Calibration Strategy to Size and Localize Multi-Shaped Nanoparticles in Tissue Sections Using LA-spICP-MS.

In the field of nanotoxicology, the detection and size characterization of nanoparticles (NPs) in biological tissues become increasingly important. To gain information on both particle size and particle distribution in histological sections, laser ablation and single particle inductively coupled plasma-mass spectrometry (LA-spICP-MS) was used in combination with a liquid calibration of dissolved metal standards via a pneumatic nebulizer. In the first step, the particle size distribution of Ag NPs embedded in matrix-matched gelatine standards introduced via LA was compared with that of Ag NPs in a suspension and nebulizer-based ICP-MS. The data show that the particles remained intact by the ablation process as confirmed by transmission electron microscopy. Moreover, the optimized method was applied to CeO2 NPs that are highly relevant for (eco-)toxicological research but, unlike Ag NPs, are multi-shaped and have a broad particle size distribution. Upon analyzing the particle size distribution of CeO2 NPs in cryosections of rat spleen, CeO2 NPs were found to remain unchanged in size over 3 h, 3 d, and 3 weeks post-intratracheal instillation, with the fraction of smaller particles reaching the spleen first. Overall, LA-spICP-MS combined with a calibration based on dissolved metal standards is a powerful tool to simultaneously localize and size NPs in histological sections in the absence of particle standards.

Surface Treatment With Hydrophobic Coating Reagents (Organosilanes) Strongly Reduces the Bioactivity of Synthetic Amorphous Silica in vitro.

Synthetic amorphous silica (SAS) is industrially relevant material whose bioactivity in vitro is strongly diminished, for example, by protein binding to the particle surface. Here, we investigated the in vitro bioactivity of fourteen SAS (pyrogenic, precipitated, or colloidal), nine of which were surface-treated with organosilanes, using alveolar macrophages as a highly sensitive test system. Dispersion of the hydrophobic SAS required pre-wetting with ethanol and extensive ultrasonic treatment in the presence of 0.05% BSA (Protocol 1). Hydrophilic SAS was suspended by moderate ultrasonic treatment (Protocol 2) and also by Protocol 1. The suspensions were administered to NR8383 alveolar macrophages under serum-free conditions for 16 h, and the release of LDH, GLU, H2O2, and TNFα was measured in cell culture supernatants. While seven surface-treated hydrophobic SAS exhibited virtually no bioactivity, two materials (AEROSIL® R 504 and AEROSIL® R 816) had minimal effects on NR8383 cells. In contrast, non-treated SAS elicited considerable increases in LDH, GLU, and TNFα, while the release of H2O2 was low except for CAB-O-SIL® S17D Fumed Silica. Dispersing hydrophilic SAS with Protocol 1 gradually reduced the bioactivity but did not abolish it. The results show that hydrophobic coating reagents, which bind covalently to the SAS surface, abrogate the bioactivity of SAS even under serum-free in vitro conditions. The results may have implications for the hazard assessment of hydrophobic surface-treated SAS in the lung.

Evaluating nanobiomaterial-induced DNA strand breaks using the alkaline comet assay.

Due to their unique chemical and physical properties, nanobiomaterials (NBMs) are extensively studied for applications in medicine and drug delivery. Despite these exciting properties, their small sizes also make them susceptible to toxicity. Whilst nanomaterial immunotoxicity and cytotoxicity are studied in great depth, there is still limited data on their potential genotoxicity or ability to cause DNA damage. In the past years, new medical device regulations, which came into place in 2020, were developed, which require the assessment of long-term NBM exposure; therefore, in recent years, increased attention is being paid to genotoxicity screening of these materials. In this article, and through an interlaboratory comparison (ILC) study conducted within the Horizon 2020 REFINE project, we assess five different NBM formulations, each with different uses, namely, a bio-persistent gold nanoparticle (AuNP), an IR-780 dye-loaded liposome which is used in deep tissue imaging (LipImage™815), an unloaded PACA polymeric nanoparticle used as a drug delivery system (PACA), and two loaded PACA NBMs, i.e. the cabazitaxel drug-loaded PACA (CBZ-PACA) and the NR668 dye-loaded PACA (NR668 PACA) for their potential to cause DNA strand breaks using the alkaline comet assay and discuss the current state of genotoxicity testing for nanomaterials. We have found through our interlaboratory comparison that the alkaline comet assay can be suitably applied to the pre-clinical assessment of NBMs, as a reproducible and repeatable methodology for assessing NBM-induced DNA damage. Workflow for assessing the applicability of the alkaline comet assay to determine nanobiomaterial (NBM)-induced DNA strand breaks, through an interlaboratory comparison study (ILC).

