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Lab Notes on: the Bioeffects of Nanoplastics on Animal Neurons and Behavior

Plastic pollution has become an increasingly larger issue, with about 10% of annual plastic production ending up in aquatic environments. Plastics degrade into smaller fragments, leading to the formation of microplastics and nanoplastics. These particles are a growing concern due to their potential health risks. Studies have shown that microplastics can be internalized by macrophages and transferred to tissues, causing toxic effects. Direct contact with nanoplastics can induce neurotoxicity in fish and bivalves through the food chain. Early research suggests that microplastic ingestion can lead to tissue accumulation and behavioral disturbances in animals.


Nanoplastics have been found in the plant Arabidopsis thaliana, affecting growth and generating reactive oxygen species. However, limited information exists about the effects of inhalable nanoplastics on mammals, particularly their nervous systems. The study “Bioeffects of Inhaled Nanoplastics on Neurons and Alteration of Animal Behaviors through Deposition in the Brain” investigated the pathways of nanoplastic uptake in mammals through inhalation, focusing on various polystyrene nanoparticles (PS-NPs).


The study utilized two types of functionalized fluorescent polystyrene nanoplastics of varying sizes: PS-COOH and PS-NH2. Standard fluorescence intensity-concentration curves were established to facilitate subsequent research involving cellular uptake and animal studies.


Standard fluorescence intensity-concentration curves are graphical representations that illustrate the relationship between the intensity of fluorescence emitted by a substance and its concentration. These curves are commonly used in fluorescence spectroscopy, which is a technique used to analyze the fluorescence properties of molecules, such as dyes, fluorophores, or nanoparticles. Standard fluorescence intensity-concentration curves serve as a reference for quantifying unknown concentrations of the fluorescent substance. By comparing the fluorescence intensity of an unknown sample to the curve, you can estimate its concentration. This is a fundamental technique used in various fields, including chemistry, biology, and environmental science, for tasks such as measuring the concentration of specific molecules or nanoparticles in a solution.


A microfluidic gradient chip was designed to achieve controlled nanoplastic gradient dilution for cellular uptake experiments. TEM analysis showcased different nanoplastic concentrations at distinct chip outlets. This chip streamlined the generation of gradient concentrations of polystyrene nanoparticles (PS-NPs) in a single step, enabling automatic assessment of PS-NP effects on neurons within the cell chamber. This approach offers advantages over conventional methods, enhancing nanoparticle study efficiency.


Transmission Electron Microscopy (TEM) is a high-resolution imaging technique. It works by passing a beam of electrons through an ultra-thin specimen, allowing for detailed visualization of its internal structure at the nanoscale. TEM offers exceptional resolution, enabling scientists to observe individual nanoparticles, crystal lattices, and biological structures. It's widely used in materials science, biology, and nanotechnology research to examine the fine details of various samples, providing crucial insights into their properties and structures.


Neurons were cultivated in the microfluidic chip's cell chamber and verified using immunofluorescence analysis. Nanoplastics were introduced into the chip, reaching different cell chambers based on concentration. High-content analysis showed that smaller nanoplastics were preferred by neurons, with NH2-modified ones being more readily internalized than pristine PS and COOH-modified types of the same size. Uptake increased with higher nanoplastics exposure. Among the studied nanoplastics, 80 nm PS-NH2 displayed the highest cellular uptake rate.


Immunofluorescence analysis is a laboratory technique used to detect and visualize specific proteins or antigens within biological samples, like cells or tissues. It involves using fluorescently labeled antibodies that bind to the target protein. When exposed to specific wavelengths of light, these bound antibodies emit fluorescent signals, indicating the presence and location of the protein of interest. Immunofluorescence is vital in biological and medical research for studying cellular structures, protein localization, and disease markers, providing valuable insights into various biological processes and pathologies.


High-content analysis (HCA) is an automated imaging and quantitative analysis technique used in biology and drug discovery. It combines advanced microscopy with computer-assisted analysis to rapidly examine and measure multiple cellular features within large datasets. HCA provides detailed information on cell morphology, protein expression, and cellular processes, allowing researchers to study complex biological phenomena and screen for potential drug candidates. This technology accelerates research in fields like cell biology, pharmacology, and toxicology by providing high-throughput and multiparametric analysis of cells and tissues.


In mice, 80 nm pristine PS, PS-NH2, and PS-COOH were selected for further study. Aerosol inhalation was used to expose mice to nanoplastics, demonstrating their penetration of the blood-brain barrier (BBB) into brains and livers. Nanoplastics accumulated in specific brain regions and were associated with possible inflammation, brain disorders, and cognitive issues. Serum accumulation was observed too. The experiment suggested two potential direct routes for nanoplastics' brain entry: the trigeminal (a cranial nerve) and olfactory nerve pathways. Inhalation of nanoplastics also affected mice behavior, reducing their average speed and altering anxiety-like behavior. Moreover, the study showed that nanoplastics inhibited acetylcholinesterase (AChE) activity, indicating potential neurotoxicity. This research underscores the need to investigate nanoplastics' effects on human health, particularly the nervous system.


Acetylcholinesterase is an enzyme found in the nervous system that regulates the neurotransmitter acetylcholine by breaking it down. This enzymatic action is critical in terminating nerve signals at synapses, allowing muscles to relax and preventing overstimulation. Inhibition of acetylcholinesterase can lead to excess acetylcholine, causing nerve and muscle dysfunction, and is a target in treatments for conditions like Alzheimer's and nerve agent poisoning.


The study investigated the effects of different-sized nanoplastics (80 nm) with varying surface ligands on mammalian brains. Previous research had only explored nanoplastic toxicity in fish brains, leaving mammalian effects unstudied. The designed inhalation system allowed nanoplastics to bypass the blood-brain barrier, reaching the brains, livers, and organs of mice. Fluorescence imaging revealed nanoplastic distribution primarily in the olfactory tubercle, cerebrum, cerebellum, and pons. Cellular uptake correlated with surface ligands, with NH2-modified nanoplastics showing higher effectiveness than COOH-modified ones. This accumulation was linked to inflammation, suggesting brain disorders and cognitive issues. Behavioral changes were observed in mice, possibly due to nanoplastic neurotoxicity. AChE activity inhibition and increased inflammatory factors indicated neurotoxic effects. Nanoplastics primarily entered the brain, as indicated by imaging and histological analysis. Overall, this study uncovered the direct entry of nanoplastics into mammalian brains through inhalation, raising concerns about their potential toxicological impacts.


A surface ligand refers to a molecule or structure on the surface of a cell or particle that can bind or interact with other molecules, such as receptors or proteins. These interactions play crucial roles in various biological processes, including cell signaling, immune responses, and cellular recognition. In this case, the surface ligands tested were NH2 and COOH.


The study emphasizes that nanoplastic properties significantly influence neuronal uptake and animal behavior. A microfluidic chip generates varying PS-NP concentrations, enabling high-throughput analysis of their interaction with neurons. All PS-NPs are taken up, with smaller ones inducing more uptake and neurotoxicity. PS-NH2 shows higher uptake than PS-COOH at the same size. Inhalation of PS-NPs reduces mouse activity compared to water droplets. The research highlights brain deposition of inhaled nanoplastics affecting animal behavior. It serves as a foundation for investigating nanoplastics' impact on mammal brains and aims to block potential pathways for brain deposition. The study's microfluidic system offers quick screening for nanoparticle-cell interactions, aiding nanoparticle toxicity research and environmental studies. It holds promise for broader nanoparticle toxicity studies. Future research will explore strategies to prevent brain deposition.

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