Occupational Inhalation Exposures to Nanoparticles at Six Singapore Printing Centers.

Laser printers emit high levels of nanoparticles (PM0.1) during operation. Although it is well established that toners contain multiple engineered nanomaterials (ENMs), little is known about inhalation exposures to these nanoparticles and work practices in printing centers. In this report, we present a comprehensive inhalation exposure assessment of indoor microenvironments at six commercial printing centers in Singapore, the first such assessment outside of the United States, using real-time personal and stationary monitors, time-integrated instrumentation, and multiple analytical methods. Extensive presence of ENMs, including titanium dioxide, iron oxide, and silica, was detected in toners and in airborne particles collected from all six centers studied. We document high transient exposures to emitted nanoparticles (peaks of ∼500 000 particles/cm3, lung-deposited surface area of up to 220 µm2/cm3, and PM0.1 up to 16 µg/m3) with complex PM0.1 chemistry that included 40-60 wt % organic carbon, 10-15 wt % elemental carbon, and 14 wt % trace elements. We also record 271.6-474.9 pmol/mg of Environmental Protection Agency-priority polycyclic aromatic hydrocarbons. These findings highlight the potentially high occupational inhalation exposures to nanoparticles with complex compositions resulting from widespread usage of nano-enabled toners in the printing industry, as well as inadequate ENM-specific exposure control measures in these settings.

Decoupling the Direct and Indirect Biological Effects of ZnO Nanoparticles Using a Communicative Dual Cell-Type Tissue Construct.

While matter at the nanoscale can be manipulated, the knowledge of the interactions between these nanoproducts and the biological systems remained relatively laggard. Current nanobiology study is rooted on in vitro study using conventional 2D cell culture model. A typical study employs monolayer cell culture that simplifies the real context of which to measure any nanomaterial effect; unfortunately, this simplification also demonstrated the limitations of 2D cell culture in predicting the actual biological response of some tissues. In fact, some of the characteristics of tissue such as spatial arrangement of cells and cell-cell interaction, which are simplified in 2D cell culture model, play important roles in how cells respond to a stimulus. To more accurately recapitulate the features and microenvironment of tissue for nanotoxicity assessments, an improved organotypic-like in vitro multicell culture system to mimic the kidney endoepithelial bilayer is introduced. Results showed that important nano-related parameters such as the diffusion, direct and indirect toxic effects of ZnO nanoparticles can be studied by combining this endoepithelial bilayer tissue model and traditional monolayer culture setting.

Mechanistic Investigation of the Biological Effects of SiO2, TiO2, and ZnO Nanoparticles on Intestinal Cells.

Silicon dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) are currently among the most widely used nanoparticles (NPs) in the food industry. This could potentially lead to unintended exposure of the gastrointestinal tract to these NPs. This study aims to investigate the potential side-effects of these food-borne NPs on intestinal cells and to mechanistically understand the observed biological responses. Among the panel of tested NPs, ZnO NPs are the most toxic. Consistently in all three tested intestinal cell models, ZnO NPs invoke the most inflammatory responses from the cells and induce the highest intracellular production of reactive oxygen species (ROS). The elevated ROS levels induce significant damage to the DNA of the cells, resulting in cell-cycle arrest and subsequently cell death. In contrast, both SiO2 and TiO2 NPs elicit minimum biological responses from the intestinal cells. Overall, the study showcases the varying capability of the food-borne NPs to induce a cellular response in the intestinal cells. In addition to physicochemical differences in the NPs, the genetic landscape of the intestinal cell models governs the toxicology profile of these food-borne NPs.

Biomimicry 3D gastrointestinal spheroid platform for the assessment of toxicity and inflammatory effects of zinc oxide nanoparticles.

