Light-induced modulation of the mitochondrial respiratory chain activity: possibilities and limitations.

Biological effects of high fluence low-power (HFLP) lasers have been reported for some time, yet the molecular mechanisms procuring cellular responses remain obscure. A better understanding of the effects of HFLP lasers on living cells will be instrumental for the development of new experimental and therapeutic strategies. Therefore, we investigated sub-cellular mechanisms involved in the laser interaction with human hepatic cell lines. We show that mitochondria serve as sub-cellular "sensor" and "effector" of laser light non-specific interactions with cells. We demonstrated that despite blue and red laser irradiation results in similar apoptotic death, cellular signaling and kinetic of biochemical responses are distinct. Based on our data, we concluded that blue laser irradiation inhibited cytochrome c oxidase activity in electron transport chain of mitochondria. Contrary, red laser triggered cytochrome c oxidase excessive activation. Moreover, we showed that Bcl-2 protein inhibited laser-induced toxicity by stabilizing mitochondria membrane potential. Thus, cells that either overexpress or have elevated levels of Bcl-2 are protected from laser-induced cytotoxicity. Our findings reveal the mechanism how HFLP laser irradiation interfere with cell homeostasis and underscore that such laser irradiation permits remote control of mitochondrial function in the absence of chemical or biological agents.

Expression of Interferons Lambda 3 and 4 Induces Identical Response in Human Liver Cell Lines Depending Exclusively on Canonical Signaling.

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.

Critical Analysis of Non-Thermal Plasma-Driven Modulation of Immune Cells from Clinical Perspective.

The emerged field of non-thermal plasma (NTP) shows great potential in the alteration of cell redox status, which can be utilized as a promising therapeutic implication. In recent years, the NTP field considerably progresses in the modulation of immune cell function leading to promising in vivo results. In fact, understanding the underlying cellular mechanisms triggered by NTP remains incomplete. In order to boost the field closer to real-life clinical applications, there is a need for a critical overview of the current state-of-the-art. In this review, we conduct a critical analysis of the NTP-triggered modulation of immune cells. Importantly, we analyze pitfalls in the field and identify persisting challenges. We show that the identification of misconceptions opens a door to the development of a research strategy to overcome these limitations. Finally, we propose the idea that solving problems highlighted in this review will accelerate the clinical translation of NTP-based treatments.

Non-Thermal Plasma, as a New Physicochemical Source, to Induce Redox Imbalance and Subsequent Cell Death in Liver Cancer Cell Lines.

BACKGROUND/AIMS: Alteration of cancer cell redox status has been recognized as a promising therapeutic implication. In recent years, the emerged field of non-thermal plasma (NTP) has shown considerable promise in various biomedical applications, including cancer therapy. However, understanding the molecular mechanisms procuring cellular responses remains incomplete. Thus, the aim of this study was a rigorous biochemical analysis of interactions between NTP and liver cancer cells. METHODS: The concept was validated using three different cell lines. We provide several distinct lines of evidence to support our findings; we use various methods (epifluorescent and confocal microscopy, clonogenic and cytotoxicity assays, Western blotting, pharmacological inhibition studies, etc.). RESULTS: We assessed the influence of NTP on three human liver cancer cell lines (Huh7, Alexander and HepG2). NTP treatment resulted in higher anti-proliferative effect against Alexander and Huh7 relative to HepG2. Our data clearly showed that the NTP-mediated alternation of mitochondrial membrane potential and dynamics led to ROS-mediated apoptosis in Huh7 and Alexander cells. Interestingly, plasma treatment resulted in p53 down-regulation in Huh7 cells. High levels of Bcl-2 protein expression in HepG2 resulted in their resistance in response to oxidative stress- mediated by plasma. CONCLUSION: We show thoroughly time- and dose-dependent kinetics of ROS accumulation in HCC cells. Furthermore, we show nuclear compartmentalization of the superoxide anion triggered by NTP. NTP induced apoptotic death in Huh7 liver cancer cells via simultaneous downregulation of mutated p53, pSTAT1 and STAT1. Contrary, hydrogen peroxide treatment results in autophagic cell death. We disclosed detailed mechanisms of NTP-mediated alteration of redox signalling in liver cancer cells.

Iron oxide nanoparticles trigger endoplasmic reticulum damage in steatotic hepatic cells.

