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  • 1.
    Ahlinder, Linnea
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper. Totalförsvarets forskningsinstitut.
    Raman Spectroscopy and Hyperspectral Analysis of Living Cells Exposed to Nanoparticles2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Nanoparticles, i.e. particles with at least one dimension smaller than 100 nm, are present in large quantities in ambient air and can also be found in an increasing amount of consumer products. It is known that many nanomaterials have physicochemical properties that differ from physicochemical properties of the same material in bulk size. It is therefore important to characterize nanoparticles and to evaluate their toxicity. To understand mechanisms behind nanotoxicity, it is important to study the uptake of nanoparticles, and how they are accumulated. For these purposes model studies of cellular uptake are useful. In this thesis metal oxide and carbon-based nanoparticles have been studied in living cells using Raman spectroscopy. Raman spectroscopy is a method that facilitates a non-destructive analysis without using any fluorescent labels, or any other specific sample preparation. It is possible to collect Raman images, i.e. images where each pixel corresponds to a Raman spectrum, and to use the spectral information to detect nanoparticles, and to identify organelles in cells. In this thesis the question whether or not nanoparticles can enter the cell nucleus of lung epithelial cells has been addressed using hyperspectral analysis. It is shown that titanium dioxide nanoparticles and iron oxide nanoparticles are taken up by cells, and also in the cell nucleus. In contrast, graphene oxide nanoparticles are mainly found attached on the outside of the cell membrane and very few nanoparticles are found in the cell, and none have been detected in the nucleus. It is concluded that graphene oxide nanoparticles are not cytotoxic. However, a comparison of Raman spectra of biomolecules in cells exposed to graphene oxide, unexposed cells and apoptotic cells, shows that the graphene oxide nanoparticles do affect lipid and protein structures. In this thesis, several multivariate data analysis methods have been used to analyze Raman spectra and Raman images. In addition, super-resolution algorithms, which originally have been developed to improve the resolution in photographic images, were optimized and applied to Raman images of cells exposed to submicron polystyrene particles in living cells.

    Delarbeid
    1. Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification
    Åpne denne publikasjonen i ny fane eller vindu >>Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification
    2013 (engelsk)Inngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, nr 2, s. 310-319Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    It is a challenging task to characterize the biodistribution of nanoparticles in cells and tissue on a subcellular level. Conventional methods to study the interaction of nanoparticles with living cells rely on labeling techniques that either selectively stain the particles or selectively tag them with tracer molecules. In this work, Raman imaging, a label-free technique that requires no extensive sample preparation, was combined with multivariate classification to quantify the spatial distribution of oxide nanoparticles inside living lung epithelial cells (A549). Cells were exposed to TiO2 (titania) and/or alpha-FeO(OH) (goethite) nanoparticles at various incubation times (4 or 48 h). Using multivariate classification of hyperspectral Raman data with partial least-squares discriminant analysis, we show that a surprisingly large fraction of spectra, classified as belonging to the cell nucleus, show Raman bands associated with nanoparticles. Up to 40% of spectra from the cell nucleus show Raman bands associated with nanoparticles. Complementary transmission electron microscopy data for thin cell sections qualitatively support the conclusions.

