Extracellular Vesicles as Next-Generation Diagnostics and Advanced Therapy Medicinal Products.

Extracellular vesicles (EVs) hold great promise for clinical application as new diagnostic and therapeutic modalities. This paper describes major GMP-based upstream and downstream manufacturing processes for EV large-scale production, also focusing on post-processing technologies such as surface bioengineering and uploading studies to yield novel EV-based diagnostics and advanced therapy medicinal products. This paper also focuses on the quality, safety, and efficacy issues of the bioengineered EV drug candidates before first-in-human studies. Because clinical trials involving extracellular vesicles are on the global rise, this paper encompasses different clinical studies registered on clinical-trial register platforms, with varying levels of advancement, highlighting the growing interest in EV-related clinical programs. Navigating the regulatory affairs of EVs poses real challenges, and obtaining marketing authorization for EV-based medicines remains complex due to the lack of specific regulatory guidelines for such novel products. This paper discusses the state-of-the-art regulatory knowledge to date on EV-based diagnostics and medicinal products, highlighting further research and global regulatory needs for the safe and reliable implementation of bioengineered EVs as diagnostic and therapeutic tools in clinical settings. Post-marketing pharmacovigilance for EV-based medicinal products is also presented, mainly addressing such topics as risk assessment and risk management.

Embryonic Stem Cells Can Generate Oral Epithelia under Matrix Instruction.

We aimed to investigate whether molecular clues from the extracellular matrix (ECM) can induce oral epithelial differentiation of pluripotent stem cells. Mouse embryonic stem cells (ESC) of the feeder-independent cell line E14 were used as a model for pluripotent stem cells. They were first grown in 2D on various matrices in media containing vitamin C and without leukemia inhibitory factor (LIF). Matrices investigated were gelatin, laminin, and extracellular matrices (ECM) synthesized by primary normal oral fibroblasts and keratinocytes in culture. Differentiation into epithelial lineages was assessed by light microscopy, immunocytochemistry, and flow cytometry for cytokeratins and stem cell markers. ESC grown in 2D on various matrices were afterwards grown in 3D organotypic cultures with or without oral fibroblasts in the collagen matrix and examined histologically and by immunohistochemistry for epithelial (keratin pairs 1/10 and 4/13 to distinguish epidermal from oral epithelia and keratins 8,18,19 to phenotype simple epithelia) and mesenchymal (vimentin) phenotypes. ECM synthesized by either oral fibroblasts or keratinocytes was able to induce, in 2D cultures, the expression of cytokeratins of the stratified epithelial phenotype. When grown in 3D, all ESC developed into two morphologically distinct cell populations on collagen gels: (i) epithelial-like cells organized in islands with occasional cyst- or duct-like structures and (ii) spindle-shaped cells suggestive of mesenchymal differentiation. The 3D culture on oral fibroblast-populated collagen matrices was necessary for further differentiation into oral epithelia. Only ESC initially grown on 2D keratinocyte or fibroblast-synthesized matrices reached full epithelial maturation. In conclusion, ESC can generate oral epithelia under matrix instruction.

Predicting intention of Norwegian dental health-care workers to use nanomaterials: An application of the augmented theory of planned behavior.

Due to the rapid development of nanotechnology and its integration into dentistry, there is a need for information on the factors influencing the decision of dental health-care workers to use nanomaterials. Based on a national survey among Norwegian dentists and dental hygienists, this study applied the theory of planned behavior (TPB), augmented with past behavior and perceived risk, to predict the intention to use dental nanomaterials in the future and to assess whether an augmented TPB model operates equivalently across professional groups. Structural equation modelling was used to assess whether the hypothesized model fits the data. Of 1792 eligible participants, 851 responded to an electronic survey. Attitudes and perceived behavioral control had the strongest effect on intention, followed by past behavior and subjective norms. Risk perceptions had an indirect effect on intention. Multigroup comparison confirmed invariance of the model across professional groups. This study supports the validity of the augmented TPB model to explain the intention of Norwegian dentists and dental hygienists to use nanomaterials. The strongest influence on intention is given by the attitudes toward nanomaterials and perceived confidence in their use. The findings of the study have implications for management of the use of nanomaterials in dentistry by policy makers.

Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally.

Thermodynamic Parameters at Bio-Nano Interface and Nanomaterial Toxicity: A Case Study on BSA Interaction with ZnO, SiO2, and TiO2.

