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1.
Biomater Sci ; 11(13): 4675-4683, 2023 Jun 27.
Article in English | MEDLINE | ID: mdl-37219049

ABSTRACT

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.


Subject(s)
Breast Neoplasms , Catechin , Humans , Female , Micelles , Trastuzumab , Tea
2.
Arch Toxicol ; 90(11): 2793-2808, 2016 Nov.
Article in English | MEDLINE | ID: mdl-26612367

ABSTRACT

The kidney is a major target for xenobiotics, which include drugs, industrial chemicals, environmental toxicants and other compounds. Accurate methods for screening large numbers of potentially nephrotoxic xenobiotics with diverse chemical structures are currently not available. Here, we describe an approach for nephrotoxicity prediction that combines high-throughput imaging of cultured human renal proximal tubular cells (PTCs), quantitative phenotypic profiling, and machine learning methods. We automatically quantified 129 image-based phenotypic features, and identified chromatin and cytoskeletal features that can predict the human in vivo PTC toxicity of 44 reference compounds with ~82 % (primary PTCs) or 89 % (immortalized PTCs) test balanced accuracies. Surprisingly, our results also revealed that a DNA damage response is commonly induced by different PTC toxicants that have diverse chemical structures and injury mechanisms. Together, our results show that human nephrotoxicity can be predicted with high efficiency and accuracy by combining cell-based and computational methods that are suitable for automation.


Subject(s)
Chromatin Assembly and Disassembly/drug effects , Cytoskeleton/drug effects , Kidney Tubules, Proximal/drug effects , Models, Molecular , Mutagens/toxicity , Xenobiotics/toxicity , Automation, Laboratory , Cell Death/drug effects , Cell Line, Transformed , Cells, Cultured , Computational Biology , DNA Damage , Drug Evaluation, Preclinical , Feasibility Studies , High-Throughput Screening Assays , Humans , Kidney Tubules, Proximal/cytology , Machine Learning , Molecular Structure , Mutagens/chemistry , Osmolar Concentration , Small Molecule Libraries , Xenobiotics/chemistry
3.
Sci Rep ; 5: 12337, 2015 Jul 27.
Article in English | MEDLINE | ID: mdl-26212763

ABSTRACT

The renal proximal tubule is a main target for drug-induced toxicity. The prediction of proximal tubular toxicity during drug development remains difficult. Any in vitro methods based on induced pluripotent stem cell-derived renal cells had not been developed, so far. Here, we developed a rapid 1-step protocol for the differentiation of human induced pluripotent stem cells (hiPSC) into proximal tubular-like cells. These proximal tubular-like cells had a purity of >90% after 8 days of differentiation and could be directly applied for compound screening. The nephrotoxicity prediction performance of the cells was determined by evaluating their responses to 30 compounds. The results were automatically determined using a machine learning algorithm called random forest. In this way, proximal tubular toxicity in humans could be predicted with 99.8% training accuracy and 87.0% test accuracy. Further, we studied the underlying mechanisms of injury and drug-induced cellular pathways in these hiPSC-derived renal cells, and the results were in agreement with human and animal data. Our methods will enable the development of personalized or disease-specific hiPSC-based renal in vitro models for compound screening and nephrotoxicity prediction.


Subject(s)
Drug Evaluation, Preclinical/methods , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/pathology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/pathology , Toxicity Tests/methods , Acute Kidney Injury , Biological Assay/methods , Cell Differentiation , Cell Survival/drug effects , Cells, Cultured , Humans , Machine Learning , Pattern Recognition, Automated/methods , Reproducibility of Results , Sensitivity and Specificity
4.
Mol Pharm ; 11(7): 1933-48, 2014 Jul 07.
Article in English | MEDLINE | ID: mdl-24502545

ABSTRACT

The kidney is a major target for drug-induced toxicity. Drug-induced nephrotoxicity remains a major problem in the clinical setting, where the use of nephrotoxic drugs is often unavoidable. This leads frequently to acute kidney injury, and current problems are discussed. One strategy to avoid such problems would be the development of drugs with decreased nephrotoxic potential. However, the prediction of nephrotoxicity during preclinical drug development is difficult and nephrotoxicity is typically detected only late. Also, the nephrotoxic potential of newly approved drugs is often underestimated. Regulatory approved or validated in vitro models for the prediction of nephrotoxicity are currently not available. Here, we will review current approaches on the development of such models. This includes a discussion of three-dimensional and microfluidic models and recently developed stem cell based approaches. Most in vitro models have been tested with a limited number of compounds and are of unclear predictivity. However, some studies have tested larger numbers of compounds and the predictivity of the respective in vitro model had been determined. The results showed that high predictivity can be obtained by using primary or stem cell derived human renal cells in combination with appropriate end points.


