Mark Geppert

Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes

Magnetic iron oxide nanoparticles (Fe-NP) are currently considered for various diagnostic and therapeutic applications in the brain. However, little is known on the accumulation and biocompatibility of such particles in brain cells. We have synthesized and characterized dimercaptosuccinic acid (DMSA) coated Fe-NP and have investigated their uptake by cultured brain astrocytes. DMSA-coated Fe-NP that were dispersed in physiological medium had an average hydrodynamic diameter of about 60 nm. Incubation of cultured astrocytes with these Fe-NP caused a time- and concentration-dependent accumulation of cellular iron, but did not lead within 6 h to any cell toxicity. After 4 h of incubation with 100-4000 µM iron supplied as Fe-NP, the cellular iron content reached levels between 200 and 2000 nmol mg⁻¹ protein. The cellular iron content after exposure of astrocytes to Fe-NP at 4 °C was drastically lowered compared to cells that had been incubated at 37 °C. Electron microscopy revealed the presence of Fe-NP-containing vesicles in cells that were incubated with Fe-NP at 37 °C, but not in cells exposed to the nanoparticles at 4 °C. These data demonstrate that cultured astrocytes efficiently take up DMSA-coated Fe-NP in a process that appears to be saturable and strongly depends on the incubation temperature.

Treatment with iron oxide nanoparticles induces ferritin synthesis but not oxidative stress in oligodendroglial cells.

Magnetic iron oxide nanoparticles (IONPs) have been used for a variety of neurobiological applications, although little is yet known as to the fate of such particles in brain cells. To address these questions, we have exposed oligodendroglial OLN-93 cells to dimercaptosuccinate-coated IONPs. Treatment of the cells strongly increased the specific cellular iron content proportional to the IONP concentrations applied (0-1000 μM total iron as IONPs) up to 300-fold, but did not cause any acute cytotoxicity or induce oxidative stress. To investigate the potential of OLN-93 cells to liberate iron from the accumulated IONPs, we have studied the upregulation of the iron storage protein ferritin and the cell proliferation as cellular processes that depend on the availability of low-molecular-weight iron. The presence of IONPs caused a concentration-dependent increase in the amount of cellular ferritin and partially bypassed the inhibition of cell proliferation by the iron chelator deferoxamine. These data demonstrate that viable OLN-93 cells efficiently take up IONPs and suggest that these cells are able to use iron liberated from accumulated IONPs for their metabolism.

Ferritin up-regulation and transient ROS production in cultured brain astrocytes after loading with iron oxide nanoparticles

To investigate the cellular consequences of a prolonged cellular presence of large amounts of iron oxide nanoparticles (IONPs) as well as the fate of such particles in brain cells, cultured primary astrocytes were loaded for 4h with dimercaptosuccinate-coated IONPs. Subsequently, the IONP-treated cells were incubated for up to 7 days in IONP-free medium and the cell viability, metabolic parameters and iron metabolism of the cells were investigated. Despite an up to 100-fold elevated specific cellular iron content, IONP-loaded cells remained viable throughout the 7 day main incubation and did not show any substantial alteration in glucose and glutathione metabolism. During the incubation, the high cellular iron content of IONP-loaded astrocytes remained almost constant. Electron microscopy revealed that after 7 days of incubation most of the cellular iron was still present in IONP-filled vesicles. However, the transient appearance of reactive oxygen species (ROS) as well as a strong increase in cellular levels of the iron storage protein ferritin suggest that at least some low-molecular-weight iron was liberated from the accumulated IONPs. These results demonstrate that even the prolonged presence of large amounts of accumulated IONPs does not harm astrocytes and that these cells store IONP-derived iron in ferritin.

Handling of Iron Oxide and Silver Nanoparticles by Astrocytes

Metal-containing nanoparticles (NPs) are currently used for various biomedical applications. Since such NPs are able to enter the brain, the cells of this organ have to deal with NPs and with NP-derived metal ions. In brain, astrocytes are considered to play a key function in regulating metal homeostasis and in protecting other brain cells against metal toxicity. Thus, among the different types of brain cells, especially astrocytes are of interest regarding the uptake and the handling of metal-containing NPs. This article summarizes the current knowledge on the consequences of an exposure of astrocytes to NPs. Special focus will be given to magnetic iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), since the biocompatibility of these NPs has been studied for astrocytes in detail. Cultured astrocytes efficiently accumulate IONPs and AgNPs in a time-, concentration- and temperature-dependent manner by endocytotic processes. Astrocytes are neither acutely damaged by the exposure to high concentrations of NPs nor by the prolonged intracellular presence of large amounts of accumulated NPs. Although metal ions are liberated from accumulated NPs, NP-derived iron and silver ions are not exported from astrocytes but are rather stored in proteins such as ferritin and metallothioneins which are synthesized in NP-treated astrocytes. The efficient accumulation of large amounts of metal-containing NPs and the upregulation of proteins that safely store NP-derived metal ions suggest that astrocytes protect the brain against the potential toxicity of metal-containing NPs.

Magnetic field-induced acceleration of the accumulation of magnetic iron oxide nanoparticles by cultured brain astrocytes.

