Size-dependent Uptake Research Articles

Evaluation of size-dependent uptake, transport and cytotoxicity of polystyrene microplastic in a blood-brain barrier (BBB) model

Microplastics, particularly those in the micrometer scale, have been shown to enter the human body through ingestion, inhalation, and dermal contact. Recent research indicates that microplastics can potentially impact the central nervous system (CNS) by crossing the blood-brain barrier (BBB). However, the exact mechanisms of their transport, uptake, and subsequent toxicity at BBB remain unclear. In this study, we evaluated the size-dependent uptake and cytotoxicity of polystyrene microparticles using an engineered BBB model. Our findings demonstrate that 0.2 μm polystyrene microparticles exhibit significantly higher uptake and transendothelial transport compared to 1.0 μm polystyrene microparticles, leading to increased permeability and cellular damage. After 24 h of exposure, permeability increased by 15.6-fold for the 0.2 μm particles and 2-fold for the 1.0 μm particles compared to the control. After 72 h of exposure, permeability further increased by 27.3-fold for the 0.2 μm particles and a 4.5-fold for the 1.0 μm particles compared to the control. Notably, microplastics administration following TNF-α treatment resulted in enhanced absorption and greater BBB damage compared to non-stimulated conditions. Additionally, the size-dependent toxicity observed differently between 2D cultured cells and 3D BBB models, highlighting the importance of testing models in evaluating environmental toxicity.Graphical

Size dependent uptake and trophic transfer of polystyrene microplastics in unicellular freshwater eukaryotes

Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat posed by MP, the possible uptake and trophic transfer in lower trophic levels of freshwater ecosystems remains understudied. This study aims to investigate the internalization and potential trophic transfer of fluorescent polystyrene (PS) beads (0.5 μm, 3.6 × 108 particles/mL; 6 μm, 2.1 × 105 particles/mL) and fragments (<30 μm, 5 × 103 particles/mL) in three unicellular eukaryotes. This study focuses on the size-dependent uptake of MP by two freshwater Ciliophora, Tetrahymena pyriformis, Paramecium caudatum and one Amoebozoa, Amoeba proteus, serving also as predator for experiments on potential trophic transfer. Size-dependent uptake of MP in all three unicellular eukaryotes was shown. P. caudatum is able to take up MP fragments up to 27.7 μm, while T. pyriformis ingests particles up to 10 μm. In A. proteus, small MP (PS0.5μm and PS6μm) were taken up via pinocytosis and were detected in the cytoplasm for up to 14 days after exposure. Large PS-MP (PS<30μm) were detected in A. proteus only after predation on MP-fed Ciliophora. These results indicate that A. proteus ingests larger MP via predation on Ciliophora (PS<30μm), which would not be taken up otherwise. This study shows trophic transfer of MP at the base of the aquatic food web and serves as basis to study the impact of MP in freshwater ecosystems.

Complex intestinal and hepatic in vitro barrier models reveal information on uptake and impact of micro-, submicro- and nanoplastics

Plastic particles are found almost ubiquitously in the environment and can get ingested orally by humans. We have used food-relevant microplastics (2 µm polylactic acid), submicroplastics (250 nm polylactic acid and 366 nm melamine formaldehyde resin) and nanoplastics (25 nm polymethylmethacrylate) to study material- and size-dependent uptake and transport across the human intestinal barrier and liver. Therefore, different Transwell™-based in vitro (co-)culture models were used: Differentiated Caco-2 cells mimicking the intestinal enterocyte monolayer, an M-cell model complementing the Caco-2 monoculture with antigen uptake-specialized cells, a mucus model complementing the barrier with an intestinal mucus layer, and an intestinal-liver co-culture combining differentiated Caco-2 cells with differentiated HepaRG cells. Using these complex barrier models, uptake and transport of particles were analyzed based on the fluorescence of the particles using confocal microscopy and a fluorescence-based quantification method. Additionally, the results were verified by Time-of-Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) analysis. Furthermore, an effect screening at the mRNA level was done to investigate oxidative stress response, inflammation and changes to xenobiotic metabolism in intestinal and hepatic cells after exposure to plastic particles. Oxidative stress and inflammation were additionally analyzed using a flow-cytometric assay for reactive oxygen species and cytokine measurements. The results reveal a noteworthy uptake into and transport of microplastic and submicroplastic particles across the intestinal epithelium. Particularly, we show a pronounced uptake of particles into liver cells after crossing of the intestinal epithelium, using the intestinal-liver co-culture. The particles evoke some alterations in xenobiotic metabolism, but did not cause increased oxidative stress or inflammatory response on protein level. Taken together, these complex barrier models can be applied on micro-, submicro- and nanoplastics and reveal information in particle uptake, transport and cellular impact.

