Novel concept of interaction-based nanoparticle dose and its modulation by lung surfactant : doctoral dissertation
Interakcijska doza nanodelcev - uvedba novega koncepta in študij vpliva surfaktantov na novo definirano dozo

english

doktorska dela

2023

dissertations   dose   fluorescence   fluorescent microscopy   high-resolution microscopy   image analysis   in vitro model   interactive dose   local dose   local interactive dose   nano-bio interactions   nanoparticles   nanotoxicity   pulmonary surfactant   pulmonary surfactant proteins   stimulated emission microscopy   surface dose   titanium dioxide   titanium dioxide nanotubes   toxicology

Univerza v Mariboru, Fakulteta za naravoslovje in matematiko

We are daily exposed to nanosized particulate matter (nanoparticles) with inherent toxicity via inhalation, digestion and skin abrasion. Degradation, pollution and production originating air-born nanoparticle (NP) inhalation is especially problematic, because it is difficult to avoid and can lead to severe adverse outcomes such as cardiovascular diseases, inflammation, lung cancer and brain damage. The correct determination of NP toxicity is thus of paramount importance for the successful regulation of NP production.Toxicity of inhaled NPs in the field of nanotoxicity is defined with respect to the cumulative dose administered to the system at which adverse effects are observed. Current most widely accepted definition of the surface dose, defined as the ratio between total NP surface administered to the system and total expected membrane/tissue surface, omits local interactions and location of the dose in the sample, which consequently determine the actual effect of delivered dose to the system. Existing norm to determine NP toxic dose is weeks long exposure of animals to NPs, while cheaper and faster alternatives in form of advanced in vitro models with model cell lines emerged, many of them missing out an important component – the pulmonary surfactant.To advance the dose definition and its understanding we used a simplified model consisting of lung epithelial cells, lung surfactant and titanium dioxide nanotubes. To mimic inhalation exposure, we nebulised surfactant on top of cells and then NPs on top of surfactant layer. We built a custom stage-top incubator capable of nebulisation exposure and imaging of our in vitro system under physiological conditions from minutes to several days after exposure directly under our high-resolution capable microscope. By exposure of cells to several NPs at the same cell normalized dose we demonstrated that current dose definition inadequately describes the actual dose effect within biological system.To advance the lacking dose definition, we introduced a new concept of interactive surface and interactive dose where the interactions between membrane surface and NPs were directly considered. Newly introduced concept enabled us to evaluate and visualize the interactive dose pixel-by-pixel in each image. Newly defined parameters, the average interactive dose and average local interactive dose, gave a much better dose distribution estimation, considering non-homogeneity of the dose distribution besides the local interactions. By monitoring of time evolution of the average local interactive dose, we were able to determine that the dose is modulated by the biological system in the absence of pulmonary surfactant.We further verified that the dose modulation changes by the introduction of pulmonary surfactant: the dose dilution-quarantining phase moves to earlier times, followed by a newly introduced dose dilution phase, absent without surfactant, which enables the dose re-distribution further in time and space.To track the surfactant proteins, NPs and lipids together right after nebulisation we developed a fluorescence lifetime based three-channel imaging. We determined that 1) hydrophobic surfactant proteins aggregate together with titanium dioxide nanotubes, 2) that the nanotubes penetrate the physiological relevant thickness of surfactant in a matter of seconds and 3) that transient holes appear in the surfactant during this process, allowing also uncoated NPs to pass the surfactant barrier.Finally, three-channel imaging revealed that the dose of NP depends on its local environment, e. g. the border between the cell nuclei and cytoplasm, and the presence of bio-molecules, enabling us the development of a novel concept of a NP dose as a bio-molecule dependent vector. This concept has the potential for discovery of toxicity mechanisms and their propagation which could lead to better understanding of key molecular events that are basis for the successful animal-free predictive toxicology.

08.08.2023