Transmission electron microscopy artifacts in characterization of the nanomaterial-cell interactions.

We investigated transmission electron microscopy artifacts obtained using standard sample preparation protocols applied to the investigation of Escherichia coli cells exposed to common nanomaterials, such as TiO2, Ag, ZnO, and MgO. While the common protocols for some nanomaterials result only in known issues of nanomaterial-independent generation of anomalous deposits due to fixation and staining, for others, there are reactions between the nanomaterial and chemicals used for post-fixation or staining. Only in the case of TiO2 do we observe only the known issues of nanomaterial-independent generation of anomalous deposits due to exceptional chemical stability of this material. For the other three nanomaterials, different artifacts are observed. For each of those, we identify causes of the observed problems and suggest alternative sample preparation protocols to avoid artifacts arising from the sample preparation, which is essential for correct interpretation of the obtained images and drawing correct conclusions on cell-nanomaterial interactions. Finally, we propose modified sample preparation and characterization protocols for comprehensive and conclusive investigations of nanomaterial-cell interactions using electron microscopy and for obtaining clear and unambiguous revelation whether the nanomaterials studied penetrate the cells or accumulate at the cell membranes. In only the case of MgO and ZnO, the unambiguous presence of Zn and Mg could be observed inside the cells.

Toxicity of ZnO and TiO2 to Escherichia coli cells.

We performed a comprehensive investigation of the toxicity of ZnO and TiO2 nanoparticles using Escherichia coli as a model organism. Both materials are wide band gap n-type semiconductors and they can interact with lipopolysaccharide molecules present in the outer membrane of E. coli, as well as produce reactive oxygen species (ROS) under UV illumination. Despite the similarities in their properties, the response of the bacteria to the two nanomaterials was fundamentally different. When the ROS generation is observed, the toxicity of nanomaterial is commonly attributed to oxidative stress and cell membrane damage caused by lipid peroxidation. However, we found that significant toxicity does not necessarily correlate with up-regulation of ROS-related proteins. TiO2 exhibited significant antibacterial activity, but the protein expression profile of bacteria exposed to TiO2 was different compared to H2O2 and the ROS-related proteins were not strongly expressed. On the other hand, ZnO exhibited lower antibacterial activity compared to TiO2, and the bacterial response involved up-regulating ROS-related proteins similar to the bacterial response to the exposure to H2O2. Reasons for the observed differences in toxicity and bacterial response to the two metal oxides are discussed.

Metal oxide nanoparticles with low toxicity.

A number of different nanomaterials produced and incorporated into various products are rising. However, their environmental hazards are frequently unknown. Here we consider three different metal oxide compounds (SnO2, In2O3, and Al2O3), which have not been extensively studied and are expected to have low toxicity. This study aimed to comprehensively characterize the physicochemical properties of these nanomaterials and investigate their toxicity on bacteria (Escherichia coli) under UV illumination and in the dark, as well as on a marine diatom (Skeletonema costatum) under ambient illumination/dark (16-8h) cycles. The material properties responsible for their low toxicity have been identified based on comprehensive experimental characterizations and comparison to a metal oxide exhibiting significant toxicity under illumination (anatase TiO2). The metal oxide materials investigated exhibited significant difference in surface properties and interaction with the living organisms. In order for a material to exhibit significant toxicity, it needs to be able to both form a stable suspension in the culture medium and to interact with the cell walls of the test organism. Our results indicated that the observed low toxicities of the three nanomaterials could be attributed to the limited interaction between the nanoparticles and cell walls of the test organisms. This could occur either due to the lack of significant attachment between nanoparticles and cell walls, or due to their tendency to aggregate in solution.

Mechanisms of antibacterial activity of MgO: non-ROS mediated toxicity of MgO nanoparticles towards Escherichia coli.

The toxicity of metal oxide nanomaterials and their antimicrobial activity is attracting increasing attention. Among these materials, MgO is particularly interesting as a low cost, environmentally-friendly material. The toxicity of MgO, similar to other metal oxide nanomaterials, is commonly attributed to the production of reactive oxygen species (ROS). We investigated the toxicity of three different MgO nanoparticle samples, and clearly demonstrated robust toxicity towards Escherichia coli bacterial cells in the absence of ROS production for two MgO nanoparticle samples. Proteomics data also clearly demonstrate the absence of oxidative stress and indicate that the primary mechanism of cell death is related to the cell membrane damage, which does not appear to be due to lipid peroxidation.

Antibacterial and photocatalytic activity of TiO2 and ZnO nanomaterials in phosphate buffer and saline solution.

We studied antibacterial and photocatalytic activity of anatase TiO2 and ZnO in phosphate buffer and saline solution. We found that the different anions in the suspension medium (chloride and phosphate) significantly affected the following suspension properties: the stability of nanoparticle suspension, the release of metal ions from the nanoparticles, and the production of the reactive oxygen species by the nanoparticles. As a result, antibacterial activity and photocatalytic dye degradation were also affected. However, the effect of the suspension medium was different for ZnO and TiO2. Obtained results are discussed.

Combination of hyaluronic acid hydrogel scaffold and PLGA microspheres for supporting survival of neural stem cells.

PURPOSE: To develop a biomaterial composite for promoting proliferation and migration of neural stem cells (NSCs), as well as angiogenesis on the materials, to rescue central nervous system (CNS) injuries. METHODS: A delivery system was constructed based on cross-linked hyaluronic acid (HA) hydrogels, containing embedded BDNF and VEGF-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for controlled delivery and support for NSCs in the CNS. The surface morphologies were evaluated by SEM and AFM, mechanical property was investigated by rheological tests, and release kinetics were performed by ELISA. Bioactivity of released BDNF and VEGF was assessed by neuron and endothelial cell culture, respectively. Compatibility with NSCs was studied by immunofluorescent staining. RESULTS: Release kinetics showed the delivery of BDNF and VEGF from PLGA microspheres and HA hydrogel composite were sustainable and stable, releasing ~20-30% within 150 h. The bioactivities preserved well to promote survival and growth of the cells. Evaluation of structure and mechanical properties showed the hydrogel composite possessed an elastic scaffold structure. Biocompatibility assay showed NSCs adhered and proliferated well on the hydrogel. CONCLUSIONS: Our created HA hydrogel/PLGA microsphere systems have a good potential for controlled delivery of varied biofactors and supporting NSCs for brain repair and implantation.