatively more pronounced cytotoxic effect in comparison to samples stabilized with only collagen or only polyvinylpyrrolidone. The minimum inhibitory concentrations for nanoparticles were less than 1 µg/mL for various types of tumor cells. It was found that neuroblastoma (SH-SY5Y) is the most susceptible, and ovarian cancer (SKOV-3) is the most resistant to the action of silver nanoparticles. The activity of the AgNPs formulation prepared with a mixture of PVP and PH studied in this work was higher that activity of other AgNPs formulations reported in the literature by about 50 times. CONCLUSIONS: The results indicate that the AgNPs formulations synthesized with an electron beam and stabilized with polyvinylpyrrolidone and protein hydrolysate deserve deep study for their further use in selective cancer treatment without harming healthy cells in the patient organism.

Silver nanoparticles induce a non-immunogenic tumor cell death.

Immunogenic cell death (ICD) is a form of cell death characterized by the release of danger signals required to trigger an adaptive immune response against tumor-associated antigens. Silver nanoparticles (AgNP) display anti-proliferative and cytotoxic effects in tumor cells, but it has not been previously studied whether AgNP act as an ICD inductor. The present study evaluated the in vitro release of calreticulin as a damage-associated molecular pattern (DAMP) associated with the cytotoxicity of AgNP and their in vivo anti-cancer effects. In vitro, mouse CT26 colon carcinoma and MCA205 fibrosarcoma cells were exposed to AgNP and then cell proliferation, adhesion, and release of calreticulin were determined. The results indicated there were time- and concentration-related anti-proliferative effects of AgNP in both the CT26 and MCA205 lines. Concurrently, changes in cell adhesion were detected mainly in the CT26 cells. Regarding DAMP detection, a significant increase in calreticulin was observed only in CT26 cells treated with doxorubicin and AgNP; however, no differences were found in the MCA205 cells. In vivo, the survival and growth of subcutaneous tumors were monitored after vaccination of mice with cell debris from tumor cells treated with AgNP or after intra-tumoral administration of AgNP to established tumors. Consequently, anti-tumoral prophylactic immunization with AgNP-dead cells failed to protect mice from tumor re-challenge; intra-tumor injection of AgNP did not induce a significant effect. In conclusion, there was a noticeable anti-tumoral effect of AgNP in vitro in both CT26 and MCA205 cell lines, accompanied by the release of calreticulin in CT26 cells. In vivo, immunization with cell debris derived from AgNP-treated tumor cells failed to induce a protective immune response in the cancer model mice. Clearly, further research is needed to determine if one could combine AgNP with other ICD inducers to improve the anti-tumor effect of these nanoparticles in vivo.

Targeted Enzymatic VLP-Nanoreactors with ß-Glucocerebrosidase Activity as Potential Enzyme Replacement Therapy for Gaucher's Disease.

Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme ß-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme ß-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29 mM) and the functionalized nanoreactors (KM =0.32 mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pH 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.

Brome mosaic virus-like particles as siRNA nanocarriers for biomedical purposes.

There is an increasing interest in the use of plant viruses as vehicles for anti-cancer therapy. In particular, the plant virus brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are novel potential nanocarriers for different therapies in nanomedicine. In this work, BMV and CCMV were loaded with a fluorophore and assayed on breast tumor cells. The viruses BMV and CCMV were internalized into breast tumor cells. Both viruses, BMV and CCMV, did not show cytotoxic effects on tumor cells in vitro. However, only BMV did not activate macrophages in vitro. This suggests that BMV is less immunogenic and may be a potential carrier for therapy delivery in tumor cells. Furthermore, BMV virus-like particles (VLPs) were efficiently loaded with small interfering RNA (siRNA) without packaging signal. The gene silencing was demonstrated by VLPs loaded with siGFP and tested on breast tumor cells that constitutively express the green fluorescent protein (GPF). After VLP-siGFP treatment, GFP expression was efficiently inhibited corroborating the cargo release inside tumor cells and the gene silencing. In addition, BMV VLP carring siAkt1 inhibited the tumor growth in mice. These results show the attractive potential of plant virus VLPs to deliver molecular therapy to tumor cells with low immunogenic response.

