Stable nanoparticle aggregates/agglomerates of different sizes and the effect of their size on hemolytic cytotoxicity

Abstract

To study the toxicity of nanoparticles under relevant conditions, it is critical to disperse nanoparticles reproducibly in different agglomeration states in aqueous solutions compatible with cell-based assays. Here, we disperse gold, silver, cerium oxide, and positively-charged polystyrene nanoparticles in cell culture media, using the timing between mixing steps to control agglomerate size in otherwise identical media. These protein-stabilized dispersions are generally stable for at least two days, with mean agglomerate sizes of ～23 nm silver nanoparticles ranging from 43–1400 nm and average relative standard deviations of less than 10%. Mixing rate, timing between mixing steps and nanoparticle concentration are shown to be critical for achieving reproducible dispersions. We characterize the size distributions of agglomerated nanoparticles by further developing dynamic light scattering theory and diffusion limited colloidal aggregation theory. These theories frequently affect the estimated size by a factor of two or more. Finally, we demonstrate the importance of controlling agglomeration by showing that large agglomerates of silver nanoparticles cause significantly less hemolytic toxicity than small agglomerates.     

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

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.     

Agglomerate behaviour of fluticasone propionate within dry powder inhaler formulations

Due to their small size, the respirable drug particles tend to form agglomerates which prevent flowing and aerosolisation. A carrier is used to be mixed with drug in one hand to facilitate the powder flow during manufacturing, in other hand to help the fluidisation upon patient inhalation. Depending on drug concentration, drug agglomerates can be formed in the mixture. The aim of this work was to study the agglomeration behaviour of fluticasone propionate (FP) within interactive mixtures for inhalation. The agglomerate phenomenon of fluticasone propionate after mixing with different fractions of lactose without fine particles of lactose (smaller than 32 μm) was demonstrated by the optical microscopy observation. A technique measuring the FP size in the mixture was developed, based on laser diffraction method. The FP agglomerate sizes were found to be in a linear correlation with the pore size of the carrier powder bed (R(2)=0.9382). The latter depends on the particle size distribution of carrier. This founding can explain the role of carrier size in de-agglomeration of drug particles in the mixture. Furthermore, it gives more structural information of interactive mixture for inhalation that can be used in the investigation of aerosolisation mechanism of powder. According to the manufacturing history, different batches of FP show different agglomeration intensities which can be detected by Spraytec, a new laser diffraction method for measuring aerodynamic size. After mixing with a carrier, Lactohale LH200, the most cohesive batch of FP, generates a lower fine particle fraction. It can be explained by the fact that agglomerates of fluticasone propionate with very large size was detected in the mixtures. By using silica-gel beads as ball-milling agent during the mixing process, the FP agglomerate size decreases accordingly to the quantity of mixing aid. The homogeneity and the aerodynamic performance of the mixtures are improved. The mixing aid based on ball-milling effect could be used to ameliorate the quality of inhalation mixture of cohesive drug, such as fluticasone propionate. However, there is a threshold where an optimal amount of mixing aids should be used. Not only the drug des-aggregation reaches its peak but the increase in drug-carrier adhesion due to high energy input should balance the de-agglomeration capacity of mixing process. This approach provides a potential alternative in DPI formulation processing.     

Investigation of melt agglomeration process with a hydrophobic binder in combination with sucrose stearate

The melt agglomeration process of lactose powder with hydrogenated cottonseed oil (HCO) as the hydrophobic meltable binder was investigated by studying the physicochemical properties of molten HCO modified by sucrose stearates S170, S770 and S1570. The size, size distribution, micromeritic and adhesion properties of agglomerates as well as surface tension, contact angle, viscosity and specific volume of molten HCO, with and without sucrose stearates, were examined. The viscosity, specific volume and surface tension of molten HCO were found to be modified to varying extents by sucrose stearates which are available in different HLB values and melt properties. The growth of melt agglomerates was promoted predominantly by an increase in viscosity, an increase in specific volume or a decrease in surface tension of the molten binding liquid. The agglomerate growth propensity was higher with an increase in inter-particulate binding strength, agglomerate surface wetness and extent of agglomerate consolidation which enhanced the liquid migration from agglomerate core to periphery leading to an increased surface plasticity for coalescence. The inclusion of high concentrations of completely meltable sucrose stearate S170 greatly induced the growth of agglomerates through increased specific volume and viscosity of the molten binding liquid. On the other hand, the inclusion of incompletely meltable sucrose stearates S770 and S1570 promoted the agglomeration mainly via the reduction in surface tension of the molten binding liquid with declining agglomerate growth propensity at high sucrose stearate concentrations. In addition to being an agglomeration modifier, sucrose stearate demonstrated anti-adherent property in melt agglomeration process. The properties of molten HCO and melt agglomerates were dependent on the type and concentration of sucrose stearate added.     

