In vivo toxicological evaluation of polymeric nanocapsules after intradermal administration

Polymeric nanocarriers have shown great promise as delivery systems. An alternative strategy has been to explore new delivery routes, such as intradermal (i.d.), that can be used for vaccines and patch-based drug delivery. Despite their many advantages, there are few toxicity studies, especially in vivo. We report a safety assessment of biodegradable poly(ɛ-caprolactone) lipid-core nanocapsules (LNC) with a mean size of 245 ± 10 nm following single and repeated intradermal injections to Wistar rats. Suspensions were prepared by interfacial deposition of polymer. The animals (n = 6/group) received a single-dose of saline solution (1.2 ml/kg) or LNC (7.2 × 10¹² LNC/kg), or repeated-doses of two controls, saline solution or Tween 80 (0.9 ml/kg), or three different concentrations of LNC (1.8, 3.6, and 5.4 × 10¹² LNC/kg) for 28 consecutive days. Clinical and physiological signs and mortality were observed. Samples of urine, blood, and tissue were used to perform toxicological evaluation. There were no clinical signs of toxicity or mortality, but there was a slight decrease in the relative body weights in the Tween 80–treated group (p < 0.01) after repeated administration. No histopathological alterations were observed in tissues or significant changes in blood and urinary biomarkers for tissue damage. Mild alterations in white blood cells count with increases in granulocytes in the Tween-80 group (p < 0.05) were found. Genotoxicity was evaluated through the comet assay, and no statistical difference was observed among the groups. Therefore, we conclude that, under the conditions of these experiments, biodegradable LNC did not present appreciable toxicity after 28 consecutive days of intradermal administration and is promising for its future application in vaccines and patch-based devices for enhancing the delivery of drugs.     

There's plenty of room at the forum: Potential risks and safety assessment of engineered nanomaterials

The celebrated physicist and Nobel laureate Richard Feynman was the first to predict the opportunities presented by the manipulation of matter at the level of individual atoms and molecules. Today, almost 50 years after his classic lecture on the wonders of the small world, the evolving nanotechnologies have the potential to bring about major changes in the lives of citizens. However, the very same properties that make engineered nanomaterials so promising from a technological perspective, such as their high degree of reactivity and the ability to cross biological barriers, could also make these novel materials harmful to human health and the environment. Therefore, exploitation of the full potential of the nanotechnologies requires close attention to safety issues. The 1st Nobel Forum mini-symposium on nanotoxicology was recently held in Stockholm, Sweden, and the program was devoted to the topic of definitions and standardization in nanotoxicological research, as well as nano-specific risk assessment and regulatory/legislative issues. Examples of recent and ongoing studies of carbon-based nanomaterials, including single-walled carbon nanotubes, using a wide range of in vitro and in vivo model systems were also presented. The current review will provide some highlights and conclusions from this exciting meeting.     

Flow cytometry evaluation of in vitro cellular necrosis and apoptosis induced by silver nanoparticles

Particles possess unique properties in the nanoscale, e.g., enhanced catalytic activity, high surface area, and light emission/absorption properties, that might result in interference with colorimetric in vitro cytotoxicity assays such as MTT, XTT or MTS. Alternatively, assays that do not use spectrophotometric detection, such as trypan blue exclusion or flow cytometry (FC) based assays, are less likely to be influenced by nanoparticle interference. The aim of this study was to evaluate FC assays to assess the cytotoxicity of three different sizes (10, 100, or 200 nm) of silver nanoparticles (AgNPs) at different mass concentrations (1, 25, or 50 ug/ml) in L-929 fibroblast cells. After 4 h and 24 h exposure, cell necrosis and apoptosis were assessed using 7-AAD and Annexin V dyes, respectively, with FC. The data indicate that cell necrosis and apoptosis in AgNP-exposed fibroblasts depends on dose, exposure time, and AgNP size. The data indicate that AgNPs produced a dose- and time-dependent decrease in cell viability; however, 10 nm AgNPs were significantly more toxic than larger-sized particles. Thus, standard FC assays can be utilized to assess apoptosis and necrosis in response to nanomaterial exposure.     

