Detection of submicron- and nanoplastics spiked in environmental fresh- and saltwater with Raman spectroscopy.

Detection of small plastic particles in environmental water samples has been a topic of increasing interest in recent years. A multitude of techniques, such as variants of Raman spectroscopy, have been employed to facilitate their analysis in such complex sample matrices. However, these studies are often conducted for a limited number of plastic types in matrices with relatively little additional materials. Thus, much remains unknown about what parameters influence the detection limits of Raman spectroscopy for more environmentally relevant samples. To address this, this study utilizes Raman spectroscopy to detect six plastic particle types; 161 and 33 nm polystyrene, < 450 nm and 36 nm poly(ethylene terephthalate), 121 nm polypropylene, and 126 nm polyethylene; spiked into artificial saltwater, artificial freshwater, North Sea, Thames River, and Elbe River water. Overall, factors such as plastic particle properties, water matrix composition, and experimental setup were shown to influence the final limits of detection.

Does the surface charge of the nanoparticles drive nanoparticle-cell membrane interactions?

Classical Coulombic interaction, characterized by electrostatic interactions mediated through surface charges, is often regarded as the primary determinant in nanoparticles' (NPs) cellular association and internalization. However, the intricate physicochemical properties of particle surfaces, biomolecular coronas, and cell surfaces defy this oversimplified perspective. Moreover, the nanometrological techniques employed to characterize NPs in complex physiological fluids often exhibit limited accuracy and reproducibility. A more comprehensive understanding of nanoparticle-cell membrane interactions, extending beyond attractive forces between oppositely charged surfaces, necessitates the establishment of databases through rigorous physical, chemical, and biological characterization supported by nanoscale analytics. Additionally, computational approaches, such as in silico modeling and machine learning, play a crucial role in unraveling the complexities of these interactions.

Theoretical evaluation of the impact of diverse treatment conditions by calculation of the tumor control probability (TCP) of simulated cervical cancer Hyperthermia-Radiotherapy (HT-RT) treatments in-silico.

INTRODUCTION: Hyperthermia (HT) induces various cellular biological processes, such as repair impairment and direct HT cell killing. In this context, in-silico biophysical models that translate deviations in the treatment conditions into clinical outcome variations may be used to study the extent of such processes and their influence on combined hyperthermia plus radiotherapy (HT + RT) treatments under varying conditions. METHODS: An extended linear-quadratic model calibrated for SiHa and HeLa cell lines (cervical cancer) was used to theoretically study the impact of varying HT treatment conditions on radiosensitization and direct HT cell killing effect. Simulated patients were generated to compute the Tumor Control Probability (TCP) under different HT conditions (number of HT sessions, temperature and time interval), which were randomly selected within margins based on reported patient data. RESULTS: Under the studied conditions, model-based simulations suggested a treatment improvement with a total CEM43 thermal dose of approximately 10 min. Additionally, for a given thermal dose, TCP increased with the number of HT sessions. Furthermore, in the simulations, we showed that the TCP dependence on the temperature/time interval is more correlated with the mean value than with the minimum/maximum value and that comparing the treatment outcome with the mean temperature can be an excellent strategy for studying the time interval effect. CONCLUSION: The use of thermoradiobiological models allows us to theoretically study the impact of varying thermal conditions on HT + RT treatment outcomes. This approach can be used to optimize HT treatments, design clinical trials, and interpret patient data.

Submicron- and nanoplastic detection at low micro- to nanogram concentrations using gold nanostar-based surface-enhanced Raman scattering (SERS) substrates.

