Concentration-dependent Uptake Research Articles

Tracking Small Extracellular Vesicles Using a Minimally Invasive PicoGreen Labeling Strategy.

Extracellular vesicles (EVs) are cell-secreted lipid bilayer delimited particles that mediate cellular communication. These tiny sacs of cellular information play an important role in cell communication and alter the physiological process under both normal and pathological conditions. As such, tracking EVs can provide valuable information regarding the basic understanding of cell communication, the onset of early malignancy, and biomarker discovery. Most of the current EV-tracking strategies are invasive, altering the natural characteristics of EVs by modifying the lipid bilayer with lipophilic dyes or surface proteins with fluorescent reporters. The invasive labeling strategies could alter the natural processes of EVs and thereby have major limitations for functional studies. Here, we report an alternative minimally invasive EV labeling strategy using PicoGreen (PG), a small molecule that fluoresces at 520 nm when bound to dsDNA. We show that PG binds to dsDNA associated with small EVs (50-200 nm), forming a stable and highly fluorescent PG-DNA complex in EVs (PG-EVs). In both 2D cell culture and 3D organoid models, PG-EV showed efficient tracking properties, including a high signal-to-noise ratio, time- and concentration-dependent uptake, and the ability to traverse a 3D environment. We further validated PG-EV tracking using dual-labeled EVs following two orthogonal labeling strategies: (1) Bioconjugation via surface amine labeling and (2) donor cell engineering via endogenously expressing mCherry-tetraspanin (CD9/CD63/CD81) reporter proteins. Our study has shown the feasibility of using PG-EV as an effective EV tracking strategy that can be applied for studying the functional role of EVs across multiple model systems.

Involvement of Proton-Coupled Organic Cation Antiporter in Human Blood-Brain Barrier Transport of Mesoridazine and Metoclopramide.

Mesoridazine and metoclopramide are cationic drugs that are distributed in the human brain despite being substrates of multidrug resistance protein 1 (MDR1), an efflux transporter expressed at the blood-brain barrier (BBB). We investigated their transport mechanisms at the BBB using hCMEC/D3, a human cerebral microvascular endothelial cell line often used as an in vitro BBB model. The cells exhibited time- and concentration-dependent uptake of mesoridazine and metoclopramide, with Km values of 34 and 277 µM, respectively. The uptake of both drugs significantly decreased in the presence of typical inhibitors and/or substrates of the H+-coupled organic cation (H+/OC) antiporter but not in the presence of inhibitors or substrates of organic cation transporters (OCTs), OCTN2, OATPs, SLC35F2, or the plasma membrane monoamine transporter (PMAT). Furthermore, metoclopramide uptake by hCMEC/D3 cells was pH- and energy-dependent, whereas mesoridazine uptake was unaffected by intracellular acidification and treatment with metabolic inhibitors. These results suggest that the H+/OC antiporter is involved in the influx of mesoridazine and metoclopramide into the brain across the BBB.

Quantitative analysis of transferrin uptake into living cells at single-molecule level

Endocytosis, apart from providing a mechanism for cells to take up nutrients, is the principal route of entry for many nanomedicines. The evaluation of therapeutic delivery efficiency without providing quantitative parameters, like the number of molecules entering the cell and the time of their entry, is very limited. Despite advances in single-molecule methods for in-cell quantitative measurements, they have not become widely used in the study of endocytosis. Their application, however, is challenging owing to the appropriate experimental design and applicable analysis methods. Here, by using an integrated approach based on the multidimensional time-correlated single-photon counting (TCSPC) technique, we demonstrate the quantitative method to measure the time- and concentration-dependent uptake of TRITC-transferrin into living cells with single-molecule sensitivity. The methodology opens possibilities for quantitatively assessing the delivery efficiency of fluorescent molecules into living cells.

