Norman J. Karin

Macrophage Responses to Silica Nanoparticles are Highly Conserved Across Particle Sizes

Concerns about the potential adverse health effects of engineered nanoparticles stems in part from the possibility that some materials display unique chemical and physical properties at nanoscales which could exacerbate their biological activity. However, studies that have assessed the effect of particle size across a comprehensive set of biological responses have not been reported. Using a macrophage cell model, we demonstrate that the ability of unopsonized amorphous silica particles to stimulate inflammatory protein secretion and induce macrophage cytotoxicity scales closely with the total administered particle surface area across a wide range of particle diameters (7-500 nm). Whole genome microarray analysis of the early gene expression changes induced by 10- and 500-nm particles showed that the magnitude of change for the majority of genes affected correlated more tightly with particle surface area than either particle mass or number. Gene expression changes that were particle size-specific were also identified. However, the overall biological processes represented by all gene expression changes were nearly identical, irrespective of particle diameter. Direct comparison of the cell processes represented in the 10- and 500-nm particle gene sets using gene set enrichment analysis revealed that among 1009 total biological processes, none were statistically enriched in one particle size group over the other. The key mechanisms involved in silica nanoparticle-mediated gene regulation and cytotoxicity have yet to be established. However, our results suggest that on an equivalent nominal surface area basis, common biological modes of action are expected for nano- and supranano-sized silica particles.

A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins.

Subcellular localization of protein synthesis provides a means to regulate the protein composition in far reaches of a cell. This localized protein synthesis gives neuronal processes autonomy to rapidly respond to extracellular stimuli. Locally synthesized axonal proteins enable neurons to respond to guidance cues and can help to initiate regeneration after injury. Most studies of axonal mRNA translation have concentrated on cytoplasmic proteins. While ultrastructural studies suggest that axons do not have rough endoplasmic reticulum or Golgi apparatus, mRNAs for transmembrane and secreted proteins localize to axons. Here, we show that growing axons with protein synthetic activity contain ER and Golgi components needed for classical protein synthesis and secretion. Isolated axons have the capacity to traffic locally synthesized proteins into secretory pathways and inhibition of Golgi function attenuates translation-dependent axonal growth responses. Finally, the capacity for secreting locally synthesized proteins in axons appears to be increased by injury.

Iron oxide nanoparticle agglomeration influences dose rates and modulates oxidative stress-mediated dose–response profiles in vitro

Abstract Spontaneous agglomeration of engineered nanoparticles (ENPs) is a common problem in cell culture media which can confound interpretation of in vitro nanotoxicity studies. The authors created stable agglomerates of iron oxide nanoparticles (IONPs) in conventional culture medium, which varied in hydrodynamic size (276 nm–1.5 μm) but were composed of identical primary particles with similar surface potentials and protein coatings. Studies using C10 lung epithelial cells show that the dose rate effects of agglomeration can be substantial, varying by over an order of magnitude difference in cellular dose in some cases. Quantification by magnetic particle detection showed that small agglomerates of carboxylated IONPs induced greater cytotoxicity and redox-regulated gene expression when compared with large agglomerates on an equivalent total cellular IONP mass dose basis, whereas agglomerates of amine-modified IONPs failed to induce cytotoxicity or redox-regulated gene expression despite delivery of similar cellular doses. Dosimetry modelling and experimental measurements reveal that on a delivered surface area basis, large and small agglomerates of carboxylated IONPs have similar inherent potency for the generation of ROS, induction of stress-related genes and eventual cytotoxicity. The results suggest that reactive moieties on the agglomerate surface are more efficient in catalysing cellular ROS production than molecules buried within the agglomerate core. Because of the dynamic, size and density-dependent nature of ENP delivery to cells in vitro, the biological consequences of agglomeration are not discernible from static measures of exposure concentration (μg/ml) alone, highlighting the central importance of integrated physical characterisation and quantitative dosimetry for in vitro studies. The combined experimental and computational approach provides a quantitative framework for evaluating relationships between the biocompatibility of nanoparticles and their physical and chemical characteristics.

Injectable and thermosensitive PLGA-g-PEG hydrogels containing hydroxyapatite: preparation, characterization and in vitro release behavior

Here we report the design and characterization of injectable and thermosensitive hydrogel composites comprised of poly(lactic acid-co-glycolic acid)-g-poly(ethylene glycol)(PLGA-g-PEG) containing hydroxyapatite (HA) for potential application in bone tissue engineering. Inclusion of HA into the hydrogels would provide both enhanced mechanical properties and bioactivity to the composites. The effects of HA on the properties of the hydrogels were investigated in terms of storage modulus, sol-gel transition properties, pH and in vitro dye release behavior. The hydrogel composites were also studied by scanning electron microscopy (SEM), x-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The results revealed that hydrogel composites preserved their sol-gel transition properties in the presence of HA. The storage modulus of the hydrogels was enhanced in a HA-content dependent manner, and the acidic pH environment of the hydrogel was neutralized by HA, both representing great advantages over the hydrogel alone. SEM images showed that HA particles were well dispersed and distributed within the hydrogel matrix. The composites showed a sustained release of a small molecule model dye for up to two weeks with slight increase of release with addition of HA. This work demonstrates the formation of novel thermogelling composites of PLGA-g-PEG and HA that are injectable and promote controlled release.

