Single-particle tracking (SPT) is an experimental technique that allows one to follow the dynamics of individual molecules in biological membranes with unprecedented precision. Given the importance of lipid and membrane protein diffusion in the formation of nanoscale functional complexes, it is critical to understand what exactly is measured in SPT experiments. To clarify this issue, we employed nanoscale computer simulations designed to match SPT experiments that exploit streptavidin-functionalized Au nanoparticles (AuNPs). The results show that lipid labeling interferes critically with the diffusion process; thus, the diffusion measured in SPT is a far more complex process than what has been assumed. It turns out that the influence of AuNP-based labels on the dynamics of probe lipids includes not only the AuNP-induced viscous drag that is the more significant the larger the NP but, more importantly, also the effects related to the interactions of the streptavidin linker with membrane lipids. Due to these effects, the probe lipid moves in a concerted manner as a complex with the linker protein and numerous unlabeled lipids, which can slow down the motion of the probe by almost an order of magnitude. Furthermore, our simulations show that nonlinker streptavidin tetramers on the AuNP surface are able to interact with the membrane lipids, which could potentially lead to multivalent labeling of the NPs by the probe lipids. Our results further demonstrate that in the submicrosecond time domain the motion of the probe lipid is uncorrelated with the motion of the AuNP, showing that there is a 1 μs limit for the temporal resolution of the SPT technique. However, this limit for the temporal resolution depends on the nanoparticle size and increases rapidly with growing AuNPs. Overall, the results provide a molecular-scale framework to accurately interpret SPT data and to design protocols that minimize label-induced artifacts.

• MeSH
• difuze * MeSH
• kovové nanočástice chemie   MeSH
• membránové lipidy chemie   MeSH
• simulace molekulární dynamiky MeSH
• zlato chemie   MeSH
• zobrazení jednotlivé molekuly * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové lipidy MeSH
• zlato MeSH

Magnetic resonance imaging (MRI) of superparamagnetic iron oxide-labeled cells can be used as a non-invasive technique to track stem cells after transplantation. The aim of this study was to (1) evaluate labeling efficiency of D-mannose-coated maghemite nanoparticles (D-mannose(γ-Fe2O3)) in neural stem cells (NSCs) in comparison to the uncoated nanoparticles, (2) assess nanoparticle utilization as MRI contrast agent to visualize NSCs transplanted into the mouse brain, and (3) test nanoparticle biocompatibility. D-mannose(γ-Fe2O3) labeled the NSCs better than the uncoated nanoparticles. The labeled cells were visualized by ex vivo MRI and their localization subsequently confirmed on histological sections. Although the progenitor properties and differentiation of the NSCs were not affected by labeling, subtle effects on stem cells could be detected depending on dose increase, including changes in cell proliferation, viability, and neurosphere diameter. D-mannose coating of maghemite nanoparticles improved NSC labeling and allowed for NSC tracking by ex vivo MRI in the mouse brain, but further analysis of the eventual side effects might be necessary before translation to the clinic.

• Klíčová slova
• brain, maghemite, magnetic resonance imaging, mouse, nanoparticles, neural stem cells,
• MeSH
• buněčný tracking metody   MeSH
• magnetická rezonanční tomografie metody   MeSH
• magnetické nanočástice chemie   MeSH
• mannosa chemie   MeSH
• mozek cytologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nervové kmenové buňky cytologie   transplantace   MeSH
• železité sloučeniny chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferric oxide MeSH Prohlížeč
• magnetické nanočástice MeSH
• mannosa MeSH
• železité sloučeniny MeSH

OBJECTIVE: Cell therapies have emerged as a promising approach in medicine. The basis of each therapy is the injection of 1-100×10(6) cells with regenerative potential into some part of the body. Mesenchymal stromal cells (MSCs) are the most used cell type in the cell therapy nowadays, but no gold standard for the labeling of the MSCs for magnetic resonance imaging (MRI) is available yet. This work evaluates our newly synthesized uncoated superparamagnetic maghemite nanoparticles (surface-active maghemite nanoparticles - SAMNs) as an MRI contrast intracellular probe usable in a clinical 1.5 T MRI system. METHODS: MSCs from rat and human donors were isolated, and then incubated at different concentrations (10-200 μg/mL) of SAMN maghemite nanoparticles for 48 hours. Viability, proliferation, and nanoparticle uptake efficiency were tested (using fluorescence microscopy, xCELLigence analysis, atomic absorption spectroscopy, and advanced microscopy techniques). Migration capacity, cluster of differentiation markers, effect of nanoparticles on long-term viability, contrast properties in MRI, and cocultivation of labeled cells with myocytes were also studied. RESULTS: SAMNs do not affect MSC viability if the concentration does not exceed 100 μg ferumoxide/mL, and this concentration does not alter their cell phenotype and long-term proliferation profile. After 48 hours of incubation, MSCs labeled with SAMNs show more than double the amount of iron per cell compared to Resovist-labeled cells, which correlates well with the better contrast properties of the SAMN cell sample in T2-weighted MRI. SAMN-labeled MSCs display strong adherence and excellent elasticity in a beating myocyte culture for a minimum of 7 days. CONCLUSION: Detailed in vitro tests and phantom tests on ex vivo tissue show that the new SAMNs are efficient MRI contrast agent probes with exclusive intracellular uptake and high biological safety.