Subject(s)
Acute Kidney Injury/chemically induced , Drug-Related Side Effects and Adverse Reactions/prevention & control , Kidney/drug effects , Animals , Drug Approval/methods , Drug Evaluation, Preclinical/methods , Humans , In Vitro Techniques/methods
5.
Adv Healthc Mater ; 2(9): 1188-97, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23713066

ABSTRACT

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.


Subject(s)
Cell Shape , Stem Cells/cytology , Adipocytes/cytology , Animals , Cell Differentiation , Cell Lineage , Cell Movement , Cellular Microenvironment , Cytoskeleton , Humans , Mesenchymal Stem Cells/cytology , Myocytes, Cardiac/cytology
6.
Toxicology ; 304: 132-40, 2013 Feb 08.
Article in English | MEDLINE | ID: mdl-23295712

ABSTRACT

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.


Subject(s)
Macrophages/drug effects , Sunscreening Agents/toxicity , Titanium/toxicity , Ultraviolet Rays , Animals , Cell Line , Hydroxyl Radical/metabolism , Macrophages/pathology , Mice , Particle Size , Sunscreening Agents/chemistry , Surface Properties , Time Factors , Titanium/chemistry
7.
J Biomed Mater Res A ; 101(3): 633-40, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22927021

ABSTRACT

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.


Subject(s)
DNA Damage , Fibroblasts/metabolism , Gadolinium , Nanoparticles/chemistry , Polyglactin 910 , Skin/metabolism , Titanium , Cell Proliferation , Cells, Cultured , Cytotoxins/chemistry , Cytotoxins/pharmacology , Fibroblasts/cytology , Gadolinium/chemistry , Gadolinium/pharmacology , Gene Expression Regulation , Histones/biosynthesis , Humans , Male , Materials Testing , Polyglactin 910/chemistry , Polyglactin 910/pharmacology , Skin/cytology , Titanium/chemistry , Titanium/pharmacology
8.
Arch Toxicol ; 87(1): 99-109, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22885792

ABSTRACT

To uncover the size influence of TiO(2) nanoparticles on their potential toxicity, the cytotoxicity of different-sized TiO(2) nanoparticles with and without photoactivation was tested. It was demonstrated that without photoactivation, TiO(2) nanoparticles were inert up to 100 µg/ml. On the contrary, with photoactivation, the toxicity of TiO(2) nanoparticles significantly increased, which correlated well with the specific surface area of the particles. Our results also suggest that the generation of hydroxyl radicals and reactive oxygen species (ROS)-mediated damage to the surface-adsorbed biomolecules could be the two major reasons for the cytotoxicity of TiO(2) nanoparticles after photoactivation. Higher ROS generation from smaller particles was detected under both biotic and abiotic conditions. Smaller particles could adsorb more proteins, which was confirmed by thermogravimetric analysis. To further investigate the influence of the generation of hydroxyl radicals and adsorption of protein, poly (ethylene-alt-maleic anhydride) (PEMA) and chitosan were used to coat TiO(2) nanoparticles. The results confirmed that surface coating of TiO(2) nanoparticles could reduce such toxicity after photoactivation, by hindering adsorption of biomolecules and generation of hydroxyl radical (·OH) during photoactivation.


Subject(s)
Dermatitis, Phototoxic , Metal Nanoparticles/toxicity , Particle Size , Adsorption , Animals , Cell Line/drug effects , Chitosan/chemistry , Chitosan/pharmacology , Coated Materials, Biocompatible/toxicity , Hydroxyl Radical/metabolism , Maleates/chemistry , Maleates/pharmacology , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Mice , Microscopy, Electron, Scanning , Polyethylenes/chemistry , Polyethylenes/pharmacology , Proteins/metabolism , Reactive Oxygen Species/metabolism , Toxicity Tests/methods , Ultraviolet Rays
9.
Arch Toxicol ; 87(6): 1075-86, 2013 Jun.
Article in English | MEDLINE | ID: mdl-22983807

ABSTRACT

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.


Subject(s)
Epithelial Cells/drug effects , Lactic Acid/toxicity , Lung/drug effects , Macrophages/drug effects , Metal Nanoparticles/toxicity , Polyglycolic Acid/toxicity , Titanium/toxicity , Adsorption , Animals , Calcium Signaling/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/pathology , Cell Survival/drug effects , Dose-Response Relationship, Drug , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Inflammation Mediators/metabolism , Lactic Acid/metabolism , Lung/immunology , Lung/metabolism , Lung/pathology , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Membrane Potential, Mitochondrial/drug effects , Mice , Oxidative Stress/drug effects , Particle Size , Polyglycolic Acid/metabolism , Polylactic Acid-Polyglycolic Acid Copolymer , Protein Binding , Reactive Oxygen Species/metabolism , Serum Albumin, Bovine/metabolism , Time Factors , Titanium/metabolism , Tumor Necrosis Factor-alpha/metabolism
10.
Stem Cells Dev ; 22(1): 136-47, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-22765653

ABSTRACT

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.