Our current mechanistic understanding on the effects of engineered nanoparticles (NPs) on cellular physiology is derived mainly from 2D cell culture studies. However, conventional monolayer cell culture may not accurately model the mass transfer gradient that is expected in 3D tissue physiology and thus may lead to artifactual experimental conclusions. Herein, using a micropatterned agarose hydrogel platform, the effects of ZnO NPs (25 nm) on 3D colon cell spheroids of well-defined sizes are examined. The findings show that cell dimensionality plays a critical role in governing the spatiotemporal cellular outcomes like inflammatory response and cytotoxicity in response to ZnO NPs treatment. More importantly, ZnO NPs can induce different modes of cell death in 2D and 3D cell culture systems. Interestingly, the outer few layers of cells in 3D model could only protect the inner core of cells for a limited time and periodically slough off from the spheroids surface. These findings suggest that toxicological conclusions made from 2D cell models might overestimate the toxicity of ZnO NPs. This 3D cell spheroid model can serve as a reproducible platform to better reflect the actual cell response to NPs and to study a more realistic mechanism of nanoparticle-induced toxicity.

Nanoparticles strengthen intracellular tension and retard cellular migration.

Nanoparticles can have profound effects on cell biology. Here, we show that after TiO2, SiO2, and hydroxyapatite nanoparticles treatment, TR146 epithelial cell sheet displayed slower migration. Cells after exposure to the nanoparticles showed increased cell contractility with significantly impaired wound healing capability however without any apparent cytotoxicity. We showed the mechanism is through nanoparticle-mediated massive disruption of the intracellular microtubule assembly, thereby triggering a positive feedback that promoted stronger substrate adhesions thus leading to limited cell motility.

Cellulose Nanofiber Platform for Pesticide Sequestration in the Gastrointestinal Tract.

Exploitation of nature-derived materials is an important approach to promote environmental sustainability. Among these materials, cellulose is of particular interest due to its abundance and relative ease of access. As a food ingredient, cellulose nanofibers (CNFs) have found interesting applications as emulsifiers and modulators of lipid digestion and absorption. In this report, we show that CNFs can also be modified to modulate the bioavailability of toxins, such as pesticides, in the gastrointestinal tract (GIT) by forming inclusion complexes and promoting interaction with surface hydroxyl groups. CNFs were successfully functionalized with (2-hydroxypropyl)-ß-cyclodextrin (HPBCD) using citric acid as a crosslinker via esterification. Functionally, the potential for pristine and functionalized CNFs (FCNFs) to interact with a model pesticide, boscalid, was tested. Based on direct interaction studies, adsorption of boscalid saturated at around 3.09% on CNFs and at 12.62% on FCNFs. Using an in vitro GIT simulation platform, the adsorption of boscalid on CNFs/FCNFs was also studied. The presence of a high-fat food model was found to have a positive effect in binding boscalid in a simulated intestinal fluid environment. In addition, FCNFs were found to have a greater effect in retarding triglyceride digestion than CNFs (61% vs 30.6%). Overall, FCNFs were demonstrated to evoke synergistic effects of reducing fat absorption and pesticide bioavailability through inclusion complex formation and the additional binding of the pesticide onto surface hydroxyl groups on HPBCD. By adopting food-compatible materials and processes for production, FCNFs have the potential to be developed into a functional food ingredient for modulating food digestion and the uptake of toxins.

Printer center nanoparticles alter the DNA repair capacity of human bronchial airway epithelial cells.

Nano-enabled, toner-based printing equipment emit nanoparticles during operation. The bioactivity of these nanoparticles as documented in a plethora of published toxicological studies raises concerns about their potential health effects. These include pro-inflammatory effects that can lead to adverse epigenetic alterations and cardiovascular disorders in rats. At the same time, their potential to alter DNA repair pathways at realistic doses remains unclear. In this study, size-fractionated, airborne particles from a printer center in Singapore were sampled and characterized. The PM0.1 size fraction (particles with an aerodynamic diameter less than 100 nm) of printer center particles (PCP) were then administered to human lung adenocarcinoma (Calu-3) or lymphoblastoid (TK6) cells. We evaluated plasma membrane integrity, mitochondrial activity, and intracellular reactive oxygen species (ROS) generation. Moreover, we quantified DNA damage and alterations in the cells' capacity to repair 6 distinct types of DNA lesions. Results show that PCP altered the ability of Calu-3 cells to repair 8oxoG:C lesions and perform nucleotide excision repair, in the absence of acute cytotoxicity or DNA damage. Alterations in DNA repair capacity have been correlated with the risk of various diseases, including cancer, therefore further genotoxicity studies are needed to assess the potential risks of PCP exposure, at both occupational settings and at the end-consumer level.

