Sijie Lin

Use of Metal Oxide Nanoparticle Band Gap To Develop a Predictive Paradigm for Oxidative Stress and Acute Pulmonary Inflammation

We demonstrate for 24 metal oxide (MOx) nanoparticles that it is possible to use conduction band energy levels to delineate their toxicological potential at cellular and whole animal levels. Among the materials, the overlap of conduction band energy (E(c)) levels with the cellular redox potential (-4.12 to -4.84 eV) was strongly correlated to the ability of Co(3)O(4), Cr(2)O(3), Ni(2)O(3), Mn(2)O(3), and CoO nanoparticles to induce oxygen radicals, oxidative stress, and inflammation. This outcome is premised on permissible electron transfers from the biological redox couples that maintain the cellular redox equilibrium to the conduction band of the semiconductor particles. Both single-parameter cytotoxic as well as multi-parameter oxidative stress assays in cells showed excellent correlation to the generation of acute neutrophilic inflammation and cytokine responses in the lungs of C57 BL/6 mice. Co(3)O(4), Ni(2)O(3), Mn(2)O(3), and CoO nanoparticles could also oxidize cytochrome c as a representative redox couple involved in redox homeostasis. While CuO and ZnO generated oxidative stress and acute pulmonary inflammation that is not predicted by E(c) levels, the adverse biological effects of these materials could be explained by their solubility, as demonstrated by ICP-MS analysis. These results demonstrate that it is possible to predict the toxicity of a large series of MOx nanoparticles in the lung premised on semiconductor properties and an integrated in vitro/in vivo hazard ranking model premised on oxidative stress. This establishes a robust platform for modeling of MOx structure-activity relationships based on band gap energy levels and particle dissolution. This predictive toxicological paradigm is also of considerable importance for regulatory decision-making about this important class of engineered nanomaterials.

Uptake, translocation, and transmission of carbon nanomaterials in rice plants.

Recent development of nanotechnology has reshaped the landscape of modern science and technology, while in the meantime raised concerns about the adverse effects of nanomaterials on biological systems and the environment. Owing to their mutual interaction, carbon-based nanomaterials readily aggregate and are not considered potential contaminants in the liquid phase. However, when discharged into the environment, the hydrophobicity of nanomaterials can be averted through their interaction with natural organic matter (NOM), a heterogeneous mixture of decomposed animals and plants and a major pollutant carrier in nature. Consequently, mobile NOM-modified nanomaterials may pose a threat to ecological terrestrial species through further physical, chemical, and biological processes. The impact of nanomaterials on high plants has scantly been examined in the current literature. Among the studies available, none have used major food crops or carbon nanoparticles (a major class of nanomaterials) for their evaluations. Although both enhanced and inhibited growth have been reported for vegetations exposed to nanomaterials at various developmental stages, including seed germina-

Codelivery of an optimal drug/siRNA combination using mesoporous silica nanoparticles to overcome drug resistance in breast cancer in vitro and in vivo.

We used a multifunctional mesoporous silica nanoparticle (MSNP) carrier to overcome doxorubicin (Dox) resistance in a multidrug resistant (MDR) human breast cancer xenograft by codelivering Dox and siRNA that targets the P-glycoprotein (Pgp) drug exporter. The Pgp siRNA selection from among a series of drug resistance targets was achieved by performing high throughput screening in a MDR breast cancer cell line, MCF-7/MDR. Following the establishment of a MCF-7/MDR xenograft model in nude mice, we demonstrated that a 50 nm MSNP, functionalized by a polyethyleneimine-polyethylene glycol (PEI-PEG) copolymer, provides protected delivery of stably bound Dox and Pgp siRNA to the tumor site. The effective biodistribution and reduced reticuloendothelial uptake, as a result of our nanocarrier design, allowed us to achieve an 8% enhanced permeability and retention effect at the tumor site. Compared to free Dox or the carrier loaded with either drug or siRNA alone, the dual delivery system resulted in synergistic inhibition of tumor growth in vivo. Analysis of multiple xenograft biopsies demonstrated significant Pgp knockdown at heterogeneous tumor sites that correspond to the regions where Dox was released intracellularly and induced apoptosis. We emphasize that the heterogeneity originates in the tumor microenvironment, which influences the vascular access, rather than heterogeneous Pgp expression in the MDR cells. Taken together, these data provide proof-of-principle testing of the use of a dual drug/siRNA nanocarrier to overcome Dox resistance in a xenograft. The study also provides the first detailed analysis of the impact of heterogeneity in the tumor microenvironment on the efficacy of siRNA delivery in vivo.