The presence of submicron- (1 µm-100 nm) and nanoplastic (<100 nm) particles within various sample matrices, ranging from marine environments to foods and beverages, has become a topic of increasing interest in recent years. Despite this interest, very few analytical techniques are known that allow for the detection of these small plastic particles in the low concentration ranges that they are anticipated to be present at. Research focused on optimizing surface-enhanced Raman scattering (SERS) to enhance signal obtained in Raman spectroscopy has been shown to have great potential for the detection of plastic particles below conventional resolution limits. In this study, we produce SERS substrates composed of gold nanostars and assess their potential for submicron- and nanoplastic detection. The results show 33 nm polystyrene could be detected down to 1.25 µg mL-1 while 36 nm poly(ethylene terephthalate) was detected down to 5 µg mL-1. These results confirm the promising potential of the gold nanostar-based SERS substrates for nanoplastic detection. Furthermore, combined with findings for 121 nm polypropylene and 126 nm polyethylene particles, they highlight potential differences in analytical performance that depend on the properties of the plastics being studied.

The Effect of Substrate Properties on Cellular Behavior and Nanoparticle Uptake in Human Fibroblasts and Epithelial Cells.

The delivery of nanomedicines into cells holds enormous therapeutic potential; however little is known regarding how the extracellular matrix (ECM) can influence cell-nanoparticle (NP) interactions. Changes in ECM organization and composition occur in several pathophysiological states, including fibrosis and tumorigenesis, and may contribute to disease progression. We show that the physical characteristics of cellular substrates, that more closely resemble the ECM in vivo, can influence cell behavior and the subsequent uptake of NPs. Electrospinning was used to create two different substrates made of soft polyurethane (PU) with aligned and non-aligned nanofibers to recapitulate the ECM in two different states. To investigate the impact of cell-substrate interaction, A549 lung epithelial cells and MRC-5 lung fibroblasts were cultured on soft PU membranes with different alignments and compared against stiff tissue culture plastic (TCP)/glass. Both cell types could attach and grow on both PU membranes with no signs of cytotoxicity but with increased cytokine release compared with cells on the TCP. The uptake of silica NPs increased more than three-fold in fibroblasts but not in epithelial cells cultured on both membranes. This study demonstrates that cell-matrix interaction is substrate and cell-type dependent and highlights the importance of considering the ECM and tissue mechanical properties when designing NPs for effective cell targeting and treatment.

Leveraging Machine Learning for Size and Shape Analysis of Nanoparticles: A Shortcut to Electron Microscopy.

Characterizing nanoparticles (NPs) is crucial in nanoscience due to the direct influence of their physiochemical properties on their behavior. Various experimental techniques exist to analyze the size and shape of NPs, each with advantages, limitations, proneness to uncertainty, and resource requirements. One of them is electron microscopy (EM), often considered the gold standard, which offers visualization of the primary particles. However, despite its advantages, EM can be expensive, less accessible, and difficult to apply during dynamic processes. Therefore, using EM for specific experimental conditions, such as observing dynamic processes or visualizing low-contrast particles, is challenging. This study showcases the potential of machine learning in deriving EM parameters by utilizing cost-effective and dynamic techniques such as dynamic light scattering (DLS) and UV-vis spectroscopy. Our developed model successfully predicts the size and shape parameters of gold NPs based on DLS and UV-vis results. Furthermore, we demonstrate the practicality of our model in situations in which conducting EM measurements presents a challenge: Tracking in situ the synthesis of 100 nm gold NPs.

Immunostimulatory nanoparticles delivering cytokines as a novel cancer nanoadjuvant to empower glioblastoma immunotherapy.

Glioblastoma (GBM) stands as a highly aggressive and deadly malignant primary brain tumor with a median survival time of under 15 months upon disease diagnosis. While immunotherapies have shown promising results in solid cancers, brain cancers are still unresponsive to immunotherapy due to immunological dysfunction and the presence of a blood-brain barrier. Interleukin-12 (IL-12) emerges as a potent cytokine in fostering anti-tumor immunity by triggering interferon-gamma production in T and natural killer cells and changing macrophages to a tumoricidal phenotype. However, systemic administration of IL-12 toxicity in clinical trials often leads to significant toxicity, posing a critical hurdle. To overcome this major drawback, we have formulated a novel nanoadjuvant composed of immunostimulatory nanoparticles (ISN) loaded with IL-12 to decrease IL-12 toxicity and enhance the immune response by macrophages and GBM cancer cells. Our in vitro results reveal that ISN substantially increase the production of pro-inflammatory cytokines in GBM cancer cells (e.g. 2.6 × increase in IL-8 expression compared to free IL-12) and macrophages (e.g. 2 × increase in TNF-α expression and 6 × increase in IL-6 expression compared to the free IL-12). These findings suggest a potential modulation of the tumor microenvironment. Additionally, our study demonstrates the effective intracellular delivery of IL-12 by ISN, triggering alterations in the levels of pro-inflammatory cytokines at both transcriptional and protein expression levels. These results highlight the promise of the nanoadjuvant as a prospective platform for resharing the GBM microenvironment and empowering immunotherapy.

