1. Title: Particle to particle and particle to cell interactions in physiologic fluids using Atomic Force Microscopy (AFM).
Investigator: Dr. Philip Demokritou, C. Blattman, J. Cohen, Georgios Pyrgiotakis
AbstractThe formation of protein corona has emerged as a key mechanism in the particle-cell and particle-particle interactions in physiological fluids. While the particle corona formation was studied using proteomics and another analytical techniques, there is a lack of data in terms of atomic forces at the molecular level and their link to bio-interactions. In this novel bottom-up approach we investigate the interactions between particles and between particles and cells in physiological fluids, utilizing the state of the art Atomic Force Microscopy (AFM). Industry relevant ENMs (SiO2, F2O3 and CeO2) were synthesized with the Harvard VENGES flame spray pyrolysis platform and were deposited on pristine Si substrate in the flame. The same ENMS were collected and characterized off-line regarding their size, crystal structure and surface area using state of the art analytical methods. The collected ENMs were also used to modify AFM tips by attaching them on the tip from an ethanol or water suspension under an inverted microscope and the use of micromanipulators. The surfaces and the tips were characterized with SEM before and after each experiment to ensure that the particles did not detach during the experiment. The interaction (adhesion) force between the surface and the tip (particle-particle interaction) was measured in various media (air, water, RPMI and RPMI with FBS) as a function of the particle material and size. Iron oxide particles were found to experience less adhesion between them compared to ceria, in water, while both of them experience the same adhesion in physiological fluid (RPMI and FBS). The measured adhesion is in accordance with the DLVO theory and was verified with the DLS particle size. In addition, the atomic level forces between the particle functionalized tip and lung epithelial cells (A549) adhered on a slide glass and immersed in physiological media were measured.
2. Title: Safe by design: Demonstrating the efficiency of a safer formulation concept for flame-generated engineered nanomaterials (ENMs
Investigator: Samuel Gass, Georgios Pyrgiotakis, Joel M. Cohen, Georgios A. Sotiriou, Sotiris. E. Pratsinis and Philip Demokritou
AbstractEngineering less toxic nanomaterials that maintain valuable functional properties is crucial to the sustainability of the nanotech industry. In this study, a safer formulation concept for flame-generated nanomaterials was demonstrated. It is based on the encapsulation of potentially toxic nanomaterials by a biologically inert nanothin amorphous SiO2 layer. The core-shell particles maintain their intrinsic optical, magnetic or plasmonic properties of the core material but exhibit surface properties of their SiO2 shell. The SiO2-coating process was performed using the recently developed flame spray pyrolysis (FSP)-based Versatile Engineered Nanomaterial Generation System (VENGES). A coating reactor was added in which core ENMs are coated in-flight by the swirl injection of hexamethyldisoloxane (HMDSO) vapor. The HMDSO vapor concentration can be adjusted to precisely control the thickness of the coating. We first demonstrate the versatility of the proposed SiO2-coating process by applying it to several ENMs (CeO2, Fe2O3, ZnO, Ag) marked by their prevalence in consumer products as well as their range in toxicity. We quantified the thickness of the SiO2-coating (TEM, XPS), evaluated its efficiency (Chemisorption) and its effect on the core material structure, composition and morphology (XRD, BET, and TEM). Furthermore, we examined the mobility and aggregation potential of SiO2-coated and uncoated ENMs in DI-water and biological media using dynamic light scattering (DLS), and compared to those of pure silica nanoparticles. Finally, we provide valuable in-vitro toxicological evidence for the safety of this novel formulation concept by evaluating the relative toxicity of SiO2-coated vs. uncoated ENMs using a number of cellular assays (MTT, LDH, fluorescence Live/Dead) and several cell-lines (A549 cancer alveolar epithelial cells and THP-1 macrophages). Our results show that the proposed method can be used to effectively and uniformly coat flame generated ENMs with a nanothin layer of amorphous SiO2 that significantly reduces their toxicological effects. This scalable method can be applied in nanomanufacturing of nanomaterials developing safer by design nanoparticles.