Subject(s)
Cell Differentiation , Focal Adhesions/metabolism , Mesenchymal Stem Cells/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cell Adhesion , Cell Culture Techniques , Cell Shape , Extracellular Matrix/physiology , Gene Expression Regulation , Humans , Integrin beta3/metabolism , Mesenchymal Stem Cells/physiology , Muscle Development , Myosin Heavy Chains/metabolism , Myosin-Light-Chain Kinase/metabolism , Protein Biosynthesis , Sarcomeres/metabolism , Transcription, Genetic , rhoA GTP-Binding Protein/metabolism
11.
Small ; 9(9-10): 1504-20, 2013 May 27.
Article in English | MEDLINE | ID: mdl-23019115

ABSTRACT

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.


Subject(s)
Environmental Exposure , Nanostructures , Humans , Stem Cells/drug effects , Tissue Engineering , Toxicity Tests
12.
Acta Biomater ; 8(3): 1267-72, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21945825

ABSTRACT

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.


Subject(s)
Acrylic Resins/chemistry , Dimethylpolysiloxanes/chemistry , Mesenchymal Stem Cells/cytology , Polyvinyl Alcohol/chemistry , Tissue Engineering/instrumentation , Tissue Engineering/methods , Humans , Surface Properties
13.
ACS Nano ; 5(9): 7284-95, 2011 Sep 27.
Article in English | MEDLINE | ID: mdl-21851096

ABSTRACT

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.


Subject(s)
Metal Nanoparticles , Oxides/toxicity , Toxicity Tests , Animals , Zebrafish/embryology
14.
Biomaterials ; 32(32): 8218-25, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21807406

ABSTRACT

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.


Subject(s)
DNA Damage , Nanoparticles/toxicity , Signal Transduction/drug effects , Tumor Suppressor Protein p53/metabolism , Zinc Oxide/toxicity , Animals , Cell Death/drug effects , Cell Line , Humans , Mice , Models, Biological , Mutagens/toxicity , Nanoparticles/ultrastructure
15.
Arch Toxicol ; 85(12): 1517-28, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21656222

ABSTRACT

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.


Subject(s)
Inflammation/chemically induced , Nanoparticles , Reactive Oxygen Species/metabolism , Zinc Oxide/toxicity , Animals , Bronchi/cytology , Bronchi/drug effects , Bronchi/metabolism , Cell Line , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Macrophages/drug effects , Macrophages/metabolism , Mice , Toxicity Tests , Zinc Oxide/administration & dosage
16.
Nanotoxicology ; 5(2): 182-94, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21609137

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 nm(2), 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.


Subject(s)
Durapatite/metabolism , Nanoparticles/chemistry , Animals , Anthraquinones/metabolism , Cell Line , Coloring Agents/metabolism , Durapatite/chemistry , Humans , Materials Testing , Mice , Particle Size , Spectroscopy, Fourier Transform Infrared , X-Ray Diffraction
17.
Biotechnol J ; 6(5): 501-8, 2011 May.
Article in English | MEDLINE | ID: mdl-21259442

ABSTRACT

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.


Subject(s)
Lactic Acid/chemistry , Nanoparticles/chemistry , Nanotechnology/methods , Polyglycolic Acid/chemistry , Animals , Biological Transport , Cell Line , Emulsions , Humans , Mice , Microscopy, Electron, Scanning , Models, Theoretical , Nanoparticles/ultrastructure , Polylactic Acid-Polyglycolic Acid Copolymer
18.
Arch Toxicol ; 85(6): 695-704, 2011 Jun.
Article in English | MEDLINE | ID: mdl-20938647

ABSTRACT

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.


Subject(s)
Epithelial Cells/drug effects , Fibroblasts/drug effects , Materials Testing/methods , Metal Nanoparticles/toxicity , Myoblasts/drug effects , Toxicity Tests , Zinc Oxide/toxicity , Animals , Cell Count , Cell Culture Techniques , Cell Line , Cell Survival/drug effects , Epithelial Cells/pathology , Fibroblasts/pathology , Humans , Kinetics , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Mice , Microscopy, Electron, Transmission , Myoblasts/pathology , Particle Size , Suspensions
19.
Food Chem Toxicol ; 48(6): 1762-6, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20412830

ABSTRACT

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.


Subject(s)
Bronchi/drug effects , Metal Nanoparticles , Zinc Oxide/toxicity , Bronchi/cytology , Bronchi/metabolism , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans
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