LA-ICP-MS and Immunohistochemical Staining with Lanthanide-Labeled Antibodies to Study the Uptake of CeO2 Nanoparticles by Macrophages in Tissue Sections.

Due to the increasing use and production of CeO2 nanoparticles (NPs), the likelihood of exposure especially via the air rapidly grows. However, the uptake of CeO2 NPs via the lung and the resulting distribution into various cell types of remote organs are not well understood because classical analytical methods provide limited spatial information. In this study, laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was combined with immunohistochemical (IHC) staining with lanthanide-labeled antibodies to investigate the distribution of intratracheally instilled CeO2 NPs from the rat lung to lymph nodes, spleen, and liver after 3 h, 3 days, and 21 days. We selected regions of interest after fast imaging using LA-ICP-MS in low-resolution mode and conducted high-resolution LA-ICP-MS in combination with IHC for cellular localization. The lanthanide labeling, which was largely congruent with conventional fluorescent labeling, allowed us to calculate the association rates of Ce to specific cell types. Major portions of Ce were found to be associated with phagocytic cells in the lung, lymph nodes, spleen, and liver. In the lung, almost 94% of the Ce was co-localized with CD68-positive alveolar macrophages after 21 days. Ce was also detected in the lymph nodes outside macrophages 3 h post instillation but shifted to macrophage-associated locations. In the liver, Ce accumulations associated with Kupffer cells (CD163-positive) were found. Ce-containing populations of metallophilic and marginal zone macrophages (both CD169-positive) as well as red pulp macrophages (CD68-positive) were identified as major targets in the spleen. Overall, high-resolution LA-ICP-MS analysis in combination with IHC staining with lanthanide-labeled antibodies is a suitable tool to quantify and localize Ce associated with specific cell types and to estimate their particle burden under in vivo conditions.

Subcellular detection of PEBCA particles in macrophages: combining darkfield microscopy, confocal Raman microscopy, and ToF-SIMS analysis.

The detection of biomedical organic nanocarriers in cells and tissues is still an experimental challenge. Here we developed an imaging strategy for the label-free detection of poly (ethylbutyl cyanoacrylate) (PEBCA) particles. Experiments were carried out with phagocytic NR8383 macrophages exposed to non-toxic and non-activating concentrations of fluorescent (PEBCA NR668 and PEBCA NR668/IR), non-fluorescent (PEBCA), and cabazitaxel-loaded PEBCA particles (PEBCA CBZ). Exposure to PEBCA NR668 revealed an inhomogeneous particle uptake similar to what was obtained with the free modified Nile Red dye (NR668). In order to successfully identify the PEBCA-loaded cells under label-free conditions, we developed an imaging strategy based on enhanced darkfield microscopy (DFM), followed by confocal Raman microscopy (CRM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Nitrile groups of the PEBCA matrix and PEBCA ions were used as suitable analytes for CRM and ToF-SIMS, respectively. Masses found with ToF-SIMS were further confirmed by Orbitrap-SIMS. The combined approach allowed to image small (< 1 µm) PEBCA-containing phagolysosomes, which were identified as PEBCA-containing compartments in NR8383 cells by electron microscopy. The combination of DFM, CRM, and ToF-SIMS is a promising strategy for the label-free detection of PEBCA particles.

Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects.

The immense diversity and constant development of nanomaterials (NMs) increase the need for a facilitated risk assessment, which requires knowledge of the modes of action (MoAs) of NMs. This necessitates a comprehensive data basis, which can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is essential to follow the 3R concept and to cope with the high number of NMs. In the present study, we aimed to compare NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data analysis strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For the in vitro investigations, rat alveolar epithelial cells (RLE-6TN) and rat alveolar macrophages (NR8383) were treated with different doses of NMs, and the results were compared with the effects on rat lungs after short-term inhalations and instillations. Since reactive oxygen species (ROS) production has been described as a critical biological effect of NMs, we focused on different levels of oxidative stress. Thus, we found opposite changes in proteins and metabolites related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, demonstrating that the MoAs of NMs depend on the model system used. Interestingly, in vivo, pathways related to inflammation were more affected than oxidative stress responses. Hence, the assignment of the observed effects to levels of oxidative stress was also different in vitro and in vivo. However, the overall classification of "active" and "passive" NMs was consistent in vitro and in vivo, suggesting that both cell lines tested are suitable for the assessment of NM toxicity. In summary, the results presented here highlight the need to carefully review model systems to decipher the extent to which they can replace in vivo assays.