Epidermal growth factor alters silica nanoparticle uptake and improves gold-nanoparticle-mediated gene silencing in A549 cells.

Introduction: Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs. Methods: We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC. Results: Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC. Discussion: These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery.

The Functions of Cholera Toxin Subunit B as a Modulator of Silica Nanoparticle Endocytosis.

The gastrointestinal tract is the main target of orally ingested nanoparticles (NPs) and at the same time is exposed to noxious substances, such as bacterial components. We investigated the interaction of 59 nm silica (SiO2) NPs with differentiated Caco-2 intestinal epithelial cells in the presence of cholera toxin subunit B (CTxB) and compared the effects to J774A.1 macrophages. CTxB can affect cellular functions and modulate endocytosis via binding to the monosialoganglioside (GM1) receptor, expressed on both cell lines. After stimulating macrophages with CTxB, we observed notable changes in the membrane structure but not in Caco-2 cells, and no secretion of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) was detected. Cells were then exposed to 59 nm SiO2 NPs and CtxB sequentially and simultaneously, resulting in a high NP uptake in J774A.1 cells, but no uptake in Caco-2 cells was detected. Flow cytometry analysis revealed that the exposure of J774A.1 cells to CTxB resulted in a significant reduction in the uptake of SiO2 NPs. In contrast, the uptake of NPs by highly selective Caco-2 cells remained unaffected following CTxB exposure. Based on colocalization studies, CTxB and NPs might enter cells via shared endocytic pathways, followed by their sorting into different intracellular compartments. Our findings provide new insights into CTxB's function of modulating SiO2 NP uptake in phagocytic but not in differentiated intestine cells.

Quantification of nanoparticles' concentration inside polymer films using lock-in thermography.

Thin nanocomposite polymer films embedding various types of nanoparticles have been the target of abundant research to use them as sensors, smart coatings, or artificial skin. Their characterization is challenging and requires novel methods that can provide qualitative as well as quantitative information about their composition and the spatial distribution of nanoparticles. In this work, we show how lock-in thermography (LIT) can be used to quantify the concentration of gold nanoparticles embedded in polyvinyl alcohol (PVA) films. LIT is an emerging and non-destructive technique that measures the thermal signature produced by an absorbing sample illuminated by modulated light with a defined frequency. Films with various concentrations of gold nanoparticles of two different sizes have been prepared by evaporation from homogeneous aqueous PVA gold nanoparticle suspensions. When the thin films were illuminated with monochromatic light at a wavelength close to the plasmonic resonance signature of the nanoparticles, the amplitude of the thermal signature emitted by the nanoparticles was recorded. The measurements have been repeated for multiple modulation frequencies of the incident radiation. We have developed a mathematical method to quantitatively relate the concentration of nanoparticles to the measured amplitude. A discussion about the conditions under which the sample thickness can be determined is provided. Furthermore, our results show how LIT measurements can easily detect the presence of concentration gradients in samples and how the model allows the measured signal to be related to the respective concentrations. This work demonstrates the successful use of LIT as a reliable and non-destructive method to quantify nanoparticle concentrations.

Rethinking of TEER measurement reporting for epithelial cells grown on permeable inserts.