Magnetic iron oxide nanoparticles (Fe-NPs) are considered for various biomedical and neurobiological applications that involve the presence of external magnetic fields. However, little is known on the effects of a magnetic field on the uptake of such particles by brain cells. Cultured brain astrocytes accumulated dimercaptosuccinate-coated Fe-NP in a time-, temperature-, and concentration-dependent manner. This accumulation was strongly enhanced by the presence of the magnetic field generated by a permanent neodymium iron boron magnet that had been positioned below the cells. The magnetic field-induced acceleration of the accumulation of Fe-NP increased almost proportional to the strength of the magnetic field applied, increasing the cellular-specific iron content from an initial 10 nmol/mg protein within 4 h of incubation at 37°C to up to 12,000 nmol/mg protein. However, presence of a magnetic field also increased the amounts of iron that attached to the cells during incubation with Fe-NP at 4°C. These results suggest that the presence of an external magnetic field promotes in cultured astrocytes both the binding of Fe-NP to the cell membrane and the internalization of Fe-NP.

An interlaboratory comparison of nanosilver characterisation and hazard identification: harmonising techniques for high quality data

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag(+)-species (the concentration of Ag(+)-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4±6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46-68% of soluble Ag(+)-species in stock, 123.8±12.2 nm mean z-average value in dH2O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50<0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01-1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1-10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10-100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag(+)-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag(+)-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

A novel two-compartment barrier model for investigating nanoparticle transport in fish intestinal epithelial cells

We introduce a novel in vitro rainbow trout intestinal barrier model and demonstrate its suitability for investigating nanoparticle transport across the intestinal epithelium. Rainbow trout (Oncorhynchus mykiss) intestinal cells (RTgutGC) were grown as monolayers on permeable supports leading to a two-compartment intestinal barrier model consisting of a polarized epithelium, dividing the system into an upper (apical) and a lower (basolateral) compartment, and thereby mimicking the intestinal lumen and the portal blood, respectively. The cells express the tight junction protein ZO-1 and build up a transepithelial electrical resistance comparable to the in vivo situation. Fluorescent polystyrene nanoparticles (PS-NPs; average hydrodynamic diameter: 73 ± 18 nm) were accumulated by RTgutGC cells in a time-, temperature- and concentration-dependent manner. Uptake of PS-NPs was confirmed using fluorescence microscopy. Cells formed an efficient barrier largely preventing the translocation of PS-NPs to the basolateral compartment. Taken together, these data demonstrate the suitability of the in vitro barrier model to study the effects of nanoparticles in fish intestinal epithelial cells.

A Novel Exposure System Termed NAVETTA for In Vitro Laminar Flow Electrodeposition of Nanoaerosol and Evaluation of Immune Effects in Human Lung Reporter Cells

A new prototype air-liquid interface (ALI) exposure system, a flatbed aerosol exposure chamber termed NAVETTA, was developed to investigate deposition of engineered nanoparticles (NPs) on cultured human lung A549 cells directly from the gas phase. This device mimics human lung cell exposure to NPs due to a low horizontal gas flow combined with cells exposed at the ALI. Electrostatic field assistance is applied to improve NP deposition efficiency. As proof-of-principle, cell viability and immune responses after short-term exposure to nanocopper oxide (CuO)-aerosol were determined. We found that, due to the laminar aerosol flow and a specific orientation of inverted transwells, much higher deposition rates were obtained compared to the normal ALI setup. Cellular responses were monitored with postexposure incubation in submerged conditions, revealing CuO dissolution in a concentration-dependent manner. Cytotoxicity was the result of ionic and nonionic Cu fractions. Using the optimized inverted ALI/postincubation procedure, pro-inflammatory immune responses, in terms of interleukin (IL)-8 promoter and nuclear factor kappa B (NFκB) activity, were observed within short time, i.e. One hour exposure to ALI-deposited CuO-NPs and 2.5 h postincubation. NAVETTA is a novel option for mimicking human lung cell exposure to NPs, complementing existing ALI systems.

Oscillations of ultra-weak photon emission from cancer and non-cancer cells stressed by culture medium change and TNF- α

Cells spontaneously emit photons in the UV to visible/near-infrared range (ultra-weak photon emission, UPE). Perturbations of the cells’ state cause changes in UPE (evoked UPE). The aim of the present study was to analyze the evoked UPE dynamics of cells caused by two types of cell perturbations (stressors): (i) a cell culture medium change, and (ii) application of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). Four types of human cell lines were used (squamous cell carcinoma cells, A431; adenocarcinomic alveolar basal epithelial cells, A549; p53-deficient keratinocytes, HaCaT, and cervical cancer cells, HeLa). In addition to the medium change, TNF-α was applied at different concentrations (5, 10, 20, and 40 ng/mL) and UPE measurements were performed after incubation times of 0, 30, 60, 90 min, 2, 5, 12, 24, 48 h. It was observed that (i) the change of cell culture medium (without added TNF-α) induces a cell type-specific transient increase in UPE with the largest UPE increase observed in A549 cells, (ii) the addition of TNF-α induces a cell type-specific and dose-dependent change in UPE, and (iii) stressed cell cultures in general exhibit oscillatory UPE changes.

Nanomaterials in the Context of Type 2 Immune Responses—Fears and Potentials

The type 2 immune response is an adaptive immune program involved in defense against parasites, detoxification, and wound healing, but is predominantly known for its pathophysiological effects, manifesting as allergic disease. Engineered nanoparticles (NPs) are non-self entities that, to our knowledge, do not stimulate detrimental type 2 responses directly, but have the potential to modulate ongoing reactions in various ways, including the delivery of substances aiming at providing a therapeutic benefit. We review, here, the state of knowledge concerning the interaction of NPs with type 2 immune responses and highlight their potential as a multifunctional platform for therapeutic intervention.