The Measurement of Mixture Homogeneity and Dissolution to Predict the Degree of Drug Agglomerate Breakdown Achieved Through Powder Mixing

Interactive mixing of agglomerates of small, cohesive particles with coarse carrier particles facilitate the deaggregation of agglomerates. In this study dispersion of agglomerates of microfine furosemide particles by such a mixing process was followed by measuring changes in the content uniformity and area under the dissolution curve. Interactive mixtures between agglomerates of different sized furosemide particles and coarse sodium chloride particles were prepared using different mixers, mixing times and mixer speeds. The dissolution rate of the drug from and content uniformity of the mixtures were measured, and degrees of dispersion were calculated. These degrees of dispersion were compared to the dispersion values obtained from the decrease in agglomerate size after mixing. An increase in mixing time led to an increase in dispersion. An initial fast deagglomeration, indicated by an increase in dissolution, increase in content uniformity and a decrease in particle size, was followed by substantially slower deaggregation of remaining agglomerates and smaller aggregates. For all mixtures studied the degree of dispersion estimated from dissolution measurements, when compared to equivalent content uniformity measurements, agreed closely with the degree of dispersion as indicated by the decrease in particle size. The use of the area under the dissolution curve to predict agglomerate breakdown proved useful and may find application in situations where it is impossible to follow directly deagglomeration through particle size measurements.     

Effects of interactions between powder particle size and binder viscosity on agglomerate growth mechanisms in a high shear mixer.

A study was performed in order to elucidate the effects of the interactions between powder particle size and binder viscosity on the mechanisms involved in agglomerate formation and growth. Calcium carbonates having mean particle sizes in the range of 5-214 microm and polyethylene glycols having viscosities in the range of approximately 50-100000 mPas were melt agglomerated in a high shear mixer. Agglomerate growth by nucleation and coalescence was found to dominate when agglomerating small powder particles and binders with a low viscosity. Increasing the binder viscosity increased the formation of agglomerates by immersion of powder particles in the surface of the binder droplets. With a larger powder particle size, an increasing binder viscosity was necessary in order to obtain an agglomerate strength being sufficient to avoid breakage. Due to a low agglomerate strength, a satisfying agglomeration of very large particles (214 microm) could not be obtained, even with very viscous binders. The study demonstrated that the optimum agglomerate growth occurred when the agglomerates were of an intermediate strength causing an intermediate deformability of the agglomerates. In order to produce spherical agglomerates (pellets), a low viscosity binder has to be chosen when agglomerating a powder with a small particle size, and a high viscosity binder must be applied in agglomeration of powders with large particles.     

Assessment of metal nanoparticle agglomeration, uptake, and interaction using high-illuminating system

