Gene expression profiling of immune-competent human cells exposed to engineered zinc oxide or titanium dioxide nanoparticles.

A comprehensive in vitro assessment of two commercial metal oxide nanoparticles, TiO2 and ZnO, was performed using human monocyte-derived macrophages (HMDM), monocyte-derived dendritic cells (MDDC), and Jurkat T cell leukemia-derived cell line. TiO2 nanoparticles were found to be non-toxic whereas ZnO nanoparticles caused dose-dependent cell death. Subsequently, global gene expression profiling was performed to identify transcriptional response underlying the cytotoxicity caused by ZnO nanoparticles. Analysis was done with doses 1 µg/ml and 10 µg/ml after 6 and 24 h of exposure. Interestingly, 2703 genes were significantly differentially expressed in HMDM upon exposure to 10 µg/ml ZnO nanoparticles, while in MDDCs only 12 genes were affected. In Jurkat cells, 980 genes were differentially expressed. It is noteworthy that only the gene expression of metallothioneins was upregulated in all the three cell types and a notable proportion of the genes were regulated in a cell type-specific manner. Gene ontology analysis revealed that the top biological processes disturbed in HMDM and Jurkat cells were regulating cell death and growth. In addition, genes controlling immune system development were affected. Using a panel of modified ZnO nanoparticles, we obtained an additional support that the cellular response to ZnO nanoparticles is largely dependent on particle dissolution and show that the ligand used to modify ZnO nanoparticles modulates Zn(2+) leaching. Overall, the study provides an extensive resource of transcriptional markers for mediating ZnO nanoparticle-induced toxicity for further mechanistic studies, and demonstrates the value of assessing nanoparticle responses through a combined transcriptomics and bioinformatics approach.

Stability and biocompatibility of a library of polyester dendrimers in comparison to polyamidoamine dendrimers.

Dendrimers can be designed for several biomedical applications due to their well-defined structure, functionality and dimensions. The present study focused on the in vitro biocompatibility evaluation of a library of aliphatic polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) with an overall diameter of 0.5-2 nm. In addition, dendrimers with two different chemical surfaces (neutral with hydroxyl end group and anionic with carboxylic end group) and dendrons corresponding to the structural fragments of the dendrimers were evaluated. Commercial polyamidoamine dendrimers (PAMAM) with cationic (amine) or neutral (hydroxyl) end group were also included for comparison. Cell viability studies were conducted in human cervical cancer (HeLa) and acute monocytic leukemia cells (THP.1) differentiated into macrophage-like cells as well as in primary human monocyte-derived macrophages. Excellent biocompatibility was observed for the entire hydroxyl functional bis-MPA dendrimer library, whereas the cationic, but not the neutral PAMAM exerted dose-dependent cytotoxicity in cell lines and primary macrophages. Studies to evaluate material stability as a function of pH, temperature, and time, demonstrated that the stability of the 4th generation hydroxyl functional bis-MPA dendrimer increased at acidic pH. Taken together, bis-MPA dendrimers are degradable and non-cytotoxic to human cell lines and primary cells.

Morphological properties of nanoporous folic acid materials and in vitro assessment of their biocompatibility.

BACKGROUND: Mesoporous silica-based particles are of potential interest for the development of novel therapeutic targeted delivery vehicles. Their ability to load and release large quantities of active pharmaceutical products with varying properties, combining controlled and targeted release functions make them unique amongst nanotechnology-based carrier systems. MATERIALS & METHODS: In this study, nanoporous folic acid-templated materials (NFM-1) were prepared and the synthetic strategies for the control of textural and morphology properties of NFM-1 are described. The potential biocompatibility of NFM-1 particles with different morphology (gyroid shaped, fibers and rod-shaped) was assessed using a panel of human cell lines. RESULTS: The results reveal that NFM-1 morphology has an impact on cell viability such that particles showing higher aspect ratios possess increased cytotoxicity. CONCLUSION: These studies provide useful information for the development of novel mesoporous materials for biomedical applications, including cell-specific drug delivery.

Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells.

Engineered nanoparticles are being considered for a wide range of biomedical applications, from magnetic resonance imaging to "smart" drug delivery systems. The development of novel nanomaterials for biomedical applications must be accompanied by careful scrutiny of their biocompatibility. In this regard, particular attention should be paid to the possible interactions between nanoparticles and cells of the immune system, our primary defense system against foreign invasion. On the other hand, labeling of immune cells serves as an ideal tool for visualization, diagnosis or treatment of inflammatory processes, which requires the efficient internalization of the nanoparticles into the cells of interest. Here, we compare novel monodispersed silica-coated iron oxide nanoparticles with commercially available dextran-coated iron oxide nanoparticles. The silica-coated iron oxide nanoparticles displayed excellent magnetic properties. Furthermore, they were non-toxic to primary human monocyte-derived macrophages at all doses tested whereas dose-dependent toxicity of the smaller silica-coated nanoparticles (30nm and 50nm) was observed for primary monocyte-derived dendritic cells, but not for the similarly small dextran-coated iron oxide nanoparticles. No macrophage or dendritic cell secretion of pro-inflammatory cytokines was observed upon administration of nanoparticles. The silica-coated iron oxide nanoparticles were taken up to a significantly higher degree when compared to the dextran-coated nanoparticles, irrespective of size. Cellular internalization of the silica-coated nanoparticles was through an active, actin cytoskeleton-dependent process. We conclude that these novel silica-coated iron oxide nanoparticles are promising materials for medical imaging, cell tracking and other biomedical applications.