3. Title: Acute inhalation study of realistic nano scale CeO2 using the Harvard VENGES toxicological platform
Investigator: George Pyrgiotakis, Samuel Gass, William Goldsmith, David Frazer, Jane Ma, Walter McKinney, Mark Barger, Bridget Dolash, Vincent Castranova, Philip Demokritou
AbstractCeria nanoparticles are increasingly used for a number of industrial and commercial applications including catalysis, chemical mechanical polishing, UV-shielding, and nanocomposites. As the number of consumers and factory workers exposed to CeO2 nanoparticles increases, the need for a comprehensive toxicological characterization is pressing. While most in-vitro models predict minimal toxicity for nanosized CeO2, preliminary in-vivo animal models using instillation of CeO2 nanoparticles point to fibrogenicity and inflamation. However, to date, most toxicological evidence is limited to in-vitro studies, intratracheal instillation studies which however, do not represent realistic nanoscale exposure scenarios. Here, we present the first ever whole-body systematic animal inhalation study of nano-CeO2. In addition, the use of a nanothin amorphous SiO2 coating as means of mitigating CeO2 toxicity was evaluated as a safer formulation concept. CeO2 (uncoated and SiO2-coated) nanoparticles were synthesized using the Harvard Versatile Engineered Nanomaterial Generating System (VENGES), which enables the synthesis and coating of industrially relevant nanoparticles in the aerosol phase with precise control over primary particle size, aggregation, and aerosol concentration. The generated aerosol was diluted and introduced into a customized exposure chamber, that is fully automated and maintain very stable exposure conditions. The generated CeO2 particles (SiO2 coated and uncoated) were characterized (1) in-situ with respect to aerosol size distribution and number concentration (SMPS), aggregate morphology (TEM, SEM) and (2) ex-situ with respect to crystallinity and chemical composition (XRD, XPS, EDX), surface area (BET), and morphology (TEM, SEM). Exposure atmospheres in the chamber were monitored in real time and characterized with respect to particle number concentration as a function of size (CPC, SMPS), mass concentration (Gravimetric Filter Measurements),aerosol mass size distribution (MOUDI), temperature, humidity, CO, CO2, (Q-Track) and NOx concentrations. Sprague Dawley rats (n=12/group) were exposed to either coated or uncoated CeO2 (2.7 mg/m3, 2 h/day, 4 days). Exposed animals, along with particle free- controls, were sacrificed at either 1 or 84 days post exposure. Pathophysiological analysis was performed and inflammatory and cytotoxic biomarkers were measured in the bronchoalveolar lung lavage (BAL) of the animals. Preliminary results showed that CeO2 is associated with lung inflammation and cytotoxicity as demonstrated by elevated PMN and LDH levels in the BAL fluid. In addition, SiO2 coatings revealed a significant reduction of toxicity, a clear indication of the effectiveness of this safe by design concept.
4. Title: A novel method for bacteria inactivation using Engineered Water Nanostructures.
Investigator: Georgios Pyrgiotakis, James McDevitt, Toshiyuki Yamauchi and Philip Demokritou
AbstractThe burden of infectious disease worldwide, related to contamination via contact with contaminated surfaces (fomites) and inhalation, is a growing issue. Apart from hospitals, the problem has also become a growing liability at places where food is prepared and handled. Herein a novel nanotechnology based method for microbial disinfection that utilizes the formation of unique Engineered Water Nanostructures (EWNS) generated via the electrospraying of water is presented. Electropsray is a method widely, used to generate aerosols . The objectives of this work are twofold: 1) showcase the proof of concept that these EWNS can be potentially used for the inactivation of pathogens from both surfaces and in the air; 2) Characterize the physico-chemical and morphological properties of EWNS and understand their formation and transport mechamisms. Bacteria Inactivation on surfaces: The inactivation of bacteria on surfaces by the EWNS was assessed both quantitatively and qualitatively. Different types of bacteria were used in order to cover a wide range of potential applications including Serratia Marcescens (gram-negative), Staphylococcus Aureus (gram-positive) and Bacillus Atrophaeus (spore forming). For the Serratia Marcescens the results showed that there is more than a 2-log10 reduction in 90 minutess of exposure. Similarly, the results for Staphylococcus Aureus showed nearly an 1-log10 reduction for same dose. The spore forming bacteria although exposed to the EWNS for 24 hours were not affected. Air disinfection: Serratia Marcescens was aerosolized in an environmental chamber and mixed with a controlled concentration EWNS aerosol. The potential of the EWNS to inactivate bacteria in the air was evaluated using a culture system approach under steady state and decay scenarios. The bioaerosol experimets showed the ability of EWNS to deactivate in a dose dependent matter, the suspended in the air Serratia Marcescens bacteria, by achieving 50% reduction at steady state and complete removal at 45 min under the decay scenario. EWNS synthesis and properties: The synthesis process and the properties of the generated EWNS, including size distribution, charge and reactive oxygen species were assessed. The size and particle charge were measure using atomic force microscopy (AFM)and an electrometer, respectively. Our results show that the EWNS have a size of approximately 25 nm, which is stable over time (hours), and carry an average charge of 10 electrons per particle. EPR was utilized to characterize the present of chemical species and showed that the EWNS are loaded with primarily with OH• radicals and secondary with O2•.