Overcoming bacterial physical defenses with molecule-like ultrasmall antimicrobial gold nanoclusters.

The size of metal nanoparticles (NPs) is crucial in their biomedical applications. Although abundant studies on the size effects of metal NPs in the range of 2-100 nm have been conducted, the exploration of the ultrasmall metal nanoclusters (NCs) of ~1 nm in size with unique features is quite limited. We synthesize three different sized gold (Au) NCs of different Au atom numbers and two bigger sized Au NPs protected by the same ligand to study the size influence on antimicrobial efficacy. The ultrasmall Au NCs can easily traverse the cell wall pores to be internalized inside bacteria, inducing reactive oxygen species generation to oxidize bacterial membrane and disturb bacterial metabolism. This explains why the Au NCs are antimicrobial while the Au NPs are non-antimicrobial, suggesting the key role of size in antimicrobial ability. Moreover, in contrast to the widely known size-dependent antimicrobial properties, the Au NCs of different atom numbers demonstrate molecule-like instead of size-dependent antimicrobial behavior with comparable effectiveness, indicating the unique molecule-like feature of ultrasmall Au NCs. Overcoming the bacterial defenses at the wall with ultrasmall Au NCs changes what was previously believed to harmless to the bacteria instead to a highly potent agent against the bacteria.

BiOClBr-coated fabrics with enhanced antimicrobial properties under ambient light.

This study demonstrates the fabrication of ambient light enabled antimicrobial functional fabrics by coating flower-like bismuth oxyhalide i.e. BiOCl0.875Br0.125, with the use of poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) as binders for improved coating robustness and durability. The uniformity of the microparticles was ensured with simultaneous probe sonication during the stages of crystal nucleation and growth. The polymeric binders not only strongly anchor the particle on the fabric, but also serve as an ultra-thin protective layer on the BiOClBr that mitigates bismuth leaching. The efficacy of inhibiting bacteria was investigated over the BiOClBr-coated fabrics i.e. cotton and polyester, and the results showed that the coated fabrics could effectively inhibit both Gram-positive and Gram-negative bacteria, i.e. S. aureus and E. coli. In comparison with fabrics coated with other photocatalytic materials including bismuth oxide (Bi2O3) and zinc oxide (ZnO), an exceptionally better antimicrobial efficacy was observed for BiOClBr-coated fabrics. The BiOClBr-coated cotton showed ∼5.0 and ∼6.8 times higher disinfection efficacy towards E. coli compared to that of ZnO and Bi2O3-coated cotton with the same particle weight percentage, respectively. Further elucidation of the probable mechanism by BiOClBr-coated fabrics is related to the excess amount of reactive oxygen species (ROS). Overall, BiOClBr has been shown to be a promising material to fabricate cost-effective antimicrobial functional surfaces for both environmental and biomedical applications e.g. protective laboratory and factory clothing.

Mesoporous Silica Nanoparticles as an Antitumoral-Angiogenesis Strategy.

Tumors depend heavily on angiogenesis for nutrient derivation and their subsequent metastasis. Targeting tumor induced angiogenesis per se can address both tumor growth and progression simultaneously. Here, we show that we could elegantly restrict the endothelial cells angiogenic behavior through digital size control of mesoporous silica nanoparticle (MSN). This antiangiogenesis effect was derived from the particle size dependent uptake and production of intracellular reactive oxygen species (ROS) that directly interfered with p53 tumor suppressor pathway. The resulting signaling cascade wrestled back the tumoral control of endothelial cells' migration, invasion, and proliferation. Overall, a mere control over the size of a highly oxidative reactive surfaced nanoparticle could provide an alternative strategy to curb the tumor induced angiogenesis process in a conventional drug-free manner.