Understanding nanomaterial (NM)-protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM-protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO2; NM-101), and silica (SiO2; NM-203). The thermodynamic signature associated with the NM interaction with an abundant protein occurring in most cell culture media, bovine serum albumin (BSA), has been investigated by isothermal titration and differential scanning calorimetry. Circular dichroism spectroscopy offers additional information concerning adsorption-induced protein conformational changes. The BSA adsorption onto NMs is enthalpy-controlled, with the enthalpic character (favorable interaction) decreasing as follows: ZnO (NM-110) > SiO2 (NM-203) > TiO2 (NM-101). The binding of BSA is spontaneous, as revealed by the negative free energy, ΔG, for all systems. The structural stability of the protein decreased as follows: TiO2 (NM-101) > SiO2 (NM-203) > ZnO (NM-110). As protein binding may alter NM reactivity and thus the toxicity, we furthermore assessed its putative influence on DNA damage, as well as on the expression of target genes for cell death (RIPK1, FAS) and oxidative stress (SOD1, SOD2, CAT, GSTK1) in the A549 human alveolar basal epithelial cell line. The enthalpic component of the BSA-NM interaction, corroborated with BSA structural stability, matched the ranking for the biological alterations, i.e., DNA strand breaks, oxidized DNA lesions, cell-death, and antioxidant gene expression in A549 cells. The relative and total content of BSA in the protein corona was determined using mass-spectrometry-based proteomics. For the present case study, the thermodynamic parameters at bio/nano interface emerge as key descriptors for the dominant contributions determining the adsorption processes and NMs toxicological effect.

Use of nanomaterials in dentistry: covariates of risk and benefit perceptions among dentists and dental hygienists in Norway.

Objective: Focusing dentists and dental hygienists employed in the Public Dental Health Services (PDHSs) in Norway, this study set out to assess whether socio-demographic factors, familiarity with nanotechnology and social trust are associated with dental health care workers' perceived risks and benefits of use of nanomaterials in dentistry and whether those associations varied according to professional status. It was hypothesized that increased knowledge, trust in stakeholders and familiarity with nanomaterials would decrease the risk and increase benefit perceptions among dental health care workers.Methods: Electronic questionnaires were administered to a census of 1792 dentists and dental hygienists.Results: About 64% and 69% of respondents perceived respectively, risk and benefits associated with use of nanomaterials. Multiple variable logistic regression revealed that dentists were more likely than hygienists to perceive risks (OR = 1.9, 95% CI 1.1-3.3) and benefits (OR = 3.6, 95% CI 2.1-6.2). Having experience with dental nanomaterials (OR = 2.2, 95% CI 1.3-3.7) and feeling safe (OR = 6.6, 95% CI 3.1-14.2) increased perceived benefits. Having moderate or much correct knowledge about nanotechnology (OR = 2.3, 95% CI 1.5-3.5) increased the likelihood of perceived risk.Conclusions: Policy makers should consider the factors that influence dental health care workers' risk and benefit perceptions associated with the use of nanomaterials in dentistry.

Knowledge about nanotechnology and intention to use nanomaterials: A comparative study among dental students in Norway and Romania.

BACKGROUND: The introduction of innovative nanotechnologies in medicine and dentistry may initiate a need for curriculum revision at the universities. The aim of this study was to assess dental students' knowledge and attitudes related to nanotechnology. Covariates of students' intention to use nanomaterials in their future dental practice were evaluated using the theory of planned behaviour (TPB). METHODS: Dental students at Norwegian and Romanian Universities were invited to participate. A self-administered structured questionnaire including socio-demographics and Ajzen's TPB components was used. FINDINGS: A total of 212 out of 732 dental students participated in the survey: 52 Norwegian and 160 Romanian. Most students reported to have little knowledge about nanotechnology (Norwegians = 44.2% vs Romanians = 46.9%, P < .05). More than 90% of the students in both countries reported that they wanted to get more information about nanotechnology. Mean knowledge score was similar for Norwegian and Romanian students (4.4 ± 1.7 vs 4.2 ± 1.4, P > .05). Romanian students had more positive attitude, stronger subjective norms and stronger perceived behavioural control towards nanotechnology compared to their Norwegian counterparts. Intention to use nanomaterials in the total sample was most strongly influenced by attitude towards the use of dental nanomaterials (beta = 0.42, P < .001). CONCLUSION: Dental students in Norway and Romania demonstrated limited knowledge about nanotechnology. Intention to use nanomaterials was primarily influenced by attitudes. A clear desire for more information about the application of nanotechnology in dentistry was expressed by the respondents indicating a need for curriculum modification.

Surface-Bioengineered Extracellular Vesicles Seeking Molecular Biotargets in Lung Cancer Cells.