Transepithelial electrical resistance (TEER) measures electrical resistance across epithelial tissue barriers involving confluent layer(s) of cells. TEER values act as a prerequisite for determining the barrier integrity of cells, which play a key role in evaluating the transport of drugs, materials or chemicals of interest across an epithelial barrier. The measurements can be performed non-invasively by measuring ohmic resistance across a defined area. Thus, the TEER values are reported in Ω·cm2. In vitro epithelial models are typically assembled on semi-permeable inserts providing two-chamber compartments, and the majority of the studies use inserts with polyethylene terephthalate (PET) membranes. Recently, new inserts with different membrane types and properties have been introduced. However, the TEER values presented so far did not allow a direct comparison. This study presents the characterization of selected epithelial tissues, i.e., lung, retina, and intestine, grown on an ultra-thin ceramic microporous permeable insert (SiMPLI) and PET membranes with different properties, i.e., thickness, material, and pore numbers. We verified the epithelial cell growth on both inserts via phase-contrast and confocal laser scanning microscope imaging. Barrier characteristics were assessed by TEER measurements and also by evaluating the permeability of fluorescein isothiocyanate through cell layers. The findings indicated that background TEER value calculations and the available surface area for cell growth must be thoroughly assessed when new inserts are introduced, as the values cannot be directly compared without re-calculations. Finally, we proposed electrical circuit models highlighting the contributors to TEER recordings on PET and SiMPLI insert membranes. This study paves the way for making the ohmic-based evaluation of epithelial tissues' permeability independent of the material and geometry of the insert membrane used for cell growth.

Correlative Light, Electron Microscopy and Raman Spectroscopy Workflow To Detect and Observe Microplastic Interactions with Whole Jellyfish.

Many researchers have turned their attention to understanding microplastic interaction with marine fauna. Efforts are being made to monitor exposure pathways and concentrations and to assess the impact such interactions may have. To answer these questions, it is important to select appropriate experimental parameters and analytical protocols. This study focuses on medusae of Cassiopea andromeda jellyfish: a unique benthic jellyfish known to favor (sub-)tropical coastal regions which are potentially exposed to plastic waste from land-based sources. Juvenile medusae were exposed to fluorescent poly(ethylene terephthalate) and polypropylene microplastics (<300 µm), resin embedded, and sectioned before analysis with confocal laser scanning microscopy as well as transmission electron microscopy and Raman spectroscopy. Results show that the fluorescent microplastics were stable enough to be detected with the optimized analytical protocol presented and that their observed interaction with medusae occurs in a manner which is likely driven by the microplastic properties (e.g., density and hydrophobicity).

The Influence of Liquid Menisci on Nanoparticle Dosimetry in Submerged Cells.

Understanding the interaction between cells and nanoparticles (NPs) is vital to understand the hazard associated with nanoparticles. This requires quantifying and interpreting dose-response relationships. Experiments with cells cultured in vitro and exposed to particle dispersions mainly rely on mathematical models that estimate the received nanoparticle dose. However, models need to consider that aqueous cell culture media wets the inner surface of hydrophilic open wells, which results in a curved liquid-air interface called the meniscus. Here the impact of the meniscus on nanoparticle dosimetry is addressed in detail. Experiments and build an advanced mathematical model, to demonstrate that the presence of the meniscus may bring about systematic errors that must be considered to advance reproducibility and harmonization is presented. The script of the model is co-published and can be adapted to any experimental setup. Finally, simple and practical solutions to this problem, such as covering the air-liquid interface with a permeable lid or soft rocking of the cell culture well plate is proposed.

Cellular Uptake of Silica Particles Influences EGFR Signaling Pathway and is Affected in Response to EGF.

