21 results on '"Maja Garvas"'
Search Results
2. Single cell temperature probed by Eu+3 doped TiO2 nanoparticles luminescence
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Maja Garvas, Selene Acosta, Iztok Urbančič, Tilen Koklič, Janez Štrancar, Luiz A. O. Nunes, Peter Guttmann, Polona Umek, and Carla Bittencourt
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luminescence ,nanoparticles ,synchrotron radiation ,TiO2 ,Materials of engineering and construction. Mechanics of materials ,TA401-492 - Abstract
Abstract Temperature is a critical parameter in biology, affecting the speed of reactions that occur in living systems. Nevertheless, measuring temperature with subcellular resolution (micrometric scale) and reliability remains a challenge to overcome. In this perspective, luminescence nanothermometry is a non‐contact technique which aims to measure temperature with a sub‐micrometric spatial resolution through the use of nanomaterials whose luminescence is affected solely by changes in temperature. Here, TiO2 nanoparticles doped with Eu+3 ions (Eu+3‐TiO2) are used for sensing temperature differences within single living cells. XRD, XPS, SEM, TEM and NEXAFS analysis allow the determination of the physicochemical characteristics of the Eu+3‐TiO2 nanoparticles and, the variation of the luminescence intensity of the Eu+3‐TiO2 nanoparticles with their temperature is investigated. The successful internalization of Eu+3‐TiO2 nanoparticles in different types of cells is observed. The luminescence of nanoparticles internalized in L929 fibroblast cells is measured when the system is heated in a biological relevant temperature range. Making use of an appropriate calibration curve the temperature variation inside the cells is determined with sensitivity of 0.5 K per 1% of luminosity change when heated.
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- 2021
- Full Text
- View/download PDF
3. Fluorescent Membrane Probes Based on a Coumarin-Thiazole Scaffold
- Author
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Stane Pajk, Maja Garvas, and Janez Štrancar
- Subjects
fluorophore ,microscopy ,coumarin ,photobleaching ,membrane ,Chemistry ,QD1-999 - Abstract
Biological functions of cell membranes and their correlation to the heterogeneity of the latter’s lipid composition are still poorly understood. Fluorescence provides one of the most versatile tools for studying biological membranes. However, few bright and photostable fluorescent probes for labeling plasma membranes are available. We have designed and synthesized two such probes, 8 and 9, that are based on the thiazole-coumarin scaffold. Both are environment sensitive and exhibit similar shifts of emission spectra in a variety of solvents as probes based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). In particular, the second, positively charged probe 9 labels the plasma membrane selectively with limited redistribution to other membranes of the cell. Unfortunately, compared to the other two probes tested, 8 and 6-NBD-PC, it exhibits the highest rate of photobleaching. Nevertheless, these new thiazole-coumarin based membrane probes provide a viable approach to the design of novel membrane probes.
- Published
- 2019
- Full Text
- View/download PDF
4. Protein Corona Prevents TiO2 Phototoxicity.
- Author
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Maja Garvas, Anze Testen, Polona Umek, Alexandre Gloter, Tilen Koklic, and Janez Strancar
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Medicine ,Science - Abstract
Background & aimTiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.Methods & resultsFluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles' surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes' surface.ConclusionThese results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired - as for efficient photodynamic cancer therapy.
- Published
- 2015
- Full Text
- View/download PDF
5. How to control fluorescent labeling of metal oxide nanoparticles for artefact-free live cell microscopy
- Author
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Maja Garvas, Iztok Urbančič, Katarina Petra van Midden, Polona Umek, Stane Pajk, Tilen Koklic, Christian Eggeling, Janez Štrancar, Hana Kokot, and Boštjan Kokot
- Subjects
Materials science ,Biomedical Engineering ,Metal Nanoparticles ,Reproducibility of Results ,Nanoparticle ,Oxides ,Nanotechnology ,Fluorescence correlation spectroscopy ,Toxicology ,Fluorescence ,Nanomaterials ,Microscopy, Fluorescence ,Microscopy ,Fluorescence microscope ,Zeta potential ,Nanoparticles ,Surface charge ,Artifacts ,Fluorescent Dyes - Abstract
Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles-the largest subpopulation of nanoparticles by industrial production and applications-and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials.