• Klíčová slova
• magnetic resonance imaging, mesenchymal stromal cells, stem cell labeling, stem cell tracking, superparamagnetic iron oxide nanoparticles,
• MeSH
• buněčný tracking metody   MeSH
• dextrany chemie   farmakokinetika   toxicita   MeSH
• fyziologie buňky účinky léků   MeSH
• kontrastní látky chemie   farmakokinetika   toxicita   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• magnetická rezonanční tomografie MeSH
• magnetické nanočástice chemie   toxicita   MeSH
• mezenchymální kmenové buňky chemie   cytologie   účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• dextrany MeSH
• ferumoxides MeSH Prohlížeč
• kontrastní látky MeSH
• magnetické nanočástice MeSH

Coprecipitation of FeCl2 and FeCl3 with aqueous ammonia was used to prepare iron oxide nanoparticles dispersible in aqueous medium. Oxidation of the particles with sodium hypochlorite then yielded maghemite (γ-Fe2 O3 ) nanoparticles which were coated with two types of coating -d-mannose or poly(l-lysine) (PLL) as confirmed by FTIR analysis. The particles were <10 nm according to transmission electron microscopy. Their hydrodynamic particle size was ∼180 nm (by dynamic light scattering). The d-mannose-, PLL-coated, and neat γ-Fe2 O3 particles as well as commercial Resovist® were used to label rat macrophages. The viability and contrast properties of labeled macrophages were compared. PLL-coated γ-Fe2 O3 nanoparticles were found optimal. The labeled macrophages were injected to rats monitored in vivo by magnetic resonance imaging up to 48 h. Transport of macrophages labeled with PLL-γ-Fe2 O3 nanoparticles in rats was confirmed. Tracking of macrophages using the developed particles can be used for monitoring of inflammations and cell migration in cell therapy.

• Klíčová slova
• MRI, iron oxide, labeling, macrophages, nanoparaticles,
• MeSH
• buněčný tracking metody   MeSH
• kontrastní látky * chemie   farmakologie   MeSH
• krysa rodu Rattus MeSH
• magnetická rezonanční tomografie metody   MeSH
• makrofágy diagnostické zobrazování   MeSH
• nanočástice chemie   MeSH
• polylysin * chemie   farmakologie   MeSH
• radiografie MeSH
• velikost částic MeSH
• železité sloučeniny * chemie   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferric oxide MeSH Prohlížeč
• kontrastní látky * MeSH
• polylysin * MeSH
• železité sloučeniny * MeSH

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

• Klíčová slova
• exosomes, microscopy techniques, nanoparticle tracking analysis, prostate cancer, self-assembled monolayer, surface plasmon resonance,
• MeSH
• exozómy * chemie   MeSH
• karcinom * metabolismus   patologie   MeSH
• lektiny analýza   metabolismus   MeSH
• lidé MeSH
• polysacharidy analýza   metabolismus   MeSH
• tekutá biopsie MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lektiny MeSH
• polysacharidy MeSH

An important issue in the context of both potenial toxicity of iron oxide nanoparticles (IONP) and their medical applications is tracking of the internalization process of these nanomaterials into living cells, as well as their localization and fate within them. The typical methods used for this purpose are transmission electron microscopy, confocal fluorescence microscopy as well as light-scattering techniques including dark-field microscopy and flow cytometry. All the techniques mentioned have their advantages and disadvantages. Among the problems it is necessary to mention complicated sample preparation, difficult interpretation of experimental data requiring qualified and experienced personnel, different behavior of fluorescently labeled IONP comparing to those label-free or finally the lack of possibility of chemical composition characteristics of nanomaterials. The purpose of the present investigation was the assessment of the usefulness of Raman microscopy for the tracking of the internalization of IONP into cells, as well as the optimization of this process. Moreover, the study focused on identification of the potential differences in the cellular fate of superparamagnetic nanoparticles having magnetite and maghemite core. The Raman spectra of U87MG cells which internalized IONP presented additional bands which position depended on the used laser wavelength. They occurred at the wavenumber range 1700-2400 cm-1 for laser 488 nm and below the wavenumber of 800 cm-1 in case of laser 532 nm. The intensity of the mentioned Raman bands was higher for the green laser (532 nm) and their position, was independent and not characteristic on the primary core material of IONP (magnetite, maghemite). The obtained results showed that Raman microscopy is an excellent, non-destructive and objective technique that allows monitoring the process of internalization of IONP into cells and visualizing such nanoparticles and/or their metabolism products within them at low exposure levels. What is more, the process of tracking IONP using the technique may be further improved by using appropriate wavelength and power of the laser source.

