Sijing Xiong

The role of the tumor suppressor p53 pathway in the cellular DNA damage response to zinc oxide nanoparticles.

In this paper, we explored how ZnO nanoparticles cross-interact with a critical tumor suppressive pathway centered around p53, which is one of the most important known tumor suppressors that protects cells from developing cancer phenotypes through its control over major pathways like apoptosis, senescence and cell cycle progression. We showed that the p53 pathway was activated in BJ cells (skin fibroblasts) upon ZnO nanoparticles treatment with a concomitant decrease in cell numbers. This suggests that cellular responses like apoptosis in the presence of ZnO nanoparticles require p53 as the molecular master switch towards programmed cell death. This also suggests that in cells without robust p53, protective response can be tipped towards carcinogenesis when stimulated by DNA damage inducing agents like ZnO nanoparticles. We observed this precarious tendency in the same BJ cells with p53 knocked down using endogeneous expressing shRNA. These p53 knocked down BJ cells became more resistant to ZnO nanoparticles induced cell death and increased cell progression. Collectively, our results suggest that cellular response towards specific nanoparticle induced cell toxicity and carcinogenesis is not only dependent on specific nanoparticle properties but also (perhaps more importantly) the endogenous genetic, transcriptomic and proteomic landscape of the target cells.

High Content Screening in Zebrafish Speeds up Hazard Ranking of Transition Metal Oxide Nanoparticles

Zebrafish is an aquatic organism that can be used for high content safety screening of engineered nanomaterials (ENMs). We demonstrate, for the first time, the use of high content bright-field and fluorescence-based imaging to compare the toxicological effect of transition metal oxide (CuO, ZnO, NiO, and Co(3)O(4)) nanoparticles in zebrafish embryos and larvae. High content bright-field imaging demonstrated potent and dose-dependent hatching interference in the embryos, with the exception of Co(3)O(4) which was relatively inert. We propose that the hatching interference was due to the shedding of Cu and Ni ions, compromising the activity of the hatching enzyme, ZHE1, similar to what we previously proposed for Zn(2+). This hypothesis is based on the presence of metal-sensitive histidines in the catalytic center of this enzyme. Co-introduction of a metal ion chelator, diethylene triamine pentaacetic acid (DTPA), reversed the hatching interference of Cu, Zn, and Ni. While neither the embryos nor larvae demonstrated morphological abnormalities, high content fluorescence-based imaging demonstrated that CuO, ZnO, and NiO could induce increased expression of the heat shock protein 70:enhanced green fluorescence protein (hsp70:eGFP) in transgenic zebrafish larvae. Induction of this response by CuO required a higher nanoparticle dose than the amount leading to hatching interference. This response was also DTPA-sensitive. We demonstrate that high content imaging of embryo development, morphological abnormalities, and HSP70 expression can be used for hazard ranking and determining the dose-response relationships leading to ENM effects on the development of the zebrafish embryo.

Toxicity of zinc oxide (ZnO) nanoparticles on human bronchial epithelial cells (BEAS-2B) is accentuated by oxidative stress.

Although several studies reported that cytotoxic effects of various nanoparticles are partially due to induction of oxidative stress, it is unclear how oxidative state of the cell per se could influence its sensitivity to cytotoxic nanoparticles. This is of clinical significance because certain pathological conditions such as inflammation is associated with elevated oxidative stress and this may alter sensitivity of cells and tissues to cytotoxic nanoparticles. Hence, this study investigated how initial exposure of BEAS-2B human bronchial epithelial cells to oxidative stress influences subsequent response to cytotoxic challenge with zinc oxide (ZnO) nanoparticles (approximately 10nm). Oxidative stress was induced by exposing BEAS-2B cells to 5 and 10 microM of H(2)O(2) for 45 min in PBS (with Ca(2+)). Subsequently, the H(2)O(2) solutions were washed off and the cells were exposed to varying concentrations (5-25 microg/ml) of ZnO nanoparticles in culture media for 24h, followed by cell viability assessment with the WST-8 assay. The results demonstrated that initial transient exposure of cells to oxidative stress accentuated cytotoxicity of ZnO nanoparticles. In the negative control unexposed to H(2)O(2), >99% of cells remained viable up to a ZnO nanoparticle concentration of 10 microg/ml, but displayed a steep decrease in viability above 10 microg/ml ZnO. By contrast, cells that were initially exposed to 5 and 10 microM of H(2)O(2), displayed a sharp drop in viability even at concentrations below 10 microg/ml ZnO. At 10 microg/ml ZnO, cells initially exposed to 10 microM H(2)O(2) displayed a viability of 40.6+/-2.0%, which is significantly lower than the corresponding values of 72.8+/-2.0% and 99.9+/-1.1% obtained for initial exposure to 5 microM H(2)O(2) and the negative control, respectively. Hence, initial exposure of BEAS-2B cells to oxidative stress sensitized their subsequent response to cytotoxic challenge with ZnO nanoparticles.

