Bryan Harper

Comparative toxicological assessment of PAMAM and thiophosphoryl dendrimers using embryonic zebrafish

Dendrimers are well-defined, polymeric nanomaterials currently being investigated for biomedical applications such as medical imaging, gene therapy, and tissue targeted therapy. Initially, higher generation (size) dendrimers were of interest because of their drug carrying capacity. However, increased generation was associated with increased toxicity. The majority of studies exploring dendrimer toxicity have focused on a small range of materials using cell culture methods, with few studies investigating the toxicity across a wide range of materials in vivo. The objective of the present study was to investigate the role of surface charge and generation in dendrimer toxicity using embryonic zebrafish (Danio rerio) as a model vertebrate. Due to the generational and charge effects observed at the cellular level, higher generation cationic dendrimers were hypothesized to be more toxic than lower generation anionic or neutral dendrimers with the same core composition. Polyamidoamine (PAMAM) dendrimers elicited significant morbidity and mortality as generation was decreased. No significant adverse effects were observed from the suite of thiophosphoryl dendrimers studied. Exposure to ≥50 ppm cationic PAMAM dendrimers G3-amine, G4-amine, G5-amine, and G6-amine caused 100% mortality by 24 hours post-fertilization. Cationic PAMAM G6-amine at 250 ppm was found to be statistically more toxic than both neutral PAMAM G6-amidoethanol and anionic PAMAM G6-succinamic acid at the same concentration. The toxicity observed within the suite of varying dendrimers provides evidence that surface charge may be the best indicator of dendrimer toxicity. Dendrimer class and generation are other potential contributors to the toxicity of dendrimers. Further studies are required to better understand the relative role each plays in driving the toxicity of dendrimers. To the best of our knowledge, this is the first in vivo study to address such a broad range of dendrimers.

Predictive modeling of nanomaterial exposure effects in biological systems

Background Predictive modeling of the biological effects of nanomaterials is critical for industry and policymakers to assess the potential hazards resulting from the application of engineered nanomaterials. Methods We generated an experimental dataset on the toxic effects experienced by embryonic zebrafish due to exposure to nanomaterials. Several nanomaterials were studied, such as metal nanoparticles, dendrimer, metal oxide, and polymeric materials. The embryonic zebrafish metric (EZ Metric) was used as a screening-level measurement representative of adverse effects. Using the dataset, we developed a data mining approach to model the toxic endpoints and the overall biological impact of nanomaterials. Data mining techniques, such as numerical prediction, can assist analysts in developing risk assessment models for nanomaterials. Results We found several important attributes that contribute to the 24 hours post-fertilization (hpf) mortality, such as dosage concentration, shell composition, and surface charge. These findings concur with previous studies on nanomaterial toxicity using embryonic zebrafish. We conducted case studies on modeling the overall effect/impact of nanomaterials and the specific toxic endpoints such as mortality, delayed development, and morphological malformations. The results show that we can achieve high prediction accuracy for certain biological effects, such as 24 hpf mortality, 120 hpf mortality, and 120 hpf heart malformation. The results also show that the weighting scheme for individual biological effects has a significant influence on modeling the overall impact of nanomaterials. Sample prediction models can be found at http://neiminer.i-a-i.com/nei_models. Conclusion The EZ Metric-based data mining approach has been shown to have predictive power. The results provide valuable insights into the modeling and understanding of nanomaterial exposure effects.

The Impact of Surface Ligands and Synthesis Method on the Toxicity of Glutathione-Coated Gold Nanoparticles