Personalized medicine is a new approach to modern oncology. Here, to facilitate the application of extracellular vesicles (EVs) derived from lung cancer cells as potent advanced therapy medicinal products in lung cancer, the EV membrane was functionalized with a specific ligand for targeting purposes. In this role, the most effective heptapeptide in binding to lung cancer cells (PTHTRWA) was used. The functionalization process of EV surface was performed through the C- or N-terminal end of the heptapeptide. To prove the activity of the EVs functionalized with PTHTRWA, both a model of lipid membrane mimicking normal and cancerous cell membranes as well as human adenocarcinomic alveolar basal epithelial cells (A549) and human normal bronchial epithelial cells (BEAS-2B) have been exposed to these bioconstructs. Magnetic resonance imaging (MRI) showed that the as-bioengineered PTHTRWA-EVs loaded with superparamagnetic iron oxide nanoparticle (SPIO) cargos reach the growing tumor when dosed intravenously in NUDE Balb/c mice bearing A549 cancer. Molecular dynamics (MD) in silico studies elucidated a high affinity of the synthesized peptide to the α5ß1 integrin. Preclinical safety assays did not evidence any cytotoxic or genotoxic effects of the PTHTRWA-bioengineered EVs.

Isolation of TiNbN wear particles from a coated metal-on-metal bearing: Morphological characterization and in vitro evaluation of cytotoxicity in human osteoblasts.

To improve the wear resistance of articulating metallic joint endoprostheses, the surfaces can be coated with titanium niobium nitride (TiNbN). Under poor tribological conditions or malalignment, wear can occur on these implant surfaces in situ. This study investigated the biological response of human osteoblasts to wear particles generated from TiNbN-coated hip implants. Abrasive particles were generated in a hip simulator according to ISO 14242-1/-2 and extracted with Proteinase K. Particle characteristics were evaluated by electron microscopy and energy dispersive x-ray spectroscopy (EDS), inductively coupled plasma mass spectrometry (ICP-MS) and dynamic light scattering (DLS) measurements. Human osteoblasts were exposed to different particle dilutions (1:20, 1:50, and 1:100), and cell viability and gene expression levels of osteogenic markers and inflammatory mediators were analyzed after 4 and 7 days. Using ICP-MS, EDS, and DLS measurements, ~70% of the particles were identified as TiNbN, ranging from 39 to 94 nm. The particles exhibited a flat and subangular morphology. Exposure to particles did not influence cell viability and osteoblastic differentiation capacity. Protein levels of collagen type 1, osteoprotegerin, and receptor activator of nuclear factor κB ligand were almost unaffected. Moreover, the pro-inflammatory response via interleukins 6 and 8 was minor induced after particle contact. A high number of TiNbN wear particles only slightly affected osteoblasts' differentiation ability and inflammatory response compared to metallic particles. Nevertheless, further studies should investigate the role of these particles in peri-implant bone tissue, especially concerning other cell types.

From papers to RDF-based integration of physicochemical data and adverse outcome pathways for nanomaterials.

Adverse Outcome Pathways (AOPs) have been proposed to facilitate mechanistic understanding of interactions of chemicals/materials with biological systems. Each AOP starts with a molecular initiating event (MIE) and possibly ends with adverse outcome(s) (AOs) via a series of key events (KEs). So far, the interaction of engineered nanomaterials (ENMs) with biomolecules, biomembranes, cells, and biological structures, in general, is not yet fully elucidated. There is also a huge lack of information on which AOPs are ENMs-relevant or -specific, despite numerous published data on toxicological endpoints they trigger, such as oxidative stress and inflammation. We propose to integrate related data and knowledge recently collected. Our approach combines the annotation of nanomaterials and their MIEs with ontology annotation to demonstrate how we can then query AOPs and biological pathway information for these materials. We conclude that a FAIR (Findable, Accessible, Interoperable, Reusable) representation of the ENM-MIE knowledge simplifies integration with other knowledge. SCIENTIFIC CONTRIBUTION: This study introduces a new database linking nanomaterial stressors to the first known MIE or KE. Second, it presents a reproducible workflow to analyze and summarize this knowledge. Third, this work extends the use of semantic web technologies to the field of nanoinformatics and nanosafety.

Lack of cytotoxic and genotoxic effects of mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium despite cellular uptake in cancerous and noncancerous lung cells.