Classes of organic pigments meet tentative PSLT criteria and lack toxicity in short-term inhalation studies.

Here, we present a non-animal testing battery to identify PSLT (poorly soluble, low toxicity) substances based on their solubility in phagolysosomal lung fluid simulant, surface reactivity and effects on alveolar macrophages in vitro. This is exemplified by eleven organic pigments belonging to five chemical classes that cover a significant share of the European market. Three of the pigments were tested as both, nanoform and non-nanoform. The results obtained in this integrated non-animal testing battery qualified two pigments as non PSLT, one pigment as poorly soluble and eight pigments as poorly soluble and low toxicity in vitro. The low toxic potency of the eight PSLT and the one poorly soluble pigment was corroborated by short-term inhalation studies with rats. These pigments did not elicit apparent toxic effects at 10 mg/m3 (systemic and in the respiratory tract). One of the pigments, Diarylide Pigment Yellow 83 transparent, however, caused minimal infiltration of neutrophils; hence its low toxicity is ambiguous and needs further verification or falsification. The present test battery provides an opportunity to identify PSLT-properties of test substances to prioritise particles for further development. Thus, it can help to reduce animal testing and steer product development towards safe applications.

Spatially and size-resolved analysis of gold nanoparticles in rat spleen after intratracheal instillation by laser ablation-inductively coupled plasma-mass spectrometry.

In a dual approach, laser ablation-inductively coupled plasma-mass spectrometry was applied to investigate spleen samples of rats after intratracheal instillation of polyvinylpyrrolidone-coated gold nanoparticles. First, spatially resolved imaging analysis was deployed to investigate gold translocation from the lungs to the spleen and to investigate the distribution pattern of gold in the spleen parenchyma itself. Using the same instrumental setup, laser ablation-inductively coupled plasma-mass spectrometry in single particle mode was applied to determine the species of translocated gold. Single particle analysis allows the determination of particle size distributions and therefore to distinguish between ionic species, intact nanoparticles, and agglomerates. A translocation of instilled gold from the lungs to the spleen was demonstrated for gold nanoparticles of 30 and 50 nm diameter. Furthermore single particle analysis revealed the translocation of intact gold nanoparticles in a non-agglomerated state.

Serum Lowers Bioactivity and Uptake of Synthetic Amorphous Silica by Alveolar Macrophages in a Particle Specific Manner.

Various cell types are compromised by synthetic amorphous silica (SAS) if they are exposed to SAS under protein-free conditions in vitro. Addition of serum protein can mitigate most SAS effects, but it is not clear whether this is solely caused by protein corona formation and/or altered particle uptake. Because sensitive and reliable mass spectrometric measurements of SiO2 NP are cumbersome, quantitative uptake studies of SAS at the cellular level are largely missing. In this study, we combined the comparison of SAS effects on alveolar macrophages in the presence and absence of foetal calf serum with mass spectrometric measurement of 28Si in alkaline cell lysates. Effects on the release of lactate dehydrogenase, glucuronidase, TNFα and H2O2 of precipitated (SIPERNAT® 50, SIPERNAT® 160) and fumed SAS (AEROSIL® OX50, AEROSIL® 380 F) were lowered close to control level by foetal calf serum (FCS) added to the medium. Using a quantitative high resolution ICP-MS measurement combined with electron microscopy, we found that FCS reduced the uptake of particle mass by 9.9% (SIPERNAT® 50) up to 83.8% (AEROSIL® OX50). Additionally, larger particle agglomerates were less frequent in cells in the presence of FCS. Plotting values for lactate dehydrogenase (LDH), glucuronidase (GLU) or tumour necrosis factor alpha (TNFα) against the mean cellular dose showed the reduction of bioactivity with a particle sedimentation bias. As a whole, the mitigating effects of FCS on precipitated and fumed SAS on alveolar macrophages are caused by a reduction of bioactivity and by a lowered internalization, and both effects occur in a particle specific manner. The method to quantify nanosized SiO2 in cells is a valuable tool for future in vitro studies.

Aerogels are not regulated as nanomaterials, but can be assessed by tiered testing and grouping strategies for nanomaterials.