Gold nanoparticles (AuNPs) are increasingly used in biomedical applications, hence understanding the processes that affect their biocompatibility and stability are of significant interest. In this study, we assessed the stability of peptide-capped AuNPs and used the embryonic zebrafish (Danio rerio) as a vertebrate system to investigate the impact of synthesis method and purity on their biocompatibility. Using glutathione (GSH) as a stabilizer, Au-GSH nanoparticles with identical core sizes were terminally modified with Tryptophan (Trp), Histidine (His) or Methionine (Met) amino acids and purified by either dialysis or ultracentrifugation. Au-GSH-(Trp)₂ purified by dialysis elicited significant morbidity and mortality at 200 µg/mL, Au-GSH-(His)₂ induced morbidity and mortality after purification by either method at 20 and 200 µg/mL, and Au-GSH-(Met)₂ caused only sublethal responses at 200 µg/mL. Overall, toxicity was significantly reduced and ligand structure was improved by implementing ultracentrifugation purifications at several stages during the multi-step synthesis and surface modification of Au-GSH nanoparticles. When carefully synthesized at high purity, peptide-functionalized AuNPs showed high biocompatibility in biological systems.Gold nanoparticles (AuNPs) are increasingly used in biomedical applications, hence understanding the processes that affect their biocompatibility and stability are of significant interest. In this study, we assessed the stability of peptide-capped AuNPs and used the embryonic zebrafish (Danio rerio) as a vertebrate system to investigate the impact of synthesis method and purity on their biocompatibility. Using glutathione (GSH) as a stabilizer, Au-GSH nanoparticles with identical core sizes were terminally modified with Tryptophan (Trp), Histidine (His) or Methionine (Met) amino acids and purified by either dialysis or ultracentrifugation. Au-GSH-(Trp)2 purified by dialysis elicited significant morbidity and mortality at 200 µg/mL, Au-GSH-(His)2 induced morbidity and mortality after purification by either method at 20 and 200 µg/mL, and Au-GSH-(Met)2 caused only sublethal responses at 200 µg/mL. Overall, toxicity was significantly reduced and ligand structure was improved by implementing ultracentrifugation purifications at several stages during the multi-step synthesis and surface modification of Au-GSH nanoparticles. When carefully synthesized at high purity, peptide-functionalized AuNPs showed high biocompatibility in biological systems.

Impacts of chemical modification on the toxicity of diverse nanocellulose materials to developing zebrafish.

Abstract Cellulose is an abundant and renewable resource currently being investigated for utility in nanomaterial form for various promising applications ranging from medical and pharmaceutical uses to mechanical reinforcement and biofuels. The utility of nanocellulose and wide implementation ensures increasing exposure to humans and the environment as nanocellulose-based technologies advance. Here, we investigate how differences in aspect ratio and changes to surface chemistry, as well as synthesis methods, influence the biocompatibility of nanocellulose materials using the embryonic zebrafish. Investigations into the toxicity of neutral, cationic and anionic surface functionalities revealed that surface chemistry had a minimal influence on the overall toxicity of nanocellulose materials. Higher aspect ratio cellulose nanofibers produced by mechanical homogenization were, in some cases, more toxic than other cellulose-based nanofibers or nanocrystals produced by chemical synthesis methods. Using fluorescently labeled nanocellulose we were able to show that nanocellulose uptake did occur in embryonic zebrafish during development. We conclude that the benign nature of nanocellulose materials makes them an ideal platform to systematically investigate the inherent surface features driving nanomaterial toxicity in order to create safer design principles for engineered nanoparticles.

Comparative hazard analysis and toxicological modeling of diverse nanomaterials using the embryonic zebrafish (EZ) metric of toxicity

Abstract The integration of rapid assays, large datasets, informatics, and modeling can overcome current barriers in understanding nanomaterial structure–toxicity relationships by providing a weight-of-the-evidence mechanism to generate hazard rankings for nanomaterials. Here, we present the use of a rapid, low-cost assay to perform screening-level toxicity evaluations of nanomaterials in vivo. Calculated EZ Metric scores, a combined measure of morbidity and mortality in developing embryonic zebrafish, were established at realistic exposure levels and used to develop a hazard ranking of diverse nanomaterial toxicity. Hazard ranking and clustering analysis of 68 diverse nanomaterials revealed distinct patterns of toxicity related to both the core composition and outermost surface chemistry of nanomaterials. The resulting clusters guided the development of a surface chemistry-based model of gold nanoparticle toxicity. Our findings suggest that risk assessments based on the size and core composition of nanomaterials alone may be wholly inappropriate, especially when considering complex engineered nanomaterials. Research should continue to focus on methodologies for determining nanomaterial hazard based on multiple sub-lethal responses following realistic, low-dose exposures, thus increasing the availability of quantitative measures of nanomaterial hazard to support the development of nanoparticle structure–activity relationships.