Background: The human epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is involved in several key cellular processes, such as cell proliferation and differentiation, and it has been linked to the development and progression of various cancers (e.g., breast and lung). Researchers have attempted to improve current cancer-targeted therapies by conjugating molecules on the surface of (nano)particles to efficiently target and inhibit EGFR. However, very few in vitro studies have investigated the effect of particles per se on EGFR signaling and dynamics. Furthermore, the impact of concomitant exposure of particles and EGFR ligands, such as epidermal growth factor (EGF) on cellular uptake efficiency has received little attention. Purpose: The purpose of this research was to determine the effects of silica (SiO2) particles on EGFR expression and intracellular signaling pathways in A549 lung epithelial cells, in the presence or absence of epidermal growth factor (EGF). Results: We showed that A549 cells are able to internalize SiO2 particles with core diameters of 130 nm and 1 µm without affecting cell proliferation or migration. However, both SiO2 particles interfere with the EGFR signaling pathway by raising the endogenous levels of extracellular signal-regulated kinase (ERK) 1/2. Furthermore, both in the presence and absence of SiO2 particles, the addition of EGF increased cell migration. EGF also stimulated cellular uptake of 130 nm SiO2 particles but not 1 µm particles. The increased uptake is primarily associated with EGF-stimulated macropinocytosis. Conclusion: This study shows that SiO2 particle uptake interferes with cellular signaling pathways and can be boosted by concurrent exposure to the bioactive molecule EGF. SiO2 particles, both alone and in combination with the ligand EGF, interfere with EGFR signaling pathway in a size-dependent manner.

Comparing species-different responses in pulmonary fibrosis research: Current understanding of in vitro lung cell models and nanomaterials.

Pulmonary fibrosis (PF) is a chronic, irreversible lung disease that is typically fatal and characterized by an abnormal fibrotic response. As a result, vast areas of the lungs are gradually affected, and gas exchange is impaired, making it one of the world's leading causes of death. This can be attributed to a lack of understanding of the onset and progression of the disease, as well as a poor understanding of the mechanism of adverse responses to various factors, such as exposure to allergens, nanomaterials, environmental pollutants, etc. So far, the most frequently used preclinical evaluation paradigm for PF is still animal testing. Nonetheless, there is an urgent need to understand the factors that induce PF and find novel therapeutic targets for PF in humans. In this regard, robust and realistic in vitro fibrosis models are required to understand the mechanism of adverse responses. Over the years, several in vitro and ex vivo models have been developed with the goal of mimicking the biological barriers of the lung as closely as possible. This review summarizes recent progress towards the development of experimental models suitable for predicting fibrotic responses, with an emphasis on cell culture methods, nanomaterials, and a comparison of results from studies using cells from various species.

A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis.

Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.

Layer-by-Layer siRNA Particle Assemblies for Localized Delivery of siRNA to Epithelial Cells through Surface-Mediated Particle Uptake.

Localized delivery of small interfering RNA (siRNA) is a promising approach for spatial control of cell responses at biomaterial interfaces. Layer-by-layer (LbL) assembly of siRNA with cationic polyelectrolytes has been used in film and nanoparticle vectors for transfection. Herein, we combine the ability of particles to efficiently deliver siRNA with the ability of film polyelectrolyte multilayers to act locally. LbL particles were prepared with alternating layers of poly(l-arginine) and siRNA and capped with hyaluronic acid. Negatively charged LbL particles were subsequently assembled on the poly(l-lysine)-functionalized substrate to form a LbL particle-decorated surface. Cells grown in contact with the particle-decorated surface were able to survive, internalize particles, and undergo gene silencing. This work shows that particle-decorated surfaces can be engineered by using electrostatic interactions and used to deliver therapeutic payloads for cell-instructive biointerfaces.

Human lung cell models to study aerosol delivery - considerations for model design and development.

Human lung tissue models range from simple monolayer cultures to more advanced three-dimensional co-cultures. Each model system can address the interactions of different types of aerosols and the choice of the model and the mode of aerosol exposure depends on the relevant scenario, such as adverse outcomes and endpoints of interest. This review focuses on the functional, as well as structural, aspects of lung tissue from the upper airway to the distal alveolar compartments as this information is relevant for the design of a model as well as how the aerosol properties determine the interfacial properties with the respiratory wall. The most important aspects on how to design lung models are summarized with a focus on (i) choice of appropriate scaffold, (ii) selection of cell types for healthy and diseased lung models, (iii) use of culture condition and assembly, (iv) aerosol exposure methods, and (v) endpoints and verification process. Finally, remaining challenges and future directions in this field are discussed.

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials.

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.