- Published
- 2021
6. A method for targeting a specified segment of DNA to a bacterial microorganelle
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Jan Otoničar, Maja Hostnik, Maja Grundner, Rok Kostanjšek, Tajda Gredar, Maja Garvas, Zoran Arsov, Zdravko Podlesek, Cene Gostinčar, Jernej Jakše, Stephen J W Busby, and Matej Butala
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biokemija ,Bacterial Proteins ,Bacteria ,physical characterisation ,molekularna biologija ,represor LacI ,udc:577 ,Genetics ,bakterijski mikrorazdelek Pdu ,DNA ,biochemical characterisation ,Propylene Glycol ,Twin-Strep-tag - Abstract
Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.
- Published
- 2022
7. Bio-integrated microcavity probes for simultaneous sensing, tracking and imaging
- Author
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Matjaž Humar, Aljaž Kavčič, Maja Garvas, Dmitry Richter, Matevž Marinčič, and Gregor Pirnat
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- 2022
8. Nanoroughening of sandblasted 3Y-TZP surface by alumina coating deposition for improved osseointegration and bacteria reduction
- Author
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Urška Gabor, Jasna Cotič, Andraž Kocjan, Anže Abram, Miha Čekada, Irena Zdovc, Karolina Ivičak-Kocjan, Aleš Dakskobler, Maja Garvas, and Juliane Moritz
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010302 applied physics ,Materials science ,technology, industry, and agriculture ,chemistry.chemical_element ,02 engineering and technology ,Adhesion ,Surface finish ,engineering.material ,021001 nanoscience & nanotechnology ,01 natural sciences ,Osseointegration ,Coating ,chemistry ,0103 physical sciences ,Materials Chemistry ,Ceramics and Composites ,Zeta potential ,engineering ,Cubic zirconia ,Wetting ,Composite material ,0210 nano-technology ,Titanium - Abstract
Zirconia-based (3Y-TZP) dental implants are being introduced as an alternative to established titanium counterparts. As 3Y-TZP’s bioinertness poses a challenge to osseointegration, modifications of the implants’ surface are necessary. Roughening is a common method to improve the cell response and should ideally combine micro- and nano-scale features. Here we show a simple synthesis procedure for the nano-roughening of sandblasted 3Y-TZP by precipitation of a nanostructured alumina coating. As-modified surfaces were characterised regarding their morphology and phase composition, roughness, wetting behaviour and zeta potential, and correlated to the behaviour of human osteoblasts and Staphylococcus aureus bacteria. Osteoblasts were successfully cultured on all surfaces regardless of their modification. Surfaces exhibiting a combination of micro- and nano-roughening performed best regarding initial attachment and proliferation of osteoblasts. Alumina-coated surfaces also limited the bacterial adhesion.
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- 2019
9. Deep tissue localization and sensing using optical microcavity probes
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Aljaž Kavčič, Maja Garvas, Matevž Marinčič, Katrin Unger, Anna Maria Coclite, Boris Majaron, and Matjaž Humar
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Multidisciplinary ,Optical Imaging ,Physics::Optics ,General Physics and Astronomy ,General Chemistry ,General Biochemistry, Genetics and Molecular Biology - Abstract
Optical microcavities and microlasers were recently introduced as probes inside living cells and tissues. Their main advantages are spectrally narrow emission lines and high sensitivity to the environment. Despite numerous novel methods for optical imaging in strongly scattering biological tissues, imaging at single-cell resolution beyond the ballistic light transport regime remains very challenging. Here, we show that optical microcavity probes embedded inside cells enable three-dimensional localization and tracking of individual cells over extended time periods, as well as sensing of their environment, at depths well beyond the light transport length. This is achieved by utilizing unique spectral features of the whispering-gallery modes, which are unaffected by tissue scattering, absorption, and autofluorescence. In addition, microcavities can be functionalized for simultaneous sensing of various parameters, such as temperature or pH value, which extends their versatility beyond the capabilities of standard fluorescent labels.