The influence of size on the toxicity of an encapsulated pesticide: a comparison of micron- and nano-sized capsules

Encapsulation technology involves entrapping a chemical active ingredient (a.i.) inside a hollow polymeric shell and has been applied to commercial pesticide manufacturing for years to produce capsule suspension (CS) formulations with average particle sizes in the micron-scale. The few literature sources that investigate the environmental fate and toxicity to non-target organisms of encapsulated commercially available pesticide products with regard to capsule size report on average sizes between 20 and 50 μm. Here, we have identified a CS formulation with an average capsule size of approximately 2 μm with some capsules extending into the nanometer scale (~200 nm). Determining how carrier size influences toxicity is important to understanding if current pesticide risk assessments are sufficient to protect against products that incorporate encapsulation technology. Here, a commercial pyrethroid CS pesticide with lambda-cyhalothrin (λ-Cy) as the a.i. was separated into two suspensions, a fraction consisting of nano-sized capsules (~250 nm) and a fraction of micron-sized capsules (~2200 nm) in order to investigate the influence of capsule size on toxicity to embryonic zebrafish, Danio rerio. Toxicity was evaluated 24h after exposure to equivalent amounts of a.i. by the presence and severity of pyrethroid-specific tremors, 14 sublethal developmental impacts and mortality. Fish exposed to greater than 20 μg a.i. L(-1) technical λ-Cy or formulated product experienced curvature of the body axis, pericardial edema, craniofacial malformations, and mortality. Exposure to the unfractionated formulation, micro fraction, nano fraction and technical a.i. resulted in no significant differences in the occurrence of sublethal impacts or mortality; however, the technical a.i. exposure resulted in significantly less fish experiencing tremors and shorter tremors compared to any of the formulated product exposures. This suggests that the capsule size does not influence the toxic response of the entrapped λ-Cy, but the presence or absence of the capsules does. Testing across other encapsulated products is needed to determine if size does not have influence on toxicity regardless of encapsulation technology.

Influence of surface chemical properties on the toxicity of engineered zinc oxide nanoparticles to embryonic zebrafish

Summary Zinc oxide nanoparticles (ZnO NPs) are widely used in a variety of products, thus understanding their health and environmental impacts is necessary to appropriately manage their risks. To keep pace with the rapid increase in products utilizing engineered ZnO NPs, rapid in silico toxicity test methods based on knowledge of comprehensive in vivo and in vitro toxic responses are beneficial in determining potential nanoparticle impacts. To achieve or enhance their desired function, chemical modifications are often performed on the NPs surface; however, the roles of these alterations play in determining the toxicity of ZnO NPs are still not well understood. As such, we investigated the toxicity of 17 diverse ZnO NPs varying in both size and surface chemistry to developing zebrafish (exposure concentrations ranging from 0.016 to 250 mg/L). Despite assessing a suite of 19 different developmental, behavioural and morphological endpoints in addition to mortality in this study, mortality was the most common endpoint observed for all of the ZnO NP types tested. ZnO NPs with surface chemical modification, regardless of the type, resulted in mortality at 24 hours post-fertilization (hpf) while uncoated particles did not induce significant mortality until 120 hpf. Using eight intrinsic chemical properties that relate to the outermost surface chemistry of the engineered ZnO nanoparticles, the highly dimensional toxicity data were converted to a 2-dimensional data set through principal component analysis (PCA). Euclidean distance was used to partition different NPs into several groups based on converted data (score) which were directly related to changes in the outermost surface chemistry. Kriging estimations were then used to develop a contour map based on mortality data as a response. This study illustrates how the intrinsic properties of NPs, including surface chemical modifications and capping agents, are useful to separate and identify ZnO NP toxicity to zebrafish (Danio rerio).

Differential dissolution and toxicity of surface functionalized silver nanoparticles in small-scale microcosms: impacts of community complexity