Aspergillus terreus mediated green synthesis of cerium oxide nanoparticles and its neuroprotective activity against rotenone-induced cytotoxicity in SH-SY5Y cells

Cerium oxide nanoparticles (CeO2 NPs) are being widely explored as therapeutic agents against neurodegenerative diseases due to their regenerative and strong antioxidant activity. The current study aims to investigate the protective effect of green synthesized CeO2 NPs using Aspergillus terreus (A. terreus) filtrate against rotenone-induced cytotoxicity in SH-SY5Y cells as an in-vitro Parkinson’s model. The CeO2 NPs were characterized using powder X-ray diffraction (PXRD), Fourier transform infrared (FTIR) spectroscopy, UV–visible (UV–vis) spectroscopy, Scanning electron microscopy (SEM), and High-resolution transmission electron microscopy (HR-TEM). PXRD data revealed the strain and the crystal grain size of the CeO2 NPs were 3.488 x 10-4 and ∼ 15 nm respectively. FTIR spectra exhibited the metal–oxygen bond (Ce–O) at 848 and 558 cm−1. The absorption peak at 352 nm obtained through UV–vis spectroscopy is because of the charge transfer from the valance band to the conduction band. SEM and HR-TEM analysis disclosed the formation of spherical-shaped CeO2 NPs within the size range of 8–12 nm. Our results revealed that CeO2 NPs dose-dependently attenuated the rotenone toxicity and reduced LDH release. Additionally, the flow cytometric studies revealed the concentration-dependent cellular uptake of CeO2 NPs. Further, the CeO2 NPs significantly attenuated the rotenone toxicity by suppressing ROS generation, and modulating apoptosis. Hence, our study suggests that the CeO2 NPs hold promising potential as nanotherapeutics against Parkinson’s disease.

Comparative Phytochemical, In-vitro Anti-Hyperglycemic Activities of Napoleona vogelii Hook & Planch Stem Bark and Cassia occidentalis L. Root Extracts

This work investigated the comparative phytochemical, proximate and anti-hyperglycemic activities of stem bark of Napoleonaea vogelii and root of Cassia occidentals extracts. Stem bark and N. vogelii root of C. occidentalis were cold extracted with methanol, ethyl acetate, and n-hexane. The extracts were subjected to proximate, phytochemical analyses and anti-hyperglycemic assays. The result of proximate analysis revealed that stem bark of N. vogelii was made up of 40.28 ± 0.49 % crude fibre, 37.05 ± 0.05 % carbohydrates, 7.45 ± 0.45 % protein and more than 6.00 % oil content. whereas 22.33±0.08 % crude fibre, 44.70± 0.93 % carbohydrates, 10.72± 0.05 % protein and 11.00± 0.00 % oil contents in roots of C. occidentalis. Alkaloids and terpenoids were present in all the extracts of N. vogelii and C. occidentalis. Flavonoids were also found in all extracts of N. vogelii, whereas tannins and glycosides were also found in all extracts of C. occidentalis. In vitro antidiabetic screening of the methanol extracts indicated that both plants showed a good concentration-dependent percentage glucose uptake. In all concentrations, extracts of C. occidentalis exhibited better glucose uptake than N. vogelii extracts. comparable to the activity of the standard drug- metronidazole (68.44±0.00 %), From 0.10 – 0.50 mg/mL, the methanol extracts of both plants also showed a dose-dependent α-amylase inhibitory effect. But the inhibition decreased as the concentration was increased to 1.00 mg/mL. At 0.50 mg/mL, C. occidentalis and N. vogelii showed percentage inhibitory effect of 50.02±0.035 and35.61±0.01. Both plants’ extracts showed good anti-hyperglycemic activity, which may be due to the presence of alkaloids, terpenoids, saponins and tannins in the extracts, validating the folkloric use of both plants in the treatment of diabetes, and thus are good anti-diabetic agents.

Microplastics role in cell migration and distribution during cancer cell division

Amidst the global plastic pollution crisis, the gastrointestinal tract serves as the primary entry point for daily exposure to micro- and nanoplastics. We investigated the complex dynamics between polystyrene micro- and nanoplastics (PS-MNPs) and four distinct human colorectal cancer cell lines (HT29, HCT116, SW480, and SW620). Our findings revealed a significant size- and concentration dependent uptake of 0.25, 1, and 10 μm PS-MNPs across all cell lines, with HCT116 cells exhibiting the highest uptake rates. During cell division, particles were distributed between mother and daughter cells. Interestingly, we observed no signs of elimination from the cells. Short-term exposure to 0.25 μm particles significantly amplified cell migration, potentially leading to pro-metastatic effects. Particles demonstrated high persistence in 2D and 3D cultures, and accumulation in non-proliferating parts of spheroids, without interfering with cell proliferation or division. Our study unveils the disturbing fact of the persistence and bioaccumulation of MNPs in colorectal cancer cell lines, key toxicological traits under REACH (Regulation concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals). Our observations underscore the potential of MNPs as hidden catalysts for tumor progression, particularly through enhancing cell migration and possibly fueling metastasis - a finding that sheds light on a significant and previously underexplored area of concern.

