Time-Dependent Internalization of Polymer-Coated Silica Nanoparticles in Brain Endothelial Cells and Morphological and Functional Effects on the Blood-Brain Barrier

Nanoparticle (NP)-assisted procedures including laser tissue soldering (LTS) offer advantages compared to conventional microsuturing, especially in the brain. In this study, effects of polymer-coated silica NPs used in LTS were investigated in human brain endothelial cells (ECs) and blood-brain barrier models. In the co-culture setting with ECs and pericytes, only the cell type directly exposed to NPs displayed a time-dependent internalization. No transfer of NPs between the two cell types was observed. Cell viability was decreased relatively to NP exposure duration and concentration. Protein expression of the nuclear factor k-light-chain-enhancer of activated B cells and various endothelial adhesion molecules indicated no initiation of inflammation or activation of ECs after NP exposure. Differentiation of CD34+ ECs into brain-like ECs co-cultured with pericytes, blood-brain barrier (BBB) characteristics were obtained. The established endothelial layer reduced the passage of integrity tracer molecules. NP exposure did not result in alterations of junctional proteins, BBB formation or its integrity. In a 3-dimensional setup with an endothelial tube formation and tight junctions, barrier formation was not disrupted by the NPs and NPs do not seem to cross the blood-brain barrier. Our findings suggest that these polymer-coated silica NPs do not damage the BBB

Do nanoparticles used in laser tissue soldering interfere with endothelial cells and the blood-brain barrier?

Introduction: Over the past few decades, nanotechnology has been gaining importance, further broadening the scope of application for a wide range of nanomaterials, involving areas from all parts of life. The utilization of nanomaterials for medical purposes has led to the rise of nanomedicine. Improved diagnostics and advanced or new treatment methods such as enhanced and targeted drug delivery or use in transplants or biodegradable implants as in laser tissue soldering (LTS) were made possible. LTS provides a novel treatment method for ruptured cerebrovascular aneurysms that offers several advantages compared to conventional microsuturing. The chromophore indocyanine green (ICG) allows for a targeted and focused effect by transducing the laser light into heat leading to tissue fusion. Incorporating nanoparticles (NPs) in the solder has enabled circumvention of the poor stability and proneness to bleaching of the ICG, further enhancing the technique whilst reducing damage to the surrounding tissue. However, due to the biodegradability of the solder, NPs are slowly released into the brain tissue over time where they might elicit adverse reactions. Even though inert or biocompatible materials are used for the production of NPs for medical purposes, they are foreign to the body and might induce morphological and functional disruption of cells of the brain or the vessels and the blood-brain barrier. Hence, detailed assessment of the interactions of NPs with tissues they come in contact with is essential. Aims: This PhD project is aimed at identifying and characterizing suitable in vitro models of brain endothelial cells and the blood-brain barrier (I) to study potential uptake and subsequent effects of polymer-coated NPs designed for LTS (II). First, cell viability and several regulatory cell-signaling pathways of endothelial cells of the brain were investigated. Second, possible effects of the NPs on mitochondrial respiration and integrity were assessed as both are critical parameters for proper cellular function. Furthermore, a potential impact of these NPs on the integrity and function of the blood-brain barrier (III) was examined whilst adapting the culture settings to get closer to the in vivo situation. Methods: The uptake and possible effects of polymer-coated and gold NPs on brain endothelial cells were assessed in a simple monoculture model of immortalized rat brain capillary endothelial cells (rBCEC4). These findings were evaluated in a co-culture model consisting of primary human brain-like endothelial cells and bovine pericytes and then verified in a 3-dimensional (3D) model. Immunofluorescence (IF) and transmission electron microscopy as well as high-content analysis were employed to study and quantify NP uptake into endothelial cells and evaluate the cells' morphology. Expression levels of key markers of various signaling pathways were analyzed by means of Western blotting and IF and potential cytotoxic effects were examined with respective assays. Studies of the transendothelial 7 8 electrical resistance and permeability to defined tracers of the cell monolayer allowed drawing conclusions on the impact of NPs on the integrity and function of the blood-brain barrier. Results: The studies revealed that the polymer-coated NPs were taken up quickly and to a high extent by rat and human endothelial cells and were found within membrane-surrounded vesicles in the cytoplasm whereas gold NPs were hardly internalized and co-localized solely with heterolysosomes. Depicting overall similar behavior, all types of NPs led to a time- and concentration-dependent reduction of cell viability, which might be due to disruption of mitochondrial function and network regulation. Polymer-coated NPs were shown to affect mitochondrial respiration during the stress test in maximally stimulated endothelial cells only. The NPs reduced the expression of proteins involved in fusion and fission processes whereas adenosine triphosphate content and mitochondrial morphology remained unaltered. The addition of another stressor led to a shift towards a stressed phenotype in the endothelial cells. Neither polymer-coated nor gold NPs interfered with regulatory signaling pathways or induced inflammation or activation of the endothelial cells in the models used. Both types of endothelial cells expressed various tight and adherens junctions and showed restricted passage of defined tracer molecules across an endothelial monolayer. NP exposure did not disrupt the junction proteins. Furthermore, the electrical resistance across and the permeability of the established endothelial cell barrier were not affected by the different types of NPs. The same result was seen when endothelial cells or pericytes were exposed to NPs during formation of the blood-brain barrier. Preliminary data on the 3D model showed that the cell types involved in the co-culture model can be transferred and adapted to a 3D setting. Endothelial cells formed tight junctions and the leakage of fluorescent tracers was reduced in the presence of an endothelial barrier. Conclusions: Validation of the models used for investigating potential effects of NPs is crucial. A simple monoculture with cells of rat origin aided in obtaining a basic understanding of the interactions between NPs and brain endothelial cells. The data could be verified in a co-culture model with human endothelial cells and bovine pericytes. NP - cell interactions could be assessed in a setting closer to the in vivo situation due to the nature and number of cell types contained in this model. Finally, adapting the co-culture model to a 3D setting enabled the structure of an in vivo vessel to be taken into account.Overall, the findings of this PhD project indicate that polymer-coated or gold NPs are suitable for use in LTS as the effects on the mitochondrial respiration and integrity of brain cells were seen only at maximal respiration and at concentrations that are much higher than the ones expected to be present in the brain tissue during degradation of the solder. However, further investigations on possible long-term effects on mitochondrial health have to be performed to 8 9 rule out lasting effects resulting in complete impairment of mitochondrial function and detrimental consequences for the cells