    HSV kategori
    Forskningsprogram
    Teknisk fysik med inriktning mot fasta tillståndets fysik
    Identifikatorer
    urn:nbn:se:uu:diva-206593 (URN)10.1016/j.bpj.2013.06.017 (DOI)000321941700006 ()
    Tilgjengelig fra: 2013-09-02 Laget: 2013-09-02 Sist oppdatert: 2017-12-06bibliografisk kontrollert
    2. Super-resolution Raman mapping of living cells exposed to submicron polystyrene particles
    Åpne denne publikasjonen i ny fane eller vindu >>Super-resolution Raman mapping of living cells exposed to submicron polystyrene particles
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-251333 (URN)
    Tilgjengelig fra: 2015-04-15 Laget: 2015-04-15 Sist oppdatert: 2015-05-18
    3. Evidence of nuclear uptake and increased DNA damage in human lung epithelial cells after low dose exposure to reactive titanium dioxide nanoparticles
    Åpne denne publikasjonen i ny fane eller vindu >>Evidence of nuclear uptake and increased DNA damage in human lung epithelial cells after low dose exposure to reactive titanium dioxide nanoparticles
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-251336 (URN)
    Tilgjengelig fra: 2015-04-15 Laget: 2015-04-15 Sist oppdatert: 2015-05-18
    4. Graphene oxide nanoparticle attachment and its toxicity on living lung epithelial cells
    Åpne denne publikasjonen i ny fane eller vindu >>Graphene oxide nanoparticle attachment and its toxicity on living lung epithelial cells
    Vise andre…
    2015 (engelsk)Inngår i: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 5, nr 73, s. 59447-59457Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Since its discovery, graphene and its oxidized form, graphene oxide (GO), have attracted interest in a wide range of technical applications. Concerns about their potential toxicity calls for scrutinized studies, but hitherto conflicting results have been reported which partly may be due to variations of synthesis and exposure procedures. Here we report on the attachment and toxicity of contamination-free graphene oxide nanoparticles (GONP) in living lung epithelial cells. The synthesis of chemically pure GONP was made by an improvement of the Hummer's method based on graphene exfoliated from graphite using high-intensity ultrasonication, resulting in two dimensional sheets with a lateral dimension in the range 200 nm to 3 mu m and thickness of 0.9 nm. Confocal Raman spectroscopy combined with multivariate analysis was used to study the interaction of GONP and living cells. It is shown that overlapping Raman bands due to GONPs and biomolecules in the cells can clearly be separated with this approach. Orthogonal partial least squares discriminant analysis was used to compare spectral data collected from cells exposed to GONP with spectral data collected from non-exposed control cells, and spectral data from cells exposed to a surfactant known to induce apoptosis. Our analyses show that GONP readily attach to the cells, forming sheets which cover a large fraction of the cell surfaces, and induce small chemical changes. In particular, chemical modifications of proteins and lipids in lung epithelial cells are inferred. GONPs do not, however, decrease cell viability. In contrast, enhanced cell proliferation is observed. Our results shed new light on the interactions of GO, and in contrast to some previous reports, suggest that GO is not toxic. The hyperspectral Raman spectroscopy analysis employed here should be applicable for other fields in nanomedicine as a label-free non-perturbing analytical method.

    sted, utgiver, år, opplag, sider
    Royal Society of Chemistry, 2015
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-251338 (URN)10.1039/C5RA09351A (DOI)000357961800060 ()
    Tilgjengelig fra: 2015-04-15 Laget: 2015-04-15 Sist oppdatert: 2017-12-04bibliografisk kontrollert
    5. Polymorph and size dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells
    Åpne denne publikasjonen i ny fane eller vindu >>Polymorph and size dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells
    Vise andre…
    2011 (engelsk)Inngår i: Small, ISSN 1613-6810, Vol. 7, nr 4, s. 514-523Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The cellular uptake and distribution of five types of well-characterized anatase and rutile TiO(2) nanoparticles (NPs) in A549 lung epithelial cells is reported. Static light scattering (SLS), in-vitro Raman microspectroscopy (mu-Raman) and transmission electron spectroscopy (TEM) reveal an intimate correlation between the intrinsic physicochemical properties of the NPs, particle agglomeration, and cellular NP uptake. It is shown that mu-Raman facilitates chemical-, polymorph-, and size-specific discrimination of endosomal-particle cell uptake and the retention of particles in the vicinity of organelles, including the cell nucleus, which quantitatively correlates with TEM and SLS data. Depth-profiling mu-Raman coupled with hyperspectral data analysis confirms the location of the NPs in the cells and shows that the NPs induce modifications of the biological matrix. NP uptake is found to be kinetically activated and strongly dependent on the hard agglomeration size-not the primary particle size-which quantitatively agrees with the measured intracellular oxidative stress. Pro-inflammatory responses are also found to be sensitive to primary particle size.

