Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of ...this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.
With the development of stem cell engineering, extrcellular vesicles promise to deliver next generation tools in regenerative medicine and tissue engineering, as well as in diagnostics. A vibrant and ...promising field, this book provides the first resource to the field.
Food synergy concept is suggested to explain observations that isolated antioxidants are less bioactive than real foods containing them. However, mechanisms behind this discrepancy were hardly ...studied. Here, we demonstrate the profound impact of interactions between two common food flavonoids (individual: aglycones quercetin-Q and naringenin-N- or their glycosides rutin-R and naringin-N+ vs. mixed: QN- and RN+) on their electrochemical properties and redox-related bioactivities. N- and N+ seemed weak antioxidants individually, yet in both chemical and cellular tests (DPPH and CAA, respectively), they increased reducing activity of mixtures synergistically. In-depth measurements (differential pulse voltammetry) pointed to kinetics of oxidation reaction as decisive factor for antioxidant power. In cellular (HT29 cells) tests, the mixtures exhibited properties of a new substance rather than those of components. Pure flavonoids did not influence proliferation; mixtures stimulated cell growth. Individual flavonoids tended to decrease global DNA methylation with growing concentration; this effect was more pronounced for mixtures, but not concentration-dependent. In nutrigenomic studies, expression of gene set affected by QN- differed entirely from common genes modulated by individual components. These results question the current approach of predicting bioactivity of mixtures based on research with isolated antioxidants.
We investigated the influence of nanoparticles' shape on the physiological responses of cells, when they were fed with spherical and needle-shaped PLGA-PEG nanoparticles (the volume of the ...nanoparticles had been chosen as the fixed parameter). We found that both types of NPs entered cells via endocytosis and upon internalization they stayed in membrane bounded vesicles. Needle-shaped, but not the spherical-shaped NPs were found to induce significant cytotoxicity in the cell lines tested. Our study evidenced that the cytotoxicity of needle-shaped NPs was induced through the lysosome disruption. Lysosome damage activated the signaling pathways for cell apoptosis, and eventually caused DNA fragmentation and cell death. The present work showed that physiological response of the cells can be very different when the shape of the fed nanoparticles changed from spherical to needle-like. The finding suggests that the toxicity of nanomaterials also depends on their shape.
Nanomedicine seeks to apply nanoscale materials for the therapy and diagnosis of diseased and damaged tissues. Recent advances in nanotechnology have made a major contribution to the development of ...multifunctional nanomaterials, which represents a paradigm shift from single purpose to multipurpose materials. Multifunctional nanomaterials have been proposed to enable simultaneous target imaging and on-demand delivery of therapeutic agents only to the specific site. Most advanced systems are also responsive to internal or external stimuli. This approach is particularly important for highly potent drugs (e.g. chemotherapeutics), which should be delivered in a discreet manner and interact with cells/tissues only locally. Both advances in imaging and precisely controlled and localized delivery are critically important in cancer treatment, and the use of such systems - theranostics - holds great promise to minimise side effects and boost therapeutic effectiveness of the treatment. Among others, mesoporous silica nanoparticles (MSNPs) are considered one of the most promising nanomaterials for drug delivery. Due to their unique intrinsic features, including tunable porosity and size, large surface area, structural diversity, easily modifiable chemistry and suitability for functionalization, and biocompatibility, MSNPs have been extensively utilized as multifunctional nanocarrier systems. The combination or hybridization with biomolecules, drugs, and other nanoparticles potentiated the ability of MSNPs towards multifunctionality, and even smart actions stimulated by specified signals, including pH, optical signal, redox reaction, electricity and magnetism. This paper provides a comprehensive review of the state-of-the-art of multifunctional, smart drug delivery systems centered on advanced MSNPs, with special emphasis on cancer related applications.
A number of medicines are currently under investigation for the treatment of COVID-19 disease including anti-viral, anti-malarial, and anti-inflammatory agents. While these treatments can improve ...patient's recovery and survival, these therapeutic strategies do not lead to unequivocal restoration of the lung damage inflicted by this disease. Stem cell therapies and, more recently, their secreted extracellular vesicles (EVs), are emerging as new promising treatments, which could attenuate inflammation but also regenerate the lung damage caused by COVID-19. Stem cells exert their immunomodulatory, anti-oxidant, and reparative therapeutic effects likely through their EVs, and therefore, could be beneficial, alone or in combination with other therapeutic agents, in people with COVID-19. In this review article, we outline the mechanisms of cytokine storm and lung damage caused by SARS-CoV-2 virus leading to COVID-19 disease and how mesenchymal stem cells (MSCs) and their secreted EVs can be utilized to tackle this damage by harnessing their regenerative properties, which gives them potential enhanced clinical utility compared to other investigated pharmacological treatments. There are currently 17 clinical trials evaluating the therapeutic potential of MSCs for the treatment of COVID-19, the majority of which are administered intravenously with only one clinical trial testing MSC-derived exosomes via inhalation route. While we wait for the outcomes from these trials to be reported, here we emphasize opportunities and risks associated with these therapies, as well as delineate the major roadblocks to progressing these promising curative therapies toward mainstream treatment for COVID-19.
