Optimization of micelle-encapsulated extremely small sized iron oxide nanoparticles as a T1 contrast imaging agent: biodistribution and safety profile.

BACKGROUND: Iron oxide nanoparticles (IONPs) have been cleared by the Food and Drug Administration (FDA) for various clinical applications, such as tumor-targeted imaging, hyperthermia therapy, drug delivery, and live-cell tracking. However, the application of IONPs as T1 contrast agents has been restricted due to their high r2 values and r2/r1 ratios, which limit their effectiveness in T1 contrast enhancement. Notably, IONPs with diameters smaller than 5 nm, referred to as extremely small-sized IONPs (ESIONs), have demonstrated potential in overcoming these limitations. To advance the clinical application of ESIONs as T1 contrast agents, we have refined a scale-up process for micelle encapsulation aimed at improving the hydrophilization of ESIONs, and have carried out comprehensive in vivo biodistribution and preclinical toxicity assessments. RESULTS: The optimization of the scale-up micelle-encapsulation process, specifically employing Tween60 at a concentration of 10% v/v, resulted in ESIONs that were uniformly hydrophilized, with an average size of 9.35 nm and a high purification yield. Stability tests showed that these ESIONs maintained consistent size over extended storage periods and dispersed effectively in blood and serum-mimicking environments. Relaxivity measurements indicated an r1 value of 3.43 mM- 1s- 1 and a favorable r2/r1 ratio of 5.36, suggesting their potential as T1 contrast agents. Biodistribution studies revealed that the ESIONs had extended circulation times in the bloodstream and were primarily cleared via the hepatobiliary route, with negligible renal excretion. We monitored blood clearance and organ distribution using positron emission tomography and magnetic resonance imaging (MRI). Additionally, MRI signal variations in a dose-dependent manner highlighted different behaviors at varying ESIONs concentrations, implying that optimal dosages might be specific to the intended imaging application. Preclinical safety evaluations indicated that ESIONs were tolerable in rats at doses up to 25 mg/kg. CONCLUSIONS: This study effectively optimized a scale-up process for the micelle encapsulation of ESIONs, leading to the production of hydrophilic ESIONs at gram-scale levels. These optimized ESIONs showcased properties conducive to T1 contrast imaging, such as elevated r1 relaxivity and a reduced r2/r1 ratio. Biodistribution study underscored their prolonged bloodstream presence and efficient clearance through the liver and bile, without significant renal involvement. The preclinical toxicity tests affirmed the safety of the ESIONs, supporting their potential use as T1 contrast agent with versatile clinical application.

The effect of a 4-week, remotely administered, post-exercise passive leg heating intervention on determinants of endurance performance.

PURPOSE: Post-exercise passive heating has been reported to augment adaptations associated with endurance training. The current study evaluated the effect of a 4-week remotely administered, post-exercise passive leg heating protocol, using an electrically heated layering ensemble, on determinants of endurance performance. METHODS: Thirty recreationally trained participants were randomly allocated to either a post-exercise passive leg heating (PAH, n = 16) or unsupervised training only control group (CON, n = 14). The PAH group wore the passive heating ensemble for 90-120 min/day, completing a total of 20 (16 post-exercise and 4 stand-alone leg heating) sessions across 4 weeks. Whole-body (peak oxygen uptake, gas exchange threshold, gross efficiency and pulmonary oxygen uptake kinetics), single-leg exercise (critical torque and NIRS-derived muscle oxygenation), resting vascular characteristics (flow-mediated dilation) and angiogenic blood measures (nitrate, vascular endothelial growth factor and hypoxia inducible factor 1-α) were recorded to characterize the endurance phenotype. All measures were assessed before (PRE), at 2 weeks (MID) and after (POST) the intervention. RESULTS: There was no effect of the intervention on test of whole-body endurance capacity, vascular function or blood markers (p > 0.05). However, oxygen kinetics were adversely affected by PAH, denoted by a slowing of the phase II time constant; τ (p = 0.02). Furthermore, critical torque-deoxygenation ratio was improved in CON relative to PAH (p = 0.03). CONCLUSION: We have demonstrated that PAH had no ergogenic benefit but instead elicited some unfavourable effects on sub-maximal exercise characteristics in recreationally trained individuals.

