Huiguang Zhu

Antioxidant Properties of Cerium Oxide Nanocrystals as a Function of Nanocrystal Diameter and Surface Coating

This work examines the effect of nanocrystal diameter and surface coating on the reactivity of cerium oxide nanocrystals with H2O2 both in chemical solutions and in cells. Monodisperse nanocrystals were formed in organic solvents from the decomposition of cerium precursors, and subsequently phase transferred into water using amphiphiles as nanoparticle coatings. Quantitative analysis of the antioxidant capacity of CeO2-x using gas chromatography and a luminol test revealed that 2 mol of H2O2 reacted with every mole of cerium(III), suggesting that the reaction proceeds via a Fenton-type mechanism. Smaller diameter nanocrystals containing more cerium(III) were found to be more reactive toward H2O2. Additionally, the presence of a surface coating did not preclude the reaction between the nanocrystal surface cerium(III) and hydrogen peroxide. Taken together, the most reactive nanoparticles were the smallest (e.g., 3.8 nm diameter) with the thinnest surface coating (e.g., oleic acid). Moreover, a benchmark test of their antioxidant capacity revealed these materials were 9 times more reactive than commercial antioxidants such as Trolox. A unique feature of these antioxidant nanocrystals is that they can be applied multiple times: over weeks, cerium(IV) rich particles slowly return to their starting cerium(III) content. In nearly all cases, the particles remain colloidally stable (e.g., nonaggregated) and could be applied multiple times as antioxidants. These chemical properties were also observed in cell culture, where the materials were able to reduce oxidative stress in human dermal fibroblasts exposed to H2O2 with efficiency comparable to their solution phase reactivity. These data suggest that organic coatings on cerium oxide nanocrystals do not limit the antioxidant behavior of the nanocrystals, and that their redox cycling behavior can be preserved even when stabilized.

Bilayers as phase transfer agents for nanocrystals prepared in nonpolar solvents.

The effective water dispersion of highly uniform nanoparticles synthesized in organic solvents is a major issue for their broad applications. In an effort to overcome this problem, iron oxide and cadmium selenide nanocrystals were surrounded by lipid bilayers to create stable, aqueous dispersions. The core inorganic particles were originally generated in oleic acid and 1-octadecene. When these organic solutions were mixed with water and a sparing amount of excess fatty acid, up to 70% of the nanoparticles transferred into the aqueous phase. This simple approach was applied to two different nanocrystal types, and nanocrystal diameters ranging from 5 to 15 nm. In all cases, the resulting materials were stable, nonaggregated suspensions that retained their original magnetic and optical properties. The phase transfer efficiency is maximum when very little oleic acid is added (e.g. 0.2 w/w %). At higher concentrations, above the critical micelle concentration, the formation of micelles begins to compete with bilayer generation leading to less effective phase transfer. Unlike other approaches for water dispersion that rely on amphiphiles with significant water solubility, the fatty acids used in this work are only sparingly soluble in water. As a result, there is minimal dynamic exchange between free and bound surface agents and the resulting aqueous solutions contain little residual free organic carbon. Thermogravimetric analysis (TGA) confirmed the presence of bilayers around the nanocrystal cores. The particle size, size distribution, process yield, and colloidal stability were found using a suite of methods including transmission electron microscopy, small angle X-ray scattering, dynamic light scattering, inductively coupled plasma-optical emission spectroscopy, and ultraviolet-visible spectroscopy. Bilayer-nanocrystal complexes possess many of the same size-dependent features as the original materials, and as such offer new avenues for exploring and exploiting the interface between nanocrystals and biology.

Toxicity of Quantum Dots and Cadmium Salt to Caenorhabditis elegans after Multigenerational Exposure

To fully understand the biological and environmental impacts of nanomaterials requires studies that address both sublethal end points and multigenerational effects. Here, we use a nematode to examine these issues as they relate to exposure to two different types of quantum dots, core (CdSe) and core-shell (CdSe/ZnS), and to compare the effect to those observed after cadmium salt exposures. The strong fluorescence of the core-shell QDs allowed for the direct visualization of the materials in the digestive track within a few hours of exposure. Multiple end points, including both developmental and locomotive, were examined at QD exposures of low (10 mg/L Cd), medium (50 mg/L Cd), and high concentrations (100 mg/L Cd). While the core-shell QDs showed no effect on fitness (lifespan, fertility, growth, and three parameters of motility behavior), the core QDs caused acute effects similar to those found for cadmium salts, suggesting that biological effects may be attributed to cadmium leaching from the more soluble QDs. Over multiple generations, we commonly found that for lower life-cycle exposures to core QDs the parents response was generally a poor predictor of the effects on progeny. At the highest concentrations, however, biological effects found for the first generation were commonly similar in magnitude to those found in future generations.

