Microfluidics for studying metastatic patterns of lung cancer.

The incidence of lung cancer continues to rise worldwide. Because the aggressive metastasis of lung cancer cells is the major drawback of successful therapies, the crucial challenge of modern nanomedicine is to develop diagnostic tools to map the molecular mechanisms of metastasis in lung cancer patients. In recent years, microfluidic platforms have been given much attention as tools for novel point-of-care diagnostic, an important aspect being the reconstruction of the body organs and tissues mimicking the in vivo conditions in one simple microdevice. Herein, we present the first comprehensive overview of the microfluidic systems used as innovative tools in the studies of lung cancer metastasis including single cancer cell analysis, endothelial transmigration, distant niches migration and finally neoangiogenesis. The application of the microfluidic systems to study the intercellular crosstalk between lung cancer cells and surrounding tumor microenvironment and the connection with multiple molecular signals coming from the external cellular matrix are discussed. We also focus on recent breakthrough technologies regarding lab-on-chip devices that serve as tools for detecting circulating lung cancer cells. The superiority of microfluidic systems over traditional in vitro cell-based assays with regard to modern nanosafety studies and new cancer drug design and discovery is also addressed. Finally, the current progress and future challenges regarding printable and paper-based microfluidic devices for personalized nanomedicine are summarized.

Label-Free Monitoring of Uptake and Toxicity of Endoprosthetic Wear Particles in Human Cell Cultures.

The evaluation of the biological effects of endoprosthetic wear particles on cells in vitro relies on a variety of test assays. However, most of these methods are susceptible to particle-induced interferences; therefore, label-free testing approaches emerge as more reliable alternatives. In this study, impedance-based real-time monitoring of cellular viability and metabolic activity were performed following exposure to metallic and ceramic wear particles. Moreover, label-free imaging of particle-exposed cells was done by high-resolution darkfield microscopy (HR-ODM) and field emission scanning electron microscopy (FESEM). The isolated human fibroblasts were exposed to CoCr28Mo6 and alumina matrix composite (AMC) ceramic particles. HR-ODM and FESEM revealed ingested particles. For impedance measurements, cells were seeded on gold-plated microelectrodes. Cellular behavior was monitored over a period of 48 h. CoCr28Mo6 and AMC particle exposure affected cell viability in a concentration-dependent manner, i.e., 0.01 mg/mL particle solutions led to small changes in cell viability, while 0.05 mg/mL resulted in a significant reduction of viability. The effects were more pronounced after exposure to CoCr28Mo6 particles. The results were in line with light and darkfield microcopy observations indicating that the chosen methods are valuable tools to assess cytotoxicity and cellular behavior following exposure to endoprosthetic wear particles.

TiO2 nanoparticles disrupt cell adhesion and the architecture of cytoskeletal networks of human osteoblast-like cells in a size dependent manner.

Human exposure to titanium dioxide nanoparticles (nano-TiO2 ) is increasing. An internal source of nano-TiO2 is represented by titanium-based orthopedic and dental implants can release nanoparticles (NPs) upon abrasion. Little is known about how the size of NPs influences their interaction with cytoskeletal protein networks and the functional/homeostatic consequences that might follow at the implant-bone interface with regard to osteoblasts. We investigated the effects of size of anatase nano-TiO2 on SaOS-2 human osteoblast-like cells exposed to clinically relevant concentrations (0.05, 0.5, 5 mg/L) of 5 and 40 nm spherical nano-TiO2 . Cell viability and proliferation, adhesion, spread and migration were assessed, as well as the orientation of actin and microtubule cytoskeletal networks. The phosphorylation of focal adhesion kinase (p-FAKY397 ) and the expression of vinculin in response to nano-TiO2 were also assessed. Treatment with nano-TiO2 disrupted the actin and microtubule cytoskeletal networks leading to morphological modifications of SaOS-2 cells. The phosphorylation of p-FAKY397 and the expression of vinculin were also modified depending on the particle size, which affected cell adhesion. Consequently, the cell migration was significantly impaired in the 5 nm-exposed cells compared to unexposed cells. The present work shows that the orientation of cytoskeletal networks and the focal adhesion proteins and subsequently the adhesion, spread and migration of SaOS-2 cells were affected by the selected nano-TiO2 in a size dependent manner. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2582-2593, 2018.