Aerogels contribute to an increasing number of novel applications due to many unique properties, such as high porosity and low density. They outperform most other insulation materials, and some are also useful as carriers in food or pharma applications. Aerogels are not nanomaterials by the REACH definition but retain properties of nanoscale structures. Here we applied a testing strategy in three tiers. In Tier 1, we examined a panel of 19 aerogels (functionalized chitosan, alginate, pyrolyzed carbon, silicate, cellulose, polyurethane) for their biosolubility, and oxidative potential. Biosolubility was very limited except for some alginate and silicate aerogels. Oxidative potential, as by the ferric reduction ability of human serum (FRAS), was very low except for one chitosan and pyrolyzed carbon, both of which were <10% of the positive control Mn2O3. Five aerogels were further subjected to the Tier 2 alveolar macrophage assay, which revealed no in vitro cytotoxicity, except for silicate and polyurethane that induced increases in tumor necrosis factor α. Insufficiently similar aerogels were excluded from a candidate group, and a worst case identified. In the Tier 3 in vivo instillation, polyurethane (0.3 to 2.4 mg) elicited dose-dependent but reversible enzyme changes in lung lavage fluid on day 3, but no significant inflammatory effects. Overall, the results show a very low inherent toxicity of aerogels and support a categorization based on similarities in Tier 1 and Tier 2. This exemplifies how nanosafety concepts and methods developed on particles can be applied to specific concerns on advanced materials that contain or release nanostructures.

Topiramate Decelerates Bicarbonate-Driven Acid-Elimination of Human Neocortical Neurons: Strategic Significance for its Antiepileptic, Antimigraine and Neuroprotective Properties.

BACKGROUND: Mammalian central neurons regulate their intracellular pH (pHi) strongly and even slight pHi-fluctuations can influence inter-/intracellular signaling, synaptic plasticity and excitability. OBJECTIVE: For the first time, we investigated topiramate´s (TPM) influence on pHi-behavior of human central neurons representing a promising target for anticonvulsants and antimigraine drugs. METHODS: In slice-preparations of tissue resected from the middle temporal gyrus of five adults with intractable temporal lobe epilepsy, BCECF-AM-loaded neocortical pyramidal-cells were investigated by fluorometry. The pHi-regulation was estimated by using the recovery-slope from intracellular acidification after an Ammonium-Prepulse (APP). RESULTS: Among 17 pyramidal neurons exposed to 50 µM TPM, seven (41.24%) responded with an altered resting-pHi (7.02±0.12), i.e., acidification of 0.01-0.03 pH-units. The more alkaline the neurons, the greater the TPM-related acidifications (r=0.7, p=0.001, n=17). The recovery from APPacidification was significantly slowed under TPM (p<0.001, n=5). Further experiments using nominal bicarbonate-free (n=2) and chloride-free (n=2) conditions pointed to a modulation of the HCO3 -- driven pHi-regulation by TPM, favoring a stimulation of the passive Cl-/HCO3 --antiporter (CBT) - an acid-loader predominantly in more alkaline neurons. CONCLUSION: TPM modulated the bicarbonate-driven pHi-regulation, just as previously described in adult guinea-pig hippocampal neurons. We discussed the significance of the resulting subtle acidifications for beneficial antiepileptic, antimigraine and neuroprotective effects as well as for unwanted cognitive deficits.

Metabolomics profiling to investigate nanomaterial toxicity in vitro and in vivo.

Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO2 variants, differing in structure, size and surface charge. TiO2 NM-105 served as a benchmark. Responses were studied in vitro in rat lung epithelial cells (RLE-6TN) and alveolar macrophages (NR8383) and compared to in vivo responses in rat lung tissues obtained from in vivo instillation and short-term inhalation studies (STIS). Time- and concentration-dependent changes were observed in both in vitro models but with cell-type specific responses. Overall, the levels of lipids and biogenic amines (BAs) tended to increase in epithelial cells but decreased in macrophages. Many identified metabolites like Met-SO, hydroxy-Pro and spermidine were related to oxidative stress, indicating that oxidative stress contributes to the MoA for the selected NMs. Several biomarker candidates such as Asp, Asn, Ser, Pro, spermidine, putrescine and LysoPCaC16:1 were identified in vitro and verified in vivo. In this study, we successfully applied a metabolomics workflow for in vitro and in vivo samples, which proved to be well suited to identify potential biomarkers, to gain insights into NM structure-activity relationship and into the underlying MoA.

Revealing Silver Nanoparticle Uptake by Macrophages Using SR-µXRF and LA-ICP-MS.