Conventional extraction of fucoidan from Irish brown seaweed Fucus vesiculosus followed by ultrasound-assisted depolymerization.

Fucoidan has attracted considerable attention from scientists and pharmaceutical companies due to its antioxidant, anticoagulant, anti-inflammatory, anti-tumor, and health-enhancing properties. However, the extraction of fucoidan from seaweeds often involves the use of harsh chemicals, which necessitates the search for alternative solvents. Additionally, the high viscosity and low cell permeability of high molecular weight (Mw) fucoidan can limit its effectiveness in drug action, while lower Mw fractions exhibit increased biological activity and are also utilized as dietary supplements. The study aimed to (1) extract fucoidan from the seaweed Fucus vesiculosus (FV) using an environmentally friendly solvent and compare it with the most commonly used extraction solvent, hydrochloric acid, and (2) assess the impact of ultrasound-assisted depolymerization on reducing the molecular weight of the fucoidan extracts and examine the cytotoxic effect of different molecular weight fractions. The findings indicated that the green depolymerization solvent, in conjunction with a brief ultrasound treatment, effectively reduced the molecular weight. Moreover, a significant decrease in cell viability was observed in selected samples, indicating potential anticancer properties. As a result, ultrasound was determined to be an effective method for depolymerizing crude fucoidan from Fucus Vesiculosus seaweed.

Investigating STEAP2 as a potential therapeutic target for the treatment of aggressive prostate cancer.

The expression of six transmembrane epithelial antigen of the prostate (STEAP2) is increased in prostate cancer when compared to normal tissue, suggesting a role for STEAP2 in disease progression. This study aimed to determine whether targeting STEAP2 with an anti-STEAP2 polyclonal antibody (pAb) or CRISPR/Cas9 knockout influenced aggressive prostate cancer traits. Gene expression analysis of the STEAP gene family was performed in a panel of prostate cancer cell lines; C4-2B, DU145, LNCaP and PC3. The highest increases in STEAP2 gene expression were observed in C4-2B and LNCaP cells (p<0.001 and p<0.0001 respectively) when compared to normal prostate epithelial PNT2 cells. These cell lines were treated with an anti-STEAP2 pAb and their viability assessed. CRISPR/Cas9 technology was used to knockout STEAP2 from C4-2B and LNCaP cells and viability, proliferation, migration and invasion assessed. When exposed to an anti-STEAP2 pAb, cell viability significantly decreased (p<0.05). When STEAP2 was knocked out, cell viability and proliferation was significantly decreased when compared to wild-type cells (p<0.001). The migratory and invasive potential of knockout cells were also decreased. These data suggest that STEAP2 has a functional role in driving aggressive prostate cancer traits and could provide a novel therapeutic target for the treatment of prostate cancer.

Assessing the transferability and reproducibility of 3D in vitro liver models from primary human multi-cellular microtissues to cell-line based HepG2 spheroids.

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 µg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.

Enhanced pyrazolopyrimidinones cytotoxicity against glioblastoma cells activated by ROS-Generating cold atmospheric plasma.

Pyrazolopyrimidinones are fused nitrogen-containing heterocyclic systems, which act as a core scaffold in many pharmaceutically relevant compounds. Pyrazolopyrimidinones have been demonstrated to be efficient in treating several diseases, including cystic fibrosis, obesity, viral infection and cancer. In this study using glioblastoma U-251MG cell line, we tested the cytotoxic effects of 15 pyrazolopyrimidinones, synthesised via a two-step process, in combination with cold atmospheric plasma (CAP). CAP is an adjustable source of reactive oxygen and nitrogen species as well as other unique chemical and physical effects which has been successfully tested as an innovative cancer therapy in clinical trials. Significantly variable cytotoxicity was observed with IC50 values ranging from around 11 µM to negligible toxicity among tested compounds. Interestingly, two pyrazolopyrimidinones were identified that act in a prodrug fashion and display around 5-15 times enhanced reactive-species dependent cytotoxicity when combined with cold atmospheric plasma. Activation was evident for direct CAP treatment on U-251MG cells loaded with the pyrazolopyrimidinone and indirect CAP treatment of the pyrazolopyrimidinone in media before adding to cells. Our results demonstrated the potential of CAP combined with pyrazolopyrimidinones as a programmable cytotoxic therapy and provide screened scaffolds that can be used for further development of pyrazolopyrimidinone prodrug derivatives.