• Klíčová slova
• Internalization into cells, Iron oxide nanoparticles, Magnetite and maghemite core, Multivariate methods, Raman spectroscopy and imaging,
• MeSH
• lidé MeSH
• magnetické nanočástice oxidů železa * chemie   MeSH
• mikroskopie metody   MeSH
• nádorové buněčné linie MeSH
• Ramanova spektroskopie * metody   MeSH
• železité sloučeniny chemie   analýza   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• železité sloučeniny MeSH

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

• Klíčová slova
• Arabidopsis thaliana, EVs, NTA, PTI, Pto DC3000, extracellular vesicles, nanoparticle tracking analysis, pattern-triggered immunity, proteomics,
• MeSH
• bakteriální proteiny metabolismus   MeSH
• extracelulární vezikuly * metabolismus   MeSH
• flagelin metabolismus   MeSH
• lidé MeSH
• nemoci rostlin mikrobiologie   MeSH
• proteomika MeSH
• Pseudomonas syringae genetika   metabolismus   MeSH
• Solanum lycopersicum * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• flagelin MeSH

Single-molecule (digital) immunoassays provide the ability to detect much lower protein concentrations than conventional immunoassays. As photon-upconversion nanoparticles (UCNPs) can be detected without optical background interference, they are excellent labels for so-called single-molecule upconversion-linked immunosorbent assays (ULISAs). We have introduced a UCNP label design based on streptavidin-PEG-neridronate and a two-step detection scheme involving a biotinylated antibody that efficiently reduces nonspecific binding on microtiter plates. In a microtiter plate immunoassay, individual sandwich immune complexes of the cancer marker prostate-specific antigen (PSA) are detected and counted by wide-field epiluminescence microscopy (digital readout). The digital detection is 16× more sensitive than the respective analogue readout and thus expands the limit of detection to the sub-femtomolar concentration range (LOD: 23 fg mL-1, 800 aM). The single molecule ULISA shows excellent correlation with an electrochemiluminescence reference method. Although the analogue readout can routinely measure PSA concentrations in human serum samples, very low concentrations have to be monitored after radical prostatectomy. Combining the digital and analogue readout covers a dynamic range of more than 3 orders of magnitude in a single experiment.

• MeSH
• bisfosfonáty MeSH
• dermatoskopie metody   MeSH
• fotony MeSH
• imunoanalýza metody   MeSH
• imunosorpční techniky * MeSH
• lidé MeSH
• nanočástice chemie   MeSH
• polyethylenglykoly MeSH
• prostatický specifický antigen krev   MeSH
• streptavidin MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 6-amino-1-hydroxyhexane-1,1-diphosphonate MeSH Prohlížeč
• bisfosfonáty MeSH
• polyethylenglykoly MeSH
• prostatický specifický antigen MeSH
• streptavidin MeSH

Cellular fate of nanoparticles is vital to application of nanoparticles to cell imaging, bio-sensing, drug delivery, suppression of drug resistance, gene delivery, and cytotoxicity analysis. However, the current studies on cellular fate of nanoparticles have been controversial due to complications of interplay between many possible factors. By well-controlled experiments, we demonstrated unambiguously that the morphology of nanoparticles independently determined their cellular fate. We found that nanoparticles with sharp shapes, regardless of their surface chemistry, size, or composition, could pierce the membranes of endosomes that carried them into the cells and escape to the cytoplasm, which in turn significantly reduced the cellular excretion rate of the nanoparticles. Such features of sharp-shaped nanoparticles are essential for drug delivery, gene delivery, subcellular targeting, and long-term tracking. This work opens up a controllable, purely geometrical and hence safe, degree of freedom for manipulating nanoparticle-cell interaction, with numerous applications in medicine, bio-imaging, and bio-sensing.

• MeSH
• biologický transport MeSH
• buněčné linie MeSH
• intracelulární prostor metabolismus   MeSH
• lidé MeSH
• nanočástice chemie   metabolismus   ultrastruktura   MeSH
• povrchové vlastnosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. MATERIALS AND METHODS: We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. RESULTS: Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. CONCLUSION: Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

• Klíčová slova
• cell differentiation, ferrites, magnetic resonance imaging, neural precursors, superparamagnetic iron oxide nanoparticles,
• MeSH
• buněčná diferenciace * MeSH
• fibroblasty cytologie   MeSH
• imunoenzymatické techniky MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• kontrastní látky chemie   MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• lysin chemie   MeSH
• magnetická rezonanční tomografie metody   MeSH
• magnetické nanočástice chemie   MeSH
• neurony cytologie   MeSH
• plíce cytologie   MeSH
• plod cytologie   MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kontrastní látky MeSH
• lysin MeSH
• magnetické nanočástice MeSH