Iron oxide nanoparticles (IONPs) are being actively researched in various biomedical applications, particularly as magnetic resonance imaging (MRI) contrast agents for diagnosing various liver pathologies like nonalcoholic fatty liver diseases, nonalcoholic steatohepatitis, and cirrhosis. Emerging evidence suggests that IONPs may exacerbate hepatic steatosis and liver injury in susceptible livers such as those with nonalcoholic fatty liver disease. However, our understanding of how IONPs may affect steatotic cells at the sub-cellular level is still fragmented. Generally, there is a lack of studies identifying the molecular mechanisms of potential toxic and/or adverse effects of IONPs on "non-heathy" in vitro models. In this study, we demonstrate that IONPs, at a dose that does not cause general toxicity in hepatic cells (Alexander and HepG2), induce significant toxicity in steatotic cells (cells loaded with non-toxic doses of palmitic acid). Mechanistically, co-treatment with PA and IONPs resulted in endoplasmic reticulum (ER) stress, accompanied by the release of cathepsin B from lysosomes to the cytosol. The release of cathepsin B, along with ER stress, led to the activation of apoptotic cell death. Our results suggest that it is necessary to consider the interaction between IONPs and the liver, especially in susceptible livers. This study provides important basic knowledge for the future optimization of IONPs as MRI contrast agents for various biomedical applications.

Mechanical Regulation of Mitochondrial Dynamics and Function in a 3D-Engineered Liver Tumor Microenvironment.

It has become evident that physical stimuli of the cellular microenvironment transmit mechanical cues regulating key cellular functions, such as proliferation, migration, and malignant transformation. Accumulating evidence suggests that tumor cells face variable mechanical stimuli that may induce metabolic rewiring of tumor cells. However, the knowledge of how tumor cells adapt metabolism to external mechanical cues is still limited. We therefore designed soft 3D collagen scaffolds mimicking a pathological mechanical environment to decipher how liver tumor cells would adapt their metabolic activity to physical stimuli of the cellular microenvironment. Here, we report that the soft 3D microenvironment upregulates the glycolysis of HepG2 and Alexander cells. Both cell lines adapt their mitochondrial activity and function under growth in the soft 3D microenvironment. Cells grown in the soft 3D microenvironment exhibit marked mitochondrial depolarization, downregulation of mitochondrially encoded cytochrome c oxidase I, and slow proliferation rate in comparison with stiff monolayer cultures. Our data reveal the coupling of liver tumor glycolysis to mechanical cues. It is proposed here that soft 3D collagen scaffolds can serve as a useful model for future studies of mechanically regulated cellular functions of various liver (potentially other tissues as well) tumor cells.

Lysosomal nanotoxicity: Impact of nanomedicines on lysosomal function.

Although several nanomedicines got clinical approval over the past two decades, the clinical translation rate is relatively small so far. There are many post-surveillance withdrawals of nanomedicines caused by various safety issues. For successful clinical advancement of nanotechnology, it is of unmet need to realize cellular and molecular foundation of nanotoxicity. Current data suggest that lysosomal dysfunction caused by nanoparticles is emerging as the most common intracellular trigger of nanotoxicity. This review analyzes prospect mechanisms of lysosomal dysfunction-mediated toxicity induced by nanoparticles. We summarized and critically assessed adverse drug reactions of current clinically approved nanomedicines. Importantly, we show that physicochemical properties have great impact on nanoparticles interaction with cells, excretion route and kinetics, and subsequently on toxicity. We analyzed literature on adverse reactions of current nanomedicines and hypothesized that adverse reactions might be linked with lysosomal dysfunction caused by nanomedicines. Finally, from our analysis it becomes clear that it is unjustifiable to generalize safety and toxicity of nanoparticles, since different particles possess distinct toxicological properties. We propose that the biological mechanism of the disease progression and treatment should be central in the optimization of nanoparticle design.

Sensitivity of endogenous autofluorescence in HeLa cells to the application of external magnetic fields.

Dramatically increased levels of electromagnetic radiation in the environment have raised concerns over the potential health hazards of electromagnetic fields. Various biological effects of magnetic fields have been proposed. Despite decades of intensive research, the molecular mechanisms procuring cellular responses remain largely unknown. The current literature is conflicting with regards to evidence that magnetic fields affect functionality directly at the cellular level. Therefore, a search for potential direct cellular effects of magnetic fields represents a cornerstone that may propose an explanation for potential health hazards associated with magnetic fields. It has been proposed that autofluorescence of HeLa cells is magnetic field sensitive, relying on single-cell imaging kinetic measurements. Here, we investigate the magnetic field sensitivity of an endogenous autofluorescence in HeLa cells. Under the experimental conditions used, magnetic field sensitivity of an endogenous autofluorescence was not observed in HeLa cells. We present a number of arguments indicating why this is the case in the analysis of magnetic field effects based on the imaging of cellular autofluorescence decay. Our work indicates that new methods are required to elucidate the effects of magnetic fields at the cellular level.