Surface functionalization can minimize nanoparticle agglomeration and expand their applications; however, these modifications can also alter particle stability, dissolution, bioavailability and toxicity. Here we investigated how silver nanoparticles (AgNPs) with different surface chemistries affect community health, and how increased trophic complexity affects the interactions between organisms and nanomaterials. We compared AgNP exposures in simple microcosms comprised of algae (Chlamydomonas reinhardtii) and bacteria (Escherichia coli) to increasingly complex microcosms containing predatory invertebrates (Daphnia magna) and developing vertebrates (Danio rerio). Each microcosm was exposed to one of three 70 nm AgNPs [polyethylene glycol (PEG–AgNP), silica (Si–AgNP), or aminated silica-coated AgNP (Ami-Si–AgNP)] at 0, 0.1, 1, and 5 mg L−1 to investigate the relative influence of surface charge, composition and dissolution on organismal uptake and toxicity. All three AgNPs released more dissolved Ag into solution when organisms were present than was measured in the same media without organisms. PEG–AgNPs had the highest overall toxicity in all scenarios, followed by Si–AgNPs, and lastly Ami-Si–AgNPs. Toxicity correlated with the amount of Ag measured in the exposure media and the amount taken up by the organisms. Our findings indicate that surface functionalization plays an important role in determining dissolution, uptake and toxicity of AgNPs. Increasing trophic complexity decreased organismal susceptibility under the same AgNP concentration exposures, likely due to the change in bioavailable Ag that each organism experienced. This implies that tests using individual species provide conservative estimates of environmental impacts, while exposure may be mitigated in more realistic multi-species scenarios like those found in nature.

Uptake and toxicity of CuO nanoparticles to Daphnia magna varies between indirect dietary and direct waterborne exposures

Research examining the direct and indirect ecological effects of nanomaterials in aquatic ecosystems is important for developing a more realistic understanding of the environmental implications of nanotechnology. Copper oxide nanoparticles (CuO NPs) are being used extensively in many industries but are considered highly toxic to aquatic species residing in surface waters. Few studies have addressed whether CuO NPs can be transferred through the aquatic food chain, and if such indirect exposure to nanomaterials impacts their toxicity. We investigated the uptake and trophic transfer of CuO NPs from the algae Chlorella vulgaris to the crustacean Daphnia magna and assessed bio-partitioning and resulting toxicity. We hypothesized that CuO NPs can be associated with algal cells and be transported to predators through feeding, and that the chronic toxicity can be altered in comparison to direct CuO NP exposure. For the indirect feeding exposure, algae pre-incubated with CuO NPs (Cu-algae) were washed to remove loose NPs and fed to D. magna while Cu uptake and toxicity were evaluated. For the direct waterborne exposures, a parallel group of D. magna were exposed to equivalent concentrations of CuO NPs while being fed unexposed algae. Using hyperspectral imaging we observed strong surface associations between pre-incubated CuO NPs and algae used in the feeding exposure, and quantified the average Cu content (0.15mg Cu/L) with ICP-OES. Cu accumulated in daphnid bodies to a greater extent in direct exposures, whereas molted carapaces and neonate offspring had more copper following the indirect feeding exposure, implying that D. magna may regulate internal Cu differently depending on the method of CuO NP delivery. Significantly higher D. magna mortality was observed following direct exposure relative to feeding exposure, and neonate production from adult daphnids exposed indirectly to CuO NPs was significantly reduced. Thus, nanoparticle interaction with biota at one trophic level may alter the biological response at the next trophic level in a way that is dependent on the delivery scenario. This study highlights the importance of evaluating potential ecological impacts of nanomaterials in more relevant, complex exposure scenarios.

Visualization tool for correlating nanomaterial properties and biological responses in zebrafish

The complexity of engineered nanomaterials with regard to their structure and system-dependent properties, and limits of instrumentation to fully characterize nanomaterials in aqueous suspensions or biological media make it difficult to understand how material structure invokes biological response. In this work, a data visualization tool was developed to explore the results of 151 zebrafish assays stored in the Nanomaterial-Biological Interactions Knowledgebase. Visualizations generated using the tool indicated that some nanomaterials exhibited a tendency to cause death, others, sublethal abnormalities. The visualizations also showed that combinations of characteristics, such as the material of the core, shell, and surface, more than any individual characteristic, influenced toxicity. Notably, the size of the nanoparticle did not appear significant in determining toxicity across studies. There was an indication that surface charge could affect toxicity, but a distinct relationship between charge and biological response was not identified. Through exploration of the Knowledgebase using the tool, it was determined that it is possible to alter the toxicity of a nanomaterial of a certain core composition by adding different combinations of a shell and/or a functional outer surface, suggesting that proper design choices, as required to achieve a specific function of a material, could mitigate or exacerbate toxicity.