    HSV kategori
    Forskningsprogram
    Teknisk fysik med inriktning mot fasta tillståndets fysik
    Identifikatorer
    urn:nbn:se:uu:diva-139730 (URN)10.1002/smll.201001832 (DOI)000288080400013 ()
    Tilgjengelig fra: 2010-12-29 Laget: 2010-12-29 Sist oppdatert: 2016-04-19bibliografisk kontrollert
  • 2.
    Ahlinder, Linnea
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Fasta tillståndets fysik.
    Ekstrand-Hammarstrom, Barbro
    Geladi, Paul
    Österlund, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Fasta tillståndets fysik.
    Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification2013Inngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, nr 2, s. 310-319Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    It is a challenging task to characterize the biodistribution of nanoparticles in cells and tissue on a subcellular level. Conventional methods to study the interaction of nanoparticles with living cells rely on labeling techniques that either selectively stain the particles or selectively tag them with tracer molecules. In this work, Raman imaging, a label-free technique that requires no extensive sample preparation, was combined with multivariate classification to quantify the spatial distribution of oxide nanoparticles inside living lung epithelial cells (A549). Cells were exposed to TiO2 (titania) and/or alpha-FeO(OH) (goethite) nanoparticles at various incubation times (4 or 48 h). Using multivariate classification of hyperspectral Raman data with partial least-squares discriminant analysis, we show that a surprisingly large fraction of spectra, classified as belonging to the cell nucleus, show Raman bands associated with nanoparticles. Up to 40% of spectra from the cell nucleus show Raman bands associated with nanoparticles. Complementary transmission electron microscopy data for thin cell sections qualitatively support the conclusions.

  • 3.
    Ahlinder, Linnea
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Fasta tillståndets fysik.
    Henych, Jiří
    Wiklund Lindström, Susanne
    Ekstrand-Hammarström, Barbro
    Stengl, Václav
    Österlund, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Fasta tillståndets fysik.
    Graphene oxide nanoparticle attachment and its toxicity on living lung epithelial cells2015Inngår i: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 5, nr 73, s. 59447-59457Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Since its discovery, graphene and its oxidized form, graphene oxide (GO), have attracted interest in a wide range of technical applications. Concerns about their potential toxicity calls for scrutinized studies, but hitherto conflicting results have been reported which partly may be due to variations of synthesis and exposure procedures. Here we report on the attachment and toxicity of contamination-free graphene oxide nanoparticles (GONP) in living lung epithelial cells. The synthesis of chemically pure GONP was made by an improvement of the Hummer's method based on graphene exfoliated from graphite using high-intensity ultrasonication, resulting in two dimensional sheets with a lateral dimension in the range 200 nm to 3 mu m and thickness of 0.9 nm. Confocal Raman spectroscopy combined with multivariate analysis was used to study the interaction of GONP and living cells. It is shown that overlapping Raman bands due to GONPs and biomolecules in the cells can clearly be separated with this approach. Orthogonal partial least squares discriminant analysis was used to compare spectral data collected from cells exposed to GONP with spectral data collected from non-exposed control cells, and spectral data from cells exposed to a surfactant known to induce apoptosis. Our analyses show that GONP readily attach to the cells, forming sheets which cover a large fraction of the cell surfaces, and induce small chemical changes. In particular, chemical modifications of proteins and lipids in lung epithelial cells are inferred. GONPs do not, however, decrease cell viability. In contrast, enhanced cell proliferation is observed. Our results shed new light on the interactions of GO, and in contrast to some previous reports, suggest that GO is not toxic. The hyperspectral Raman spectroscopy analysis employed here should be applicable for other fields in nanomedicine as a label-free non-perturbing analytical method.