Understanding the impact of more realistic low-dose, prolonged engineered nanomaterial exposure on genotoxicity using 3D models of the human liver.

BACKGROUND: With the continued integration of engineered nanomaterials (ENMs) into everyday applications, it is important to understand their potential for inducing adverse human health effects. However, standard in vitro hazard characterisation approaches suffer limitations for evaluating ENM and so it is imperative to determine these potential hazards under more physiologically relevant and realistic exposure scenarios in target organ systems, to minimise the necessity for in vivo testing. The aim of this study was to determine if acute (24 h) and prolonged (120 h) exposures to five ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) would have a significantly different toxicological outcome (cytotoxicity, (pro-)inflammatory and genotoxic response) upon 3D human HepG2 liver spheroids. In addition, this study evaluated whether a more realistic, prolonged fractionated and repeated ENM dosing regime induces a significantly different toxicity outcome in liver spheroids as compared to a single, bolus prolonged exposure. RESULTS: Whilst it was found that the five ENMs did not impede liver functionality (e.g. albumin and urea production), induce cytotoxicity or an IL-8 (pro-)inflammatory response, all were found to cause significant genotoxicity following acute exposure. Most statistically significant genotoxic responses were not dose-dependent, with the exception of TiO2. Interestingly, the DNA damage effects observed following acute exposures, were not mirrored in the prolonged exposures, where only 0.2-5.0 µg/mL of ZnO ENMs were found to elicit significant (p ≤ 0.05) genotoxicity. When fractionated, repeated exposure regimes were performed with the test ENMs, no significant (p ≥ 0.05) difference was observed when compared to the single, bolus exposure regime. There was < 5.0% cytotoxicity observed across all exposures, and the mean difference in IL-8 cytokine release and genotoxicity between exposure regimes was 3.425 pg/mL and 0.181%, respectively. CONCLUSION: In conclusion, whilst there was no difference between a single, bolus or fractionated, repeated ENM prolonged exposure regimes upon the toxicological output of 3D HepG2 liver spheroids, there was a difference between acute and prolonged exposures. This study highlights the importance of evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

Anti-carcinogenic effects of exercise-conditioned human serum: evidence, relevance and opportunities.

Regular physical activity reduces the risk of several site-specific cancers in humans and suppresses tumour growth in animal models. The mechanisms through which exercise reduces tumour growth remain incompletely understood, but an intriguing and accumulating body of evidence suggests that the incubation of cancer cells with post-exercise serum can have powerful effects on key hallmarks of cancer cell behaviour in vitro. This suggests that exercise can impact tumour biology through direct changes in circulating proteins, RNA molecules and metabolites. Here, we provide a comprehensive narrative overview of what is known about the effects of exercise-conditioned sera on in vitro cancer cell behaviour. In doing so, we consider the key limitations of the current body of literature, both from the perspective of exercise physiology and cancer biology, and we discuss the potential in vivo physiological relevance of these findings. We propose key opportunities for future research in an area that has the potential to identify key anti-oncogenic protein targets and optimise physical activity recommendations for cancer prevention, treatment and survivorship.

Ursolic Acid Inhibits Collective Cell Migration and Promotes JNK-Dependent Lysosomal Associated Cell Death in Glioblastoma Multiforme Cells.

Ursolic acid (UA) is a bioactive compound which has demonstrated therapeutic efficacy in a variety of cancer cell lines. UA activates various signalling pathways in Glioblastoma multiforme (GBM) and offers a promising starting point in drug discovery; however, understanding the relationship between cell death and migration has yet to be elucidated. UA induces a dose dependent cytotoxic response demonstrated by flow cytometry and biochemical cytotoxicity assays. Inhibitor and fluorescent probe studies demonstrate that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM collective cell migration in a time dependent manner that is independent of the JNK signalling pathway. Cytotoxicity induced by UA results in the formation of acidic vesicle organelles (AVOs), speculating the activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs. Confocal microscopy and isosurface visualisation determined co-localisation of lysosomes with the previously identified AVOs, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death. Collectively, our data identify that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM collective cell migration.

In Vitro Three-Dimensional Liver Models for Nanomaterial DNA Damage Assessment.