The interactions between DNA nanostructures and cells: A critical overview from a cell biology perspective.

DNA nanotechnology has yielded remarkable advances in  composite materials with diverse applications in biomedicine. The specificity and predictability of building 3D structures at the nanometer scale make DNA nanotechnology a promising tool for uses in biosensing, drug delivery, cell modulation, and bioimaging. However, for successful translation of DNA nanostructures to real-world applications, it is crucial to understand how they interact with living cells, and the consequences of such interactions. In this review, we summarize the current state of knowledge on the interactions of DNA nanostructures with cells. We identify key challenges, from a cell biology perspective, that influence progress towards the clinical translation of DNA nanostructures. We close by providing an outlook on what questions must be addressed to accelerate the clinical translation of DNA nanostructures. STATEMENT OF SIGNIFICANCE: Self-assembled DNA nanostructures (DNs) offers unique opportunities to overcome persistent challenges in the nanobiotechnology field. However, the interactions between engineered DNs and living cells are still not well defined. Critical systematization of current cellular models and biological responses triggered by DNs is a crucial foundation for the successful clinical translation of DNA nanostructures. Moreover, such an analysis will identify the pitfalls and challenges that are present in the field, and provide a basis for overcoming those challenges.

Protein Corona Inhibits Endosomal Escape of Functionalized DNA Nanostructures in Living Cells.

DNA nanostructures (DNs) can be designed in a controlled and programmable manner, and these structures are increasingly used in a variety of biomedical applications, such as the delivery of therapeutic agents. When exposed to biological liquids, most nanomaterials become covered by a protein corona, which in turn modulates their cellular uptake and the biological response they elicit. However, the interplay between living cells and designed DNs are still not well established. Namely, there are very limited studies that assess protein corona impact on DN biological activity. Here, we analyzed the uptake of functionalized DNs in three distinct hepatic cell lines. Our analysis indicates that cellular uptake is linearly dependent on the cell size. Further, we show that the protein corona determines the endolysosomal vesicle escape efficiency of DNs coated with an endosome escape peptide. Our study offers an important basis for future optimization of DNs as delivery systems for various biomedical applications.

Analyzing the mechanisms of iron oxide nanoparticles interactions with cells: A road from failure to success in clinical applications.

Iron oxide nanoparticles (IONPs) were the first generation of nanomaterials that reached real clinic use. Particularly, several IONPs-based magnetic resonance imaging contrast agents gained approval by US Food and Drug Administration (FDA). However, latter body of evidence revealed the overlooked side effects of IONPs, resulting in their withdrawal. Emerging evidence suggests that this happened due to poor understanding of the mechanisms by which IONPs act at the cellular and sub-cellular levels. Recent studies indicate that better understanding of fundamental signal modulations induced by nanomaterials is essential to overcome the clinical problems with nanoparticles. Therefore, in this article we critically review potential mechanisms of IONPs-cell interactions and challenges related with their identification. We describe mechanisms of IONPs-induced toxicity. Ultimately, we demonstrate that knowledge of cellular mechanisms of IONPs action helped to overcome certain translation problems in nanomedicine - we explore potential causes and challenges associated with poor clinical performance of IONPs and propose outlook of how to overcome problems in the field. Our critical analysis implies that a clear understanding of molecular mechanisms of IONPs-cell interactions will provide a basement to increase the likelihood for clinical success of IONPs.

Iron Oxide Nanoparticle-Induced Autophagic Flux Is Regulated by Interplay between p53-mTOR Axis and Bcl-2 Signaling in Hepatic Cells.

Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies.

Functionalizable Antifouling Coatings as Tunable Platforms for the Stress-Driven Manipulation of Living Cell Machinery.

Cells are continuously sensing their microenvironment and subsequently respond to different physicochemical cues by the activation or inhibition of different signaling pathways. To study a very complex cellular response, it is necessary to diminish background environmental influences and highlight the particular event. However, surface-driven nonspecific interactions of the abundant biomolecules from the environment influence the targeted cell response significantly. Yes-associated protein (YAP) translocation may serve as a marker of human hepatocellular carcinoma (Huh7) cell responses to the extracellular matrix and surface-mediated stresses. Here, we propose a platform of tunable functionable antifouling poly(carboxybetain) (pCB)-based brushes to achieve a molecularly clean background for studying arginine, glycine, and aspartic acid (RGD)-induced YAP-connected mechanotransduction. Using two different sets of RGD-functionalized zwitterionic antifouling coatings with varying compositions of the antifouling layer, a clear correlation of YAP distribution with RGD functionalization concentrations was observed. On the other hand, commonly used surface passivation by the oligo(ethylene glycol)-based self-assembled monolayer (SAM) shows no potential to induce dependency of the YAP distribution on RGD concentrations. The results indicate that the antifouling background is a crucial component of surface-based cellular response studies, and pCB-based zwitterionic antifouling brush architectures may serve as a potential next-generation easily functionable surface platform for the monitoring and quantification of cellular processes.

