Zhaoxia Ji

Stability and Aggregation of Metal Oxide Nanoparticles in Natural Aqueous Matrices

There is a pressing need for information on the mobility of nanoparticles in the complex aqueous matrices found in realistic environmental conditions. We dispersed three different metal oxide nanoparticles (TiO(2), ZnO and CeO(2)) in samples taken from eight different aqueous media associated with seawater, lagoon, river, and groundwater, and measured their electrophoretic mobility, state of aggregation, and rate of sedimentation. The electrophoretic mobility of the particles in a given aqueous media was dominated by the presence of natural organic matter (NOM) and ionic strength, and independent of pH. NOM adsorbed onto these nanoparticles significantly reduces their aggregation, stabilizing them under many conditions. The transition from reaction to diffusion limited aggregation occurs at an electrophoretic mobility from around -2 to -0.8 microm s(-1) V(-1) cm. These results are key for designing and interpreting nanoparticle ecotoxicity studies in various environmental conditions.

Engineered Design of Mesoporous Silica Nanoparticles to Deliver Doxorubicin and P-Glycoprotein siRNA to Overcome Drug Resistance in a Cancer Cell Line

Overexpression of drug efflux transporters such as P-glycoprotein (Pgp) protein is one of the major mechanisms for multiple drug resistance (MDR) in cancer cells. A new approach to overcome MDR is to use a co-delivery strategy that utilizes a siRNA to silence the expression of efflux transporter together with an appropriate anticancer drug for drug resistant cells. In this paper, we report that mesoporous silica nanoparticles (MSNP) can be functionalized to effectively deliver a chemotherapeutic agent doxorubicin (Dox) as well as Pgp siRNA to a drug-resistant cancer cell line (KB-V1 cells) to accomplish cell killing in an additive or synergistic fashion. The functionalization of the particle surface with a phosphonate group allows electrostatic binding of Dox to the porous interior, from where the drug could be released by acidification of the medium under abiotic and biotic conditions. In addition, phosphonate modification also allows exterior coating with the cationic polymer, polyethylenimine, which endows the MSNP to contemporaneously deliver Pgp siRNA. The dual delivery of Dox and siRNA in KB-V1 cells was capable of increasing the intracellular as well as intranuclear drug concentration to levels exceeding that of free Dox or the drug being delivered by MSNP in the absence of siRNA codelivery. These results demonstrate that it is possible to use the MSNP platform to effectively deliver a siRNA that knocks down gene expression of a drug exporter that can be used to improve drug sensitivity to a chemotherapeutic agent.

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.

Use of a Rapid Cytotoxicity Screening Approach to Engineer a Safer Zinc Oxide Nanoparticle through Iron Doping

The establishment of verifiably safe nanotechnology requires the development of assessment tools to identify hazardous nanomaterial properties that could be modified to improve nanomaterial safety. While there is a lot of debate of what constitutes appropriate safety screening methods, one approach is to use the assessment of cellular injury pathways to collect knowledge about hazardous material properties that could lead to harm to humans and the environment. We demonstrate the use of a multiparameter cytotoxicity assay that evaluates toxic oxidative stress to compare the effects of titanium dioxide (TiO(2)), cerium oxide (CeO(2)), and zinc oxide (ZnO) nanoparticles in bronchial epithelial and macrophage cell lines. The nanoparticles were chosen on the basis of their volume of production and likelihood of spread to the environment. Among the materials, dissolution of ZnO nanoparticles and Zn(2+) release were capable of ROS generation and activation of an integrated cytotoxic pathway that includes intracellular calcium flux, mitochondrial depolarization, and plasma membrane leakage. These responses were chosen on the basis of the compatibility of the fluorescent dyes that contemporaneously assess their response characteristics by a semiautomated epifluorescence procedure. Purposeful reduction of ZnO cytotoxicity was achieved by iron doping, which changed the material matrix to slow Zn(2+) release. In summary, we demonstrate the utility of a rapid throughput, integrated biological oxidative stress response pathway to perform hazard ranking of a small batch of metal oxide nanoparticles, in addition to showing how this assay can be used to improve nanosafety by decreasing ZnO dissolution through Fe doping.

Use of Size and a Copolymer Design Feature To Improve the Biodistribution and the Enhanced Permeability and Retention Effect of Doxorubicin-Loaded Mesoporous Silica Nanoparticles in a Murine Xenograft Tumor Model

A key challenge for improving the efficacy of passive drug delivery to tumor sites by a nanocarrier is to limit reticuloendothelial system uptake and to maximize the enhanced permeability and retention effect. We demonstrate that size reduction and surface functionalization of mesoporous silica nanoparticles (MSNP) with a polyethyleneimine-polyethylene glycol copolymer reduces particle opsonization while enhancing the passive delivery of monodispersed, 50 nm doxorubicin-laden MSNP to a human squamous carcinoma xenograft in nude mice after intravenous injection. Using near-infrared fluorescence imaging and elemental Si analysis, we demonstrate passive accumulation of ∼12% of the tail vein-injected particle load at the tumor site, where there is effective cellular uptake and the delivery of doxorubicin to KB-31 cells. This was accompanied by the induction of apoptosis and an enhanced rate of tumor shrinking compared to free doxorubicin. The improved drug delivery was accompanied by a significant reduction in systemic side effects such as animal weight loss as well as reduced liver and renal injury. These results demonstrate that it is possible to achieve effective passive tumor targeting by MSNP size reduction as well as by introducing steric hindrance and electrostatic repulsion through coating with a copolymer. Further endowment of this multifunctional drug delivery platform with targeting ligands and nanovalves may further enhance cell-specific targeting and on-demand release.

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.

Role of Fe doping in tuning the band gap of TiO2 for the photo-oxidation-induced cytotoxicity paradigm.

UV-light-induced electron-hole (e(-)/h(+)) pair generation with free radical production in TiO(2)-based nanoparticles is a major conceptual paradigm for biological injury. However, to date, this hypothesis has been difficult to experimentally verify due to the high energy of UV light that is intrinsically highly toxic to biological systems. Here, a versatile flame spray pyrolysis (FSP) synthetic process has been exploited to synthesize a library of iron-doped (0-10 wt%) TiO(2) nanoparticles. These particles have been tested for photoactivation-mediated cytotoxicity using near-visible light exposure. The reduction in TiO(2) band gap energy with incremental levels of Fe loading maintained the nanoparticle crystalline structure in spite of homogeneous Fe distribution (demonstrated by XRD, HRTEM, SAED, EFTEM, and EELS). Photochemical studies showed that band gap energy was reciprocally tuned proportional to the Fe content. The photo-oxidation capability of Fe-doped TiO(2) was found to increase during near-visible light exposure. Use of a macrophage cell line to evaluate cytotoxic and ROS production showed increased oxidant injury and cell death in parallel with a decrease in band gap energy. These findings demonstrate the importance of band gap energy in the phototoxic response of the cell to TiO(2) nanoparticles and reflect the potential of this material to generate adverse effects in humans and the environment during high-intensity light exposure.