Carboxyl-modified Polystyrene Nanoparticles Research Articles

Impact of mucus modulation by N-acetylcysteine on nanoparticle toxicity

Human respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.

In vitro wheat protoplast cytotoxicity of polystyrene nanoplastics

Nanoplastics are an emerging environmental pollutant, having a potential risk to the terrestrial ecosystem. In the natural environment, almost all the micro-or nano-plastics will be aged by many factors and their characterizations of the surface will be modified. However, the toxicity and mechanism of the modified polystyrene nanoparticles (PS-NPs) to plant cells are not clear. In the study, the amino- and carboxyl-modified PS-NPs with different sizes (20 and 200 nm) were selected as the typical representatives to investigate their effects on protoplast cell viability, reactive oxygen species (ROS) production in the cell and the leakage of cell-inclusion and apoptosis. The results indicated that the 20 nm amino-modified PS-NPs (PS-20A) could significantly damage the structure of the cell, especially the cell membrane, chloroplast and mitochondrion. After being modified by amino group, smaller size nanoplastics had the potential to cause more severe damage. In addition, compared with carboxyl-modified PS-NPs, the amino-modified PS-NPs induced more ROS production and caused higher membrane permeability/lactate dehydrogenase (LDH) leakage. Apoptosis assay indicated that the proportion of viable cells in the PS-20A treatment decreased significantly, and the proportion of necrotic cells increased by four times. This study provides new insights into the toxicity and damage mechanism of PS-NPs to terrestrial vascular plants at the cellular level, and guides people to pay attention to the quality and safety of agricultural products caused by nanoplastics.

Interplay Between Nanoplastics and the Immune System of the Mediterranean Sea Urchin Paracentrotus lividus

The present study highlights for the first time the interplay between model nanoplastics, such as the carboxyl-modified polystyrene nanoparticles (PS-COOH, 60 nm) NPs and the coelomocytes of the sea urchin Paracentrotus lividus, a benthic grazer widely distributed in Mediterranean coastal area, upon acute in vitro exposure (4 h) (5 and 25 μg mL–1). Insight into PS-COOH trafficking (uptake and clearance) and effects on immune cell functions (i.e., cell viability, lysosomal membrane stability, and phagocytosis) are provided. Dynamic Light Scattering analysis reveals that PS NP suspensions in CF undergo a quick agglomeration, more pronounced for PS-COOH (608.3 ± 43 nm) compared to PS-NH2 (329.2 ± 5 nm). However, both PS NPs are still found as nano-scale agglomerates in CF after 4 h of exposure, as shown by the polydispersity index > 0.3 associated with the presence of different PS NP size populations in the CF. The observed changes in ζ-potential upon suspension in CF (–11.1 ± 3 mV and –12.1 ± 4 mV for PS-COOH and PS-NH2, respectively) confirm the formation of a bio-corona on both PS NPs. Optical fluorescence microscopy and fluorimetric analyses using fluorescently labeled PS-COOH (60 nm) reveal a fast uptake of PS-COOH primarily by phagocytes within 1 h of exposure. Upon transfer to PS NP-free CF, a significant decrease in fluorescence signal is observed, suggesting a fast cell clearance. No effect on cell viability is observed after 4 h of exposure to PS-COOH, however a significant decrease in lysosomal membrane stability (23.7 ± 4.8%) and phagocytic capacity (63.43 ± 3.4%) is observed at the highest concentration tested. Similarly, a significant reduction in cell viability, lysosomal membrane stability and phagocytosis is found upon exposure to PS-NH2 (25 μg mL–1), which confirms the important role of surface charges in triggering immunotoxicity. Overall, our results show that, although being quickly internalized, PS-COOH can be easily eliminated by the coelomocytes but may still be able to trigger an immune response upon long-term exposure scenarios. Taking into account that sediments along Mediterranean coasts are a sink for micro- and nanoplastics, the latter can reach concentrations able to exceed toxicity-thresholds for marine benthic species.

