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

Bittner, Aniela (2020). Do nanoparticles used in laser tissue soldering interfere with endothelial cells and the blood-brain barrier? (Unpublished). (Dissertation)

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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.

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