Three Human Cell Types Respond to Multi-Walled Carbon Nanotubes and Titanium Dioxide Nanobelts with Cell-Specific Transcriptomic and Proteomic Expression Patterns.

Abstract The growing use of engineered nanoparticles (NPs) in commercial and medical applications raises the urgent need for tools that can predict NP toxicity. Global transcriptome and proteome analyses were conducted on three human cell types, exposed to two high aspect ratio NP types, to identify patterns of expression that might indicate high versus low NP toxicity. Three cell types representing the most common routes of human exposure to NPs, including macrophage-like (THP-1), small airway epithelial and intestinal (Caco-2/HT29-MTX) cells, were exposed to TiO2 nanobelts (TiO2-NB; high toxicity) and multi-walled carbon nanotubes (MWCNT; low toxicity) at low (10 µg/mL) and high (100 µg/mL) concentrations for 1 and 24 h. Unique patterns of gene and protein expressions were identified for each cell type, with no differentially expressed (p < 0.05, 1.5-fold change) genes or proteins overlapping across all three cell types. While unique to each cell type, the early response was primarily independent of NP type, showing similar expression patterns in response to both TiO2-NB and MWCNT. The early response might, therefore, indicate a general response to insult. In contrast, the 24 h response was unique to each NP type. The most significantly (p < 0.05) enriched biological processes in THP-1 cells indicated TiO2-NB regulation of pathways associated with inflammation, apoptosis, cell cycle arrest, DNA replication stress and genomic instability, while MWCNT-regulated pathways indicated increased cell proliferation, DNA repair and anti-apoptosis. These two distinct sets of biological pathways might, therefore, underlie cellular responses to high and low NP toxicity, respectively.

Automated time-lapse microscopy and high-resolution tracking of cell migration.

We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in “scratch” or “wound healing” assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.

A Role for G Protein‐Coupled Lysophospholipid Receptors in Sphingolipid‐Induced Ca2+ Signaling in MC3T3‐E1 Osteoblastic Cells

Sphingolipids have been proposed to modulate cell function by acting as intracellular second messengers and through binding to plasma membrane receptors. Exposure of MC3T3‐E1 osteoblastic cells to sphingosine (SPH), sphingosine‐1‐phosphate (SPP), or sphingosylphosphorylcholine (SPC) led to the release of Ca2+ from the endoplasmic reticulum (ER) and acute elevations in cytosolic‐free Ca2+ ([Ca2+]i). Desensitization studies suggest that SPP and SPC bind plasma membrane endothelial differentiation gene (Edg) receptors for lysophosphatidic acid (LPA). Consistent with the coupling of Edg receptors to G proteins, SPP‐ and SPC‐induced Ca2+ signaling was inhibited by pretreatment of the cells with pertussis toxin (PTx). Of the Edg receptors known to bind SPH derivatives in other cell types, MC3T3‐E1 cells were found to express transcripts encoding Edg‐1 and Edg‐5 but not Edg‐3, Edg‐6, or Edg‐8. In contrast to SPP and SPC, the ability of SPH to elicit [Ca2+]i elevations was affected neither by prior exposure of cells to LPA nor by PTx treatment. However, LPA‐induced Ca2+ signaling was blocked in MC3T3‐E1 cells previously exposed to SPH. Elevations in [Ca2+]i were not evoked by SPP or SPC in cells treated with 2‐aminoethoxydiphenylborate (2‐APB), an inhibitor of inositol 1,4,5‐trisphosphate (IP3)‐gated Ca2+ channels in the ER. No effect of 2‐APB was observed on SPH‐ or LPA‐induced [Ca2+]i elevations. The data support a model in which SPP and SPC bind Edg‐1 and/or Edg‐5 receptors in osteoblasts leading to the release of Ca2+ from the ER through IP3‐gated channels.