Cellular and Transcriptional Response of Pseudomonas stutzeri to Quantum Dots under Aerobic and Denitrifying Conditions

Pseudomonas stutzeri was exposed to quantum dots (QDs) with three different surface coatings (anionic polymaleic anhydride-alt-1-octadecene (PMAO), cationic polyethylenimine (PEI), and carboxyl QDs) under both aerobic and anaerobic (denitrifying) conditions. Under aerobic conditions, toxicity (assessed per growth inhibition) increased from PMAO to carboxyl to PEI QDs. The positive charge of PEI facilitated direct contact with negatively charged bacteria, which was verified by TEM analysis. Both PMAO and PEI QDs hindered energy transduction (indicated by a decrease in cell membrane potential), and this effect was most pronounced with PEI QDs under denitrifying conditions. Up-regulation of denitrification genes (i.e., nitrate reductase narG, periplasmic nitrate reductase napB, nitrite reductase nirH, and NO reductase norB) occurred upon exposure to subinhibitory PEI QD concentrations (1 nM). Accordingly, denitrification activity (assessed per respiratory nitrate consumption in the presence of ammonia) increased during sublethal PEI QD exposure. However, cell viability (including denitrification) was hindered at 10 nM or higher PEI QD concentrations. Efflux pump genes czcB and czcC were induced by PEI QDs under denitrifying conditions, even though Cd and Se dissolution from QDs did not reach toxic levels (exposure was at pH 7 to minimize hydrolysis of QD coatings and the associated release of metal constituents). Up-regulation of the superoxide dismutase (stress) gene sodB occurred only under aerobic conditions, likely due to intracellular production of reactive oxygen species (ROS). The absence of ROS under denitrifying conditions suggests that the antibacterial activity of QDs was not due to ROS production alone. Overall, this work forewarns about unintended potential impacts to denitrification as a result of disposal and incidental releases of QDs, especially those with positively charged coatings (e.g., PEI QDs).

Quantification of water solubilized CdSe/ZnS quantum dots in Daphnia magna.

The relative transparency of Daphnia magna (daphnia) and the unique optical properties of quantum dots (QDs) were paired to study the accumulation potential and surface coating effects on uptake of amphiphilic polymer coated CdSe/ZnS QDs. Fluorescence confocal laser scanning microscopy was used to visualize and spectrally distinguish QDs from competing autofluorescent signals arising from the daphnia themselves and their food sources. QDs were found to accumulate within the digestive tracts of daphnia, as well as, in some cases, adhere to the carapace, antennae, and thoracic appendages. After 48 h of gut clearance with and without feeding, QD fluorescence signal was still apparent in the digestive tracts of daphnia, and inductively coupled plasma mass spectrometry (ICP-MS) measurements confirmed that 36-53% of the initial uptake was retained. As surface charge and pegylation can influence the uptake of nanoparticles, uptake of QDs coated with two different amphililic polymers and their polyethylene glycol (PEG) coated counterparts was also examined. Fluorescence microscopy and ICP-MS measurements revealed differences in uptake after 24 h of exposure which were attributed to particle surface coating and stability.

Fluorescence Reports Intact Quantum Dot Uptake into Roots and Translocation to Leaves of Arabidopsis thaliana and Subsequent Ingestion by Insect Herbivores

We explored the impact of quantum dot (QD) coat characteristics on NP stability, uptake, and translocation in Arabidopsis thaliana, and subsequent transfer to primary consumers, Trichoplusia ni (T. ni). Arabidopsis was exposed to CdSe/CdZnS QDs with three different coatings: Poly(acrylic acid-ethylene glycol) (PAA-EG), polyethylenimine (PEI) and poly(maleic anhydride-alt-1-octadecene)-poly(ethylene glycol) (PMAO-PEG), which are anionic, cationic, and relatively neutral, respectively. PAA-EG-coated QDs were relatively stable and taken up from a hydroponic medium through both Arabidopsis leaf petioles and roots, without apparent aggregation, and showed generally uniform distribution in leaves. In contrast, PEI- and PMAO-PEG-coated QDs displayed destabilization in the hydroponic medium, and generated particulate fluorescence plant tissues, suggesting aggregation. PAA-EG QDs moved faster than PEI QDs through leaf petioles; however, 8-fold more cadmium accumulated in PEI QD-treated leaves than in those exposed to PAA-EG QDs, possibly due to PEI QD dissolution and direct metal uptake. T. ni caterpillars that fed on Arabidopsis exposed to QDs had reduced performance, and QD fluorescence was detected in both T. ni bodies and frass, demonstrating trophic transfer of intact QDs from plants to insects. Overall, this paper demonstrates that QD coat properties influence plant nanoparticle uptake and translocation and can impact transfer to herbivores.

Measuring the grafting density of nanoparticles in solution by analytical ultracentrifugation and total organic carbon analysis.

Many of the solution phase properties of nanoparticles, such as their colloidal stability and hydrodynamic diameter, are governed by the number of stabilizing groups bound to the particle surface (i.e., grafting density). Here, we show how two techniques, analytical ultracentrifugation (AUC) and total organic carbon analysis (TOC), can be applied separately to the measurement of this parameter. AUC directly measures the density of nanoparticle-polymer conjugates while TOC provides the total carbon content of its aqueous dispersions. When these techniques are applied to model gold nanoparticles capped with thiolated poly(ethylene glycol), the measured grafting densities across a range of polymer chain lengths, polymer concentrations, and nanoparticle diameters agree to within 20%. Moreover, the measured grafting densities correlate well with the polymer content determined by thermogravimetric analysis of solid conjugate samples. Using these tools, we examine the particle core diameter, polymer chain length, and polymer solution concentration dependence of nanoparticle grafting densities in a gold nanoparticle-poly(ethylene glycol) conjugate system.