Cytotoxic and genotoxic characterization of titanium dioxide, gadolinium oxide, and poly(lactic-co-glycolic acid) nanoparticles in human fibroblasts†

Engineered nanomaterials have become prevalent in our everyday life. While the popularity of using nanomaterials in consumer products continues to rise, increasing awareness of nanotoxicology has also fuelled efforts to accelerate our understanding of the ill effects that different nanomaterials can bring to biological systems. In this study, we investigated the potential cytotoxicity and genotoxicity of three nanoparticles: titanium dioxide (TiO(2)), terbium-doped gadolinium oxide (Tb-Gd(2)O(3)), and poly(lactic-co-glycolic acid) (PLGA). To evaluate nanoparticle-induced genotoxicity more realistically, a human skin fibroblast cell line (BJ) with less mutated genotype compared with cancer cell line was used. The nanoparticles were first characterized by size, morphology, and surface charge. Cytotoxicity effects of the nanoparticles were then evaluated by monitoring the proliferation of treated BJ cells. Genotoxic influence was ascertained by profiling DNA damage via detection of γH2AX expression. Our results suggested that both TiO(2) and Tb-Gd(2)O(3) nanoparticles induced cytotoxicity in a dose dependent way on BJ cells. These two nanomaterials also promoted genotoxicity via DNA damage. On the contrary, PLGA nanoparticles did not induce significant cytotoxic or genotoxic effects on BJ cells.

In vitro assessment of cellular responses to rod-shaped hydroxyapatite nanoparticles of varying lengths and surface areas

Abstract Rod-shaped hydroxyapatite nanoparticles of varying dimensions (≈ 60 ± 10, 120 ± 15, 240 ± 30 nm in length, labeled respectively as nHA60, nHA120 and nHA240) with specific surface areas (47.02, 23.33, 46.12 nm2, respectively), were synthesized and their effects on cell viability, reactive oxygen species generation and cellular interaction with BEAS-2B, RAW264.7 and HepG2 were investigated. In vitro exposure of these cell lines to rod shape nHA particles within a range of 10–300 μg/ml for 24 h did not significantly alter cell viability studied by the WST-8 assay. A significant increase in reactive oxygen species (ROS) generation was however observed with the dihydrofluorescein diacetate (DFDA) assay after 4 h incubation with these nanoparticles. The lowest level of ROS generation was observed with nHA120 (with the smallest specific surface area); whereas nHA60 and nHA240 exhibited comparable ROS generation. Subsequently, the Alizarin Red-S (ARS) assay indicated a weaker association of calcium with cells compared to nHA60 and nHA240. The results thus suggest that high surface area may increase cell-particle interaction, which in turn influenced ROS generation. The combined results from all the cell lines thus indicated high biocompatibility of rod-shaped nHA.