Correlation between cellular uptake and cytotoxicity of polystyrene micro/nanoplastics in HeLa cells: A size-dependent matter.

The cytotoxicity of micro/nanoplastics (MNPs) is known to be strongly influenced by particle size, but the mechanism is not clear so far. We reported the ability of polystyrene MNPs to be internalized by HeLa cells could be a reason for the size dependent cytotoxicity of MNPs. We found that small MNPs (10 nm and 15 nm in radius) could be efficiently internalized by HeLa cells, MNPs of 25 nm in radius could be slightly internalized by the cells, and larger MNPs could not enter the cells at all. We showed that only MNPs, which could be internalized by cells, had a toxic effect on cell activity in a dose-dependent manner. In contrast, MNPs, which could not be internalized by cells, showed no cytotoxicity even if at extremely high concentrations. We attributed the correlation between the size-dependent uptake of MNPs and the size-dependent cytotoxicity of MNPs to the enhanced reactive oxygen species (ROS) level and abnormal gene expression. Our study pointed out that cellular uptake is one of the most fundamental mechanisms for the cytotoxicity of MNPs.

Size variations of mesoporous silica nanoparticle control uptake efficiency and delivery of AC2-derived dsRNA for protection against tomato leaf curl New Delhi virus.

We report the size dependent uptake of dsRNA loaded MSNPs into the leaves and roots of Nicotiana benthamiana plants and accessed for their relative reduction in Tomato leaf curl New Delhi viral load. A non-GMO method of RNA interference (RNAi) has been recently in practice through direct delivery of double stranded RNA into the plant cells. Tomato leaf curl New Delhi virus (ToLCNDV), a bipartitie begomovirus, is a significant viral pathogen of many crops in the Indian subcontinent. Conventional RNAi cargo delivery strategies for instance uses viral vectors and Agrobacterium-facilitated delivery, exhibiting specific host responses from the plant system. In the present study, we synthesized three different sizes of amine-functionalized mesoporous silica nanoparticles (amino-MSNPs) to mediate the delivery of dsRNA derived from the AC2 (dsAC2) gene of ToLCNDV and showed that these dsRNA loaded nanoparticles enabled effective reduction in viral load. Furthermore, we demonstrate that amino-MSNPs protected the dsRNA molecules from nuclease degradation, while the complex was efficiently taken up by the leaves and roots of Nicotiana benthamiana. The real time gene expression evaluation showed that plants treated with nanoparticles of different sizes ~ 10nm (MSNPDEA), ~ 32nm (MSNPTEA) and ~ 66nm (MSNPNH3) showed five-, eleven- and threefold reduction of ToLCNDV in N. benthamiana, respectively compared to the plants treated with naked dsRNA. This work clearly demonstrates the size dependent internalization of amino-MSNPs and relative efficacy in transporting dsRNA into the plant system, which will be useful in convenient topical treatment to protect plants against their pathogens including viruses. Mesoporous silica nanoparticles loaded with FITC, checked for its uptake into Nicotiana benthamiana.