The Nuclear Factor-κB Pathway Regulates Cytochrome P450 3A4 Protein Stability

We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 were degraded by the proteasome, it seemed likely that there might be another protein susceptible to proteasomal degradation that regulated CYP3A4 expression. In this study, we evaluated whether the nuclear factor-κB (NF-κB) pathway was involved in that process. Our model system used an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, the inhibition of the proteasome with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) suppresses CYP3A4 protein levels. We also found that MG132 treatment had a broad affect on the NF-κB pathway, including down-regulation of NF-κB DNA binding activity and IκB kinase (IKK)α levels and up-regulation of IKKβ and inhibitory κB levels. Treatment of the HepG2 cells with several structurally distinct NF-κB inhibitors also suppressed CYP3A4 protein levels. When the HepG2 cells were treated with cycloheximide, a general inhibitor of protein synthesis, the loss of CYP3A4 protein was accelerated by cotreatment with either proteasome or NF-κB inhibitors. These results indicate that NF-κB activity regulated CYP3A4 protein stability, and they suggest that the NF-κB pathway was responsible for the decrease in CYP3A4 protein levels that resulted from the proteasomal inhibition.

Lysophosphatidic acid-induced ERK activation and chemotaxis in MC3T3-E1 preosteoblasts are independent of EGF receptor transactivation.

Bone‐forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of tissue injury. LPA is a potent inducer of bone cell chemotaxis, proliferation and survival in vitro, and this lipid factor is an attractive candidate to facilitate preosteoblast migration during skeletal regeneration in vivo. In this study we sought to more clearly define the intracellular signaling pathways mediating the effects of LPA on bone cells. LPA‐treated MC3T3‐E1 preosteoblastic cells exhibited a bimodal activation of extracellular signal‐related kinase (ERK1/2) with maximal phosphorylation at 5 and 60 min. MEK1/2 activation was detected within 2.5 min of LPA exposure and remained elevated for at least an hour. ERK1/2 phosphorylation was not coupled to Ras activation or to LPA‐induced elevations in cytosolic Ca2+. While LPA exposure transactivates the EGF receptor in many cell types, LPA‐stimulated ERK1/2 activation in MC3T3‐E1 cells was unaffected by the inhibition of EGF receptor function. ERK isoforms can function as transcription factors and ERK1/2 rapidly accumulated in the nuclei of LPA‐treated cells, a process that was blocked if ERK1/2 phosphorylation was prevented. Blocking ERK1/2 phosphorylation also led to significant decreases in LPA‐induced MC3T3‐E1 cell chemotaxis, while the inhibition of EGF receptor function had no effect on the stimulation of preosteoblast motility by LPA. Our results identify ERK1/2 activation as a mediator of LPA‐stimulated MC3T3‐E1 cell migration that may be relevant to preosteoblast motility and gene expression during bone repair in vivo. J. Cell. Physiol. 219: 716–723, 2009.

Comparative iron oxide nanoparticle cellular dosimetry and response in mice by the inhalation and liquid cell culture exposure routes

BackgroundToxicity testing the rapidly growing number of nanomaterials requires large scale use of in vitro systems under the presumption that these systems are sufficiently predictive or descriptive of responses in in vivo systems for effective use in hazard ranking. We hypothesized that improved relationships between in vitro and in vivo models of experimental toxicology for nanomaterials would result from placing response data in vitro and in vivo on the same dose scale, the amount of material associated with cells.MethodsBalb/c mice were exposed nose-only to an aerosol (68.6 nm CMD, 19.9 mg/m3, 4 hours) generated from of 12.8 nm superparamagnetic iron oxide particles (SPIO). Target cell doses were calculated, histological evaluations conducted, and biomarkers of response were identified by global transcriptomics. Representative murine epithelial and macrophage cell types were exposed in vitro to the same material in liquid suspension for four hours and levels of nanoparticle regulated cytokine transcripts identified in vivo were quantified as a function of measured nanoparticle cellular dose.ResultsTarget tissue doses of 0.009-0.4 μg SPIO/cm2 in lung led to an inflammatory response in the alveolar region characterized by interstitial inflammation and macrophage infiltration. In vitro, higher target tissue doses of ~1.2-4 μg SPIO/ cm2 of cells were required to induce transcriptional regulation of markers of inflammation, CXCL2 & CCL3, in C10 lung epithelial cells. Estimated in vivo macrophage SPIO nanoparticle doses ranged from 1-100 pg/cell, and induction of inflammatory markers was observed in vitro in macrophages at doses of 8-35 pg/cell.ConclusionsApplication of target tissue dosimetry revealed good correspondence between target cell doses triggering inflammatory processes in vitro and in vivo in the alveolar macrophage population, but not in the epithelial cells of the alveolar region. These findings demonstrate the potential for target tissue dosimetry to enable the more quantitative comparison of in vitro and in vivo systems and advance their use for hazard assessment and extrapolation to humans. The mildly inflammogentic cellular doses experienced by mice were similar to those calculated for humans exposed to the same material at the existing permissible exposure limit of 10 mg/m3 iron oxide (as Fe).