Neuroprotective effects of polyacrylic acid (PAA) conjugated cerium oxide against hydrogen peroxide- and 6-OHDA-induced SH-SY5Y cell damage

Cerium oxide nanoparticles have been widely investigated against neurodegenerative diseases due to their antioxidant properties that aid in quenching reactive oxygen species. In this study, polyacrylic acid conjugated cerium oxide (PAA-CeO) nanoparticles were synthesized in a 50–60 nm size range with a zeta potential of − 35 mV. X-ray photoelectron spectroscopy analysis revealed a mixed valence state of Ce4+ and Ce3+. PAA-CeO nanoparticles were safe for undifferentiated (UN-) and retinoic acid-differentiated (RA-) human neuroblastoma SH-SY5Y cells and reduced the extent of cell damage evoked by hydrogen peroxide (H2O2) and 6-hydroxydopamine (6-OHDA). In the H2O2 model of cell damage PAA-CeO did not affect the caspase-3 activity (apoptosis marker) but attenuated the number of propidium iodide-positive cells (necrosis marker). In the 6-OHDA model, nanoparticles profoundly reduced necrotic changes and partially attenuated caspase-3 activity. However, we did not observe any impact of PAA-CeO on intracellular ROS formation induced by H2O2. Further, the flow cytometry analysis of fluorescein isothiocyanate-labeled PAA-CeO revealed a time- and concentration-dependent cellular uptake of nanoparticles. The results point to the neuroprotective potential of PAA-CeO nanoparticles against neuronal cell damage induced by H2O2 and 6-OHDA, which are in both models associated with the inhibition of necrotic processes and the model-dependent attenuation of activity of executor apoptotic protease, caspase-3 (6-OHDA model) but not with the direct inhibition of ROS (H2O2 model).

The impact of amine and carboxyl functionalised microplastics on the physiology of daphnids

Plastic waste is considered a major threat for terrestrial, marine and freshwater ecosystems. Ingestion of primary or secondary microparticles resulting from plastic degradation can lead to their trophic transfer raising serious health concerns. In this study, the effect of amine and carboxy functionalized polystyrene microparticles on the physiology of daphnids was investigated with a combination of phenotypic and metabolic endpoints. Carboxy functionalized microparticles showed higher toxicity in acute exposures compared to their amine counterparts. Accumulation of both microparticles in animal gut was confirmed by stereo-microscopy as well as fluorescent microscopy which showed no presence of particles in the rest of the animal. Fluorescence based quantification of microparticles extracted from animal lysates validated their concentration-dependent uptake. Additionally, exposure of daphnids to amine and carboxy functionalized microparticles resulted in increased activities of key enzymes related to metabolism and detoxification. Finally, significant metabolic perturbations were discovered following exposure to microplastics. These findings suggest that polystyrene microparticles can hinder organism performance of the freshwater species and highlight the importance of seeking for holistic and physiological endpoints for pollution assessment.

Sepia Melanin-Loaded Primary Human Gingival Keratinocytes: An In Vitro Model for Studies on Pigmented Gingiva