Rational design of phase separating peptides based on phase separating protein sequence of p53

Artificial phase-separating (PS) peptides can be used in various applications such as microreactors and drug delivery; however, the design of artificial PS peptides remains a challenge. This can be attributed to the limitation of PS-relevant residues that drive phase separation by interactions of their pairs in short peptides and the difficulty in the design involving interaction with target PS proteins. In this study, we propose a rational method to design artificial PS peptides that satisfy the requirements of liquid droplet formation and co-phase separation with target PS proteins based on the target PS protein sequence. As a proof of concept, we designed five artificial peptides from the model PS protein p53 using this method and confirmed their PS properties using differential interference contrast and fluorescence microscopy. Single-molecule fluorescent tracking demonstrated rapid diffusion of the designed peptides in their droplets compared to that of p53 in p53 droplets. In addition, size-dependent uptake of p53 oligomers was observed in the designed peptide droplets. Large oligomers were excluded from the droplet voids and localized on the droplet surface. The uptake of high-order p53 oligomers into the droplets was enhanced by the elongated linker of the designed peptides. Furthermore, we found that the designed peptide droplets recruited p53 to suppress gel-like aggregate formation. Finally, we discuss aspects that were crucial in the successful design of the artificial PS peptides.

Size-Dependent Uptake and Depuration of Nanoplastics in Tilapia (Oreochromis niloticus) and Distinct Intestinal Impacts.

Nanoplastics (NPs, <1 μm) are of great concern worldwide because of their high potential risk toward organisms in aquatic systems, while very little work has been focused on their tissue-specific toxicokinetics due to the limitations of NP quantification for such a purpose. In this study, NPs with two different sizes (86 and 185 nm) were doped with palladium (Pd) to accurately determine the uptake and depuration kinetics in various tissues (intestine, stomach, liver, gill, and muscle) of tilapia (Oreochromis niloticus) in water, and subsequently, the corresponding toxic effects in the intestine were explored. Our results revealed uptake and depuration constants of 2.70-378 L kg-1 day-1 and 0.138-0.407 day-1 for NPs in tilapia for the first time, and the NPs in tissues were found to be highly dependent on the particle size. The intestine exhibited the greatest relative accumulation of both sizes of NPs; the smaller NPs caused more severe damage than the larger NPs to the intestinal mucosal layer, while the larger NPs induced a greater impact on microbiota composition. The findings of this work explicitly indicate the size-dependent toxicokinetics and intestinal toxicity pathways of NPs, providing new insights into the ecological effects of NPs on aquatic organisms.

Macrophage specificity: Imaging and characterization of interaction between macrophages and fluorescent organosilica nanoparticles depending on surface structure and size.

Macrophages have key functions in tissue homeostasis and also diseases. Macrophages show marked heterogeneity in response to environmental stimuli. The heterogeneity of macrophages was characterized by using morphology, surface marker, single cell sequence, and so on. We are investigating macrophage specificity using our nanomedical technology. We are developing novel materials, functional organosilica nanoparticles, and performed imaging to evaluate the diversity and specificity of mouse macrophage uptake. The organosilica nanoparticles surface-functionalized with polyethylene glycol (PEGylated) were applied to time-lapse fluorescent microscopic imaging and flow cytometry analyses. Single-cell imaging and analysis revealed that PEGylated nanoparticles revealed a stealth function against most macrophages. Notably, we found that the “PEG-resistant macrophages” that could take up PEGylated nanoparticles at the same level as bare nanoparticles. In addition, size-dependent uptake of macrophages was also observed in various tissues. The size-dependent uptake of functional organosilica nanoparticles by macrophages in the mouse various organs in vivo. These results indicated imaging and characterization of interaction between macrophages and fluorescent organosilica nanoparticles depending on surface structure and size were very useful to evaluate the heterogeneity and possible specificity of macrophage.