While ultrasound is most widely known for its use in diagnostic imaging, the energy carried by ultrasound waves can be utilized to influence cell function and drug delivery. Consequently, our ability ...to use ultrasound energy at a given intensity unlocks the opportunity to use the ultrasound for therapeutic applications. Indeed, in the last decade ultrasound-based therapies have emerged with promising treatment modalities for several medical conditions. More recently, ultrasound in combination with nanomedicines, i.e., nanoparticles, has been shown to have substantial potential to enhance the efficacy of many treatments including cancer, Alzheimer disease or osteoarthritis. The concept of ultrasound combined with drug delivery is still in its infancy and more research is needed to unfold the mechanisms and interactions of ultrasound with different nanoparticles types and with various cell types. Here we present the state-of-art in ultrasound and ultrasound-assisted drug delivery with a particular focus on cancer treatments. Notably, this review discusses the application of high intensity focus ultrasound for non-invasive tumor ablation and immunomodulatory effects of ultrasound, as well as the efficacy of nanoparticle-enhanced ultrasound therapies for different medical conditions. Furthermore, this review presents safety considerations related to ultrasound technology and gives recommendations in the context of system design and operation.
By means of the in situ electrokinetic assessment of aqueous particles in conjunction with the addition of anionic adsorbates, we develop and examine a new approach to the scalable characterization ...of the specific accessible surface area of particles in water. For alumina powders of differing morphology in mildly acidic aqueous suspensions, the effective surface charge was modified by carboxylate anion adsorption through the incremental addition of oxalic and citric acids. The observed zeta potential variation as a function of the proportional reagent additive was found to exhibit inverse hyperbolic sine-type behavior predicted to arise from monolayer adsorption following the Grahame–Langmuir model. Through parameter optimization by inverse problem solving, the zeta potential shift with relative adsorbate addition revealed a nearly linear correlation of a defined surface-area-dependent parameter with the conventionally measured surface area values of the powders, demonstrating that the proposed analytical framework is applicable for the in situ surface area characterization of aqueous particulate matter. The investigated methods have advantages over some conventional surface analysis techniques owing to their direct applicability in aqueous environments at ambient temperature and the ability to modify analysis scales by variation of the adsorption cross section.
New nanocomposite membranes with high bioactivity were fabricated using the electrospinning. These nanocomposites combine a degradable polymer poly(
l
/
dl
)-lactide and bone cell signaling carbonate ...nano-hydroxyapatite (n-HAp). Chemical and physical characterization of the membranes using scanning electron microscopy, Fourier transform infrared spectroscopy and the wide angle X-ray diffraction evidenced that nanoparticles were successfully incorporated into the fibers and membrane structure. The incorporation of the n-HAp into the structure increased significantly the mineralization of the membrane in vitro. It has been demonstrated that after a 3-day incubation of composite membrane in the Simulated Body Fluid a continuous compact apatite layer was formed. In vitro experiments demonstrated that the incorporation of n-HAp significantly improved cell attachment, upregulated cells proliferation and stimulated cell differentiation quantified using Alkaline Phosphatase and OsteoImage tests. In conclusion, the results demonstrated that the addition of n-HAp provided chemical cues that were a key factor that regulated osteoblastic differentiation.
Redox homeostasis involves factors that ensure proper function of cells. The excess reactive oxygen species (ROS) leads to oxidative stress and increased risk of oxidative damage to cellular ...components. In contrast, upon reductive stress, insufficient ROS abundance may result in faulty cell signalling. It may be expected that dietary antioxidants, depending on their standard reduction potentials (E°), will affect both scenarios. In our study, for the first time, we systematically tested the relationship among E°, chemical properties, and biological effects in HT29 cells for a series of structurally different catechins and a major endogenous antioxidant – glutathione (GSH), at both physiological and dietary concentrations. Among chemical antioxidant activity tests, the strongest correlation with E° was seen using a DPPH assay. The values of E° were also highly correlated with cellular antioxidant activity (CAA) values determined in HT29 cells. Our results indicated that physiological concentrations (1–10 µM) of tested catechins stabilized the redox status of cells, which was not exhibited at higher concentrations. This stabilization of redox homeostasis was mirrored by constant, dose and E° independent CAA values, uninhibited growth of HT29 cells, modulation of hydrogen peroxide-induced DNA damage, as well as effects at the genomic level, where either up-regulation of three redox-related genes (ALB, CCL5, and HSPA1A) out of 84 in the array (1 µM) or no effect (10 µM) was observed for catechins. Higher catechin concentrations (over 10 µM) increased CAA values in a dose- and E°-dependent manner, caused cell growth inhibition, but surprisingly did not protect HT29 cells against reactive oxygen species (ROS)-induced DNA fragmentation. In conclusion, dose-dependent effects of dietary antioxidants and biological functions potentially modulated by them may become deregulated upon exposure to excessive doses.
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•Standard reduction potential (E°) is a good predictor of antioxidant activity of catechins.•Physiological doses of catechins maintain cellular redox status of cells.•Redox control by catechins includes regulation of expression of redox-related genes.•Excessive doses of catechins deregulate redox homeostasis in a dose- and E°-dependent manner.
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