Relative susceptibility and transcriptional response of nitrogen cycling bacteria to quantum dots.

Little is known about the potential impacts of accidental or incidental releases of manufactured nanomaterials to microbial ecosystem services (e.g., nutrient cycling). Here, quantum dots (QDs) coated with cationic polyethylenimine (PEI) were more toxic to pure cultures of nitrogen-cycling bacteria than QDs coated with anionic polymaleic anhydride-alt-1-octadecene (PMAO). Nitrifying bacteria (i.e., Nitrosomonas europaea) were much more susceptible than nitrogen fixing (i.e., Azotobacter vinelandii, Rhizobium etli, and Azospirillum lipoferum) and denitrifying bacteria (i.e., Pseudomonas stutzeri). Antibacterial activity was mainly exerted by the QDs rather than by their organic coating or their released QD components (e.g., Cd and Zn), which under the near-neutral pH tested (to minimize QD weathering) were released into the bacterial growth media at lower levels than their minimum inhibitory concentrations. Sublethal exposure to QDs stimulated the expression of genes associated with nitrogen cycling. QD-PEI (10 nM) induced three types of nitrogenase genes (nif, anf, and vnf) in A. vinelandii, and one ammonia monooxygenase gene (amoA) in N. europaea was up-regulated upon exposure to 1 nM QD-PEI. We previously reported up-regulation of denitrification genes in P. stutzeri exposed to low concentrations of QD-PEI. (1) Whether this surprising stimulation of nitrogen cycling activities reflects the need to generate more energy to overcome toxicity (in the case of nitrification or denitrification) or to synthesize organic nitrogen to repair or replace damaged proteins (in the case of nitrogen fixation) remains to be determined.

Low temperature synthesis of ZnS and CdZnS shells on CdSe quantum dots

Methods for synthesizing quantum dots generally rely on very high temperatures to both nucleate and grow core and core-shell semiconductor nanocrystals. In this work, we generate highly monodisperse ZnS and CdZnS shells on CdSe semiconductor nanocrystals at temperatures as low as 65 degrees C by enhancing the precursor solubility. Relatively small amounts of trioctylphosphine and trioctylphosphine oxide have marked effects on the solubility of the metal salts used to form shells; their inclusion in the precursor solutions, which use thiourea as a sulfur source, can lead to homogeneous and fully dissolved solutions. Upon addition to suspensions of quantum dot cores, these precursors deposit as uniform shells; the lowest temperature for shell growth (65 degrees C) yields the thinnest shells (d < 1 nm) while the same process at higher temperatures (180 degrees C) forms thicker shells (d approximately 1-2 nm). The growth of the shell structures, average particle size, size distribution, and shape were examined using optical spectroscopy, transmission electron microscopy, x-ray diffraction, and transmittance small angle x-ray scattering. The photoluminescence quantum yield (QY) of the as-prepared CdSe/ZnS quantum dots ranged from 26% to 46% as compared to 10% for the CdSe cores. This method was further generalized to CdZnS shells by mixing cadmium and zinc acetate precursors. The CdSe/CdZnS nanocrystals have a thicker shell and higher QY (40% versus 36%) as compared to the CdSe/ZnS prepared under similar conditions. These low temperature methods for shell growth are readily amenable to scale-up and can provide a route for economical and less energy intensive production of quantum dots.

Small and stable phosphorylcholine zwitterionic quantum dots for weak nonspecific phagocytosis and effective Tat peptide functionalization.

Facile surface modification of quantum dots (QDs) to make them water-soluble, small, stable, antibiofouling, and functional is crucial for their biological applications. This study demonstrates a simple ligand-exchange reaction to convert hydrophobic CdSe/ZnS QDs into water-soluble QDs using amphiphilic, zwitterionic 11-mercaptoundecylphosphorylcholine (HS-PC). The phosphorylcholine (PC)-modified QDs (QD-PC) possess several advantages, such as small hydrodynamic diameter, good resistance to pH variations and high salinity, excellent stability in 100% human plasma, and low protein adsorption. Importantly, the PC modification endows the QDs with very low, nonspecific interaction with cells, and strongly minimizes nonspecific phagocytosis of QDs by macrophages. In addition, cell penetrating Tat peptide functionalized QDs can be easily produced by mixing Tat with HS-PC with various ratios, which is proved to effectively enhance QD ability to enter cells and accumulate around perinuclear region. Compared to traditional mercaptoundecanoic acid (MUA) modification, PC modification not only makes the cell penetrating QDs more stable and brighter, but also provides the Tat- and PC-conjugated QDs with much lower nonspecific phagocytic uptake than the Tat- and MUA-conjugated ones. This research will provide insights into designing suitable ligands for surface modification of QDs and improving biofunctional QD performance in biological applications.