  • 4.
    Andersson, Per Ola
    et al.
    FOI, Umeå.
    Lejon, Christian
    FOI, Umeå.
    Ekstrand Hammarström, Barbro
    FOI, Umeå.
    Akfur, Christine
    FOI, Umeå.
    Ahlinder, Linnea
    FOI, Umeå.
    Bucht, Anders
    FOI, Umeå.
    Österlund, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Fasta tillståndets fysik.
    Polymorph and size dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells2011Inngår i: Small, ISSN 1613-6810, Vol. 7, nr 4, s. 514-523Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The cellular uptake and distribution of five types of well-characterized anatase and rutile TiO(2) nanoparticles (NPs) in A549 lung epithelial cells is reported. Static light scattering (SLS), in-vitro Raman microspectroscopy (mu-Raman) and transmission electron spectroscopy (TEM) reveal an intimate correlation between the intrinsic physicochemical properties of the NPs, particle agglomeration, and cellular NP uptake. It is shown that mu-Raman facilitates chemical-, polymorph-, and size-specific discrimination of endosomal-particle cell uptake and the retention of particles in the vicinity of organelles, including the cell nucleus, which quantitatively correlates with TEM and SLS data. Depth-profiling mu-Raman coupled with hyperspectral data analysis confirms the location of the NPs in the cells and shows that the NPs induce modifications of the biological matrix. NP uptake is found to be kinetically activated and strongly dependent on the hard agglomeration size-not the primary particle size-which quantitatively agrees with the measured intracellular oxidative stress. Pro-inflammatory responses are also found to be sensitive to primary particle size.

  • 5.
    Wilkinson, Kai
    et al.
    Dept of Chemistry, SLU BioCenter, Uppsala.
    Ekstrand-Hammarström, Barbro
    FOI, Umeå.
    Ahlinder, Linnea
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Fasta tillståndets fysik.
    Guldevall, Karolin
    Science for Life Laboratory, Cell Physics, Dept of Applied Physics, Albanova University center, KTH, Stockholm.
    Pazik, Robert
    Dept of Chemistry, SLU BioCenter, Uppsala.
    Kępiński, Leszek
    Inst Low Temp Struct Res, Polish Academy of Sciences, Wroclaw, Polen.
    Kvashnina, Kristina O.
    European Synchrotron Radiation Facility, Grenoble, Frankrike.
    Butorin, Segei M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Fysiska sektionen, Institutionen för fysik och astronomi.
    Brismar, Hjalmar
    Science for Life Laboratory, Cell Physics, Dept of Applied Physics, Albanova University center, KTH, Stockholm.
    Önfelt, Björn
    Science for Life Laboratory, Cell Physics, Dept of Applied Physics, Albanova University center, KTH, Stockholm.
    Österlund, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Fasta tillståndets fysik.
    Seisenbaeva, Gulaim A.
    Dept of Chemistry, SLU BioCenter, Uppsala.
    Kessler, Vadim G.
    Dept of Chemistry, SLU BioCenter, Uppsala.
    Visualization of custom-tailored iron oxide nanoparticles chemistry, uptake, and toxicity2012Inngår i: Nanoscale, ISSN 2040-3364, Vol. 4, nr 23, s. 7383-7393Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Nanoparticles of iron oxide generated by wearing of vehicles have been modelled with a tailored solution of size-uniform engineered magnetite particles produced by the Bradley reaction, a solvothermal metal–organic approach rendering hydrophilic particles. The latter does not bear any pronounced surface charge in analogy with that originating from anthropogenic sources in the environment. Physicochemical properties of the nanoparticles were thoroughly characterized by a wide range of methods, including XPD, TEM, SEM, DLS and spectroscopic techniques. The magnetite nanoparticles were found to be sensitive for transformation into maghemite under ambient conditions. This process was clearly revealed by Raman spectroscopy for high surface energy magnetite particles containing minor impurities of the hydromaghemite phase and was followed by quantitative measurements with EXAFS spectroscopy. In order to assess the toxicological effects of the produced nanoparticles in humans, with and without surface modification with ATP (a model of bio-corona formed in alveolar liquid), a pathway of potential uptake and clearance was modelled with a sequence of in vitro studies using A549 lung epithelial cells, lymphocyte 221-B cells, and 293T embryonal kidney cells, respectively. Raman microscopy unambiguously showed that magnetite nanoparticles are internalized within the A549 cells after 24 h co-incubation, and that the ATP ligand is retained on the nanoparticles throughout the uptake process. The toxicity of the nanoparticles was estimated using confocal fluorescence microscopy and indicated no principal difference for unmodified and modified particles, but revealed considerably different biochemical responses. The IL-8 cytokine response was found to be significantly lower for the magnetite nanoparticles compared to TiO2, while an enhancement of ROS was observed, which was further increased for the ATP-modified nanoparticles, implicating involvement of the ATP signalling pathway in the epithelium.

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