Synthesis of NaYF4:20% Yb3+,2% Er3+,2% Ce3+@NaYF4 nanorods and their size dependent uptake efficiency under flow condition

Lanthanide doped fluorescent nanoparticles have gained considerable attention in biomedical applications. However, the low uptake efficiency of nanoparticles by cells has limited their applications. In this work, we demonstrate how the uptake efficiency is affected by the size of nanoparticles under flow conditions. Using the same size NaYF4:20% Yb3+,2% Er3+,2% Ce3+ (the contents of rare earths elements are in molar fraction) nanoparticles as core, NaYF4:20% Yb3+,2% Er3+,2% Ce3+@NaYF4 core–shell structured nanorods (NRs) with different sizes of 60–224 nm were synthesized by thermal decomposition and hot injection method. Under excitation at 980 nm, a strong upconversion green emission (541 nm, 2H11/2 → 4I15/2 of Er3+) is observed for all samples. The emission intensity for each size nanorod was calibrated and is found to depend on the width of NRs. Under flow conditions, the nanorods with 96 nm show a maximum uptake efficiency by endothelial cells. This work demonstrates the importance of optimizing the size for improving the uptake efficiency of lanthanide-doped nanoparticles.

Body Size, Light Intensity, and Nutrient Supply Determine Plankton Stoichiometry in Mixotrophic Plankton Food Webs.

Trophic strategy determines stoichiometry of plankton. In general, heterotrophic zooplankton have lower and more stable C∶N and C∶P ratios than photoautotrophic phytoplankton, whereas mixotrophic protists, which consume prey and photosynthesize, have stoichiometry between zooplankton and phytoplankton. As trophic strategies change with cell size, body size may be a key trait influencing eukaryotic plankton stoichiometry. However, the relationship between body size and stoichiometry remains unclear. Here we measured plankton size-fractionated C∶N ratios under different intensities of light and nutrient supply in subtropical freshwater and marine systems. We found a unimodal body size-C∶N ratio pattern, with a maximum C∶N ratio at ∼50 μm diameter in marine and freshwater systems. Moreover, the variation in C∶N ratios is explained mainly by body size, followed by light intensity and nutrient concentration. To investigate the mechanisms behind this unimodal pattern, we constructed a size-based plankton food web model in which the trophic strategy and C∶N ratio are an emerging result. Our model simulations reproduce the unimodal pattern with a C∶N ratio of photoautotrophs ≤50 μm increasing with body size due to increase of photosynthetic carbon, whereas C∶N ratios of organisms >50 μm decrease with size due to decreasing photoautotrophic but increasing heterotrophic uptake. Based on our field observations and simulation, we extend the classic "light-nutrient" theory that determines plankton C∶N ratio to include body size and trophic strategy dependency. We conclude that body size and size-dependent uptake of resources (light, nutrients, and prey) determine plankton stoichiometry at various light and nutrient supplies.

Size-Dependent Uptake and Distribution of AgNPs by Silkworms

Silk is an attractive natural fiber due to its excellent mechanical properties and biocompatibility. Xenobiotic modification of pristine silk is gaining significant attention to meet specific demands. The silk payload of xenobiotics is affected by their uptake efficiency, biodistribution, metabolism, and excretion. Herein, we systematically evaluated the effect of the nanoparticle size (20–100 nm) on the biodistribution and silk production by introducing silver nanoparticles (AgNPs) into the silkworm diet. Interestingly, the silk payload of AgNPs is highly dependent on their overall size, where 50 nm AgNPs are prone to accumulate in silk glands and reveal the highest content in the produced silk, qualifying silk antibacterial activity against both Gram-positive and Gram-negative bacteria. These findings provide new insights to understand how nanoparticle properties influence the incorporation of nanomaterials into silk and can help optimize the material design.

In vivo uptake of gold nanoparticles by the diatom Stephanopyxis turris

Nanoparticle (NP) implementation in industry has increased during the last years. However, the consequences of NP release to the environment have not been fully understood yet and long-term effects are hardly predictable. Information regarding a possible NP uptake by organisms is urgently needed. Here, we investigate the uptake of presynthesized AuNPs of different diameters by living diatoms. To better understand the influence of NPs on diatoms, cells were monitored in vivo using Surface Enhanced Raman Spectroscopy (SERS). By 3D Raman imaging, AuNPs with diameters ≥50 nm could clearly be localized inside the cells, whereas smaller AuNPs were never detected in the cell interior. This indicates a size-dependent uptake mechanism that comes along with different toxicities. As diatoms present an essential source of the marine food chain, this observation is relevant not only for diatoms themselves but also for higher organisms.