In the present study, an ultrahigh-resolution system was applied as a simple and convenient technique to characterize the extent of metal nanoparticle agglomeration in solution and to visualize nanoparticle agglomeration, uptake, and surface interaction in three cell phenotypes under normal culture conditions. The experimental results demonstrated that silver (25, 80, 130 nm); aluminum (80 nm); and manganese (40 nm) particles and agglomerates were effectively internalized by rat liver cells (BRL 3A), rat alveolar macrophages (MACs), and rat neuroendocrine cells (PC-12). Individual and agglomerated nanoparticles were observed within the cells and agglomerates were observed on the cell surface membranes. The particles were initially dispersed in aqueous or physiological balanced salt solutions and agglomeration was observed using the Ultra Resolution Imaging (URI) system. Different methods, such as sonication and addition of surfactant (0.1% sodium dodecyl sulfate [SDS]) reduced agglomeration. Due to effects of SDS itself on cell viability, the surfactant could not be directly applied during cell exposure. Therefore, following addition of 0.1% SDS, the particles were washed twice with ultrapure water, which reduced agglomeration even further. Reducing the agglomeration of the nanoparticles is important for studying their uptake and in applications that benefit from individual nanoparticles such as diagnostics. In summary, this study demonstrates a simple technique to characterize the extent of nanoparticle agglomeration in solution and visualize nanoparticle (40 nm and larger) uptake and interaction with cells. Additionally, an example application of nanoparticle labeling onto the surface and neurite extensions of murine neuroblastoma cells (N2A) is presented as a potential imaging tool.     

Simultaneous screening of the stability and dosimetry of nanoparticles dispersions for in vitro toxicological studies with static multiple light scattering technique

To evaluate the nanoparticle (NP) toxicity, much efforts have been devoted for developing methods to accurately disperse NPs into aqueous suspensions prior to in vitro toxicological studies. As NP toxicity is strongly dependent on their physicochemical properties, NP characterization is a key step for any in vitro toxicological study. This study demonstrates that the static multiple light scattering (SMLS) technique allows for the simultaneous screening of the NP size, agglomeration state, stability and dosimetry in biological media. Batch dispersions of TiO₂ P25 NPs in water with various bovine serum albumin (BSA) mass fractions (from 0% to 0.5%) and dilutions of these dispersions into cell culture media were characterized with SMLS. In the batch dispersions, TiO₂ NPs are stable and well dispersed for BSA mass fraction lower than 0.2% while agglomeration and rapid settling is observed for higher BSA mass fractions. Paradoxically, when diluted in cell culture media, TiO₂ NPs are well dispersed and stable for BSA mass fractions higher than 0.2%. The TiO₂ NP dosimetry of these dilutions was evaluated experimentally with SMLS and confronted with numerical approaches. The TiO₂ NP bottom concentration evolves far more slowly in the case of the higher BSA mass fraction. Such measurements give valuable insights on the NP fate and transport in biological media to obtain in fine reliable size and dose-cytotoxicity responses.     

De-agglomeration of micronized benzodiazepines in dissolution media measured by laser diffraction particle sizing

The objective of this research was to develop a method to characterize the degree of particle agglomeration using laser diffraction particle sizing, following the addition of benzodiazepine interactive mixtures to water. Interactive mixtures of diazepam, nitrazepam and oxazepam (up to 20% w/w) were prepared by mixing micronized benzodiazepines with lactose granules (250-355 microm). Micronized sodium lauryl sulfate and cetrimide (up to 5% w/w) were added to the benzodiazepine-lactose interactive mixes to produce ternary mixtures. Particle size distributions of benzodiazepines, after addition of the interactive mixtures to water, were determined using laser diffraction particle sizing. Bimodal distributions representing dispersed particles and agglomerates were observed initially after lactose carrier dissolution. Partial agglomerate to dispersed particle transition occurred during a 60-min observation period for all mixtures, reaching a constant level of agglomeration after this time. Interactive mixtures with higher benzodiazepine concentrations displayed transition profiles with higher levels of agglomeration. The presence of surfactant in interactive mixtures dramatically decreased agglomeration. Sodium lauryl sulfate was more effective than cetrimide in dispersing agglomerates. The shape of the transition curves during de-agglomeration demonstrated the presence of stable agglomerates that remained after the initial transition; these may be important in explaining dissolution and absorption rates.     

