Toxicity and efficacy of green tea catechin derivative-based micellar nanocomplexes for anticancer protein delivery.

Toxicity towards non-tumor cells during anticancer therapy can be reduced by using nanoscale systems for anticancer drug delivery. Usually only the loaded drug has anticancer activity. Recently, micellar nanocomplexes (MNCs) comprising green tea catechin derivatives for the delivery of the anticancer proteins, such as Herceptin, have been developed. Herceptin as well as the MNCs without the drug were effective against HER2/neu-overexpressing human tumor cells and had synergistic anticancer effects in vitro and in vivo. It remained unclear which kinds of negative effects the MNCs had on tumor cells exactly, and which of their components mediated them. Also, it was unclear if MNC has any toxicity effects on the normal cells of vital human organ systems. Herein we examined the effects of Herceptin-MNCs and their individual components on human breast cancer cells and on normal primary human endothelial and kidney proximal tubular cells. We applied a novel in vitro model that predicts nephrotoxicity in humans with high accuracy, as well as high-content screening and microfluidic mono- and co-culture models to thoroughly address effects on various cell types. The results showed that MNCs alone were profoundly toxic for breast cancer cells, and induced apoptosis regardless of HER2/neu expression levels. Apoptosis was induced by both green tea catechin derivatives contained within MNCs. In contrast, MNCs were not toxic for normal human cells, and the probability was low that MNCs would be nephrotoxic in humans. Together, the results supported the hypothesis that green tea catechin derivative-based MNCs could improve efficacy and safety of therapies with anticancer proteins.

Functional morphometric analysis in cellular behaviors: shape and size matter.

Cellular morphogenesis in response to biophysical and topographical cues provides insights into cytoskeletal status, biointerface communications, and phenotypic adaptations in an incessant signaling feedback that governs cellular fate. Morphometric characterization is an important element in the study of the dynamic cellular behaviors, in their interactive response to environmental influence exerted by culture system. They collectively serve to reflect cellular proliferation, migration, and differentiation, which may serve as prognostic indices for clinical and pathological diagnosis. Various parameters are proposed to categorize morphological adaptations in relation to cellular function. In this review, the underlying principles, assumptions, and limitations of morphological characterizations are discussed. The significance, challenges, and implications of quantitative morphometric characterization of cell shapes and sizes in determining cellular functions are discussed.

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.

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 ß(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.

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.

Size influences the cytotoxicity of poly (lactic-co-glycolic acid) (PLGA) and titanium dioxide (TiO(2)) nanoparticles.

The aim of this study is to uncover the size influence of poly (lactic-co-glycolic acid) (PLGA) and titanium dioxide (TiO(2)) nanoparticles on their potential cytotoxicity. PLGA and TiO(2) nanoparticles of three different sizes were thoroughly characterized before in vitro cytotoxic tests which included viability, generation of reactive oxygen species (ROS), mitochondrial depolarization, integrity of plasma membrane, intracellular calcium influx and cytokine release. Size-dependent cytotoxic effect was observed in both RAW264.7 cells and BEAS-2B cells after cells were incubated with PLGA or TiO(2) nanoparticles for 24 h. Although PLGA nanoparticles did not trigger significantly lethal toxicity up to a concentration of 300 µg/ml, the TNF-α release after the stimulation of PLGA nanoparticles should not be ignored especially in clinical applications. Relatively more toxic TiO(2) nanoparticles triggered cell death, ROS generation, mitochondrial depolarization, plasma membrane damage, intracellular calcium concentration increase and size-dependent TNF-α release, especially at a concentration higher than 100 µg/ml. These cytotoxic effects could be due to the size-dependent interaction between nanoparticles and biomolecules, as smaller particles tend to adsorb more biomolecules. In summary, we demonstrated that the ability of protein adsorption could be an important paradigm to predict the in vitro cytotoxicity of nanoparticles, especially for low toxic nanomaterials such as PLGA and TiO(2) nanoparticles.

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.

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.

Evaluation of the cytotoxic and inflammatory potential of differentially shaped zinc oxide nanoparticles.

Zinc oxide (ZnO) nanoparticles have wide-ranging applications in a diverse array of industrial and consumer products, from ceramic manufacture and paint formulation to sunscreens and haircare products. Hence, it is imperative to rigorously characterize the health and safety aspects of human exposure to ZnO nanoparticles. This study therefore evaluated the cellular association, cytotoxic and inflammatory potential of spherical and sheet-shaped ZnO nanoparticles (of approximately the same specific surface area ≈30 cm²/g) on mouse and human cell lines (RAW-264.7 and BEAS-2B respectively), as well as with primary cultures of mouse bone marrow-derived dendritic cells (DC). The WST-8 assay demonstrated dose-dependent effects on the cytotoxicity of spherical and sheet-shaped ZnO nanoparticles on both RAW-264.7 and BEAS-2B cells, even though there was no significant effect of shape on the cytotoxicity of ZnO nanoparticles. There was however higher cellular association of spherical versus sheet-shaped ZnO nanoparticles. Measurement of reactive oxygen species (ROS) with the 2',7'-dichlorfluorescein-diacetate (DCFH-DA) assay indicated up to 4-folds increase in ROS level upon exposure to ZnO nanoparticles, but there was again no significant difference between both ZnO nanoparticle shapes. Exposure of primary dendritic cells to ZnO nanoparticles upregulated expression of CD80 and CD86 (well-known markers of DC activation and maturation) and stimulated release of pro-inflammatory cytokines--IL-6 and TNF-α, thus pointing to the potential of ZnO nanoparticles in inducing inflammation. Hence, our study indicated that ZnO nanoparticles can have potential detrimental effects on cells even at dosages where there are little or no observable cytotoxic effects.

Cytotoxicity of zinc oxide (ZnO) nanoparticles is influenced by cell density and culture format.

A parameter that has often been overlooked in cytotoxicity assays is the density and confluency of mammalian cell monolayers utilized for toxicology screening. Hence, this study investigated how different cell seeding densities influenced their response to cytotoxic challenge with ZnO nanoparticles. Utilizing the same volume (1 ml per well) and concentration range (5-40 µg/ml) of ZnO nanoparticles, contradictory results were observed with higher-density cell monolayers (BEAS-2B cells) obtained either by increasing the number of seeded cells per well (50,000 vs. 200,000 cells per well of 12-well plate) or by seeding the same numbers of cells (50,000) within a smaller surface area (12-well vs. 48-well plate, 4.8 vs. 1.2 cm(2), respectively). Further experiments demonstrated that the data may be skewed by inconsistency in the mass/number of nanoparticles per unit area of culture surface, as well as by inconsistent nanoparticle to cell ratio. To keep these parameters constant, the same number of cells (50,000 per well) were seeded on 12-well plates, but with the cells being seeded at the edge of the well for the experimental group (by tilting the plate) to form a dense confluent monolayer, as opposed to a sparse monolayer for the control group seeded in the conventional manner. Utilizing such an experimental set-up for the comparative evaluation of four different cell lines (BEAS-2B, L-929, CRL-2922 and C2C12), it was observed that the high cell density monolayer was consistently more resistant to the cytotoxic effects of ZnO nanoparticles compared to the sparse monolayer for all four different cell types, with the greatest differences being observed above a ZnO concentration of 10 µg/ml. Hence, the results of this study demonstrate the need for the standardization of cell culture protocols utilized for toxicology screening of nanoparticles, with respect to cell density and mass/number of nanoparticles per unit area of culture surface.

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.