- Published
- 2021
10. Fluorescent Membrane Probes Based on a Coumarin-Thiazole Scaffold
- Author
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Maja Garvas, Janez Štrancar, and Stane Pajk
- Subjects
Scaffold ,fluorophore ,Fluorophore ,010405 organic chemistry ,Biological membrane ,photobleaching ,01 natural sciences ,Photobleaching ,Fluorescence ,coumarin ,0104 chemical sciences ,lcsh:Chemistry ,chemistry.chemical_compound ,Membrane ,chemistry ,lcsh:QD1-999 ,Microscopy ,Biophysics ,microscopy ,General Earth and Planetary Sciences ,Thiazole ,membrane ,General Environmental Science - Abstract
Biological functions of cell membranes and their correlation to the heterogeneity of the latter%s lipid composition are still poorly understood. Fluorescence provides one of the most versatile tools for studying biological membranes. However, few bright and photostable fluorescent probes for labeling plasma membranes are available. We have designed and synthesized two such probes, 8 and 9, that are based on the thiazole-coumarin scaffold. Both are environment sensitive and exhibit similar shifts of emission spectra in a variety of solvents as probes based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). In particular, the second, positively charged probe 9 labels the plasma membrane selectively with limited redistribution to other membranes of the cell. Unfortunately, compared to the other two probes tested, 8 and 6-NBD-PC, it exhibits the highest rate of photobleaching. Nevertheless, these new thiazole-coumarin based membrane probes provide a viable approach to the design of novel membrane probes. Biološke funkcije celičnih membran in njihove korelacije s heterogenostjo njihove lipidne sestave so še vedno slabo raziskane. Fluorescenca omogoča enega izmed najbolj vsestranskih pristopov k raziskovanju bioloških membran, vendar je za označevanje plazemskih membran na voljo le malo svetlih in fotostabilnih fluorescenčnih označevalcev. Načrtovali in sintetizirali smo dve novi tovrstni fluorescenečni barvili, spojini 8 in 9, ki temeljita na tiazolo-kumarinskem ogrodju. Obe izkazujeta občutljivost na okolje in kažeta podobne premike v emisijskih spektrih v različnih topilih kot je bilo opaženo pri 7-nitrobenz-2-oksa-1,3-diazol-4-ilu (NBD). Drugo, pozitivno nabito barvilo 9, obarva plazemsko membrano selektivno in z omejenim prerazporejanjem v ostale celične membrane. Žal pa 9, za razliko od preostalih dveh testiranih barvil, torej 8 in 6-NBD-PC, zelo hitro fotobledi. Ne glede na to, razvoj dveh novih tiazolo-kumarinskih membranskih barvil predstavlja smiselen pristop k načrtovanju novih membranskih barvil.
