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Raman Spectroscopy and Hyperspectral Analysis of Living Cells Exposed to Nanoparticles
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper. Totalförsvarets forskningsinstitut.
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2015. , s. 72
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1257
Nyckelord [en]
Raman spectroscopy, Hyperspectral analysis, Multivariate data analysis, Nanotoxicology, Graphene oxide, Iron oxide, Titanium dioxide, Cells
Nationell ämneskategori
Teknik och teknologier
Identifikatorer
URN: urn:nbn:se:uu:diva-251339ISBN: 978-91-554-9250-2 (tryckt)OAI: oai:DiVA.org:uu-251339DiVA, id: diva2:805490
Disputation
2015-06-04, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2015-05-13 Skapad: 2015-04-15 Senast uppdaterad: 2015-07-07Bibliografiskt granskad
Delarbeten
1. Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification
Öppna denna publikation i ny flik eller fönster >>Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification
2013 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, nr 2, s. 310-319Artikel i tidskrift (Refereegranskat) 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.

Nationell ämneskategori
Teknik och teknologier
Forskningsämne
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 ()
Tillgänglig från: 2013-09-02 Skapad: 2013-09-02 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
2. Super-resolution Raman mapping of living cells exposed to submicron polystyrene particles
Öppna denna publikation i ny flik eller fönster >>Super-resolution Raman mapping of living cells exposed to submicron polystyrene particles
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Teknik och teknologier
Identifikatorer
urn:nbn:se:uu:diva-251333 (URN)
Tillgänglig från: 2015-04-15 Skapad: 2015-04-15 Senast uppdaterad: 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
Öppna denna publikation i ny flik eller fönster >>Evidence of nuclear uptake and increased DNA damage in human lung epithelial cells after low dose exposure to reactive titanium dioxide nanoparticles
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Teknik och teknologier
Identifikatorer
urn:nbn:se:uu:diva-251336 (URN)
Tillgänglig från: 2015-04-15 Skapad: 2015-04-15 Senast uppdaterad: 2015-05-18
4. Graphene oxide nanoparticle attachment and its toxicity on living lung epithelial cells
Öppna denna publikation i ny flik eller fönster >>Graphene oxide nanoparticle attachment and its toxicity on living lung epithelial cells
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2015 (Engelska)Ingår i: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 5, nr 73, s. 59447-59457Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Royal Society of Chemistry, 2015
Nationell ämneskategori
Annan teknik
Identifikatorer
urn:nbn:se:uu:diva-251338 (URN)10.1039/C5RA09351A (DOI)000357961800060 ()
Tillgänglig från: 2015-04-15 Skapad: 2015-04-15 Senast uppdaterad: 2017-12-04Bibliografiskt granskad
5. Polymorph and size dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells
Öppna denna publikation i ny flik eller fönster >>Polymorph and size dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells
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2011 (Engelska)Ingår i: Small, ISSN 1613-6810, Vol. 7, nr 4, s. 514-523Artikel i tidskrift (Refereegranskat) 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.

Nationell ämneskategori
Teknik och teknologier
Forskningsämne
Teknisk fysik med inriktning mot fasta tillståndets fysik
Identifikatorer
urn:nbn:se:uu:diva-139730 (URN)10.1002/smll.201001832 (DOI)000288080400013 ()
Tillgänglig från: 2010-12-29 Skapad: 2010-12-29 Senast uppdaterad: 2016-04-19Bibliografiskt granskad

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