The objective of this study is the development of an in vitro cell culture model of pigmented gingival keratinocytes to provide a unique tool to assess oral care products such as toothpaste and evaluate whether pigmented gingival cells might be less susceptible than unpigmented cells to cytotoxicity by any toothpaste. Sepia melanin at various concentrations was added to primary human gingival keratinocyte (HGK) monolayers to identify the concentration at which melanin is sufficiently phagocytosed in the absence of cytotoxicity; this concentration was subsequently used to generate pigmented HGK model. Extracts from three commercial adult toothpastes (Crest 3D White, Sensodyne, and Colgate Optic) at different dilutions were evaluated in pigmented and unpigmented HGKs for cytotoxicity over a 24 h duration by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Results demonstrated that HGKs showed a concentration-dependent uptake of sepia melanin with a significant linear correlation of pigment uptake. Moreover, the melanin was distributed perinuclearly in the cells, that was similar to the distribution of physiological gingiva in vivo. Further experiments were conducted with 25 µg/mL sepia melanin as higher concentrations induced cytotoxicity. Evaluation of three commercial toothpastes on unpigmented and pigmented HGKs showed no differential effects at any dilution. In summary, a model of pigmented HGKs with the ability to create a controlled level of pigmentation was demonstrated. Examination of extracts from three commercial toothpastes revealed similar cytotoxicity to both pigmented and unpigmented HGKs. In conclusion, this study shows that the artificially pigmented HGK model is an easy and low-cost tool that mimics the in vivo gingival pigmentation. Moreover, the evaluated toothpastes showed similar cytotoxicity to pigmented and unpigmented HGKs, suggesting that the presence of melanin did not impart any protective effects. Further studies to employ this pigmented HGK model to evaluate a large number of oral care products and include repeated exposures and longer duration are warranted.

The chemical neighborhood of cells in a diffusion-limited system.

Microorganisms follow us everywhere, and they will be essential to sustaining long-term human space exploration through applications such as vitamin synthesis, biomining, and more. Establishing a sustainable presence in space therefore requires that we better understand how stress due to the altered physical conditions of spaceflight affects our companion organisms. In microgravity environments such as orbital space stations, microorganisms likely experience the change in gravity primarily through changes in fluid mixing processes. Without sedimentation and density-driven convection, diffusion becomes the primary process governing the movement of growth substrates and wastes for microbial cells in suspension culture. Non-motile cells might therefore develop a substrate-deficient "zone of depletion" and experience stress due to starvation and/or waste build-up. This would in turn impact the concentration-dependent uptake rate of growth substrates and could be the cause of the altered growth rates previously observed in microorganisms in spaceflight and in ground-simulated microgravity. To better understand the extent of these concentration differences and their potential influence on substrate uptake rates, we used both an analytical solution and finite difference method to visualize concentration fields around individual cells. We modeled diffusion, using Fick's Second Law, and nutrient uptake, using Michaelis-Menten kinetics, and assessed how that distribution varies in systems with multiple cells and varied geometries. We determined the radius of the zone of depletion, within which cells had reduced the substrate concentration by 10%, to be 5.04 mm for an individual Escherichia coli cell in the conditions we simulated. However, we saw a synergistic effect with multiple cells near each other: multiple cells in close proximity decreased the surrounding concentration by almost 95% from the initial substrate concentration. Our calculations provide researchers an inside look at suspension culture behavior in the diffusion-limited environment of microgravity at the scale of individual cells.

Amphiphilic Cell-Penetrating Peptides Containing Arginine and Hydrophobic Residues as Protein Delivery Agents

The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2-10 μM) and [DipR]5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-β-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.

Concentration-Dependent Cellular Uptake of Graphene Oxide Quantum Dots Promotes the Odontoblastic Differentiation of Dental Pulp Cells via the AMPK/mTOR Pathway.

As zero-dimension nanoparticles, graphene oxide quantum dots (GOQDs) have broad potential for regulating cell proliferation and differentiation. However, such regulation of dental pulp cells (DPSCs) with different concentrations of GOQDs is insufficiently investigated, especially on the molecular mechanism. The purpose of this study was to explore the effect and molecular mechanism of GOQDs on the odontoblastic differentiation of DPSCs and to provide a theoretical basis for the repair of pulp vitality by pulp capping. CCK-8, immunofluorescence staining, alkaline phosphatase activity assay and staining, alizarin red staining, qRT-PCR, and western blotting were used to detect the proliferation and odontoblastic differentiation of DPSC coculturing with different concentrations of GOQDs. The results indicate that the cellular uptake of low concentration of GOQDs (0.1, 1, and 10 μg/mL) could promote the proliferation and odontoblastic differentiation of DPCSs. Compared with other concentration groups, 1 μg/mL GOQDs show better ability in such promotion. In addition, with the activation of the AMPK signaling pathway, the mTOR signaling pathway was inhibited in DPSCs after coculturing with GOQDs, which indicates that low concentrations of GOQDs could regulate the odontoblastic differentiation of DPSCs by the AMPK/mTOR signaling pathway.

Soluble Iron Enhances Extracellular Electron Uptake by Shewanella oneidensis MR-1.