Gold Nanoparticles Induced Endothelial Leakiness Depends on Particle Size and Endothelial Cell Origin.

The endothelium presents a formidable barrier for cancer nanomedicine, as the intravenously introduced nanomedicine needs to leave the blood vessel at the tumor site. Endothelial permeability and retention effect (EPR) is not dependable since it is derived from tumors. Certain nanoparticles with specific characteristics are able to induce micrometer sized gaps between endothelial cells. This effect is called "nanoparticle induced endothelial leakiness" (NanoEL). NanoEL therefore allows the nanotechnology to control access to the tumor even in the absence of any EPR effect. Morever, NanoEL can be applicable to noncancer issues, thereby expanding its usefulness in other subfields of nanomedicine. In this paper, we have shown that Gold (Au) nanoparticles within the range of 10-30 nm are good NanoEL inducing particles. As not all endothelial cells have the same permeability, we found that human mammary endothelial cells and human skin endothelial cells are sensitive to Au induced NanoEL, while human umbilical vein endothelial cells are insensitive, reflective of their innate nature of endothelial permeability. The size window and endothelial cell type sensitivity then helps the nanotechnologists to design future nanoparticles that either exploit NanoEL as a nanotechnology driven strategy to access immature tumors, which do not induce the EPR effect, or avoid NanoEL as a nanotoxic side effect.

Antimicrobial Gold Nanoclusters.

Bulk gold (Au) is known to be chemically inactive. However, when the size of Au nanoparticles (Au NPs) decreases to close to 1 nm or sub-nanometer dimensions, these ultrasmall Au nanoclusters (Au NCs) begin to possess interesting physical and chemical properties and likewise spawn different applications when working with bulk Au or even Au NPs. In this study, we found that it is possible to confer antimicrobial activity to Au NPs through precise control of their size down to NC dimension (typically less than 2 nm). Au NCs could kill both Gram-positive and Gram-negative bacteria. This wide-spectrum antimicrobial activity is attributed to the ultrasmall size of Au NCs, which would allow them to better interact with bacteria. The interaction between ultrasmall Au NCs and bacteria could induce a metabolic imbalance in bacterial cells after the internalization of Au NCs, leading to an increase of intracellular reactive oxygen species production that kills bacteria consequently.

Directing Assembly and Disassembly of 2D MoS2 Nanosheets with DNA for Drug Delivery.

Layer-by-layer (LbL) self-assembled stacked Testudo-like MoS2 superstructures carrying cancer drugs are formed from nanosheets controllably assembled with sequence-based DNA oligonucleotides. These superstructures can disassemble autonomously in response to cancer cells' heightened ATP metabolism. First, we functionalize MoS2 nanosheets (MoS2-NS) nanostructures with DNA oligonucleotides having thiol-terminated groups (DNA/MoS2-NS) via strong binding to sulfur atom defect vacancies on MoS2 surfaces. The driving force to assemble into a higher-order DNA/MoS2-NS superstructure is guided by a linker aptamer that induced interlayer assembly. In the presence of target ATP molecules, these multilayer superstructures disassembled as a consequence of stronger binding of ATP molecules with the linking aptamers. This design plays a dual role of protection and delivery by LbL stacked MoS2-NS similar in concept to a Greek Testudo. These superstructures present a protective armor-like shell of MoS2-NS, which still remains responsive to small and infiltrating ATP molecules diffusing through the protective MoS2-NS, contributing to an enhanced stimuli-responsive drug release system for targeted chemotherapy.

Tuning Endothelial Permeability with Functionalized Nanodiamonds.