Nanoparticle-plasma Membrane Interactions: Thermodynamics, Toxicity and Cellular Response.

Nanomaterials have become part of our daily lives, particularly nanoparticles contained in food, water, cosmetics, additives and textiles. Nanoparticles interact with organisms at the cellular level. The cell membrane is the first protective barrier against the potential toxic effect of nanoparticles. This first contact, including the interaction between the cell membranes -and associated proteins- and the nanoparticles is critically reviewed here. Nanoparticles, depending on their toxicity, can cause cellular physiology alterations, such as a disruption in cell signaling or changes in gene expression and they can trigger immune responses and even apoptosis. Additionally, the fundamental thermodynamics behind the nanoparticle-membrane and nanoparticle-proteins-membrane interactions are discussed. The analysis is intended to increase our insight into the mechanisms involved in these interactions. Finally, consequences are reviewed and discussed.

Effect of Ions Released and pH of Two Glass Ionomer Cements in Human Gingival Fibroblasts / Efecto de la liberación de iones y pH de dos cementos de ionómero de vidrio en fibroblastos gingivales humanos

Abstract 20. Conventional glass ionomer cements are used as dental provisional restorative materials, which present several advantages such as adhesion to the tooth mineral phase among others. On the other hand, the knowledge about biological property of glass ionomers shows various approaches and results. In this work, it was studied the in vitro biological response of human gingival fibroblasts in contact with commercial cements of glass ionomer: Mirafil® and Ionglass® and with their extracts, according to ISO 10993. The extracts of the cements, in which the cells were cultured, were adjusted at different concentrations ranging 0.1% to 100%. The cellular metabolic activity of gingival fibroblasts was measured using the Alamar Blue® reagent. The results showed a significant effect on the cellular metabolic activity correlated with the concentration of liberated ions (Al³+ and Ca²+) for both ionomers, as well as the pH variations of the culture media. This could mean that the cellular metabolic activity is substantially influenced by ions and pH of the cell culture.

Resumen 24. Los cementos de ionómero de vidrio convencionales se utilizan como materiales de restauración provisional para uso dental, los cuales presentan varias ventajas como la adhesión a la fase mineral de los dientes. Por otro lado, las propiedades biológicas de los ionómeros de vidrio muestran diversos enfoques y resultados. En éste trabajo se estudió la respuesta biológica in vitro de fibroblastos gingivales humanos en contacto con cementos comerciales de ionómero de vidrio: Mirafil® e Ionglass® y con sus respectivos extractos según la norma ISO 10993. Los extractos de los cementos en los que se cultivaron las células estaban en diferentes concentraciones: de 0.1% a 100%. La actividad metabólica celular se midió usando el reactivo Alamar Blue®. Los resultados mostraron un efecto significativo sobre la actividad metabólica celular correlacionada con la concentración de iones liberados (Al³+ y Ca²+) para ambos ionómeros, así como las variaciones de pH de los medios de cultivo. Ello podria explicar la influencia por los iones y el pH del cultivo celular en la actividad metabólica celular.

Multifunctionalized biocatalytic P22 nanoreactor for combinatory treatment of ER+ breast cancer.

BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.

Silver deposition on titanium surface by electrochemical anodizing process reduces bacterial adhesion of Streptococcus sanguinis and Lactobacillus salivarius.