- Published
- 2019
11. Controlled Fluorescent Labelling of Metal Oxide Nanoparticles for Artefact-free Live Cell Microscopy
- Author
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Boštjan Kokot, Hana Kokot, Christian Eggeling, Tilen Koklic, Iztok Urbančič, Polona Umek, Maja Garvas, Stane Pajk, Janez Štrancar, and K P Midden
- Subjects
Fluorescent labelling ,Materials science ,Labelling ,Microscopy ,Fluorescence microscope ,Nanoparticle ,Nanotechnology ,Surface charge ,Fluorescence ,Nanomaterials - Abstract
Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. They are best investigated using non-invasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labelling of nanoparticles with fluorescent dyes. When performed inadequately, unbound fluorophores and inadvertently altered chemical and physical properties of the nanoparticles can, however, result in experimental artefacts and erroneous conclusions.To prevent such unintentional errors, we here describe a minimal combination of experimental methods to enable artefact-free fluorescent labelling of metal-oxide nanoparticles – the largest subpopulation of nanoparticles by industrial production and applications – and demonstrate its application in the case of TiO2 nanotubes. We 1) characterize potential changes of the nanoparticles’ surface charge and morphology that might occur during labelling, and 2) assess stable binding of the fluorescent dye to nanomaterial, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials’ mechanism of action and will foster widespread and safe use of new nanomaterials.Abstract Figure
- Published
- 2021
12. Effects of physicochemical properties of TiO
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Pernille Høgh, Danielsen, Kristina Bram, Knudsen, Janez, Štrancar, Polona, Umek, Tilen, Koklič, Maja, Garvas, Esa, Vanhala, Sauli, Savukoski, Yaobo, Ding, Anne Mette, Madsen, Nicklas Raun, Jacobsen, Ingrid Konow, Weydahl, Trine, Berthing, Sarah Søs, Poulsen, Otmar, Schmid, Henrik, Wolff, and Ulla, Vogel
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Titanium ,Dose-Response Relationship, Drug ,Pneumonia ,Pulmonary Alveolar Proteinosis ,Nanostructures ,Mice, Inbred C57BL ,Pulmonary Alveoli ,Mice ,Microscopy, Electron ,Animals ,Female ,Acute-Phase Reaction ,Bronchoalveolar Lavage Fluid ,Lung - Abstract
Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO
- Published
- 2019
13. Effects of physicochemical properties of TiO2 nanomaterials for pulmonary inflammation, acute phase response and alveolar proteinosis in intratracheally exposed mice
- Author
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Trine Berthing, Nicklas Raun Jacobsen, Janez Štrancar, Sarah S. Poulsen, Anne Mette Madsen, Ulla Vogel, Kristina Bram Knudsen, Henrik Wolff, Maja Garvas, Polona Umek, Ingrid Elise Konow Weydahl, Otmar Schmid, Sauli Savukoski, Yaobo Ding, Esa Vanhala, Tilen Koklic, Pernille Høgh Danielsen, Medicum, Department of Pathology, and University of Helsinki
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Pulmonary toxicity ,Alveolar proteinosis ,GENOTOXICITY ,Acute phase response ,INHALATION ,DNA STRAND BREAKS ,Toxicology ,CARBON NANOTUBES ,03 medical and health sciences ,0302 clinical medicine ,INSTILLATION ,NANOPARTICLES ,medicine ,PARTICLES ,CYTOTOXICITY ,Titanium Dioxide ,Nanoparticles ,Crystal Phase ,Inflammation ,Acute Phase Response ,Pulmonary Alveolar Proteinosis ,Pharmacology ,Lung ,Inhalation ,medicine.diagnostic_test ,Chemistry ,technology, industry, and agriculture ,Acute-phase protein ,medicine.disease ,Crystal phase ,3. Good health ,Comet assay ,030104 developmental biology ,medicine.anatomical_structure ,Bronchoalveolar lavage ,030220 oncology & carcinogenesis ,CELLS ,Titanium dioxide ,3111 Biomedicine ,Pulmonary alveolar proteinosis ,LUNG - Abstract
Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO2 in hazard assessment might be challenging because of variation in physicochemical properties. We exposed C57BL/6 J mice to a single dose of four anatase TiO2 NMs with various sizes and shapes by intratracheal instillation and assessed the pulmonary toxicity 1, 3, 28, 90 or 180 days post-exposure. The quartz DQ12 was included as benchmark particle. Pulmonary responses were evaluated by histopathology, electron microscopy, bronchoalveolar lavage (BAL) fluid cell composition and acute phase response. Genotoxicity was evaluated by DNA strand break levels in BAL cells, lung and liver in the comet assay. Multiple regression analyses were applied to identify specific TiO2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO2 NMs induced similar inflammatory responses when surface area was used as dose metrics, although inflammatory and acute phase response was greatest and more persistent for the TiO2 tube. Similar histopathological changes were observed for the TiO2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO2 NMs indicated that rutile TiO2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO2 NMs are important predictors for the observed pulmonary effects of TiO2 NMs.