Size-dependent uptake of electrically neutral amphipathic polymeric nanoparticles by cell-sized liposomes and an insight into their internalization mechanism in living cells

Size-dependent uptake behaviors of electrically neutral amphipathic polymeric nanoparticles in cell-sized liposomes and living cells were investigated. Kinetic analyses and the particle size distribution suggested a size-dependent penetration mechanism (size threshold: 3.1 nm). The definite size-dependent uptake provides a new insight into the interactions between nanomaterials and living cells.

Size Dependent Uptake and Hemolytic Effect of Zinc Oxide Nanoparticles on Erythrocytes and Biomedical Potential of ZnO-Ferulic acid Conjugates

Despite zinc oxide nanoparticles (ZnONPs) being increasingly used as carriers in biomedical fields due to their multifaceted properties and therapeutic importance, better understanding of the mechanisms and cellular consequences resulting from their interaction with cells and cellular components has been warranted. In the present study, we investigate the size-dependent interaction of ZnONPs on RBCs, and its impact on cell viability, DNA damage, ROS generation and morphological changes, employing cellular and analytical methods. Size, charge, stability and solubility were confirmed by DLS, zeta potential, ICP-AES and TEM analysis. Further ICP-AES, TEM, spectroscopic observations and cell based assays showed that ZnONPs exhibited a size dependent impact on RBCs and haemoglobin (Hb), particularly size <50 nm. Conversely, ferulic acid (FA) conjugates and serum albumin significantly reduced the adverse effects exhibited by ZnONPs. The extent of DNA damage and ROS generation is comparatively low in ZnONPs-FA than in ZnONPs alone treated cells. Thus our study documents a novel conceptualization delineating the influence of size on the material properties and therapeutic potential of nanoparticle.

Mesoporous Silica Nanoparticles as an Antitumoral-Angiogenesis Strategy.

Tumors depend heavily on angiogenesis for nutrient derivation and their subsequent metastasis. Targeting tumor induced angiogenesis per se can address both tumor growth and progression simultaneously. Here, we show that we could elegantly restrict the endothelial cells angiogenic behavior through digital size control of mesoporous silica nanoparticle (MSN). This antiangiogenesis effect was derived from the particle size dependent uptake and production of intracellular reactive oxygen species (ROS) that directly interfered with p53 tumor suppressor pathway. The resulting signaling cascade wrestled back the tumoral control of endothelial cells' migration, invasion, and proliferation. Overall, a mere control over the size of a highly oxidative reactive surfaced nanoparticle could provide an alternative strategy to curb the tumor induced angiogenesis process in a conventional drug-free manner.

Distribution of Systemically Administered Nanoparticles Reveals a Size-Dependent Effect Immediately following Cardiac Ischaemia-Reperfusion Injury.

Nanoparticles represent an attractive option for systemic delivery of therapeutic compounds to the heart following myocardial infarction. However, it is well known that physicochemical properties of nanoparticles such as size, shape and surface modifications can vastly alter the distribution and uptake of injected nanoparticles. Therefore, we aimed to provide an examination of the rapid size-dependent uptake of fluorescent PEG-modified polystyrene nanoparticles administered immediately following cardiac ischaemia-reperfusion injury in mice. By assessing the biodistribution of nanoparticles with core diameters between 20 nm and 2 μm 30 minutes after their administration, we conclude that 20–200 nm diameter nanoparticles are optimal for passive targeting of the injured left ventricle.

Silver nanoparticles affect the neural development of zebrafish embryos.