Cytotoxic and genotoxic effects of novel mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium (Mg0.1-γ-Fe2O3(mPEG-silane)0.5) have been investigated on human adenocarcinomic alveolar basal epithelial (A549) and human normal bronchial epithelial (BEAS-2B) cells. In the studies several molecular and cellular targets addressing to cell membrane, cytoplasm organelles and nucleus components were served as toxicological endpoints. The as-synthesized nanoparticles were found to be stable in the cell culture media and were examined for different concentration and exposure times. No cytotoxicity of the tested nanoparticles was found although these nanoparticles slightly increased reactive oxygen species in both cell types studied. Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles did not produce any DNA strand breaks and oxidative DNA damages in A549 and BEAS-2B cells. Different concentration of Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles and different incubation time did not affect cell migration. The lung cancer cells' uptake of the nanoparticles was more effective than in normal lung cells. Altogether, the results evidence that mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium do not elucidate any deleterious effects on human normal and cancerous lung cells despite cellular uptake of these nanoparticles. Therefore, it seems reasonable to conclude that these novel biocompatible nanoparticles are promising candidates for further development towards medical applications.

A template wizard for the cocreation of machine-readable data-reporting to harmonize the evaluation of (nano)materials.

Making research data findable, accessible, interoperable and reusable (FAIR) is typically hampered by a lack of skills in technical aspects of data management by data generators and a lack of resources. We developed a Template Wizard for researchers to easily create templates suitable for consistently capturing data and metadata from their experiments. The templates are easy to use and enable the compilation of machine-readable metadata to accompany data generation and align them to existing community standards and databases, such as eNanoMapper, streamlining the adoption of the FAIR principles. These templates are citable objects and are available as online tools. The Template Wizard is designed to be user friendly and facilitates using and reusing existing templates for new projects or project extensions. The wizard is accompanied by an online template validator, which allows self-evaluation of the template (to ensure mapping to the data schema and machine readability of the captured data) and transformation by an open-source parser into machine-readable formats, compliant with the FAIR principles. The templates are based on extensive collective experience in nanosafety data collection and include over 60 harmonized data entry templates for physicochemical characterization and hazard assessment (cell viability, genotoxicity, environmental organism dose-response tests, omics), as well as exposure and release studies. The templates are generalizable across fields and have already been extended and adapted for microplastics and advanced materials research. The harmonized templates improve the reliability of interlaboratory comparisons, data reuse and meta-analyses and can facilitate the safety evaluation and regulation process for (nano) materials.

Polycrystalline Diamond Coating on Orthopedic Implants: Realization and Role of Surface Topology and Chemistry in Adsorption of Proteins and Cell Proliferation.

Polycrystalline diamond has the potential to improve the osseointegration of orthopedic implants compared to conventional materials such as titanium. However, despite the excellent biocompatibility and superior mechanical properties, the major challenge of using diamond for implants, such as those used for hip arthroplasty, is the limitation of microwave plasma chemical vapor deposition (CVD) techniques to synthesize diamond on complex-shaped objects. Here, for the first time, we demonstrate diamond growth on titanium acetabular shells using the surface wave plasma CVD method. Polycrystalline diamond coatings were synthesized at low temperatures (∼400 °C) on three types of acetabular shells with different surface structures and porosities. We achieved the growth of diamond on highly porous surfaces designed to mimic the structure of the trabecular bone and improve osseointegration. Biocompatibility was investigated on nanocrystalline diamond (NCD) and ultrananocrystalline diamond (UNCD) coatings terminated either with hydrogen or oxygen. To understand the role of diamond surface topology and chemistry in the attachment and proliferation of mammalian cells, we investigated the adsorption of extracellular matrix proteins and monitored the metabolic activity of fibroblasts, osteoblasts, and bone-marrow-derived mesenchymal stem cells (MSCs). The interaction of bovine serum albumin and type I collagen with the diamond surfaces was investigated by confocal fluorescence lifetime imaging microscopy (FLIM). We found that the proliferation of osteogenic cells was better on hydrogen-terminated UNCD than on the oxygen-terminated counterpart. These findings correlated with the behavior of collagen on diamond substrates observed by FLIM. Hydrogen-terminated UNCD provided better adhesion and proliferation of osteogenic cells, compared to titanium, while the growth of fibroblasts was poorest on hydrogen-terminated NCD and MSCs behaved similarly on all tested surfaces. These results open new opportunities for application of diamond coatings on orthopedic implants to further improve bone fixation and osseointegration.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models.

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.

A strategy towards the generation of testable adverse outcome pathways for nanomaterials.

