Anion-Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels.

The suckerin family of proteins, identified from the squid sucker ring teeth assembly, offers unique mechanical properties and potential advantages over other natural biomaterials. In this study, a small suckerin isoform, suckerin-12, is used to create enzymatically crosslinked, macro-scale hydrogels. Upon exposure to specific salt conditions, suckerin-12 hydrogels contracted into a condensed state where mechanical properties are found to be modulated by the salt anion present. The rate of contraction is found to correlate well with the kosmotropic arm of the Hofmeister anion series. However, the observed changes in hydrogel mechanical properties are better explained by the ability of the salt to neutralize charges in suckerin-12 by deprotonization or charge screening. Thus, by altering the anions in the condensing salt solution, it is possible to tune the mechanical properties of suckerin-12 hydrogels. The potential for suckerins to add new properties to materials based on naturally-derived proteins is highlighted.

Toxicological Implications of Platinum Nanoparticle Exposure: Stimulation of Intracellular Stress, Inflammatory Response, and Akt Signaling In Vitro.

Due to their distinctive physicochemical properties, platinum nanoparticles (PtNPs) have emerged as a material of interest for a number of biomedical therapeutics. However, in some instances NP exposure has been correlated to health and safety concerns, including cytotoxicity, activation of cellular stress, and modification to normal cell functionality. As PtNPs have induced differential cellular responses in vitro, the goal of this study was to further characterize the behavior and toxicological potential of PtNPs within a HepG2 liver model. This study identified that a high PtNP dosage induced HepG2 cytotoxicity. However, lower, subtoxic PtNP concentrations were able to elicit multiple stress responses, secretion of proinflammatory cytokines, and modulation of insulin-like growth factor-1 dependent signal transduction. Taken together, this work suggests that PtNPs would not be overtly toxic for acute exposures, but sustained cellular interactions might produce long term health consequences.

Implementation of physiological fluids to provide insight into the characterization, fate, and biological interactions of silver nanoparticles.

Silver nanoparticles (AgNPs) are being increasingly utilized in consumer and medical applications. However, there remains conflicting reports on their safety, which are evaluated through a combination of in vitro and in vivo exposure models. These discrepancies may arise, in part, due to the inherent differences between cell-based and animal systems. It is well established that nanotoxicological effects are highly dependent on the unique physicochemical properties and behavior of the particle set, including size, surface chemistry, agglomeration, and ionic dissolution. However, recent studies have identified that these properties vary as a function of exposure environment; providing a rationale for the contradictory results between in vitro and in vivo assessments. Artificial physiological fluids are emerging as a powerful tool as they allow for the characterization of NPs in an environment which they would likely encounter in vivo, in addition to having the experimental advantages of flexibility and consistency. Here, we demonstrated that the utilization of artificial fluids provided a mechanism to assess AgNP behavior and induced bioresponses in environments that they would likely encounter in vivo. AgNPs were introduced within an alveolar-based exposure model, which included alveolar epithelial (A549) cells incubated within artificial alveolar fluid (AF). Additionally, the particles underwent extensive characterization within both AF and lysosomal fluid, which the AgNPs would encounter following cellular internalization. Following incubation in physiological environments AgNP properties were significantly modified versus a traditional media environment, including alterations to both extent of agglomeration and rate of ionic dissolution. Moreover, when A549s were exposed to AgNPs in AF, the cells displayed lower cytotoxicity and stress rates, corresponding to a fluid-dependent drop in silver ion production. This work highlights the need for enhanced in vitro models that more closely mimic in vivo exposure environments in order to capture true NP behaviors and cellular interactions.

Immunogenicity and antimicrobial effectiveness of Pseudomonas aeruginosa specific bacteriophage in a human lung in vitro model.