Do polymer-coated nanoparticles impair mitochondrial function of brain endothelial cells?

A promising novel treatment method for cerebrovascular aneurysms and injuries of other hollow organs is laser tissue soldering. The solder contains nanomaterials that are foreign to the body, making careful examination of possible adverse effects prior to introducing this technique essential. This study aimed at analyzing the effects of different concentrations of polymer-coated silica nanoparticles (NPs) on mitochondrial function and integrity of brain endothelial cells using the rat brain capillary endothelial cell line rBCEC4. NP exposure led to a decrease in the oxygen consumption rate at maximal capacity whereas glycolysis was not affected. In combination with glucose deprivation, NPs primarily hindered glycolytic ATP generation rather than oxidative phosphorylation and caused a metabolic shift towards a stressed phenotype indicated by increased oxygen consumption rates and extracellular acidification rates compared to untreated controls. Mitochondrial mass, distribution and morphology as well as intracellular ATP content were not altered by NPs and mitochondrial membrane potential was increased after exposure to the highest NP concentration only. Merely slight changes were seen in the expression levels of proteins involved in mitochondrial dynamics. In conclusion, polymer-coated NPs did not seem to impair mitochondrial function vitally in rBCEC4 cells at steady state

Polymer-coated nanoparticles and their effects on mitochondrial function in brain endothelial cells.