Hepatic Tumor Cell Morphology Plasticity under Physical Constraints in 3D Cultures Driven by YAP-mTOR Axis.

Recent studies undoubtedly show that the mammalian target of rapamycin (mTOR) and the Hippo-Yes-associated protein 1 (YAP) pathways are important mediators of mechanical cues. The crosstalk between these pathways as well as de-regulation of their signaling has been implicated in multiple tumor types, including liver tumors. Additionally, physical cues from 3D microenvironments have been identified to alter gene expression and differentiation of different cell lineages. However, it remains incompletely understood how physical constraints originated in 3D cultures affect cell plasticity and what the key mediators are of such process. In this work, we use collagen scaffolds as a model of a soft 3D microenvironment to alter cellular size and study the mechanotransduction that regulates that process. We show that the YAP-mTOR axis is a downstream effector of 3D cellular culture-driven mechanotransduction. Indeed, we found that cell mechanics, dictated by the physical constraints of 3D collagen scaffolds, profoundly affect cellular proliferation in a YAP-mTOR-mediated manner. Functionally, the YAP-mTOR connection is key to mediate cell plasticity in hepatic tumor cell lines. These findings expand the role of YAP-mTOR-driven mechanotransduction to the control hepatic tumor cellular responses under physical constraints in 3D cultures. We suggest a tentative mechanism, which coordinates signaling rewiring with cytoplasmic restructuring during cell growth in 3D microenvironments.

Progressive lysosomal membrane permeabilization induced by iron oxide nanoparticles drives hepatic cell autophagy and apoptosis.

Iron oxide nanoparticles (IONs) are frequently used in various biomedical applications, in particular as magnetic resonance imaging contrast agents in liver imaging. Indeed, number of IONs have been withdrawn due to their poor clinical performance. Yet comprehensive understanding of their interactions with hepatocytes remains relatively limited. Here we investigated how iron oxide nanocubes (IO-cubes) and clusters of nanocubes (IO-clusters) affect distinct human hepatic cell lines. The viability of HepG2, Huh7 and Alexander cells was concentration-dependently decreased after exposure to either IO-cubes or IO-clusters. We found similar cytotoxicity levels in three cell lines triggered by both nanoparticle formulations. Our data indicate that different expression levels of Bcl-2 predispose cell death signaling mediated by nanoparticles. Both nanoparticles induced rather apoptosis than autophagy in HepG2. Contrary, IO-cubes and IO-clusters trigger distinct cell death signaling events in Alexander and Huh7 cells. Our data clarifies the mechanism by which cubic nanoparticles induce autophagic flux and the mechanism of subsequent toxicity. These findings imply that the cytotoxicity of ION-based contrast agents should be carefully considered, particularly in patients with liver diseases.

Modulation of Living Cell Behavior with Ultra-Low Fouling Polymer Brush Interfaces.

Ultra-low fouling and functionalizable coatings represent emerging surface platforms for various analytical and biomedical applications such as those involving examination of cellular interactions in their native environments. Ultra-low fouling surface platforms as advanced interfaces enabling modulation of behavior of living cells via tuning surface physicochemical properties are presented and studied. The state-of-art ultra-low fouling surface-grafted polymer brushes of zwitterionic poly(carboxybetaine acrylamide), nonionic poly(N-(2-hydroxypropyl)methacrylamide), and random copolymers of carboxybetaine methacrylamide (CBMAA) and HPMAA [p(CBMAA-co-HPMAA)] with tunable molar contents of CBMAA and HPMAA are employed. Using a model Huh7 cell line, a systematic study of surface wettability, swelling, and charge effects on the cell growth, shape, and cytoskeleton distribution is performed. This study reveals that ultra-low fouling interfaces with a high content of zwitterionic moieties (>65 mol%) modulate cell behavior in a distinctly different way compared to coatings with a high content of nonionic HPMAA. These differences are attributed mostly to the surface hydration capabilities. The results demonstrate a high potential of carboxybetaine-rich ultra-low fouling surfaces with high hydration capabilities and minimum background signal interferences to create next-generation bioresponsive interfaces for advanced studies of living objects.