Whilst the liver possesses the ability to repair and restore sections of damaged tissue following acute injury, prolonged exposure to engineered nanomaterials (ENM) may induce repetitive injury leading to chronic liver disease. Screening ENM cytotoxicity using 3D liver models has recently been performed, but a significant challenge has been the application of such in vitro models for evaluating ENM associated genotoxicity; a vital component of regulatory human health risk assessment. This review considers the benefits, limitations, and adaptations of specific in vitro approaches to assess DNA damage in the liver, whilst identifying critical advancements required to support a multitude of biochemical endpoints, focusing on nano(geno)toxicology (e.g., secondary genotoxicity, DNA damage, and repair following prolonged or repeated exposures).

Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations.

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (≤14 days). These spheroid models (≥500 µm in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the 'gold standard' micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells.

Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

Cold Atmospheric Plasma Induces ATP-Dependent Endocytosis of Nanoparticles and Synergistic U373MG Cancer Cell Death.

Gold nanoparticles (AuNP) have potential as both diagnostic and therapeutic vehicles. However, selective targeting and uptake in cancer cells remains challenging. Cold atmospheric plasma (CAP) can be combined with AuNP to achieve synergistic anti-cancer cytotoxicity. To explore synergistic mechanisms, we demonstrate both rate of AuNP uptake and total amount accumulated in U373MG Glioblastoma multiforme (GBM) cells are significantly increased when exposed to 75 kV CAP generated by dielectric barrier discharge. No significant changes in the physical parameters of AuNP were caused by CAP but active transport mechanisms were stimulated in cells. Unlike many other biological effects of CAP, long-lived reactive species were not involved, and plasma-activated liquids did not replicate the effect. Chemical effects induced by direct and indirect exposure to CAP appears the dominant mediator of enhanced uptake. Transient physical alterations of membrane integrity played a minor role. 3D-reconstruction of deconvoluted confocal images confirmed AuNP accumulation in lysosomes and other acidic vesicles, which will be useful for future drug delivery and diagnostic strategies. Toxicity of AuNP significantly increased by 25-fold when combined with CAP. Our data indicate that direct exposure to CAP activates AuNP-dependent cytotoxicity by increasing AuNP endocytosis and trafficking to lysosomes in U373MG cells.

Non-thermal atmospheric plasma induces ROS-independent cell death in U373MG glioma cells and augments the cytotoxicity of temozolomide.

BACKGROUND: Non-thermal atmospheric plasma (NTAP) is an ionised gas produced under high voltage that can generate short-lived chemically active species and induce a cytotoxic insult in cancer cells. Cell-specific resistance to NTAP-mediated cytotoxicity has been reported in the literature. The aim of this study was to determine whether resistance against NTAP could be overcome using the human glioma cell line U373MG. METHODS: Non-thermal atmospheric plasma was generated using a Dielectric Barrier Device (DBD) system with a maximum voltage output of 120 kV at 50 Hz. The viability of U373MG GBM cells and HeLa cervical carcinoma cells was determined using morphology, flow cytometry and cytotoxicity assays. Fluorescent probes and inhibitors were used to determine the mechanisms of cytotoxicity of cells exposed to the plasma field. Combinational therapy with temozolomide (TMZ) and multi-dose treatments were explored as mechanisms to overcome resistance to NTAP. RESULTS: Non-thermal atmospheric plasma decreased cell viability in a dose (time)-dependent manner. U373MG cells were shown to be resistant to NTAP treatment when compared with HeLa cells, and the levels of intracellular reactive oxygen species (ROS) quantified in U373MG cells were much lower than in HeLa cells following exposure to the plasma field. Reactive oxygen species inhibitor N-acetyl cysteine (NAC) only alleviated the cytotoxic effects in HeLa cells and not in the relatively NTAP-resistant cell line U373MG. Longer exposures to NTAP induced a cell death independent of ROS, JNK and caspases in U373MG. The relative resistance of U373MG cells to NTAP could be overcome when used in combination with low concentrations of the GBM chemotherapy TMZ or exposure to multiple doses. CONCLUSIONS: For the very first time, we report that NTAP induces an ROS-, JNK- and caspase-independent mechanism of cell death in the U373MG GBM cell line that can be greatly enhanced when used in combination with low doses of TMZ. Further refinement of the technology may facilitate localised activation of cytotoxicity against GBM when used in combination with new and existing chemotherapeutic regimens.