Laser tissue soldering is a novel treatment method for injuries of hollow organs such as cerebrovascular aneurysms. Nanomaterials contained in the solder are foreign to the body. Hence, it is indispensable to carefully examine possible adverse effects prior to introducing this technique. The aim of this study was to characterize the impact of different concentrations of polymer-coated silica nanoparticles (NPs) on mitochondrial function and integrity of brain endothelial cells using the rat brain capillary endothelial cell line rBCEC4. At maximal capacity, NP exposure resulted in a decrease in the oxygen consumption rate whereas glycolysis was not affected. In combination with a stressor, i.e. lack of glucose in the medium, NP exposure interfered primarily with glycolytic ATP generation rather than oxidative phosphorylation. Furthermore, NPs caused a metabolic shift towards a stressed phenotype, exhibiting increased levels of the oxygen consumption rate and the extracellular acidification rate compared to untreated controls. Overall, mitochondrial mass, distribution and morphology as well as intracellular ATP content were not altered. The mitochondrial membrane potential was increased after exposure to the highest NP concentration and the content of proteins involved in mitochondrial dynamics was changed slightly, indicating possible modifications of the fusion / fission balance. In conclusion, PCL-NP exposure changed mitochondrial respiration, especially under glucose deprivation, but did not affect mitochondrial morphology and distribution. Further studies are needed to investigate whether the functional effects are transient or long-term as this will be crucial for the use of these NPs in laser tissue soldering

Effects of gold and PCL- or PLLA-coated silica nanoparticles on brain endothelial cells and the blood–brain barrier

Nanomedicine is a constantly expanding field, facilitating and improving diagnosis and treatment of diseases. As nanomaterials are foreign objects, careful evaluation of their toxicological and functional aspects prior to medical application is imperative. In this study, we aimed to determine the effects of gold and polymer-coated silica nanoparticles used in laser tissue soldering on brain endothelial cells and the blood–brain barrier using rat brain capillary endothelial cells (rBCEC4). All types of nanoparticles were taken up time-dependently by the rBCEC4 cells, albeit to a different extent, causing a time- and concentration-dependent decrease in cell viability. Nanoparticle exposure did not change cell proliferation, differentiation, nor did it induce inflammation. rBCEC4 cells showed blood–brain barrier characteristics including tight junctions. None of the nanoparticles altered the expression of tight junctions or impaired the blood–brain barrier permeability. The findings suggest that effects of these nanoparticles on the metabolic state of cells have to be further characterized before use for medical purposes

Effects of silica nanoparticle exposure on mitochondrial function during neuronal differentiation

Abstract Background Nanomedicine offers a promising tool for therapies of brain diseases, but potential effects on neuronal health and neuronal differentiation need to be investigated to assess potential risks. The aim of this study was to investigate effects of silica-indocyanine green/poly (ε-caprolactone) nanoparticles (PCL-NPs) engineered for laser tissue soldering in the brain before and during differentiation of SH-SY5Y cells. Considering adaptations in mitochondrial homeostasis during neuronal differentiation, metabolic effects of PCL-NP exposure before and during neuronal differentiation were studied. In addition, kinases of the PI3 kinase (PI3-K/Akt) and the MAP kinase (MAP-K/ERK) pathways related to neuronal differentiation and mitochondrial function were investigated. Results Differentiation resulted in a decrease in the cellular respiration rate and the extracellular acidification rate (ECAR). PCL-NP exposure impaired mitochondrial function depending on the time of exposure. The cellular respiration rate was significantly reduced compared to differentiated controls when PCL-NPs were given before differentiation. The shift in ECAR was less pronounced in PCL-NP exposure during differentiation. Differentiation and PCL-NP exposure had no effect on expression levels and the enzymatic activity of respiratory chain complexes. The activity of the glycolytic enzyme phosphofructokinase was significantly reduced after differentiation with the effect being more pronounced after PCL-NP exposure before differentiation. The increase in mitochondrial membrane potential observed after differentiation was not found in SH-SY5Y cells exposed to PCL-NPs before differentiation. The cellular adenosine triphosphate (ATP) production significantly dropped during differentiation, and this effect was independent of the PCL-NP exposure. Differentiation and nanoparticle exposure had no effect on superoxide levels at the endpoint of the experiments. A slight decrease in the expression of the neuronal differentiation markers was found after PCL-NP exposure, but no morphological variation was observed. Conclusions PCL-NP exposure affects mitochondrial function depending on the time of exposure before and during neuronal differentiation. PCL-NP exposure during differentiation was associated with impaired mitochondrial function, which may affect differentiation. Considering the importance of adaptations in cellular respiration for neuronal differentiation and function, further studies are needed to unravel the underlying mechanisms and consequences to assess the possible risks including neurodegeneration