The rise of antibiotic resistant bacteria is posing a serious threat to human health. For example, resistant strains of Pseudomonas aeruginosa have resulted in untreatable and potentially lethal infections in both cystic fibrosis and immunocompromised patients. Due to the growing need for alternative treatment options, bacteriophage, or phage, therapy is gaining considerable attention. While previous studies have demonstrated the effectiveness of phage in combating persistent bacterial infections, there is currently a lack of knowledge regarding the host immunological response following phage exposure. In the present study, the bioresponses of an enhanced in vitro model were characterized following exposure to either DMS3 or PEV2, P. aeruginosa targeting phages. Results demonstrated a PEV2-dependent increase in IL-6 and TNF-α production, but no changes associated with DMS3 exposure. Additionally, following the establishment of an in vitro infection model, DMS3 was found to successfully protect mammalian lung cells from P. aeruginosa. Taken together, the biocompatibility and antibacterial effectiveness distinguish DMS3 bacteriophage as a strong candidate for phage therapy. However, as DMS3 is pilin dependent and bacterial receptor expression varies significantly, this work highlights the necessity of generating phage cocktails.

Exposure to metal oxide nanoparticles in physiological fluid induced synergistic biological effects in a keratinocyte model.

Nanoparticles (NPs) possess distinctive physicochemical properties that in addition to differentiating them from their bulk counterparts can induce negative cellular consequences. Standard in vitro systems have served as the primary model for NP safety evaluations, but suffer from a lack physiological relevance. One way to overcome this limitation and evaluate NP characteristics under more accurate conditions is through the use of artificial physiological fluids, which mimic the composition of in vivo environments. In this study, we identified that copper oxide (CuO) and titanium dioxide (TiO2) NPs displayed modified behavior when dispersed in artificial interstitial fluid (IF) versus traditional media, including extensive agglomeration and increased particle deposition. When keratinocyte cells underwent CuO NP exposure, synergistic stress and toxicity responses occurred within an IF environment, correlating with augmented particle deposition. However, following IF incubation alone or concurrently with TiO2 NPs, which are not innately toxic, no combinatorial responses were identified. These results indicate that synergistic outcomes arise when toxic NPs undergo fluid-induced alterations to key physicochemical properties and behaviors. This study highlights the necessity of carrying out NP characterization and safety assessments in physiologically-representative environments; as altered behavior patterns have the potential to induce bioresponses not identified within traditional models.

Modification of the protein corona-nanoparticle complex by physiological factors.

Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications.

The role of biological fluid and dynamic flow in the behavior and cellular interactions of gold nanoparticles.

BACKGROUND: Due to their distinctive physicochemical properties, nanoparticles (NPs) have proven to be extremely advantageous for product and application development, but are also capable of inducing detrimental outcomes in biological systems. Standard in vitro methodologies are currently the primary means for evaluating NP safety, as vast quantities of particles exist that require appraisal. However, cell-based models are plagued by the fact that they are not representative of complex physiological systems. The need for a more accurate exposure model is highlighted by the fact that NP behavior and subsequent bioresponses are highly dependent upon their surroundings. Therefore, standard in vitro models will likely produce inaccurate NP behavioral analyses and erroneous safety results. As such, the goal of this study was to develop an enhanced in vitro model for NP evaluation that retained the advantages of cell culture, but implemented the key physiological variables of accurate biological fluid and dynamic flow. RESULTS: In this study, a cellular microenvironment was modeled and created after an inhalation exposure scenario. This system comprised of A549 lung epithelial cells, artificial alveolar fluid (AAF), and biologically accurate dynamic flow. Under the influence of microenvironment variables, tannic acid coated gold NPs (AuNPs) displayed modulated physicochemical characteristics, including increased agglomeration, disruption of the spectral signature, and decreased rate of ionic dissolution. Furthermore, AuNP deposition efficiency, internalization patterns, and the nano-cellular interface varied as a function of fluid composition and flow condition. AAF incubation simultaneously influenced both AuNPs and cellular behavior, through excessive NP agglomeration and alteration to A549 morphology. Dynamic flow targeted the nano-cellular interface, with differential responses including modified deposition, internalization patterns, and cellular elongation. Lastly, the biocompatibility of the system was verified to ensure cellular health following AAF exposure and fluid dynamics. CONCLUSIONS: This study confirmed the feasibility of improving standard in vitro models through the incorporation of physiological variables. Utilization of this enhanced system demonstrated that to elucidate true NP behavior and accurately gauge their cellular interactions, assessments should be carried out in a more complex and relevant biological exposure model.

At the Crossroads of Nanotoxicology in vitro: Past Achievements and Current Challenges.