Use of a High Throughput Screening Approach Coupled With In Vivo Zebrafish Embryo Screening to Develop Hazard Ranking for Engineered Nanomaterials

Because of concerns about the safety of a growing number of engineered nanomaterials (ENM), it is necessary to develop high-throughput screening and in silico data transformation tools that can speed up in vitro hazard ranking. Here, we report the use of a multiparametric, automated screening assay that incorporates sublethal and lethal cellular injury responses to perform high-throughput analysis of a batch of commercial metal/metal oxide nanoparticles (NP) with the inclusion of a quantum dot (QD1). The responses chosen for tracking cellular injury through automated epifluorescence microscopy included ROS production, intracellular calcium flux, mitochondrial depolarization, and plasma membrane permeability. The z-score transformed high volume data set was used to construct heat maps for in vitro hazard ranking as well as showing the similarity patterns of NPs and response parameters through the use of self-organizing maps (SOM). Among the materials analyzed, QD1 and nano-ZnO showed the most prominent lethality, while Pt, Ag, SiO2, Al2O3, and Au triggered sublethal effects but without cytotoxicity. In order to compare the in vitro with the in vivo response outcomes in zebrafish embryos, NPs were used to assess their impact on mortality rate, hatching rate, cardiac rate, and morphological defects. While QDs, ZnO, and Ag induced morphological abnormalities or interfered in embryo hatching, Pt and Ag exerted inhibitory effects on cardiac rate. Ag toxicity in zebrafish differed from the in vitro results, which is congruent with this materials designation as extremely dangerous in the environment. Interestingly, while toxicity in the initially selected QD formulation was due to a solvent (toluene), supplementary testing of additional QDs selections yielded in vitro hazard profiling that reflect the release of chalcogenides. In conclusion, the use of a high-throughput screening, in silico data handling and zebrafish testing may constitute a paradigm for rapid and integrated ENM toxicological screening.

Decreased Dissolution of ZnO by Iron Doping Yields Nanoparticles with Reduced Toxicity in the Rodent Lung and Zebrafish Embryos

We have recently shown that the dissolution of ZnO nanoparticles and Zn(2+) shedding leads to a series of sublethal and lethal toxicological responses at the cellular level that can be alleviated by iron doping. Iron doping changes the particle matrix and slows the rate of particle dissolution. To determine whether iron doping of ZnO also leads to lesser toxic effects in vivo, toxicity studies were performed in rodent and zebrafish models. First, we synthesized a fresh batch of ZnO nanoparticles doped with 1-10 wt % of Fe. These particles were extensively characterized to confirm their doping status, reduced rate of dissolution in an exposure medium, and reduced toxicity in a cellular screen. Subsequent studies compared the effects of undoped to doped particles in the rat lung, mouse lung, and the zebrafish embryo. The zebrafish studies looked at embryo hatching and mortality rates as well as the generation of morphological defects, while the endpoints in the rodent lung included an assessment of inflammatory cell infiltrates, LDH release, and cytokine levels in the bronchoalveolar lavage fluid. Iron doping, similar to the effect of the metal chelator, DTPA, interfered in the inhibitory effects of Zn(2+) on zebrafish hatching. In the oropharyngeal aspiration model in the mouse, iron doping was associated with decreased polymorphonuclear cell counts and IL-6 mRNA production. Doped particles also elicited decreased heme oxygenase 1 expression in the murine lung. In the intratracheal instillation studies in the rat, Fe doping was associated with decreased polymorphonuclear cell counts, LDH, and albumin levels. All considered, the above data show that Fe doping is a possible safe design strategy for preventing ZnO toxicity in animals and the environment.

Aspect Ratio Determines the Quantity of Mesoporous Silica Nanoparticle Uptake by a Small GTPase-Dependent Macropinocytosis Mechanism

Although the aspect ratio (AR) of engineered nanomaterials (ENMs) is one of the key physicochemical parameters that could determine biological outcome, not much is understood about how AR contributes to shaping biological outcome. By using a mesoporous silica nanoparticle (MSNP) library that has been constructed to cover a range of different lengths, we could demonstrate that the AR of rod-shaped particles determines the rate and abundance of MSNP uptake by a macropinocytosis process in HeLa and A549 cancer cell lines. MSNPs with an AR of 2.1-2.5 were taken up in larger quantities compared to shorter or longer length rods by a process that is sensitive to amiloride, cytochalasin D, azide, and 4 °C inhibition. The rods with intermediary AR also induced the maximal number of filopodia, actin polymerization, and activation of small GTP-binding proteins (e.g., Rac1, CDC42) that involve assembly of the actin cytoskeleton and filopodia formation. When assessing the role of AR in the delivery of paclitaxel or camptothecin, the rods with AR 2.1-2.5 were clearly more efficient for drug delivery and generation of cytotoxic killing in HeLa cells. All considered, our data suggest an active sensoring mechanism by which HeLa and A549 cells are capable of detecting AR differences in MSNP to the extent that accelerated macropinocytosis can be used to achieve more efficient drug delivery.