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- 2019
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14. Eu3+−doped titanium oxide nanoparticles for optical thermometry in the first biological window
- Author
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L. J. Borrero-González, Selene Acosta, Maja Garvas, Polona Umek, Carla Bittencourt, and Luiz Antônio de Oliveira Nunes
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Photoluminescence ,Materials science ,Analytical chemistry ,Nanoparticle ,chemistry.chemical_element ,Context (language use) ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Temperature measurement ,Inorganic Chemistry ,Electrical and Electronic Engineering ,Physical and Theoretical Chemistry ,Spectroscopy ,Organic Chemistry ,PULSO ,Atmospheric temperature range ,021001 nanoscience & nanotechnology ,Atomic and Molecular Physics, and Optics ,0104 chemical sciences ,Electronic, Optical and Magnetic Materials ,Titanium oxide ,chemistry ,0210 nano-technology ,Luminescence ,Europium - Abstract
Lanthanide-based optical nanothermometers, operating in the physiological temperature range (288–323 K), with excitation and emission in the first biological transparent window have special interest for biological applications. In this context, trivalent europium doped titanium oxide (Eu3+:TiO2) nanoparticles were prepared via a sol-gel method and their spectroscopic properties were studied. In order to assess their potential for temperature sensing, excitation and luminescence spectroscopies were performed. We observed that the intensities of the excitation bands for the 7F0→5D0 (576 nm) and 7F2→5D0 (610 nm) transitions, monitoring the 5D0→7F4 (700 nm) transition have a strong dependence on temperature. This dependence, which is explained in terms of a thermal coupling between the Eu3+:7FJ levels, was used for the construction of an optical nanothermometer. Relative sensitivity values between 1.78 and 1.41% K−1, when the temperature of the material increases from 288 to 323 K, were obtained. We show that the nanothermometer calibration can be obtained by a single luminescence room temperature measurement. Our results indicate the potential application of Eu3+:TiO2 nanoparticles for temperature sensing in the first biological window and physiological temperature range.
- Published
- 2020
15. Nanoparticles can wrap epithelial cell membranes and relocate them across the epithelial cell layer
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Miha Škarabot, Zoran Arsov, Silvia Galiani, Igor Muševič, Tilen Koklic, Boštjan Kokot, Hana Majaron, Hilary Cassidy, Janez Štrancar, Maja Garvas, Falk Schneider, Iztok Urbančič, Polona Umek, Christian Eggeling, Miran Čeh, and David Matallanas
- Subjects
0301 basic medicine ,Letter ,Proteome ,Lipid Bilayers ,Nanoparticle ,Protein Corona ,02 engineering and technology ,Signal transduction ,Cell survival ,Cell membrane ,Mice ,Cell Movement ,Surface properties ,General Materials Science ,Lipid bilayer ,Lung ,Titanium ,Nanotubes ,Chemistry ,Particle size ,Blood coagulation ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,medicine.anatomical_structure ,Membrane ,Protein corona ,0210 nano-technology ,Signal Transduction ,Lipid bilayers ,Cell Survival ,Surface Properties ,membrane disruption ,Bioengineering ,lipid wrapping ,03 medical and health sciences ,Tissue factor ,Cell movement ,STED microscopy ,medicine ,Animals ,Humans ,Particle Size ,Blood Coagulation ,coagulation cascade interference ,Mechanical Engineering ,Cell Membrane ,Epithelial Cells ,General Chemistry ,Epithelium ,030104 developmental biology ,Biophysics ,TiO2 nanoparticles ,tissue factor relocation - Abstract
Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane's disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease. European Commission Horizon 2020 Wellcome Trust Slovenian Research Agency Wolfson Foundation Oxford-internal funding
- Published
- 2018
16. Protein Corona Prevents TiO2 Phototoxicity
- Author
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Anze Testen, Tilen Koklic, Janez Štrancar, Maja Garvas, Alexandre Gloter, and Polona Umek
- Subjects
Cell Survival ,Ultraviolet Rays ,Radical ,Science ,Nanoparticle ,Protein Corona ,Cell Line ,Animals ,Humans ,Titanium ,chemistry.chemical_classification ,Reactive oxygen species ,Multidisciplinary ,Spin trapping ,Blood Proteins ,Photochemical Processes ,Blood proteins ,Fluorescence ,chemistry ,Biophysics ,Nanoparticles ,Medicine ,Cattle ,Reactive Oxygen Species ,Phototoxicity ,Research Article - Abstract
Background & aimTiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.Methods & resultsFluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles' surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes' surface.ConclusionThese results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired - as for efficient photodynamic cancer therapy.