The exponential growth in the employment of nanomaterials (NMs) has given rise to the field of nanotoxicology; which evaluates the safety of engineered NMs. Initial nanotoxicological studies were limited by a lack of both available materials and accurate biodispersion characterization tools. However, the years that followed were marked by the development of enhanced synthesis techniques and characterization technologies; which are now standard practice for nanotoxicological evaluation. Paralleling advances in characterization, significant progress was made in correlating specific physical parameters, such as size, morphology, or coating, to resultant physiological responses. Although great strides have been made to advance the field, nanotoxicology is currently at a crossroads and faces a number of obstacles and technical limitations not associated with traditional toxicology. Some of the most pressing and influential challenges include establishing full characterization requirements, standardization of dosimetry, evaluating kinetic rates of ionic dissolution, improving in vitro to in vivo predictive efficiencies, and establishing safety exposure limits. This Review will discuss both the progress and future directions of nanotoxicology: highlighting key previous research successes and exploring challenges plaguing the field today.

Dynamic characteristics of silver nanoparticles in physiological fluids: toxicological implications.

The field of nanotoxicology has made tremendous progress identifying novel and potentially adverse biological effects following nanomaterial (NM) exposure. However, one facet yet to be satisfactorily explored is how a physiological environment modifies NM physicochemical properties, thus introducing novel complexities associated with solid phase material exposures. In this study, artificial alveolar, lysosomal, and interstitial fluids were used to identify environmental-specific modulations to the properties and behavior of hydrocarbon-coated (Ag-HC) and polysaccharide-coated (Ag-PS) silver NMs. As inhalation is a common route of exposure, an alveolar macrophage cell model with deposition dosages representing approximately 2.5 months and 10 years of occupational exposure (0.5 and 25 ng/mL, respectively) were employed. Following dispersion in the artificial fluids, the Ag-HC and Ag-PS NMs demonstrated significant alterations to morphology, aggregation patterns, and particle reactivity. However, the Ag-PS also demonstrated a loss of particle coating, which elicited increased cytotoxicity, phagocytosis, and inflammation not associated with the original Ag-PS. This study demonstrated that in a physiological system NMs undergo considerable modulation, introducing a scenario where the toxicity of NMs may increase over time due to internal bioconditions. These findings highlight the critical influence that the dynamic and insoluble nature of NMs have on bioeffects and the importance of characterizing this behavior.

Slow release of ions from internalized silver nanoparticles modifies the epidermal growth factor signaling response.

Due to their distinctive physiochemical properties, including a robust antibacterial activity and plasmonic capability, hundreds of consumer and medical products contain colloidal silver nanoparticles (AgNPs). However, even at sub-toxic dosages, AgNPs are able to disrupt cell functionality, through a yet unknown mechanism. Moreover, internalized AgNPs have the potential to prolong this disruption, even after the removal of excess particles. In the present study, we evaluated the impact, mechanism of action, and continual effects of 50 nm AgNP exposure on epidermal growth factor (EGF) signal transduction within a human keratinocyte (HaCaT) cell line. After AgNP expose, EGF signaling was initially obstructed due to the dissolution of particles into silver ions. However, at longer durations, the internalized AgNPs increased EGF signaling activity. This latter behavior correlated to sustained HaCaT stress, believed to be maintained through the continual dissolution of internalized AgNPs. This study raises concerns that even after exposure ceases, the retained nanomaterials are capable of acting as a slow-release mechanism for metallic ions; continually stressing and modifying normal cellular functionality.

Less is more: long-term in vitro exposure to low levels of silver nanoparticles provides new insights for nanomaterial evaluation.

In view of the vast number of new nanomaterials (NMs) that require testing and the constraints associated with animal models, the majority of studies to elucidate nanotoxicological effects have occurred in vitro, with limited correlation and applicability to in vivo systems and realistic, occupational exposure scenarios. In this study, we developed and implemented a chronic in vitro model coupled with lower, regulatory dosages in order to provide a more realistic assessment of NM-dependent consequences and illuminate the implications of long-term NM exposure. When keratinocytes were exposed to 50 nm silver nanoparticles (Ag-NPs), we determined that chronically dosed cells operated under augmented stress and modified functionality in comparison to their acute counterparts. Specifically, Ag-NP exposure through a chronic mechanism increased p38 activation, actin disorganization, heightened ki67 expression, and extensive gene modification. Additionally, chronic Ag-NP exposure altered the way in which cells perceived and responded to epidermal growth factor stimulation, indicating a transformation of cell functionality. Most importantly, this study demonstrated that chronic exposure in the pg/mL range to Ag-NPs did not induce a cytotoxic response, but instead activated sustained stress and signaling responses, suggesting that cells are able to cope with prolonged, low levels of Ag-NP exposure. In summary, we demonstrated that through implementation of a chronic dosimetry paradigm, which more closely resembles realistic NM exposure scenarios, it is possible to illuminate long-term cellular consequences, which greatly differ from previously obtained acute assessments.