Use of Coated Silver Nanoparticles to Understand the Relationship of Particle Dissolution and Bioavailability to Cell and Lung Toxicological Potential

Since more than 30% of consumer products that include engineered nanomaterials contain nano-Ag, the safety of this material is of considerable public concern. In this study, Ag nanoparticles (NPs) are used to demonstrate that 20 nm polyvinylpyrrolidone (PVP or P) and citrate (C)-coated Ag NPs induce more cellular toxicity and oxidative stress than larger (110 nm) particles due to a higher rate of dissolution and Ag bioavailability. Moreover, there is also a higher propensity for citrate 20 nm (C20) nanoparticles to generate acute neutrophilic inflammation in the lung and to produce chemokines compared to C110. P110 has less cytotoxic effects than C110, likely due to the ability of PVP to complex released Ag(+) . In contrast to the more intense acute pulmonary effects of C20, C110 induces mild pulmonary fibrosis at day 21, likely as a result of slow but persistent Ag(+) release leading to a sub-chronic injury response. Interestingly, the released metallic Ag is incorporated into the collagen fibers depositing around airways and the lung interstitium. Taken together, these results demonstrate that size and surface coating affect the cellular toxicity of Ag NPs as well as their acute versus sub-chronic lung injury potential.

Dispersal State of Multiwalled Carbon Nanotubes Elicits Profibrogenic Cellular Responses That Correlate with Fibrogenesis Biomarkers and Fibrosis in the Murine Lung

We developed a dispersal method for multiwalled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on profibrogenic responses in tissue culture cells and in mouse lung. We demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA, and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells, and alveolar macrophages (AM) and induced more prominent TGF-β1 and IL-1β production in vitro and TGF-β1, IL-1β, and PDGF-AA production in vivo than nondispersed tubes. Moreover, there was good agreement between the profibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bioprocessed by AM and induced less prominent collagen deposition at sites of nongranulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects profibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

High Content Screening in Zebrafish Speeds up Hazard Ranking of Transition Metal Oxide Nanoparticles

Zebrafish is an aquatic organism that can be used for high content safety screening of engineered nanomaterials (ENMs). We demonstrate, for the first time, the use of high content bright-field and fluorescence-based imaging to compare the toxicological effect of transition metal oxide (CuO, ZnO, NiO, and Co(3)O(4)) nanoparticles in zebrafish embryos and larvae. High content bright-field imaging demonstrated potent and dose-dependent hatching interference in the embryos, with the exception of Co(3)O(4) which was relatively inert. We propose that the hatching interference was due to the shedding of Cu and Ni ions, compromising the activity of the hatching enzyme, ZHE1, similar to what we previously proposed for Zn(2+). This hypothesis is based on the presence of metal-sensitive histidines in the catalytic center of this enzyme. Co-introduction of a metal ion chelator, diethylene triamine pentaacetic acid (DTPA), reversed the hatching interference of Cu, Zn, and Ni. While neither the embryos nor larvae demonstrated morphological abnormalities, high content fluorescence-based imaging demonstrated that CuO, ZnO, and NiO could induce increased expression of the heat shock protein 70:enhanced green fluorescence protein (hsp70:eGFP) in transgenic zebrafish larvae. Induction of this response by CuO required a higher nanoparticle dose than the amount leading to hatching interference. This response was also DTPA-sensitive. We demonstrate that high content imaging of embryo development, morphological abnormalities, and HSP70 expression can be used for hazard ranking and determining the dose-response relationships leading to ENM effects on the development of the zebrafish embryo.

Differential Uptake of Carbon Nanoparticles by Plant and Mammalian Cells

Consequently, a body of literature over the past decade hasbeen centered on cytotoxicity, genotoxicity, and ecotoxicity ofnanomaterials, pointing to the general understanding thatnanotoxicity is often derived from the physiochemicalproperties of the nanomaterials and their interplay with thehost environment.