To better study the impact of nanoparticles on both in vitro and in vivo models, tissue distribution and cellular doses need to be described more closely. Here silver nanoparticles were visualized in alveolar macrophages by means of synchrotron radiation micro X-ray fluorescence spectroscopy (SR-µXRF) with high spatial resolution of 3 × 3 µm2. For the spatial allocation of silver signals to cells and tissue structures, additional elemental labeling was carried out by staining with eosin, which binds to protein and can be detected as bromine signal with SR-µXRF. The method was compatible with immunostaining of macrophage antigens. We found that the silver distribution obtained with SR-µXRF was largely congruent with distribution maps from a subsequent laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) of the same tissue sites. The study shows a predominant, though not exclusive uptake of silver into alveolar macrophages in the rat lung, which can be modeled by a similar uptake in cultured alveolar macrophages. Advantages and limitations of the different strategies for measuring nanoparticle uptake at the single cell level are discussed.

Lung Toxicity Analysis of Nano-Sized Kaolin and Bentonite: Missing Indications for a Common Grouping.

Kaolin and bentonite (nanoclay NM-600) are nanostructured aluminosilicates that share a similar chemical composition, platelet-like morphology, and high binding capacity for biomolecules. To investigate if these material-based criteria allow for a common grouping, we prepared particle suspensions of kaolin and bentonite with a similar hydrodynamic diameter and administered them to NR8383 alveolar macrophages in vitro and also to a rat lung using quartz DQ12 as a reference material. Bentonite was far more bioactive in vitro, indicated by a lower threshold for the release of enzymes, tumor necrosis factor α, and H2O2. In addition, in the lung, the early effects of bentonite exceeded those of kaolin and even those of quartz, due to strongly increased numbers of inflammatory cells, and elevated concentrations of total protein and fibronectin within the bronchoalveolar lavage fluid. The pro-inflammatory effects of bentonite decreased over time, although assemblies of particle-laden alveolar macrophages (CD68 positive), numerous type-2 epithelial cells (immunopositive for pro-surfactant protein C), and hypertrophic lung epithelia persisted until day 21. At this point in time, kaolin-treated lungs were completely recovered, whereas quartz DQ12 had induced a progressive inflammation. We conclude that bentonite is far more bioactive than equally sized kaolin. This argues against a common grouping of aluminosilicates, previously suggested for different kaolin qualities.

Predicting dissolution and transformation of inhaled nanoparticles in the lung using abiotic flow cells: The case of barium sulfate.

Barium sulfate (BaSO4) was considered to be poorly-soluble and of low toxicity, but BaSO4 NM-220 showed a surprisingly short retention after intratracheal instillation in rat lungs, and incorporation of Ba within the bones. Here we show that static abiotic dissolution cannot rationalize this result, whereas two dynamic abiotic dissolution systems (one flow-through and one flow-by) indicated 50% dissolution after 5 to 6 days at non-saturating conditions regardless of flow orientation, which is close to the in vivo half-time of 9.6 days. Non-equilibrium conditions were thus essential to simulate in vivo biodissolution. Instead of shrinking from 32 nm to 23 nm (to match the mass loss to ions), TEM scans of particles retrieved from flow-cells showed an increase to 40 nm. Such transformation suggested either material transport through interfacial contact or Ostwald ripening at super-saturating conditions and was also observed in vivo inside macrophages by high-resolution TEM following 12 months inhalation exposure. The abiotic flow cells thus adequately predicted the overall pulmonary biopersistence of the particles that was mediated by non-equilibrium dissolution and recrystallization. The present methodology for dissolution and transformation fills a high priority gap in nanomaterial hazard assessment and is proposed for the implementation of grouping and read-across by dissolution rates.

A multi-omics approach reveals mechanisms of nanomaterial toxicity and structure-activity relationships in alveolar macrophages.

In respect to the high number of released nanomaterials and their highly variable properties, novel grouping approaches are required based on the effects of nanomaterials. Proper grouping calls for a combination of an experimental setup with a higher number of structurally similar nanomaterials and for employing integrated omics approaches to identify the mode of action. Here, we analyzed the effects of seven well-characterized NMs comprising different chemical compositions, sizes and chemical surface modifications on the rat alveolar macrophage cell line NR8383. The NMs were investigated at three doses ranging from 2.5 to 10 µg/cm2 after 24 h incubation using an integrated multi-omics approach involving untargeted proteomics, targeted metabolomics, and src homology 2 (SH2) profiling. By using Weighted Gene Correlation Network Analysis (WGCNA) for the integrative data, we identified correlations of molecular pathways with physico-chemical properties and toxicological endpoints. The three investigated SiO2 variants induced strong alterations in all three omics approaches and were, therefore, be classified as "active." Two organic phthalocyanines showed minor responses and Mn2O3 induced a different molecular response pattern than the other NMs. WGCNA revealed that agglomerate size and surface area as well as LDH release are among the most important parameters correlating with nanotoxicology. Moreover, we identified key drivers that can serve as representative biomarker candidates, supporting the value of multi-omics approaches to establish integrated approaches to testing and assessment (IATAs).