Toxicology of engineered nanomaterials: focus on biocompatibility, biodistribution and biodegradation.

BACKGROUND: It is widely believed that engineered nanomaterials will be increasingly used in biomedical applications. However, before these novel materials can be safely applied in a clinical setting, their biocompatibility, biodistribution and biodegradation needs to be carefully assessed. SCOPE OF REVIEW: There are a number of different classes of nanoparticles that hold promise for biomedical purposes. Here, we will focus on some of the most commonly studied nanomaterials: iron oxide nanoparticles, dendrimers, mesoporous silica particles, gold nanoparticles, and carbon nanotubes. MAJOR CONCLUSIONS: The mechanism of cellular uptake of nanoparticles and the biodistribution depend on the physico-chemical properties of the particles and in particular on their surface characteristics. Moreover, as particles are mainly recognized and engulfed by immune cells special attention should be paid to nano-immuno interactions. It is also important to use primary cells for testing of the biocompatibility of nanoparticles, as they are closer to the in vivo situation when compared to transformed cell lines. GENERAL SIGNIFICANCE: Understanding the unique characteristics of engineered nanomaterials and their interactions with biological systems is key to the safe implementation of these materials in novel biomedical diagnostics and therapeutics. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

Ex vivo supplementation with nicotinic acid enhances cellular poly(ADP-ribosyl)ation and improves cell viability in human peripheral blood mononuclear cells.

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) by using NAD(+) as substrate and is directly triggered by DNA strand breaks. Under mild genotoxic stress poly(ADP-ribose) (PAR) formation plays an important role in DNA repair whereas severe genotoxic stress and the ensuing overactivation of PARP-1 induce cellular NAD(+) depletion, energy failure and ultimately cell death. We are interested in studying the consequences of moderately enhanced enzymatic activity under conditions of DNA damage. Here we chose supplementation of cells with the NAD(+) precursor nicotinic acid (NA) as a strategy. In order to reliably assess PAR accumulation in living cells we first developed a novel, sensitive flow-cytometric method for the rapid analysis of poly(ADP-ribose) accumulation (RAPARA). Our data showed that ex vivo supplementation of human peripheral blood mononuclear cells (PBMC) with low concentrations of NA significantly raised cellular NAD(+) levels by 2.1-fold. Upon X-irradiation or exposure to hydrogen peroxide or N-methyl-N'-nitro-N-nitrosoguanidine, PAR accumulation was significantly increased and sustained in NA-supplemented cells. Furthermore, NA-supplemented PBMC displayed significantly higher cell viability due to a lower rate of necrotic cell death. In summary, ex vivo supplementation of human PBMC with NA increases cellular NAD(+) levels, boosts the cellular poly(ADP-ribosyl)ation response to genotoxic treatment, and protects from DNA-damage-induced cell death.

Effect of zinc on cellular poly(ADP-ribosyl)ation capacity.

Poly(ADP-ribosyl)ation is a posttranslational protein modification, which is catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) and plays a role in DNA repair and maintenance of genomic stability. A decrease in cellular poly(ADP-ribosyl)ation has been implicated in the aging process. As PARP-1 is a zinc finger protein its decreased function might be related to age-related zinc deficiency. To test this hypothesis we assessed cellular poly(ADP-ribosyl)ation capacity in 29 donors from Greece, Italy and Poland as function of age and nutritional zinc status. Our results reveal a positive correlation between cellular poly(ADP-ribosyl)ation capacity and zinc status in human peripheral blood mononuclear cells (PBMC) (p<0.05). We could also confirm a decrease of PARP-1 activity with donor age, highlighting the role of poly(ADP-ribosyl)ation in the aging process. The results demonstrate that zinc supplementation in elderly people can increase the cellular poly(ADP-ribosyl)ation capacity of their PBMC. We speculate that this may help maintain integrity and stability of the genome more efficiently and thus contribute to an extension of healthspan.

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes.

BACKGROUND: Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD+ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions. RESULTS: This paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosyl)ation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC50 that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay. CONCLUSION: The methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosyl)ation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i) selective analysis of mononuclear cells by gating and (ii) detection of a possible heterogeneity in poly(ADP-ribosyl)ation capacity between cells of the same type.