Numerical simulations of in vitro nanoparticle toxicity – The case of poly(amido amine) dendrimers

A phenomenological rate equation model is constructed to numerically simulate nanoparticle uptake and subsequent cellular response. Polyamidoamine dendrimers (generations 4–6) are modelled and the temporal evolution of the intracellular cascade of; increased levels of reactive oxygen species, intracellular antioxidant species, caspase activation, mitochondrial membrane potential decay, tumour necrosis factor and interleukin generation is simulated, based on experimental observations.The dose and generation dependence of several of these response factors are seen to well represent experimental observations at a range of time points. The model indicates that variations between responses of different cell-lines, including murine macrophages, human keratinocytes and colon cells, can be simulated and understood in terms of different intracellular antioxidant levels, and, within a given cell-line, varying responses of different cytotoxicity assays can be understood in terms of their sensitivities to different intracellular cascade events.The model serves as a tool to interpolate and visualise the range of dose and temporal dependences and elucidate the mechanisms underlying the in vitro cytotoxic response to nanoparticle exposure and describes the interaction in terms of independent nanoparticle properties and cellular parameters, based on reaction rates. Such an approach could be a valid alternative to that of effective concentrations for classification of nanotoxicity and may lay the foundation for future quantitative structure activity relationships and predictive nanotoxicity models.     

The performance of gradient alloy quantum dots in cell labeling

The interest in using quantum dots (QDots) as highly fluorescent and photostable nanoparticles in biomedicine is vastly increasing. One major hurdle that slows down the (pre)clinical translation of QDots is their potential toxicity. Several strategies have been employed to optimize common core–shell QDots, such as the use of gradient alloy (GA)-QDots. These particles no longer have a size-dependent emission wavelength, but the emission rather depends on the chemical composition of the gradient layer. Therefore, particles of identical sizes but with emission maxima spanning the entire visible spectrum can be generated. In the present study, two types of GA-QDots are studied with respect to their cytotoxicity and cellular uptake. A multiparametric cytotoxicity approach reveals concentration-dependent effects on cell viability, oxidative stress, cell morphology and cell functionality (stem cell differentiation and neurite outgrowth), where the particles are very robust against environmentally-induced breakdown. Non-toxic concentrations are defined and compared to common core–shell QDots analyzed under identical conditions. Additionally, this value is translated into a functional value by analyzing the potential of the particles for cell visualization. Interestingly, these particles result in clear endosomal localization, where different particles result in identical intracellular distributions. This is in contrast with CdTe QDots with the same surface coating, which resulted in clearly distinct intracellular distributions as a result of differences in nanoparticle diameter. The GA-QDots are therefore ideal platforms for cell labeling studies given their high brightness, low cytotoxicity and identical sizes, resulting in highly similar intracellular particle distributions which offer a lot of potential for optimizing drug delivery strategies.     

Possible genotoxic mechanisms of nanoparticles: Criteria for improved test strategies

Abstract

We review the mechanisms and pathways whereby nanoparticles might cause genotoxicity. Primary and secondary mechanisms are discussed in relation to the general particle toxicology paradigm. We also discuss how we might improve genotoxicity assays for nanoparticles. In this context we describe the role of the dispersion and the protein corona, the most relevant metric, choice of controls and new endpoints for genotoxicity along with the need for a structure activity model of NP genotoxicity.     

Altered protein expression profile associated with phenotypic changes in lung fibroblasts co-cultured with gold nanoparticle-treated small airway epithelial cells

Despite the availability of toxicity studies on cellular exposure to gold nanoparticles (AuNPs), there is scarcity of information with regard to the bystander effects induced by AuNPs on neighboring cells not exposed to the NPs. In this study, we showed that exposure of small airway epithelial cells (SAECs) to AuNPs induced changes in protein expression associated with functional effects in neighboring MRC5 lung fibroblasts in a co-culture system. Uptake of 20 nm size AuNPs by SAECs was first verified by focused ion beam scanning electron microscopy. Subsequently, pretreated SAECs were co-cultured with unexposed MRC5 lung fibroblasts, which then underwent proteome profiling using a quantitative proteomic approach. Stable-isotope labeling by amino acids in cell culture (SILAC)–based mass spectrometry identified 109 proteins (which included 47 up-regulated and 62 down-regulated proteins) that were differentially expressed in the lung fibroblasts co-cultured with AuNP pretreated SAECs. There was altered expression of proteins such as Paxillin, breast cancer anti-estrogen resistance 1 and Caveolin-1, which are known to be involved in the cell adhesion process. Morphological studies revealed that there was a concomitant increase in cell adhesion and altered F-actin stress fiber arrangement involving vinculin in the lung fibroblasts. It is likely that phenotypic changes observed in the underlying lung fibroblasts were mediated by AuNP-induced downstream signals in the pretreated SAECs and cell–cell cross talk.     