Preliminary Study of Ge-DLC Nanocomposite Biomaterials Prepared by Laser Codeposition.

This paper deals with the synthesis and study of the properties of germanium-doped diamond-like carbon (DLC) films. For deposition of doped DLC films, hybrid laser technology was used. Using two deposition lasers, it was possible to arrange the dopant concentrations by varying the laser repetition rate. Doped films of Ge concentrations from 0 at.% to 12 at.% were prepared on Si (100) and fused silica (FS) substrates at room temperature. Film properties, such as growth rate, roughness, scanning electron microscopy (SEM) morphology, wavelength dependent X-ray spectroscopy (WDS) composition, VIS-near infrared (IR) transmittance, and biological properties (cytotoxicity, effects on cellular morphology, and ability to produce reactive oxygen species (ROS)) were studied in relation to codeposition conditions and dopant concentrations. The analysis showed that Ge-DLC films exhibit cytotoxicity for higher Ge doping.

Targeting the mTOR Signaling Pathway Utilizing Nanoparticles: A Critical Overview.

Proteins of the mammalian target of rapamycin (mTOR) signaling axis are overexpressed or mutated in cancers. However, clinical inhibition of mTOR signaling as a therapeutic strategy in oncology shows rather limited progress. Nanoparticle-based mTOR targeted therapy proposes an attractive therapeutic option for various types of cancers. Along with the progress in the biomedical applications of nanoparticles, we start to realize the challenges and opportunities that lie ahead. Here, we critically analyze the current literature on the modulation of mTOR activity by nanoparticles, demonstrate the complexity of cellular responses to functionalized nanoparticles, and underline challenges lying in the identification of the molecular mechanisms of mTOR signaling affected by nanoparticles. We propose the idea that subcytotoxic doses of nanoparticles could be relevant for the induction of subcellular structural changes with possible involvement of mTORC1 signaling. The evaluation of the mechanisms and therapeutic effects of nanoparticle-based mTOR modulation will provide fundamental knowledge which could help in developing safe and efficient nano-therapeutics.

Remote Actuation of Apoptosis in Liver Cancer Cells via Magneto-Mechanical Modulation of Iron Oxide Nanoparticles.

Lysosome-activated apoptosis represents an alternative method of overcoming tumor resistance compared to traditional forms of treatment. Pulsed magnetic fields open a new avenue for controlled and targeted initiation of lysosomal permeabilization in cancer cells via mechanical actuation of magnetic nanomaterials. In this study we used a noninvasive tool; namely, a benchtop pulsed magnetic system, which enabled remote activation of apoptosis in liver cancer cells. The magnetic system we designed represents a platform that can be used in a wide range of biomedical applications. We show that liver cancer cells can be loaded with superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs retained in lysosomal compartments can be effectively actuated with a high intensity (up to 8 T), short pulse width (~15 µs), pulsed magnetic field (PMF), resulting in lysosomal membrane permeabilization (LMP) in cancer cells. We revealed that SPION-loaded lysosomes undergo LMP by assessing an increase in the cytosolic activity of the lysosomal cathepsin B. The extent of cell death induced by LMP correlated with the accumulation of reactive oxygen species in cells. LMP was achieved for estimated forces of 700 pN and higher. Furthermore, we validated our approach on a three-dimensional cellular culture model to be able to mimic in vivo conditions. Overall, our results show that PMF treatment of SPION-loaded lysosomes can be utilized as a noninvasive tool to remotely induce apoptosis.

A Critical Review on Selected External Physical Cues and Modulation of Cell Behavior: Magnetic Nanoparticles, Non-thermal Plasma and Lasers.

Physics-based biomedical approaches have proved their importance for the advancement of medical sciences and especially in medical diagnostics and treatments. Thus, the expectations regarding development of novel promising physics-based technologies and tools are very high. This review describes the latest research advances in biomedical applications of external physical cues. We overview three distinct topics: using high-gradient magnetic fields in nanoparticle-mediated cell responses; non-thermal plasma as a novel bactericidal agent; highlights in understanding of cellular mechanisms of laser irradiation. Furthermore, we summarize the progress, challenges and opportunities in those directions. We also discuss some of the fundamental physical principles involved in the application of each cue. Considerable technological success has been achieved in those fields. However, for the successful clinical translation we have to understand the limitations of technologies. Importantly, we identify the misconceptions pervasive in the discussed fields.