Expression of cytokine-induced neutrophil chemoattractant in rat lungs by intratracheal instillation of nickel oxide nanoparticles

Since nanoparticles easily agglomerate to form larger particles, it is important to maintain the size of their agglomerates at the nano-level to evaluate the harmful effect of the nanoparticles. We prevented agglomeration of nickel oxide nanoparticles by ultrasound diffusion and filtration, established an acute exposure model using animals, and examined inflammation and chemokine expression. The mass median diameter of nickel oxide nanoparticle agglomerates suspended in distilled water for intratracheal instillation was 26?nm (8.41?nm weighted average surface primary diameter). Male Wistar rats received intratracheal instillation of nickel oxide nanoparticles at 0.1?mg (0.33?mg/kg) or 0.2?mg (0.66?mg/kg), and were dissected 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The control group received intratracheal instillation of distilled water. Three chemokines (cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2αβ, and CINC-3) in the lung tissue and bronchoalveolar lavage fluid (BALF) were determined by quantitative measurement of protein by ELISA. Both CINC-1 and CINC-2αβ concentration was elevated from day 3 to 3 months in lung tissue and from day 3 to 6 months in BALF. On the other hand, CINC-3 was elevated on day 3 in both lung tissue and BALF, and then decreased. The total cell and neutrophil counts in BALF were increased from day 3 to 3 months. In lung tissue, infiltration of mainly neutrophils and alveolar macrophages was observed from day 3 to 6 months in alveoli. These results suggest that CINC was involved in lung injury by nickel oxide nanoparticles.     

Agglomeration tendency in dry pharmaceutical granular systems.

The agglomeration tendency of dry pharmaceutical mixtures containing various concentrations of Xylitab 100 (Xylitol), calcium carbonate precipitated (CCP) and magnesium stearate (MgSt) was evaluated statistically as a function of mixing time. A Ro-Tap tester was employed to mix the three pharmaceutical components, and the agglomerates formed were measured with respect to their weight and size. An experimental design was devised and applied to structure and then statistically analyze the results. Xylitab was found not to be influential in the formation of agglomerates, but aided in deagglomeration when mixed with other components. CCP and MgSt formed agglomerates over time and showed positive interactions favouring agglomeration. The agglomerates started to fracture when they reached a critical size, at which stage the particles' attraction forces (cohesion forces) were weaker than both gravity and inertia. It has been shown and quantitatively demonstrated that the mixing time and ingredient concentrations of a three-component pharmaceutical mixture can affect agglomeration tendency.     

Effect of a melt agglomeration process on agglomerates containing solid dispersions

The purpose was to produce solid dispersions of a poorly water-soluble drug, Lu-X, by melt agglomeration in a laboratory scale rotary processor. The effect of binder type and method of manufacturing on the dissolution profile of Lu-X was investigated. Lactose monohydrate and Lu-X were melt agglomerated with Rylo MG12, Gelucire 50/13, PEG 3000, or poloxamer 188. Either a mixture of binder, drug, and excipient was heated to a temperature above the melting point of the binder (melt-in procedure) or a dispersion of drug in molten binder was sprayed on the heated excipient (spray-on procedure). The agglomerates were characterized by DSC, XRPD, SEM, and EDX-SEM. The study showed that the agglomerates containing solid dispersions had improved dissolution rates compared to physical mixtures and pure drug. The melt-in procedure gave a higher dissolution rate than the spray-on procedure with PEG 3000, poloxamer 188, and Gelucire 50/13, whereas the opposite was found with Rylo MG12. This was explained by differences in mechanisms of agglomerate formation and growth, which were dominated by immersion with PEG 3000, poloxamer 188, and Gelucire 50/13, and by distribution and coalescence with Rylo MG12. The spray-on procedure resulted in a higher content of Lu-X in the core of the agglomerates when immersion was the dominating mechanism, and in a higher content in the agglomerate surface when distribution was dominating. The melt-in procedure resulted generally in a homogeneous distribution of Lu-X in the agglomerates. The compounds in the agglomerates were found primarily to be crystalline, and the dissolution profiles were unchanged after 12 weeks storage at 25 degrees C at 50% RH.     

Melt agglomeration with polyethylene glycol beads at a low impeller speed in a high shear mixer.