In vitro cytotoxicity assessment of nanodiamond particles and their osteogenic potential.

Scaffolds functionalized with nanodiamond particles (nDP) hold great promise with regard to bone tissue formation in animal models. Degradation of the scaffolds over time may leave nDP within the tissues, raising concerns about possible long-term unwanted effects. Human SaOS-2 osteoblast-like cells and U937 monoblastoid cells were exposed to five different concentrations (0.002-2 mg/L) of nDP (size range: 2.36-4.42 nm) for 24 h. Cell viability was assessed by impedance-based methods. The differential expression of stress and toxicity-related genes was evaluated by polymerase chain reaction (PCR) super-array, while the expression of selected inflammatory and cell death markers was determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Furthermore, the expression of osteogenic genes by SaOS-2 cells, alkaline phosphatase activity and the extracellular calcium nodule deposition in response to nDP were determined in vitro. Cells responded differently to higher nDP concentrations (≥0.02 mg/L), that is, no loss of viability for SaOS-2 cells and significantly reduced viability for U937 cells. Gene expression showed significant upregulation of several cell death and inflammatory markers, among other toxicity reporter genes, indicating inflammatory and cytotoxic responses in U937 cells. Nanodiamond particles improved the osteogenicity of osteoblast-like cells with no evident cytotoxicity. However, concentration-dependent cytotoxic and inflammatory responses were seen in the U937 cells, negatively affecting osteogenicity in co-cultures. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1697-1707, 2018.

Nano-TiO2 penetration of oral mucosa: in vitro analysis using 3D organotypic human buccal mucosa models.

BACKGROUND: Oral cavity is a doorway for a variety of products containing titanium dioxide (TiO2 ) nanoparticles (NPs) (nano-TiO2 ) such as food additives, oral healthcare products and dental materials. Their potential to penetrate and affect normal human oral mucosa is not yet determined. OBJECTIVES: To evaluate the ability of nano-TiO2 to penetrate the in vitro reconstructed normal human buccal mucosa (RNHBM). METHODS: RNHBM was generated from primary normal human oral keratinocytes and fibroblasts isolated from buccal oral mucosa of healthy patients (n = 6). The reconstructed tissues were exposed after 10 days to clinically relevant concentrations of spherical or spindle rutile nano-TiO2 in suspension for short (20 min) and longer time (24 h). Ultrahigh-resolution imaging (URI) microscopy (CytoViva™ , Auburn, AL, USA) was used to assess the depth of penetration into reconstructed tissues. RESULTS: Ultrahigh-resolution imaging microscopy demonstrated the presence of nano-TiO2 mostly in the epithelium of RNHBM at both 20 min and 24-h exposure, and this was shape and doze dependent at 24 h of exposure. The depth of penetration diminished in time at higher concentrations. The exposed epithelium showed increased desquamation but preserved thickness. CONCLUSION: Nano-TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze- and time-dependent manner.

The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles.

Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein.

Role of physicochemical characteristics in the uptake of TiO2 nanoparticles by fibroblasts.

The relation between the physico-chemical properties of nanoparticles (NPs) and the degree of cellular uptake is incompletely elucidated. In this study, we investigated the influence on the cellular uptake of a wide range of fully characterized TiO2 NPs. L929 fibroblasts were exposed for 24 h to clinically relevant concentrations of nano-TiO2 and the degree of their association was assessed by ultrahigh resolution imaging microscopy (URI), scanning (SEM) and transmission (TEM) electron microscopy, as well as inductivity coupled plasma-mass spectroscopy (ICP-MS). The role of actin polymerization, a central feature of active internalization, was also studied and the results indicated that the internalization of TiO2 NPs involves a combination of actin-dependent uptake of large agglomerates as well as non actin-dependent uptake of small agglomerates. SEM and TEM revealed that the agglomerates of all NPs types were attached to the cellular membrane as well as internalized and confined inside cytoplasmic vesicles. URI and ICP-MS demonstrated that the particle association with cells was dose-dependent. The highest association was observed for spherical particles having mixed anatase-rutile crystallographic phase and the lowest for spindle-shaped rutile particles. ICP-MS revealed that the association was size-dependent in the order 5>10>40 nm for anatase spherical nanoparticles.