The nanoGRAVUR framework to group (nano)materials for their occupational, consumer, environmental risks based on a harmonized set of material properties, applied to 34 case studies.

The project nanoGRAVUR (BMBF, 2015-2018) developed a framework for grouping of nanomaterials. Different groups may result for each of the three distinct perspectives of occupational, consumer and environmental safety. The properties, methods and descriptors are harmonised between the three perspectives and are based on: Tier 1 intrinsic physico-chemical properties (what they are) or GHS classification of the non-nano-form (human tox, ecotox, physical hazards); Tier 2 extrinsic physico-chemical properties, release from nano-enabled products, in vitro assays with cells (where they go; what they do); Tier 3 case-specific tests, potentially in vivo studies to substantiate the similarity within groups or application-specific exposure testing. Amongst all properties, dissolution and transformation are least modulated by different nanoforms within one substance, whereas dustiness, dispersion stability, abiotic and especially in vitro surface reactivity vary more often between different nanoforms. The methods developed or selected by nanoGRAVUR fill several gaps highlighted in the ProSafe reviews, and are useful to implement (i) the concept of nanoforms of the European Chemicals Agency (ECHA) and (ii) the concept of discrete forms of the United States Environmental Protection Agency (EPA). One cannot assess the significance of a dissimilarity, if the dynamic range of that property is unknown. Benchmark materials span dynamic ranges that enable us to establish bands, often with order-of-magnitude ranges. In 34 case studies we observed high biological similarity within each substance when we compared different (nano)forms of SiO2, BaSO4, kaolin, CeO2, ZnO, organic pigments, especially when we compared forms that are all untreated on the surface. In contrast, different Fe2O3 or TiO2 (nano)forms differ more significantly. The same nanoforms were also integrated in nano-enabled products (NEPs) for automotive coatings, clinker-reduced cements, cosmetic sunscreen, and lightweight polymers.

Levetiracetam mediates subtle pH-shifts in adult human neocortical pyramidal cells via an inhibition of the bicarbonate-driven neuronal pH-regulation - Implications for excitability and plasticity modulation.

The intracellular pH (pHi) of mammalian central neurons is tightly regulated and small pHi-fluctuations can fine-tune inter-/intracellular signaling, excitability, and synaptic plasticity. The research-gap about the pHi-regulation of human brain neurons is addressed here by testing possible influences of the anticonvulsant levetiracetam (LEV). BCECF-AM-loaded neocortical pyramidal cells were fluorometrically investigated in slice-preparations of tissue resected from the middle temporal gyrus of five adults with intractable temporal-lobe epilepsy. Recovery-slope from intracellular acidification following an ammonium prepulse (APP) was used to measure the pHi-regulation. Among twenty pyramidal cells exposed to 50 µM LEV, the resting pHi (7.09 ±â€¯0.14) was lowered in eight (40%) neurons, on average by 0.02 ±â€¯0.011 pH-units. In three (15%) and nine (45%) neurons, a minimal alkaline shift (0.017 ±â€¯0.004 pH-units) and no pHi-shift occurred, respectively. The LEV-induced pHi-shifts were positively correlated with the resting pHi (r = 0.6, p = 0.006, n = 20). In five neurons, which all had responded on LEV with an acidification before, the recovery from APP-acidification was significantly delayed during LEV (p < 0.001). This inhibitory LEV-effect on pHi-regulation i) was similar to that of 200 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (n = 2) and ii) did not occur under nominal bicarbonate-free conditions (n = 2). Thus, LEV lowered the pHi of human neocortical pyramidal cells most likely by a weakening of the transmembrane HCO3(-)-mediated acid-extrusion. This might contribute to LEV's anticonvulsive potency. Neurons with more acidic resting pHi-values showed a minimal alkalization upon LEV providing a mechanism for paradoxical proconvulsive LEV-effects rarely observed in epilepsy patients. The significance of these subtle pHi-shifts for cortical excitability and plasticity is discussed.