Tannic acid coated gold nanorods demonstrate a distinctive form of endosomal uptake and unique distribution within cells.

One of the primary challenges associated with nanoparticle-dependent biological applications is that endosomal entrapment in a physiological environment severely limits the desired targeting and functionality of the nanoconstructs. This study sought to overcome that challenge through a systematic approach of gold nanorod (GNR) functionalization: evaluating the influence of both aspect ratio and surface chemistry on targeted cellular internalization rates and preservation of particle integrity. Owing to their unique spectral properties and enhanced surface area, GNRs possess great potential for the advancement of nanobased delivery and imaging applications. However, their ability for efficient intracellular delivery while maintaining their specific physiochemical parameters has yet to be satisfactorily explored. This study identified that longer and positively charged GNRs demonstrated a higher degree of internalization compared to their shorter and negative counterparts. Notably, of the four surface chemistries explored, only tannic acid resulted in retention of GNR integrity following endocytosis into keratinocyte cells, due to the presence of a strong protein corona matrix that served to protect the particles. Taken together, these results identify tannic acid functionalized GNRs as a potential candidate for future development in nanobased biomolecule delivery, bioimaging, and therapeutic applications.

High aspect ratio gold nanorods displayed augmented cellular internalization and surface chemistry mediated cytotoxicity.

Due to their unique properties, gold nanorods (GNRs) have shown tremendous potential for advancing bio-imaging and sensing applications. As these nanoparticles display size-dependent optical properties, high aspect ratio GNRs are of particular interest for these applications because of their increased scattering contrast. While studies are emerging that demonstrate successful synthesis of high aspect ratio GNRs, their behavior and fate in a physiological environment has yet to be investigated. The goal of this study was to evaluate the rate of cellular internalization and cytotoxicity of long GNRs (aspect ratio 32) in a human keratinocyte cell line. Additionally, the critical role of surface chemistry in extent of cellular interactions and cytotoxicity was evaluated. Through comparison with aspect ratio 3 GNRs, it was identified that high aspect ratio GNRs displayed enhanced cellular internalization. Furthermore, surface functionalization dictated the quantity of GNRs internalized, with tannic acid having a significant increase over polyethylene glycol. However, the augmented intracellular concentration identified with long, tannic acid GNRs resulted in a considerable degree of cytotoxicity, which was not associated with other GNR conditions. Therefore, while the inclusion of high aspect ratio GNRs may increase the capabilities for nano-based applications, there exist some unintentional toxicological consequences that must also be considered.

The biological impact of concurrent exposure to metallic nanoparticles and a static magnetic field.

The rapid advancement of technology has led to an exponential increase of both nanomaterial and magnetic field utilization in applications spanning a variety of sectors. While extensive work has focused on the impact of these two variables on biological systems independently, the existence of any synergistic effects following concurrent exposure has yet to be investigated. This study sought to ascertain the induced alterations to the stress and proliferation responses of the human adult low calcium, high temperature keratinocyte (HaCaT) cell line by the application of a static magnetic field (approximately 0.5 or 30 mT) in conjunction with either gold or iron oxide nanoparticles for a duration of 24 h. By evaluating targets at a cellular, protein, and genetic level a complete assessment of the HaCaT response was generated. A magnetic field-dependent proliferative effect was found (∼15%), which correlated with a decrease in reactive oxygen species and a simultaneous increase in ki67 expression, all occurring independently of nanoparticle presence. Furthermore, the application of a static magnetic field was able to counteract the cellular stress response induced by nanoparticle exposure through a combination of decreased reactive oxygen species production and modification of gene regulation. Therefore, we conclude that while these variables each introduce the potential to uniquely influence physiological events, no negative synergistic reactions were identified.