5. Title: Wound healing of human corneal epithelial cells is impacted by nanoparticles
Investigator :Enhua H Zhou, Richard Pizzo, Christa Watson, Joel Cohen, Quynh Dang, Georgios Pyrgiotakis, Joseph D. Brain, Jeffrey J Fredberg, Philip Demokritou
AbstractThe eye surface is a major site of environmental exposure and resulting irritation. However, there is a lack of understanding of the effects of engineered nanoparticles (ENPs) on the epithelium of the eye. Here we wanted to explore whether ENPs can impede the wound healing of a corneal epithelium. We developed a novel wound healing assay and focused on cells from human cornea. We tested a panel of well characterized, industrially relevant ENPs, including several commercially available ENPs and metals and metal oxides generated using our recently developed Versatile Engineered Nanomaterial Generation System (VENGES). Taken together, these studies provide a novel in-vitro model system for evaluating the physiological impact of nanoparticles on the ocular surface.
6. Title: Synthesis and Characterization of Zno and SiO2 coated ZnO Nanoparticles for Biological Applications
Investigator :Kimberly M. Murdaugh, Evelyn Hu, Joseph Brain, Philip Demokriou
AbstractZnO nanoparticles have emerged as a useful vehicle to study biointeractions as they have fluorescence properties that allow real-time tracking with confocal microscopy. However, they are highly soluble in aqueous solutions and the released ions have significant toxic implications. We present a method of using the recently developed, flame spray pyrolysis based, Harvard Versatile Engineered Nanomaterial Generation System (VENGES) to generate ZnO nanoparticles. VENGES platform s was used to synthesize ZnO nanoparticles of various particle sizes, with and without a hermetic coating of amorphous silica (SiO2).
To confirm that ZnO particles were successfully coated with a nanothin layer of SiO2, highly surface sensitive X-Ray Photoelectron Spectroscopy (XPS) was used. The crystal size was determined by X-ray diffraction, and the nanoparticles were imaged with TEM. In addition, ZnO and ZnO/SiO2 nanoparticles were dispersed and characterized in DI H2O and Survanta dispersions. Dynamic light scattering was performed to characterize the hydrodynamic diameter and zeta potential of the solutions. The effect of the SiO2 coating on the UV absorption and fluorescence was also examined.
XPS data indicate that the ZnO particles are hermetically coated with SiO2. XRD analysis indicates that the particles range from approximately 18 nm to 30 nm. Additionally, preliminary evidence suggests that the ZnO and ZnO/SiO2 nanoparticles have the same absorbance and fluorescence properties; this result is a promising indication that the SiO2 coatings doesn’t interfere with functional properties of core nanoparticles. Given the fact that amorphous SiO2 is considered a biologically inert material, this concept can also be used to reduce the toxicological effects of ZnO nanoparticles for a wide range of applications. Future animal instillation studies s will be performed to examine the pharmacokinetics and toxicity of the ZnO and ZnO/SiO2 nanoparticles.