Extracellular electron transfer (EET) is a process that microorganisms use to reduce or oxidize external insoluble electron acceptors or donors. Much of our mechanistic understanding of this process is derived from studies of transmembrane cytochrome complexes and extracellular redox shuttles that mediate outward EET to anodes and external electron acceptors. In contrast, there are knowledge gaps concerning the reverse process of inward EET from external electron donors to cells. Here, we describe a role for soluble iron (exogenous FeCl2) in enhancing EET from cathodes to the model EET bacterium Shewanella oneidensis MR-1, with fumarate serving as the intracellular electron acceptor. This iron concentration-dependent electron uptake was eradicated upon addition of an iron chelator and occurred only in the presence of fumarate reductase, confirming an electron pathway from cathodes to this periplasmic enzyme. Moreover, S. oneidensis mutants lacking specific outer membrane and periplasmic cytochromes exhibited significantly decreased current levels relative to wild-type. These results indicate that soluble iron can function as an electron carrier to the EET machinery of S. oneidensis.

Self-nanoemulsifying formulation for oral delivery of sildenafil: effect on physicochemical attributes and in vivo pharmacokinetics.

Sildenafil (SLD) is employed for the management of erectile dysfunction and pulmonary arterial hypertension. It exhibits meagre water solubility and is available in the form of citrate salt hydrate to improve the solubility. However, it still exhibits moderate solubility, high first-pass metabolism, resulting in very less oral bioavailability. The present study demonstrates the preparation of self-nanoemulsifying drug delivery system for augmenting the oral bioavailability of SLD. Oleic acid and Capmul MCM C8 blend (oil phase), Cremophor® RH40 (surfactant), and Labrafil® M1944 CS (cosurfactant) were selected as main constituents for making liquid preconcentrate based on the solubility and emulsification study. The preconcentrate upon dilution and emulsification showed droplet size 52.03 ± 13.03nm, PDI 0.143 ± 0.028, and % transmittance was 99.77 ± 1.86% with SLD load of 40mg/g of formulation. The prepared formulation was further assessed for stability, in vitro release, Caco-2 cell uptake, and in vivo pharmacokinetic performance. SLD-SNEDDS formulation was found to be robust in terms of stability against several folds dilution in the gastrointestinal tract (GIT), freeze-thaw cycles, and had a storage stability of 3months at 4°C and 25°C. SLD-SNEDDS showed ~4.7-fold and ~5-fold increase in time- and concentration-dependent cellular uptake as against SLD cultured with Caco-2 cells. In vivo pharmacokinetic study revealed ~5.8- and ~2.5-fold increase in AUC0-∞ values in case of SLD-SNEDDS as against SLD suspension and SLD citrate solution, respectively.

Omniparticle Contrast Agent for Multimodal Imaging: Synthesis and Characterization in an Animal Model.

Individual imaging modalities have certain advantages, but each suffers from drawbacks that other modalities may overcome. The goal of this study was to create a novel contrast agent suitable for various imaging modalities that after a single administration can bridge and strengthen the collaboration between the research fields as well as enrich the information obtained from any one modality. The contrast agent platform is based on dextran-coated iron oxide nanoparticles (for MRI and MPI) and synthesized using a modified co-precipitation method, followed by a series of conjugation steps with a fluorophore (for fluorescence and photoacoustic imaging), thyroxine (for CT imaging), and chelators for radioisotope labeling (for PET imaging). The fully conjugated agent was then tested in vitro in cell uptake, viability, and phantom studies and in vivo in a model of intraductal injection and in a tumor model. The agent was synthesized, characterized, and tested in vitro where it showed the ability to produce a signal on MRI/MPI/FL/PA/CT and PET images. Studies in cells showed the expected concentration-dependent uptake of the agent without noticeable toxicity. In vivo studies demonstrated localization of the agent to the ductal tree in mice after intraductal injection with different degrees of resolution, with CT being the best for this particular application. In a model of injected labeled tumor cells, the agent produced a signal with all modalities and showed persistence in tumor cells confirmed by histology. A fully functional omniparticle contrast agent was synthesized and tested in vitro and in vivo in two animal models. Results shown here point to the generation of a potent signal in all modalities tested without detrimental toxicity. Future use of this agent includes its exploration in various models of human disease including image-guided diagnostic and therapeutic applications.