Limited in-depth invasion of Fusobacterium nucleatum into in vitro reconstructed human gingiva.

OBJECTIVE: Fusobacterium nucleatum is an opportunistic pathogen with a key role in subgingival plaque formation and it is found in increased numbers in periodontally affected sites. This study aimed to investigate the potential of F. nucleatum to penetrate and induce alterations in an in vitro reconstructed human gingival mucosa model. METHODS: Three-dimensional (3D) organotypic models of human gingiva were engineered using primary gingival keratinocytes and fibroblasts. The reconstructed tissues were challenged with four different strains of fluorescently labelled F. nucleatum in suspension placed on top of epithelial layers. Confocal laser scanning was used to assess the presence of fusobacteria through the organotypic model. Apoptosis (cleaved caspase-3) and cell proliferation (Ki-67) were evaluated by the use of immunohistochemistry in 3D-tissue models. Quantitative real-time PCR was performed to investigate the mRNA expression for MMP-13 and E-cadherin in both 3D-tissues and monolayers. RESULTS: F. nucleatum invaded the superficial epithelial layers of gingival 3D-tissue models. Challenged tissues showed accentuated shedding of superficial layers and increased number of cleaved caspase-3 and Ki-67 positive cells than controls, although not statistically significant. Levels of E-cadherin and MMP-13 mRNA were not significantly perturbed in multilayer culture. A variable and disproportionate response of MMP-13 mRNA level resulted in challenged primary keratinocytes in monolayers, compared to multilayer culture. CONCLUSION: These results indicate that F. nucleatum is able to invade superficially a differentiated, stratified gingival epithelium in vitro and triggers the efficient elimination of bacterial infection through epithelial shredding without causing a permanent damage of the tissue.

Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium.

The physicochemical characterization of nanoparticles in suspension is a prerequisite for the adequate assessment of their potential biological effect. Little is known to date about the colloidal stability of TiO2 nanoparticles in cell culture medium. This study investigates the effect of particle concentration, ionic strength, pH, and the presence of fetal bovine serum (FBS) and human serum albumin (HSA) on the colloidal stability of TiO2 nanoparticles in RPMI cell culture medium, by sedimentation measurements, dynamic light scattering, and electrokinetic measurements (zeta-potential). TEM revealed that the particles were polydisperse, with diameters ranging from approximately 15 to approximately 350 nm. The agglomeration rate and sedimentation rate increased with particles' concentration. The size of the agglomerates at 100 mg/L TiO2 was significantly reduced, from 1620+/-160 to 348+/-13 and 378+/-15 nm, upon the addition of 10% (v/v) FBS and 1% (w/w) HSA, respectively. The isoelectric point of TiO2 in water was 2.9 and the measured zeta-potential in RPMI was -16+/-2 mV at pH 7.4. A slight increase in the zeta-potential of TiO2 in RPMI was observed upon the addition of FBS and HSA. The addition of FBS and HSA prevented high agglomeration, leading to a stable dispersion of TiO2 nanoparticles for at least 24 h, possibly due to steric stabilization of the particles.

Induction of cell death by TiO2 nanoparticles: studies on a human monoblastoid cell line.

The cellular responses to degradation products from titanium (Ti) implants are important indicators for the biocompatibility of these widely used implantable medical devices. The potential toxicity of nanoparticulate matter released from implants has been scarcely studied. The aim of this study was to investigate the potential of TiO2 nanoparticles to induce modifications characteristic for death by apoptosis and/or necrosis in U937 human monoblastoid cells. Suspensions of TiO2 nanoparticles with a diameter <100nm were prepared in RPMI cell culture medium at concentrations that covered a range (0.005-4mg/ml) corresponding to concentrations found in blood, plasma, or in tissues surrounding Ti implants. The cells were exposed to the nanoparticulate suspensions for 24 and 48h and the responses were evaluated by flow cytometry and transmission electron microscopy. TiO2 nanoparticles induced both apoptotic and necrotic modifications in U937 cells.