Novel platform development using an assembly of carbon nanotube, nanogold and immobilized RNA capture element towards rapid, selective sensing of bacteria.

This study examines the creation of a nano-featured biosensor platform designed for the rapid and selective detection of the bacterium Escherichia coli. The foundation of this sensor is carbon nanotubes decorated with gold nanoparticles that are modified with a specific, surface adherent ribonucleiuc acid (RNA) sequence element. The multi-step sensor assembly was accomplished by growing carbon nanotubes on a graphite substrate, the direct synthesis of gold nanoparticles on the nanotube surface, and the attachment of thiolated RNA to the bound nanoparticles. The application of the compounded nano-materials for sensor development has the distinct advantage of retaining the electrical behavior property of carbon nanotubes and, through the gold nanoparticles, incorporating an increased surface area for additional analyte attachment sites, thus increasing sensitivity. We successfully demonstrated that the coating of gold nanoparticles with a selective RNA sequence increased the capture of E. coli by 189% when compared to uncoated particles. The approach to sensor formation detailed in this study illustrates the great potential of unique composite structures in the development of a multi-array, electrochemical sensor for the fast and sensitive detection of pathogens.

Interference of silver, gold, and iron oxide nanoparticles on epidermal growth factor signal transduction in epithelial cells.

Metallic nanomaterials, including silver, gold, and iron oxide, are being utilized in an increasing number of fields and specialties. The use of nanosilver as an antimicrobial agent is becoming ever-more common, whereas gold and iron oxide nanomaterials are frequently utilized in the medical field due to their recognized "biocompatibility". Numerous reports have examined the general toxicity of these nanomaterials; however, little data exists on how the introduction of these nanomaterials, at nontoxic levels, affects normal cellular processes. In the present study the impact of low levels of 10 nm silver (Ag-NP), gold (Au-NP), and iron oxide nanoparticles (SPION) on epidermal growth factor (EGF) signal transduction within the human epithelial cell line, A-431, was investigated. Following a biocompatibility assessment, the nanoparticle-induced interference at four specific targets within the EGF signaling process was evaluated: (1) nanoparticle-EGF association, (2) Akt and Erk phosphorylation, (3) Akt activity, and (4) EGF-dependent gene regulation. For all tested nanoparticles, following cellular exposure, a disruption in the EGF signaling response transpired; however, the metallic composition determined the mechanism of alteration. In addition to inducing high quantities of ROS, Ag-NPs attenuated levels of Akt and Erk phosphorylation. Au-NPs were found to decrease EGF-dependent Akt and Erk phosphorylation as well as inhibit Akt activity. Lastly, SPIONs produced a strong alteration in EGF activated gene transcription, with targeted genes influencing cell proliferation, migration, and receptor expression. These results demonstrate that even at low doses, introduction of Ag-NPs, Au-NPs, and SPIONs impaired the A-431 cell line's response to EGF.

Combinatorial Signal Transduction Responses Mediated by Interleukin-2 and -4 Receptors in a Helper TH2 Cell Line.

The cytokines interleukin (IL)-2 and IL-4 are important regulators of the adaptive immune response, due in part to their effects on clonal expansion and differentiation of T cells. When IL-2 and IL-4 are administered together, both antagonistic and synergistic effects have been reported, but little is known in general concerning the mechanisms underlying such combinatorial effects. We found evidence for both effects in the proliferation responses of the IL-2 and IL-4 responsive T cell line, HT-2; IL-4 delays the onset of cell growth yet ultimately allows a higher cell density to be achieved in static culture. At the level of signal transduction pathways, we found that IL-4 partially inhibits IL-2 receptor-mediated pathways (PI3K/Akt, Ras/Erk, and STAT5a/b) and does not prolong their transient kinetics. This mode of antagonism, but not the effects on cell proliferation, is overcome at higher concentrations of IL-2 that are sufficient to saturate the signaling responses. By comparison, IL-4-stimulated activation of STAT6 is unaffected by IL-2 and shows sustained kinetics, and we speculate that this or another IL-4 receptor-specific pathway is responsible for the effects of IL-4 on IL-2-stimulated proliferation. A possibly related observation is that IL-4 induces a dramatic cell adhesion phenotype.