Comparative toxicokinetics and toxicity of PFOA and its replacement GenX in the early stages of zebrafish

Perand poly-fluoroalkyl substances (PFAS) are receiving attention due to their persistence, and potential adverse effects on environmental and human health. Efforts to reduce long-chained PFAS (≥C8) compounds were implemented in 2006 as a part of “PFOA Stewardship Program Initiative” (PFOA–perfluorooctanoic acid). Short-chained PFAS (<C8) were introduced as replacements, which were believed to have lower potential for environmental persistence and bioaccumulation. Little is known about the uptake and elimination, and potential toxic effects of these replacement compounds. Hence, it is important to compare toxicokinetics and toxicity of long-chain PFAS to their replacement compounds. To this end, zebrafish (ZF), Danio rerio, embryos were exposed to PFOA and its short-chain replacement perfluoro (2-methyl-3-oxahexanoic) acid (GenX) with the aim to assess uptake and elimination kinetics, hatching success, morphology, startle response, and survival. At 24 hpf, LC50 was 82 μM for PFOA and 170 μM for GenX. At 54 hpf, GenX but not PFOA showed an increase in hatching success. At 120 hpf, no statistically significant differences were seen in white light startle response below the LC50. PFAS internal concentrations were measured at 72 and 120 hpf during exposure phase, and at 168 hpf during depuration phase. GenX and PFOA internal concentrations in 120 hpf larvae exposed to highest concentration (20 μM) were 35.02 and 44.51 μM, respectively. Concentrations were eliminated almost completely at 168 hpf for GenX up to 95%, while for PFOA up to 50%. As steady-state was not reached, we estimated kinetic bioconcentration factors (BCFkin). BCFkin for GenX was lower than PFOA at equimolar concentrations. However, bioconcentration factors were higher at the lower exposure concentrations for both chemicals, suggesting a concentration-dependent uptake of PFASs. The predicted internal effect concentrations, accounting for the differences in bioconcentration factors, were 229 μM for GenX and 226 μM for PFOA, suggesting similar toxic potencies.

In-Vitro Use of Verteporfin for Photodynamic Therapy in Glioblastoma

BackgroundStummer et al. established fluorescence-guided surgery (FGS) for glioblastoma (GBM) using 5-aminolevulinic acid (5-ALA). Its metabolite, protoporphyrin IX (PPIX), is also a photosensitizer and can be used for photodynamic therapy (PDT) using a laser beam of 635 nm. The porphyrin derivate verteporfin (VP) was discovered to have properties to penetrate the brain, pharmacologically target glioma cells, and is approved for PDT of choroidal neovascularization in wet age-related macular degeneration at 689 nm. ObjectiveTo elucidate whether GBM cell lines are susceptible to PDT with second-generation photosensitizer VP. MethodsHuman glioma cell lines LN229, HSR-GBM1, and a low-passage patient-derived GBM cell line P1 were treated with variable concentrations of VP for 24 h, followed by PDT at 689 nm using a diode laser light. Cell viability was measured using the MTT assay and VP uptake was measured using a desktop cytometer. ResultsSignificantly higher cell death following PDT with VP compared to VP treatment alone or no treatment was detected in all cell models (LN229, HSR-GBM1, P1). Flowcytometric measurements revealed a concentration-dependent cellular uptake of VP after 24 h incubation up to 99% at 10 µM (HSR-GBM1). ConclusionThis study demonstrates that PDT with VP causes cell death in GBM cells at marginal concentrations. Additionally, red spectrum fluorescence was detected at therapeutic concentrations in all cell lines, validating the cellular uptake of VP in GBM cells. VP, therefore, is not only a potential drug for targeting GBM pharmacologically but can be used as an optical imaging dye in surgery and photosensitizer to make GBM susceptible to PDT.