Rapid and sensitive pathogen detection platform based on a lanthanide-labeled immunochromatographic strip test combined with immunomagnetic separation

Sensitive point-of-care pathogen detection methods are urgently required as pathogens pose a significant threat to food safety and human health. Here, we reported a platform based on a lanthanide-labeled immunochromatographic strip test combined with immunomagnetic separation for the detection of pathogens in food and clinical samples. First, immunomagnetic separation technology was employed as a pre-treatment to eliminate the effect of background in the samples. Then, the lanthanide chelate-loaded carboxyl-modified polystyrene nanoparticles, exhibiting bright luminescence, wide Stokes shift, and excellent stability, were prepared and used to label pathogens with antibody. Finally, an immunochromatographic strip test, based on a sandwich format and supported by our custom-designed portable reader, was used to rapidly quantify the result. Using Salmonella typhimurium as a model pathogen, under optimal conditions, we achieved a detection limit of 103 CFU/mL and a linear range of 104–106 CFU/mL in milk and human serum with good recovery, reproducibility, and specificity. Compared with the existing colloidal gold-based immunochromatographic strip test, our platform showed high sensitivity (100-fold improvement) and provided quantitative determination. Our detection platform was therefore demonstrated to be highly sensitive, rapid (within 1.5 h), and cost-effective, providing a promising platform for point-of-care pathogen detection in food and clinical fields.

Polystyrene Nanoparticles Reduced ROS and Inhibited Ferroptosis by Triggering Lysosome Stress and TFEB Nucleus Translocation in a Size-Dependent Manner.

Though plastic nanoparticles have already raised much concern for their potential impact on health, our understanding of their biological effects is still utterly limited. Here we demonstrate for the first time that carboxyl-modified polystyrene nanoparticles (CPS) could effectively inhibit ferroptosis as a result of reduced cellular ROS which was triggered by transcription factor EB (TFEB) nucleus translocation. In this process, CPS first entered cells via macropinocytosis, then CPS-containing macropinosomes fused with lysosomes and expanded into abnormal lysosome-like large vacuoles in vacuolar-type H+-ATPase (V-ATPase) and aquaporins (AQPs) in a dependent way. These large vacuoles were detected both in vitro and in vivo, which was found to be a sign of lysosome stress. The lysosome stress induced deactivation of mammalian target of rapamycin (mTOR) which led to nucleus translocation of TFEB. Then, TFEB-dependent enhanced expression of lysosomal proteins and superoxide dismutase (SOD) which ultimately led to ROS reduction and inhibition of ferroptosis. Knockout of TFEB-enhanced ferroptosis was triggered by Erastin and abolished the effect of CPS on ROS and ferroptosis. In summary, our results reveal a novel mechanism whereby CPS reduced ROS and inhibited ferroptosis in a TFEB-dependent way. These findings have important implications for understanding the biological effects of polystyrene nanoparticles and searching for new anti-ROS and antiferroptosis particles or reagents.

Buffer formulation affects the interaction between lysozyme and polymeric nanoparticles

The effect of the buffer formulation in terms of buffer identity and ionic strength on the interaction between chicken egg lysozyme and carboxyl-modified polystyrene nanoparticles has been systematically studied. The time evolution of the fluorescence of a reporter molecule shows that lysozyme interacts with the nanoparticles in all the studied conditions. The interaction results in changes in protein conformation and decrease of the colloidal stability of nanoparticles. In absence of a background salt the rate of adsorption is affected mainly by the ionic strength of the buffer solution, although, specific buffer effects may contribute to a certain extent. The identity of the different buffer components does not significantly alter the dynamics of the process in presence of salt at constant ionic strength. However, an increase of ionic strength leads to slower processes indicating that the adsorption is affected by the presence of increasing number of ions in solution.

Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions.

Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (t1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration.

Inactivation and adsorption of human carbonic anhydrase II by nanoparticles.

The enzymatic activity of human carbonic anhydrase II (HCAII) was studied in the presence of nanoparticles of different nature and charge. Negatively charged nanoparticles inhibit HCAII whereas no effect is seen for positively charged particles. The kinetic effects were correlated with the strength of binding of the enzyme to the particle surface as measured by ITC and adsorption assays. Moreover, conformational changes upon adsorption were observed by circular dichroism. The main initial driving force for the adsorption of HCAII to nanoparticles is of electrostatic nature whereas the hydrophobic effect is not strong enough to drive the initial binding. This is corroborated by the fact that HCAII do not adsorb on positively charged hydrophobic polystyrene nanoparticles. Furthermore, the dehydration of the particle and protein surface seems to play an important role in the inactivation of HCAII by carboxyl-modified polystyrene nanoparticles. On the other hand, the inactivation by unmodified polystyrene nanoparticles is mainly driven by intramolecular interactions established between the protein and the nanoparticle surface upon conformational changes in the protein.