Concerns regarding nano-sized titanium dioxide dermal penetration and toxicity study

Drawing accurate conclusions from toxicology studies is of critical importance, including the relatively new field of nanotoxicology, as results of these toxicity studies will likely have significant impact on regulatory decisions with regard to their use in consumer products, as well as consumer understanding of these materials. Therefore, we feel it necessary to point out our concerns with the recent report by Wu and colleagues (Wu et al., 2009).     

Impact of lithiated cobalt oxide and phosphate nanoparticles on rainbow trout gill epithelial cells

Abstract

Metal oxide and phosphate nanoparticles (NPs) are ubiquitous in emerging applications, ranging from energy storage to catalysis. Cobalt-containing NPs are particularly important, where their widespread use raises questions about the relationship between composition, structure, and potential for environmental impacts. To address this gap, we investigated the effects of lithiated metal oxide and phosphate NPs on rainbow trout gill epithelial cells, a model for environmental exposure. Lithium cobalt oxide (LCO) NPs significantly reduced cell viability at10?µg/mL, while a 10-fold higher concentration of lithiated cobalt hydroxyphosphate (LCP) NPs was required to significantly reduce viability. Exposure to Li⁺ and Co²⁺ alone, at concentrations relevant to ion released from the NPs, did not reduce cell viability and minimally impacted reactive oxygen species (ROS) levels. Both LCO- and LCP-NPs were found within membrane-bound organelles. However, only LCP-NPs underwent rapid and complete dissolution in artificial lysosomal fluid. Unlike LCP-NPs, LCO-NPs significantly increased intracellular ROS, could be found within abnormal multilamellar bodies, and induced formation of intracellular vacuoles. Increased p53 gene expression, measured in individual cells, was observed at sub-toxic concentrations of both LCO- and LCP-NPs, implicating both in inductions of cellular damage and stress at concentrations approaching predicted environmental levels. Our results implicate the intact NP, not the dissolved ions, in the observed adverse effects and show that LCO-NPs significantly impact cell viability accompanied by increase in intracellular ROS and formation of organelles indicative of cell stress, while LCP-NPs have minimal adverse effects, possibly due to their rapid dissolution in acidic organelles.     

Toxicology of nanoparticles: A historical perspective

The rapid expansion of nanotechnology promises to have great benefits for society, yet there is increasing concern that human and environmental exposure to engineered nanomaterials may result in significant adverse effects. That is why the field of nanotoxicology – dealing with effects and potential risks of particulate structures <100 nm in size – has emerged, growing significantly over the past decade from long-standing foundations of well established knowledge on the toxicology of fibrous and non-fibrous particles and the interactions of viruses with cells. This review places nanoparticles in the context of conventional particle toxicology and so includes references to other types of particles, such as silica and asbestos, which have been extensively studied and can provide useful lessons relevant to newly engineered nanoparticles (NP). Discoveries of nanoparticle-specific concepts of toxicology related to their small size and large specific surface area go back to the early parts of the past century, although the distinctive biological effects and kinetics of NP were not recognized until the last decade of the past century. Today, the propensity of NP to cross cell barriers, enter cells and interact with subcellular structures is well established, as is the induction of oxidative stress as a major mechanism of nanoparticle effects. In addition to the significance of small size and surface area of NP, uncovering the impact of many other physico-chemical characteristics – in particular NP surface properties – for initiating effects in the mammalian organism and the environment is now an active area of research. The article aims to cover hazards relevant to humans, provides an introduction to some of the newly emerging literature on fate and behavior of NP in the environment, as well as describing their ecotoxicology in a variety of species. Major milestones in the research leading to our present understanding of nanotoxicology and the potential risks of NP to humans and the environment are summarized. These risks are likely to be different for different nanomaterials, ranging from perceived and very low for most, to real and very high for some. There are many questions that remain to be addressed, and we foresee for the future a continuing extended research in nanotoxicology. A full understanding of the hazard of NP will make a major contribution to the risk assessment that is so urgently needed to ensure that products that utilize NP are made safely, are exploited to their full potential and then disposed of safely.