In vivo phytotoxic effect of yttrium-oxide nanoparticles on the growth, uptake and translocation of tomato seedlings (Lycopersicon esculentum)

The potential toxicity and ecological risks of rare-earth nanoparticles in the environment have become a concern due to their widespread application and inevitable releases. The integration of hydroponics experiments, partial least squares structural equation modeling (PLS-SEM), and Transmission Electron Microscopy (TEM) were utilized to investigate the physiological toxicity, uptake and translocation of yttrium oxide nanoparticles (Y2O3 NPs) under different hydroponic treatments (1, 5, 10, 20, 50 and 100 mg·L−1 of Y2O3 NPs, 19.2 mg·L−1 Y(NO3)3 and control) in tomato (Lycopersicon esculentum) seedlings. The results indicated that Y2O3 NPs had a phytotoxic effect on tomato seedlings' germination, morphology, physiology, and oxidative stress. The Y2O3 NPs and soluble YIII reduced the root elongation, bud elongation, root activity, chlorophyll, soluble protein content and superoxide dismutase and accelerated the proline and malondialdehyde in the plant with increasing concentrations. The phytotoxic effects of Y2O3 NPs on tomato seedlings had a higher phytotoxic effect than soluble YIII under the all treatments. The inhibition rates of different levels of Y2O3 NPs in shoot and root biomass ranged from 0.2% to 6.3% and 1.0–11.3%, respectively. The bioaccumulation and translocation factors were less than 1, which suggested that Y2O3 NPs significantly suppressed shoot and root biomass of tomato seedlings and easily bioaccumulated in the root. The observations were consistent with the process of concentration-dependent uptake and translocation factor and confirmed by TEM. Y2O3 NPs penetrate the epidermis, enter the cell wall, and exist in the intercellular space and cytoplasm of mesophyll cells of tomato seedlings by endocytic pathway. Moreover, PLS-SEM revealed that the concentration of NPs significantly negatively affects the morphology and physiology, leading to the change in biomass of plants. This study demonstrated the possible pathway of Y2O3 NPs in uptake, phytotoxicity and translocation of Y2O3 NPs in tomato seedlings.

Substrates of the Human Brain Proton-Organic Cation Antiporter and Comparison with Organic Cation Transporter 1 Activities.

Many organic cations (OCs) may be transported through membranes by a genetically still uncharacterized proton-organic cation (H + OC) antiporter. Here, we characterized an extended substrate spectrum of this antiporter. We studied the uptake of 72 drugs in hCMEC/D3 cells as a model of the human blood–brain barrier. All 72 drugs were tested with exchange transport assays and the transport of 26 of the drugs was studied in more detail concerning concentration-dependent uptake and susceptibility to specific inhibitors. According to exchange transport assays, 37 (51%) drugs were good substrates of the H + OC antiporter. From 26 drugs characterized in more detail, 23 were consistently identified as substrates of the H + OC antiporter in six different assays and transport kinetic constants could be identified with intrinsic clearances between 0.2 (ephedrine) and 201 (imipramine) mL × minute−1 × g protein−1. Excellent substrates of the H + OC antiporter were no substrates of organic cation transporter OCT1 and vice versa. Good substrates of the H + OC antiporter were more hydrophobic and had a lower topological polar surface area than non-substrates or OCT1 substrates. These data and further research on the H + OC antiporter may result in a better understanding of pharmacokinetics, drug–drug interactions and variations in pharmacokinetics.

Toxicity and bioaccumulation of copper in the oribatid mite Oppia nitens (Acari: Oribatida)

To determine the toxicity and bioaccumulation of copper, adult oribatid soil mites Oppia nitens were exposed for 28 days to LUFA 2.2 soil spiked at concentrations of 0–6400 mg Cu kg−1 dry soil. Effects on survival and reproduction were related to total and available (0.01 M CaCl2 extractable and pore water) concentrations in the soil and concentrations in the animals. The mites showed a concentration-dependent uptake of copper, which, however, decreased at toxic concentrations. Overall bioaccumulation factors were low, suggesting a low tendency for copper bioaccumulation. The estimated median lethal concentration (LC50) values were 3251 mg Cu kg−1 dry soil, 1130 mg Cu kg−1 dry soil, 1977 mg Cu L−1 pore water, and 592 mg Cu kg−1 dry body weight, and the estimated 50 % effective concentrations (EC50) for effects of copper on reproduction were 589 mg Cu kg−1 dry soil, 116 mg Cu kg−1 dry soil, 78.5 mg Cu L−1 pore water, and 413 mg Cu kg−1 dry body weight, based on measured soil total concentrations, 0.01 M CaCl2 extractable, porewater, and internal concentrations, respectively. The results show that the mite O. nitens is a suitable test organism for measuring metal bioavailability and toxicity in soil.