Cancer nanomedicine vehicles are required to cross the vascular barrier to reach the tumor site in order to ensure the successful delivery of their therapeutic load. Here, nanodiamond (ND) variants were shown to induce surface dependent vascular barrier leakiness. The ND-induced leakiness was found to be mediated by the increase in intracellular reactive oxygen species (ROS) and Ca(2+). These then in turn triggered the loss in endothelial cell-endothelial cell connections of the vascular barrier and also triggered their quasi-stable cytoskeletal remodelling. This ND driven increase in leakiness allowed more doxorubicin drug to penetrate through the vascular barrier to reach the cancer cells. This increase in the doxorubicin penetration subsequently led to an increase in the cancer killing effect. Overall, tuning the vascular barrier leakiness through ND surface group functionalization could provide an alternative strategy for the cancer nanomedicine to traverse across the vascular barrier.

Antimicrobial Cluster Bombs: Silver Nanoclusters Packed with Daptomycin.

Integration of two distinctive bactericides into one entity is a promising platform to improve the efficiency of antimicrobial agents. We report an efficient antimicrobial hybrid formed through conjugating silver nanoclusters (AgNCs) with daptomycin. The as-designed antimicrobial hybrid (D-AgNCs) inherits intrinsic properties of both bactericides with an enhanced synergistic performance. In particular, the chemically integrated D-AgNCs showed improved bacterial killing efficiency over the physically mixed daptomycin and AgNCs (D+AgNCs). More interestingly, the as-designed D-AgNCs could effectively damage the bacterial membrane. Propidium iodide (PI) stain showed bacterial membrane damage in about 85% of the bacteria population after treatment with D-AgNCs through creation of larger pores on the membrane as compared to D+AgNCs, largely due to the localization of daptomycin within the hybrid structure. These larger pores facilitated the entry of the D-AgNCs into the cell and led to more severe DNA damage of the bacterial DNA as compared to D+AgNCs in genomic DNA PAGE analysis. TUNEL assay further depicted more bacterial DNA breaks induced by D-AgNCs. The RecA gene expression level was upregulated, suggestive of DNA repair activation. The strong induced DNA damage benefited from the localization of AgNCs in the core of the antimicrobial hybrid structure, which could generate localized high ROS concentration and work as a critical ROS reservoir to continually generate ROS within the bacterium. The continual bombardments by these ROS generators restrict the ability of the bacteria to now develop resistance against this.

In vivo and ex vivo proofs of concept that cetuximab conjugated vitamin E TPGS micelles increases efficacy of delivered docetaxel against triple negative breast cancer.

In this study we examined the efficacy of our micellar system in xenograft models of triple negative breast cancers and explored the effect of the micelles on post-treatment tumours in order to elucidate the mechanism underlying the nanomedicine treatment in oncology. Here, we developed docetaxel-loaded vitamin E D-α-tocopheryl polyethylene glycol succinate (TPGS) micelles, of which the surface modified with cetuximab ligands for targeting epidermal growth factor receptors (EGFR) that are overexpressed in MDA-MB-231 breast cancer cells. The targeting micelles accumulated in the tumours immediately after the intravenous injection and retained for at least 24 h. The successful delivery of docetaxel into the tumours by the targeting micelles was shown by the greater degree of tumour growth inhibition than that for Taxotere(®) after the 15-day treatment. Furthermore, the explanted tumour culture study involving gene analysis and immunohistochemistry staining indicated that the in vivo micelle treatment induced cell cycle arrest and attenuated cell proliferation. In addition, the targeting and non-targeting micellar formulations brought about anti-angiogenesis and anti-migration effects. Overall, both the in vivo and ex vivo data increased the confidence that our micellar formulations effectively targeted and inhibited EGFR-overexpressing MDA-MB-231 tumours.

Phage based green chemistry for gold ion reduction and gold retrieval.

The gold mining industry has taken its toll on the environment, triggering the development of more environmentally benign processes to alleviate the waste load release. Here, we demonstrate the use of bacteriophages (phages) for biosorption and bioreduction of gold ions from aqueous solution, which potentially can be applied to remediate gold ions from gold mining waste effluent. Phage has shown a remarkably efficient sorption of gold ions with a maximum gold adsorption capacity of 571 mg gold/g dry weight phage. The product of this phage mediated process is gold nanocrystals with the size of 30-630 nm. Biosorption and bioreduction processes are mediated by the ionic and covalent interaction between gold ions and the reducing groups on the phage protein coat. The strategy offers a simple, ecofriendly and feasible option to recover of gold ions to form readily recoverable products of gold nanoparticles within 24 h.