- Published
- 2015
17. Fluorescence microspectroscopy as a tool to study mechanism of nanoparticles delivery into living cancer cells
- Author
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Zoran Arsov, Iztok Urbančič, Maja Garvas, Daniele Biglino, Ajasja Ljubetič, Tilen Koklič, and Janez Štrancar
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Microscopy ,ocis:(110.4234) Multispectral and hyperspectral imaging ,ocis:(230.7408) Wavelength filtering devices ,ocis:(300.6280) Spectroscopy, fluorescence and luminescence ,ocis:(170.2520) Fluorescence microscopy ,Atomic and Molecular Physics, and Optics ,Biotechnology ,ocis:(170.0170) Medical optics and biotechnology - Abstract
Lack of better understanding of nanoparticles targeted delivery into cancer cells calls for advanced optical microscopy methodologies. Here we present a development of fluorescence microspectroscopy (spectral imaging) based on a white light spinning disk confocal microscope with emission wavelength selection by a liquid crystal tunable filter. Spectral contrasting of images was used to localize polymer nanoparticles and cell membranes labeled with fluorophores that have substantially overlapping spectra. In addition, fluorescence microspectroscopy enabled spatially-resolved detection of small but significant effects of local molecular environment on the properties of environment-sensitive fluorescent probe. The observed spectral shift suggests that the delivery of suitably composed cancerostatic alkylphospholipid nanoparticles into living cancer cells might rely on the fusion with plasma cell membrane.
- Published
- 2011
18. Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells
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Urška Batista, Milan Schara, Maja Garvas, Marjana Nemec, Tilen Koklic, and Peter Veranič
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Cell Membrane Permeability ,Membrane permeability ,Cell Survival ,Membrane Fluidity ,Cell ,Cell Culture Techniques ,Cell Line ,Cyclic N-Oxides ,Cricetinae ,medicine ,Membrane fluidity ,Animals ,Trypsin ,Viability assay ,Citrates ,Cell damage ,Chemistry ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,Cell Biology ,General Medicine ,Triacetoneamine-N-Oxyl ,medicine.disease ,Trypsinization ,Kinetics ,Membrane ,medicine.anatomical_structure ,Biochemistry ,Biophysics ,Microscopy, Electron, Scanning ,Nitrogen Oxides ,Spin Labels ,Stress, Mechanical ,medicine.drug - Abstract
Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin-probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin-treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.
- Published
- 2010
19. EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity
- Author
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Katja Rebolj, a Biserka Bakrac, a Maja Garvas, b Katja Ota, a Marjeta Sentjurc, b Cristina Potrich, c,d Manuela Coraiola, c,d Rossella Tomazzolli, c,d Mauro Dalla Serra, c,d Peter Macek, a Kristina Sepcic, and a,?
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Swine ,Liquid ordered phase ,Membrane lipids ,Biophysics ,Phospholipid ,Biochemistry ,Fungal Proteins ,Hemolysin Proteins ,chemistry.chemical_compound ,Membrane Microdomains ,Spectroscopy, Fourier Transform Infrared ,Membrane fluidity ,Membrane activity ,Animals ,Steroid ,Lipid raft ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,Cell Biology ,Sphingomyelins ,Ostreolysin ,Sterols ,Cholesterol ,Membrane ,chemistry ,Liposomes ,Fourier-transformed infrared spectroscopy ,lipids (amino acids, peptides, and proteins) ,Electron paramagnetic resonance ,Sphingomyelin - Abstract
Ostreolysin is a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation. 2010 Elsevier B.V. All rights reserved.