Nanoparticle-Membrane Interactions Studied with Lipid Bilayer Arrays

Nanoparticles (NPs) have been explored for use in biomedical applications including gene and drug delivery, and recent studies have indicated that they are also cytotoxic and have significant interaction with lipid bilayer membranes. An understanding of the interactions between NPs and the cell membrane systems will help aid the design of NPs for beneficial applications and evaluate the cytotoxicity of environmental NPs. To study these interactions, we measured amine- and carboxyl-modified polystyrene NPs in arrays of 24-32 artificial lipid bilayers. The throughput possible with simultaneous electrical measurement of the bilayer arrays enabled study of NP/bilayer interactions while varying lipid composition, ionic strength, pH, voltage, and particle type. Our studies showed that amine-modified NPs interacted with bilayers whereas carboxyl-modified NPs did not. The interaction of the amine-modified NPs could be altered by changing the charge density on the bilayers, the ionic strength and pH.

Biocompatibility, endocytosis, and intracellular trafficking of mesoporous silica and polystyrene nanoparticles in ovarian cancer cells: effects of size and surface charge groups

Background and methodsNanoparticles engineered to carry both a chemotherapeutic drug and a sensitive imaging probe are valid tools for early detection of cancer cells and to monitor the cytotoxic effects of anticancer treatment simultaneously. Here we report on the effect of size (10–30 nm versus 50 nm), type of material (mesoporous silica versus polystyrene), and surface charge functionalization (none, amine groups, or carboxyl groups) on biocompatibility, uptake, compartmentalization, and intracellular retention of fluorescently labeled nanoparticles in cultured human ovarian cancer cells. We also investigated the involvement of caveolae in the mechanism of uptake of nanoparticles.ResultsWe found that mesoporous silica nanoparticles entered via caveolae-mediated endocytosis and reached the lysosomes; however, while the 50 nm nanoparticles permanently resided within these organelles, the 10 nm nanoparticles soon relocated in the cytoplasm. Naked 10 nm mesoporous silica nanoparticles showed the highest and 50 nm carboxyl-modified mesoporous silica nanoparticles the lowest uptake rates, respectively. Polystyrene nanoparticle uptake also occurred via a caveolae-independent pathway, and was negatively affected by serum. The 30 nm carboxyl-modified polystyrene nanoparticles did not localize in lysosomes and were not toxic, while the 50 nm amine-modified polystyrene nanoparticles accumulated within lysosomes and eventually caused cell death. Ovarian cancer cells expressing caveolin-1 were more likely to endocytose these nanoparticles.ConclusionThese data highlight the importance of considering both the physicochemical characteristics (ie, material, size and surface charge on chemical groups) of nanoparticles and the biochemical composition of the cell membrane when choosing the most suitable nanotheranostics for targeting cancer cells.

Quantitative Assessment of the Comparative Nanoparticle‐Uptake Efficiency of a Range of Cell Lines

The mechanism(s) of nanoparticle-cell interactions are still not understood. At present there is little knowledge of the relevant length- and timescales for nanoparticle intracellular entry and localization within cells, or the cell-specificity of nanoparticle uptake and localisation. Here, the effect of particle size on the in-vitro intracellular uptake of model fluorescent carboxyl-modified polystyrene nanoparticles is investigated in various cell lines. A range of micro- and nanoparticles of defined sizes (40 nm to 2 μm) are incubated with a series of cell types, including HeLa and A549 epithelial cells, 1321N1 astrocytes, HCMEC D3 endothelial cells, and murine RAW 264.7 macrophages. Techniques such as confocal microscopy and flow cytometry are used to study particle uptake and subcellular localisation, making significant efforts to ensure reproducibility in a semiquantitative approach. The results indicate that internalization of (nano)particles is highly size-dependent for all cell lines studied, and the kinetics of uptake for the same type of nanoparticle varies in the different cell types. Interestingly, even cells not specialized for phagocytosis are able to internalize the larger nanoparticles. Intracellular uptake of all sizes of particles is observed to be highest in RAW 264.7 cells (a specialized phagocytic cell line) and the lowest in the HeLa cells. These results suggest that (nano)particle uptake might not follow commonly defined size limits for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell types. These conclusions have important implications for the assessment of the safety of nanomaterials and for the potential biomedical applications of nanoparticles.