A novel and simple microcontact printing technique for tacky, soft substrates and/or complex surfaces in soft tissue engineering

Microcontact printing (μCP) has attracted much interest due to its simplicity and wide range of applications. However, when conventional μCP is applied to soft and/or tacky substrates, substrate sagging and difficulty in stamp removal cause non-conformance in the patterns. Moreover, it is almost impossible to apply conventional μCP on complex or wavy surfaces. In this study, we developed a novel yet simple trans-print method to create efficient micropatterning on soft and/or tacky substrates such as polydimethylsiloxane and polyacrylamide gel, and also on curved surfaces, by introducing polyvinyl alcohol film as a trans-print media. This technique is simple as it only involves one trans-print step and is also cost-effective. Most importantly, this technique is also versatile and we have proven this by printing various designs on more complex non-flat surfaces using various proteins as inks. The quality of the trans-printed pattern was excellent with high reproducibility and resolution as verified by immunostaining. Human mesenchymal stem cells cultured on these patterns displayed good conformance on the soft and tacky substrates printed using this technique. These results suggest that this novel trans-print technique can be extended to a potentially generic methodology for μCP of other proteins and biomolecules, other shapes and sizes, and cells, and will also be useful in three-dimensional micropatterning for soft tissue engineering.

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the β(3) integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and α-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering.

Specific surface area of titanium dioxide (TiO2) particles influences cyto- and photo-toxicity

The aim of this study is to examine how different specific surface areas of similar-sized titanium dioxide (TiO(2)) particles could influence both cytotoxicity and phototoxicity. TiO(2) particles of different specific surface areas were compared for their toxic effects on RAW264.7 cells in the absence and presence of UV light. From the results, TiO(2) particles with larger specific surface area were found to induce higher cyto- (UV absent) and photo-toxicity (UV activated) to cells after 24h incubation. The observed cytotoxicity from TiO(2) particles with larger surface area could be explained from their interactions with biomolecules. Upon photoactivation, a larger number of hydroxyl radicals were detected from TiO(2) particles with larger surface area, again suggesting a surface area dependent phototoxic effect. On the other hand, pre-adsorbing TiO(2) particles with extracellular proteins were found to decrease toxicity effects.

Cellular uptake of Poly‐(D,L‐lactide‐co‐glycolide) (PLGA) nanoparticles synthesized through solvent emulsion evaporation and nanoprecipitation method

Poly‐(D,L‐lactide‐co‐glycolide) (PLGA) nanoparticles have been widely studied for drug delivery. The aim of this study is to determine how cellular uptake of these nanoparticles is influenced by different surface properties, incubation time, particle concentration and cell types. Spherical coumarin‐6 loaded PLGA nanoparticles with a size of about 100 nm were synthesized through solvent emulsion evaporation and nanoprecipitation methods. In vitro cellular uptake efficiency was determined using human bronchial epithelial cells (BEAS‐2B) and murine monocyte‐derived macrophage (RAW264.7) cells. PLGA nanoparticles were incubated with these cells in a concentration range of 10‐300 μg/ml for different time periods. The results show that cellular uptake decreased for nanoparticles surface coated with PVA surfactant and was especially limited for severely aggregated particles. At higher particle concentration, the total amount of particles taken up by cells increased while the uptake efficiency decreased. In addition, cells could take up more particles with longer incubation time, although the uptake rate decreased gradually with time. Finally, RAW264.7 cells show increased uptake compared to BEAS‐2B cells. The information drawn from this study would provide important clues on how nanomaterials interact with cells and how these interactions can influence biocompatibility or toxicity.

Emerging In Vitro Models for Safety Screening of High‐Volume Production Nanomaterials under Environmentally Relevant Exposure Conditions

The rising production of nanomaterial-based consumer products has raised safety concerns. Testing these with animal and other direct models is neither ethically nor economically viable, nor quick enough. This review aims to discuss the strength of in vitro testing, including the use of 2D and 3D cultures, stem cells, and tissue constructs, etc., which would give fast and repeatable answers of a highly specific nature, while remaining relevant to in vivo outcomes. These results can then be combined and the overall toxicity predicted with relative accuracy. Such in vitro models can screen potentially toxic nanomaterials which, if required, can undergo further stringent studies in animals. The cyto- and phototoxicity of some high-volume production nanomaterials, using in vitro models, is also reviewed.