This study was performed in order to evaluate the possibility of obtaining spherical agglomerates with a high content of meltable binder by a melt agglomeration process in a high shear mixer. Lactose monohydrate was melt agglomerated with polyethylene glycol (PEG) 1500 or 6000 in a 10-l high shear mixer at an impeller speed of 400 rpm. The PEG 1500 was used as a size fraction of beads, and the PEG 6000 as a fine powder, a powder, unfractionated beads, and size fractions of beads. It was found to be possible to incorporate a high amount of PEG (28% m/m of the amount of lactose), because the rather low impeller speed applied in the present experiments caused less densification of the agglomerates. The fine powder of the PEG 6000 caused a complete adhesion of the mass to the bowl shortly after melting. A rapid agglomerate growth by coalescence was found to be the dominant growth mechanism when agglomeration was performed with the PEG 6000 powder. The PEG beads resulted in a slow and more controllable agglomerate growth, because the growth occurred primarily by an immersion of the lactose particles in the surface of the molten binder droplets. The initial shape of the agglomerates produced with the PEG beads was similar to the spherical shape of the beads. This shape could not be maintained during the process due to a breakage of the agglomerates caused by a hollow structure of the PEG beads.     

Budesonide Nanoparticle Agglomerates as Dry Powder Aerosols With Rapid Dissolution

: Nanoparticle technology represents an attractive approach for formulating poorly water-soluble pulmonary medicines. Unfortunately, nanoparticle suspensions used in nebulizers or metered dose inhalers often suffer from physical instability in the form of uncontrolled agglomeration or Ostwald ripening. In addition, processing such suspensions into dry powders can yield broad particle size distributions. To address these encumbrances, a controlled nanoparticle flocculation process has been developed. Nanosuspensions of the poorly water-soluble drug budesonide were prepared by dissolving the drug in organic solvent containing surfactants followed by rapid solvent extraction in water. Different surfactants were employed to control the size and surface charge of the precipitated nanoparticles. Nanosuspensions were flocculated using leucine and lyophilized. Selected budesonide nanoparticle suspensions exhibited an average particle size ranging from ~ 160 to 230 nm, high yield and high drug content. Flocculated nanosuspensions produced micron-sized agglomerates. Freeze-drying the nanoparticle agglomerates yielded dry powders with desirable aerodynamic properties for inhalation therapy. In addition, the dissolution rates of dried nanoparticle agglomerate formulations were significantly faster than that of stock budesonide. The results of this study suggest that nanoparticle agglomerates possess the microstructure desired for lung deposition and the nanostructure to facilitate rapid dissolution of poorly water-soluble drugs.     

Inflammatory response of mice to manufactured titanium dioxide nanoparticles: Comparison of size effects through different exposure routes

TiO₂ is a widely used manufactured nanomaterial and the opportunity for human exposure makes it necessary to study its health implications. Using murine models for inflammation, size effects of inflammatory response in instillation and acute inhalation exposures of TiO₂ nanoparticles with manufacturers’ average particles sizes of 5 and 21 nm were investigated. The properties of the primary nanoparticles, nanoparticle agglomerates aerosol and instillation solution for both sized nanoparticles were evaluated. Mice were acutely exposed in a whole-body exposure chamber or through nasal instillation and toxicity was assessed by enumeration of total and differential cells, determination of total protein, LDH activity and inflammatory cytokines in BAL fluid. Lungs were also evaluated for histopathological changes. Results show the larger TiO₂ nanoparticles were found to be moderately, but significantly, more toxic. The nanoparticles had different agglomeration states which may be a factor as important as the surface and physical characteristics of the primary nanoparticles in determining toxicity.     

Effects of droplet size and type of binder on the agglomerate growth mechanisms by melt agglomeration in a fluidised bed.

This study was performed in order to evaluate the effects of binder droplet size and type of binder on the agglomerate growth mechanisms by melt agglomeration in a fluidised bed granulator. Lactose monohydrate was agglomerated with melted polyethylene glycol (PEG) 3000 or Gelucire 50/13 (esters of polyethylene glycol and glycerol), which was atomised at different nozzle air flow rates giving rise to median droplet sizes of 40, 60, and 80 microm. Different product temperatures were investigated, below the melting range, in the middle of the melting range, and above the melting range for each binder. The agglomerates were found to be formed by initial nucleation of lactose particles immersed in the melted binder droplets. Agglomerate growth occurred by coalescence between nuclei followed by coalescence between agglomerates. Complex effects of binder droplet size and type of binder were seen at low product temperatures. Low product temperatures resulted in smaller agglomerate sizes, because the agglomerate growth was counteracted by very high binder viscosity or solidification of the binder. At higher product temperatures, neither the binder droplet size nor the type of binder had a clear effect on the final agglomerate size.     