- Published
- 2010
20. Nitroxide–fluorophore double probes: a potential tool for studying membrane heterogeneity by ESR and fluorescence
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Maja Garvas, Slavko Pečar, Janez Štrancar, and Stane Pajk
- Subjects
Nitroxide mediated radical polymerization ,Fluorophore ,Biochemistry ,Fluorescence ,Spin probe ,chemistry.chemical_compound ,Nuclear magnetic resonance ,Cell Line, Tumor ,Image Processing, Computer-Assisted ,Fluorescence microscope ,Humans ,Physical and Theoretical Chemistry ,Lipid bilayer ,Fluorescent Dyes ,Microscopy, Confocal ,Molecular Structure ,Organic Chemistry ,Electron Spin Resonance Spectroscopy ,Stereoisomerism ,Membrane ,Microscopy, Fluorescence ,chemistry ,Biophysics ,Nitrogen Oxides ,Linker - Abstract
A serious drawback of ESR, particularly in its application to cells, is the lack of information on the location of spin probes in the system. In order to realize real time tracking, a spin probe was combined with a fluorophore in a new kind of nitroxide-fluorophore double probe which, in addition to information about lipid dynamics, enables visualization by fluorescence microscopy. The two sets of probes synthesized are based on an amino-alkyne-functionalized sugar that serves as a central polar group and as a linker between the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore and the derivative of the spin labelled fatty acid. In this setting, the location of the fluorophore is restricted to the water-lipid interface, while the nitroxide is located deep in the lipid bilayer. Preliminary tests on cells show preferential localization of both probes in the plasma membrane, with a relatively slow redistribution to other membranes of the cell. We believe that such double probes would be particularly useful for studies of plasma membrane heterogeneity and associated cellular processes.
- Published
- 2011
21. Single cell temperature probed by Eu +3 doped TiO 2 nanoparticles luminescence
- Author
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Maja Garvas, Peter Guttmann, Luiz Antônio de Oliveira Nunes, Selene Acosta, Iztok Urbančič, Polona Umek, Carla Bittencourt, Tilen Koklic, and Janez Štrancar
- Subjects
Materials science ,synchrotron radiation ,Doping ,Tio2 nanoparticles ,Synchrotron radiation ,Nanoparticle ,Photochemistry ,RADIAÇÃO SINCROTRON ,luminescence ,TA401-492 ,TiO2 ,nanoparticles ,Luminescence ,Materials of engineering and construction. Mechanics of materials - Abstract
Temperature is a critical parameter in biology, affecting the speed of reactions that occur in living systems. Nevertheless, measuring temperature with subcellular resolution (micrometric scale) and reliability remains a challenge to overcome. In this perspective, luminescence nanothermometry is a non‐contact technique which aims to measure temperature with a sub‐micrometric spatial resolution through the use of nanomaterials whose luminescence is affected solely by changes in temperature. Here, TiO2 nanoparticles doped with Eu+3 ions (Eu+3‐TiO2) are used for sensing temperature differences within single living cells. XRD, XPS, SEM, TEM and NEXAFS analysis allow the determination of the physicochemical characteristics of the Eu+3‐TiO2 nanoparticles and, the variation of the luminescence intensity of the Eu+3‐TiO2 nanoparticles with their temperature is investigated. The successful internalization of Eu+3‐TiO2 nanoparticles in different types of cells is observed. The luminescence of nanoparticles internalized in L929 fibroblast cells is measured when the system is heated in a biological relevant temperature range. Making use of an appropriate calibration curve the temperature variation inside the cells is determined with sensitivity of 0.5 K per 1% of luminosity change when heated.
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