Manufactured nanomaterials (NMs) are increasingly used in a wide range of industrial applications leading to a constant increase in the market size of nano-enabled products. The increased production and use of NMs are raising concerns among different stakeholder groups with regard to their effects on human and environmental health. Currently, nanosafety hazard assessment is still widely performed using in vivo (animal) models, however the development of robust and reg­ulatory relevant strategies is required to prioritize and/or reduce animal testing. An adverse outcome pathway (AOP) is a structured representation of biological events that start from a molecular initiating event (MIE) leading to an adverse outcome (AO) through a series of key events (KEs). The AOP framework offers great advancement to risk assessment and regulatory safety assessments. While AOPs for chemicals have been more frequently reported, the AOP collection for NMs is limited. By using existing AOPs, we aimed to generate simple and testable strategies to predict if a given NM has the potential to induce a MIE leading to an AO through a series of KEs. Firstly, we identified potential MIEs or initial KEs reported for NMs in the literature. Then, we searched the identified MIE or initial KEs as keywords in the AOP-Wiki to find associated AOPs. Finally, using two case studies, we demonstrate how in vitro strategies can be used to test the identified MIE/KEs.

Label-free impedance flow cytometry for nanotoxicity screening.

The development of reliable and cost-efficient methods to assess the toxicity of nanomaterials (NMs) is critical for the proper identification of their impact on human health and for ensuring a safe progress of nanotechnology. In this study, we investigated the reliability and applicability of label-free impedance flow cytometry (IFC) for in vitro nanotoxicity screening, which avoids time-consuming labelling steps and minimizes possible NM-induced interferences. U937 human lymphoma cells were exposed for 24 h to eight different nanomaterials at five concentrations (2, 10, 20, 50, and 100 µg/mL). The NMs' effect on viability was measured using IFC and the results were compared to those obtained by trypan blue (TB) dye exclusion and conventional flow cytometry (FC). To discriminate viable from necrotic cells, the IFC measurement settings regarding signal trigger level and frequency, as well as the buffer composition, were optimised. A clear discrimination between viable and necrotic cells was obtained at 6 MHz in a sucrose-based measurement buffer. Nanomaterial-induced interferences were not detected for IFC. The IFC and TB assay results were in accordance for all NMs. The IFC was found to be robust, reliable and less prone to interferences due to the advantage of being label-free.

Epigenetics in Breast Cancer Therapy-New Strategies and Future Nanomedicine Perspectives.

Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identification, allowing for patient stratification and tailored management. In combination with new-generation epi-drugs, nanomedicine can help to overcome low therapeutic efficacy in treatment-resistant tumors. This review provides an overview of ongoing clinical trials focusing on combination therapies employing epi-drugs for breast cancer treatment and summarizes the latest nano-based targeted delivery approaches for epi-drugs. Moreover, it highlights the current limitations and obstacles associated with applying these experimental strategies in the clinics.

Fusobacterium nucleatum enters normal human oral fibroblasts in vitro.

BACKGROUND: Fusobacterium nucleatum, a commensal opportunistic oral bacterium, is capable of invading gingival epithelial cells, but the entrance into human primary oral fibroblast cells has not been documented. This study evaluated the ability of three strains of F. nucleatum (F. nucleatum ssp. nucleatum, F. nucleatum ssp. polymorphum, and F. nucleatum ssp. vincentii) to enter gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PLFs). METHODS: GFs and PLFs were cocultured for various periods of time with different strains of F. nucleatum. Scanning and transmission electron microscopy, together with confocal laser scanning microscopy, were used to visualize the entrance and presence of bacteria in host cells. Flow cytometry was performed to compare the load of internalized bacteria in GFs and PLFs exposed for 3 and 5 hours to live F. nucleatum labeled with fluorescein isothiocyanate. RESULTS: All three strains of F. nucleatum were found entering and located in the cytoplasm of GFs and PLFs after 1 hour of exposure. Flow cytometry tests revealed a significant increase in the fluorescent signal, compared to baseline, derived from bacteria internalized in fibroblasts exposed for 3 hours (P <0.001); a further increase was found at 5 hours. The greatest bacterial mass in exposed fibroblasts of both types was of F. nucleatum ssp. polymorphum; the smallest was of F. nucleatum ssp. vincentii. Although not statistically significant, PLFs had a higher bacterial load than corresponding GFs. CONCLUSION: F. nucleatum was capable of entering GFs and PLFs in a manner that is dependent on the cell type and the bacterial strain.

High throughput toxicity screening and intracellular detection of nanomaterials.

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety-preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read-across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter-experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM-cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose- and time-dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label-free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance-based monitoring, Multiplex analysis of secreted products, and genotoxicity methods-namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.