OBJECTIVES: The aim of this study was to determine the antibacterial properties of silver-doped titanium surfaces prepared with a novel electrochemical anodizing process. MATERIAL AND METHODS: Titanium samples were anodized with a pulsed process in a solution of silver nitrate and sodium thiosulphate at room temperature with stirring. Samples were processed with different electrolyte concentrations and treatment cycles to improve silver deposition. Physicochemical properties were determined by X-ray photoelectron spectroscopy, contact angle measurements, white-light interferometry, and scanning electron microscopy. Cellular cytotoxicity in human fibroblasts was studied with lactate dehydrogenase assays. The in vitro effect of treated surfaces on two oral bacteria strains (Streptococcus sanguinis and Lactobacillus salivarius) was studied with viable bacterial adhesion measurements and growth curve assays. Nonparametric statistical Kruskal-Wallis and Mann-Whitney U-tests were used for multiple and paired comparisons, respectively. Post hoc Spearman's correlation tests were calculated to check the dependence between bacteria adhesion and surface properties. RESULTS: X-ray photoelectron spectroscopy results confirmed the presence of silver on treated samples and showed that treatments with higher silver nitrate concentration and more cycles increased the silver deposition on titanium surface. No negative effects in fibroblast cell viability were detected and a significant reduction on bacterial adhesion in vitro was achieved in silver-treated samples compared with control titanium. CONCLUSIONS: Silver deposition on titanium with a novel electrochemical anodizing process produced surfaces with significant antibacterial properties in vitro without negative effects on cell viability.

Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane.

Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

S. sanguinis adhesion on rough titanium surfaces: effect of culture media.

Bacterial colonization plays a key role in dental implant failure, because they attach directly on implant surface upon implantation. Between different types of bacteria associated with the oral environment, Streptococcus sanguinis is essential in this process since it is an early colonizer. In this work the relationship between titanium surfaces modified by shot blasting treatment and S. sanguinis adhesion; have been studied in approached human mouth environment. Bacteria pre-inoculated with routinary solution were put in contact with titanium samples, shot-blasted with alumina and silicon carbide, and adhesion results were compared with those obtained when bacteria were pre-inoculated with modified artificial saliva medium and on saliva pre-coated titanium samples. Our results showed that bacterial adhesion on titanium samples was influenced by culture conditions. When S. sanguinis was inoculated in routinary culture media, colonies forming unities per square millimeter presented an increment correlated with roughness and surface energy, but separated by the type of particle used during shot-blasting treatment; whereas in modified artificial saliva only a relationship between bacteria adhered and the increment in both roughness and surface energy were observed, regardless of the particle type. Finally, on human saliva pre-coated samples no significant differences were observed among roughness, surface energy or particle.

Streptococcus sanguinis adhesion on titanium rough surfaces: effect of shot-blasting particles.

Dental implant failure is commonly associated to dental plaque formation. This problem starts with bacterial colonization on implant surface upon implantation. Early colonizers (such as Streptococcus sanguinis) play a key role on that process, because they attach directly to the surface and facilitate adhesion of later colonizers. Surface treatments have been focused to improve osseointegration, where shot-blasting is one of the most used. However the effects on bacterial adhesion on that sort of surfaces have not been elucidated at all. A methodological procedure to test bacterial adherence to titanium shot-blasted surfaces (alumina and silicon carbide) by quantifying bacterial detached cells per area unit, was performed. In parallel, the surface properties of samples (i.e., roughness and surface energy), were analyzed in order to assess the relationship between surface treatment and bacterial adhesion. Rather than roughness, surface energy correlated to physicochemical properties of shot-blasted particles appears as critical factors for S. sanguinis adherence to titanium surfaces.

STEM-HAADF electron microscopy analysis of the central dark line defect of human tooth enamel crystallites.

When human tooth enamel is observed with the Transmission Electron Microscope (TEM), a structural defect is registered in the central region of their nanometric grains or crystallites. This defect has been named as Central Dark Line (CDL) and its structure and function in the enamel structure have been unknown yet. In this work we present the TEM analysis to these crystallites using the High Angle Annular Dark Field (HAADF) technique. Our results suggest that the CDL region is the calcium richest part of the human tooth enamel crystallites.