Silver nanoparticles (AgNPs) have been widely used in commercial products. This study aims to understand the impact of AgNPs on the early developmental stages in zebrafish (Danio rerio) embryos. Embryos were exposed to two sizes of AgNPs at three dose levels, as well to free Ag(+) ions, for a range of 4-96 h post-fertilization (hpf). The acute exposure study showed that exposure to AgNPs affected the neurological development, and the exposed embryos exhibited anomalies such as small head with hypoplastic hindbrain, small eye and cardiac defects. At the molecular level, AgNPs altered the expression profiles of neural development-related genes (gfap, huC and ngn1), metal-sensitive metallothioneins and ABCC genes in exposed embryos. The expression of AhR2 and Cyp1A, which are usually considered to mediate polycyclic aromatic hydrocarbon toxicity, were also significantly changed. A size-dependent uptake of AgNPs was observed, whereby 4 nm AgNPs were more efficiently taken up compared with the 10 nm-sized particles. Importantly, the head area accumulated AgNPs more efficiently than the trunk area of exposed zebrafish embryos. No free Ag(+) ions, which can be potentially released from the AgNP solutions, were detected. This study suggests that AgNPs could affect the neural development of zebrafish embryos, and the toxicity of AgNPs may be partially attributed to the comparatively higher uptake in the head area. These results indicate the potential neurotoxicity of AgNPs and could be extended to other aquatic organisms.

Nanotoxicity of silver nanoparticles to red blood cells: size dependent adsorption, uptake, and hemolytic activity.

Silver nanoparticles (AgNPs) are increasingly being used as antimicrobial agents and drug carriers in biomedical fields. However, toxicological information on their effects on red blood cells (RBCs) and the mechanisms involved remain sparse. In this article, we examined the size dependent nanotoxicity of AgNPs using three different characteristic sizes of 15 nm (AgNPs15), 50 nm (AgNPs50), and 100 nm (AgNPs100) against fish RBCs. Optical microscopy and transmission electron microscopy observations showed that AgNPs exhibited a size effect on their adsorption and uptake by RBCs. The middle sized AgNPs50, compared with the smaller or bigger ones, showed the highest level of adsorption and uptake by the RBCs, suggesting an optimal size of ∼50 nm for passive uptake by RBCs. The toxic effects determined based on the hemolysis, membrane injury, lipid peroxidation, and antioxidant enzyme production were fairly size and dose dependent. In particular, the smallest sized AgNPs15 displayed a greater ability to induce hemolysis and membrane damage than AgNPs50 and AgNPs100. Such cytotoxicity induced by AgNPs should be attributed to the direct interaction of the nanoparticle with the RBCs, resulting in the production of oxidative stress, membrane injury, and subsequently hemolysis. Overall, the results suggest that particle size is a critical factor influencing the interaction between AgNPs and the RBCs.

Size-Dependent Nanoparticle Uptake by Endothelial Cells in a Capillary Flow System

An in vitro cell culture system is developed for studying the uptake characteristics of nanoparticles (NPs) by endothelial cells under shear stress. Results show that the smaller polystyrene nanospheres are uptaken more than larger nanospheres for sizes ranging from 100 nm to 500 nm for 12, 24, and 48 hrs delivery times. While the result is similar to that found in static cultures, the observed trend is different from NP delivery behaviors to a simple glass surface in a flow, where no clear size dependence was observed because of repulsive electrostatic force on marginating NPs. The trend is also opposite to the behavior found in another study of the adhesion of labeled particles onto endothelial cells in whole blood flow. The comparison shows that the reduced zeta potential of NPs in a serum-containing cell medium and particle removal by cells results in reduced repulsive electrostatic force on marginating NPs. Consequently, the uptake behaviors are dominated by Brownian diffusion and cell membrane deformation process, which favor the uptake of NPs with reduced sizes.

Liposomes as Nanovaccine Delivery Systems

Since the discovery of liposomes by Alec Bangham in mid-1960s, these phospholipid vesicles have been widely used as pharmaceutical carriers. Liposomes have been extensively studied in the vaccine delivery field as a carrier and an immune stimulating agent. Liposomes are usually formulated as nanoparticles, mimicking the properties of pathogens, and have the ability to induce humoral and cell-mediated immune responses. In this review, we focused on modern nanotechnology-based approaches for the improvement of liposomal vaccine delivery systems. Topics such as size-dependent uptake, processing and activation of antigen presenting cells, targeting liposomes and route of administration are discussed.