Novel theranostic DNA nanoscaffolds for the simultaneous detection and killing of Escherichia coli and Staphylococcus aureus.

A novel theranostic platform is made by utilizing a self-assembled DNA nanopyramid (DP) as scaffold for incorporation of both detection and therapeutic moieties to combat bacterial infection. Red-emissive glutathione-protected gold nanoclusters (GSH-Au NCs) were used for bacterial detection. Actinomycin D (AMD) that was intercalated on the DP scaffold was used as therapeutic agent. This results in the formation of theranostic DPAu/AMD. Model bacteria Escherichia coli and Staphylococcus aureus were found to be readily taken in the DPAu/AMD and be susceptible to its killing effect. In addition, DPAu/AMD was observed to outperform the free AMD in killing infectious bacteria. The degradation of the DP structure by DNase was found to be responsible for the release of AMD and the effective killing effect of the infectious bacteria. This novel strategy presents a basic platform for future improvements to detect infectious bacteria and treatment.

Effect of zinc oxide nanomaterials-induced oxidative stress on the p53 pathway.

Excessive production of reactive oxygen species (ROS) is a hallmark feature in nanomaterials (NMs) induced cellular toxicity. However, the inter-relationship between NMs induced ROS generation and the cells innate ability to regulate intracellular ROS level in effecting a particular cellular outcome is currently underexplored. Here, using a BJ fibroblast p53 knockdown system, we showed that p53 may be implicated in playing a dual regulatory role to determine cell survivability in response to oxidative stress induced by ZnO NMs. At low level of ZnO NMs induced ROS, p53 triggers expression of antioxidant genes such as SOD2, GPX1, SESN1, SESN2 and ALDH4A1 to restore oxidative homeostasis while at high concentration of ZnO NMs, the elevated level of intracellular ROS activated the apoptotic pathway through p53. The implication of our finding that p53 can function as an important regulator in determining ZnO induced cytotoxicity is highlighted by the differential action of ZnO on p53 deficient and proficient colorectal cell lines. p53 deficient cells cancer cells such as DLD-1 and SW480 are more susceptible to ZnO induced cell death compared to p53 proficient cells such as colon epithelial cells NCM460 and HCT116 cells in a ROS dependent manner. Collectively, our findings showcased a role p53 plays in the context of nanotoxicity and highlights the need to consider the interplay of physicochemical properties of NMs and cell biology.

The role of the tumor suppressor p53 pathway in the cellular DNA damage response to zinc oxide nanoparticles.

In this paper, we explored how ZnO nanoparticles cross-interact with a critical tumor suppressive pathway centered around p53, which is one of the most important known tumor suppressors that protects cells from developing cancer phenotypes through its control over major pathways like apoptosis, senescence and cell cycle progression. We showed that the p53 pathway was activated in BJ cells (skin fibroblasts) upon ZnO nanoparticles treatment with a concomitant decrease in cell numbers. This suggests that cellular responses like apoptosis in the presence of ZnO nanoparticles require p53 as the molecular master switch towards programmed cell death. This also suggests that in cells without robust p53, protective response can be tipped towards carcinogenesis when stimulated by DNA damage inducing agents like ZnO nanoparticles. We observed this precarious tendency in the same BJ cells with p53 knocked down using endogeneous expressing shRNA. These p53 knocked down BJ cells became more resistant to ZnO nanoparticles induced cell death and increased cell progression. Collectively, our results suggest that cellular response towards specific nanoparticle induced cell toxicity and carcinogenesis is not only dependent on specific nanoparticle properties but also (perhaps more importantly) the endogenous genetic, transcriptomic and proteomic landscape of the target cells.