Phosphate-enhanced cytotoxicity of zinc oxide nanoparticles and agglomerates

Zinc oxide (ZnO) nanoparticles (NPs) have been found to readily react with phosphate ions to form zinc phosphate (Zn₃(PO₄)₂) crystallites. Because phosphates are ubiquitous in physiological fluids as well as waste water streams, it is important to examine the potential effects that the formation of Zn₃(PO₄)₂ crystallites may have on cell viability. Thus, the cytotoxic response of NIH/3T3 fibroblast cells was assessed following 24 h of exposure to ZnO NPs suspended in media with and without the standard phosphate salt supplement. Both particle dosage and size have been shown to impact the cytotoxic effects of ZnO NPs, so doses ranging from 5 to 50 μg/mL were examined and agglomerate size effects were investigated by using the bioinert amphiphilic polymer polyvinylpyrrolidone (PVP) to generate water-soluble ZnO ranging from individually dispersed 4 nm NPs up to micron-sized agglomerates. Cell metabolic activity measures indicated that the presence of phosphate in the suspension media can led to significantly reduced cell viability at all agglomerate sizes and at lower ZnO dosages. In addition, a reduction in cell viability was observed when agglomerate size was decreased, but only in the phosphate-containing media. These metabolic activity results were reflected in separate measures of cell death via the lactate dehydrogenase assay. Our results suggest that, while higher doses of water-soluble ZnO NPs are cytotoxic, the presence of phosphates in the surrounding fluid can lead to significantly elevated levels of cell death at lower ZnO NP doses. Moreover, the extent of this death can potentially be modulated or offset by tuning the agglomerate size. These findings underscore the importance of understanding how nanoscale materials can interact with the components of surrounding fluids so that potential adverse effects of such interactions can be controlled.     

Influence of rheological behaviour of particulate/polymer dispersions on liquid-filling characteristics for hard gelatin capsules

Lactose/poloxamer dispersions were prepared by mixing under vacuum to achieve a de-aerated mix with good capsule filling properties and disperse phase uniformity at 70 degrees C. Satisfactory capsule filling of molten dispersions was achieved up to a limiting concentration of disperse phase, dependent on particle size distribution and continuous phase viscosity. Lactose/poloxamer dispersions exhibited thixotropic shear thinning behaviour with an abrupt increase in apparent viscosity above a limiting concentration of disperse phase. There was a good correlation between satisfactory filling of molten dispersions into capsules and apparent viscosity of the formulation, whereas, the pronounced increase in apparent viscosity resulted in unsatisfactory filling above a critical concentration of disperse phase. The rheological data was analysed in detail using empirical models and also used to identify capsule filling problems at extrudate shear rates for flow from hopper to pump (12 s(-1)) and from nozzle to capsule (340 s(-1)).     

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20 nm and 200 nm) and titanium dioxide (21 nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact.     

The effect of capping agents on the toxicity of silver nanoparticles to Danio rerio embryos

Addition of capping agents like surfactants and polymers during the synthesis of nanoparticles may affect the stability and toxicity of dispersions of nanoparticles. This study revealed the impact of anionic, cationic, and amphoteric surfactants and a cationic polymer on the physical and chemical properties, stability and behavior of silver nanomaterials, as well as on the toxicity of nanosized silver particles with respect to zebrafish embryos. Some of the stabilizers applied were shown to significantly affect embryos of Danio rerio. Colloidal dispersions of stabilized silver nanoparticles were demonstrated to induce a complex mechanism of toxicity with respect to embryos of D. rerio, which is mainly explained by the toxicity of the organic ligand, while other parameters are somewhat inferior. The newly generated data on the toxicity of nanoparticles and their stabilizers with respect to D. rerio embryos reveal the complexity of the toxicity mechanism of nanoparticles impacting living systems.