| 1. |
- Bauer, Brigitte, 1978, et al.
(author)
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Modification and expulsion of keratins by human epidermal keratinocytes upon hapten exposure in vitro.
- 2011
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In: Chemical research in toxicology. - : American Chemical Society (ACS). - 1520-5010 .- 0893-228X. ; 24:5, s. 737-43
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Journal article (peer-reviewed)abstract
- Allergic contact dermatitis is the most prevalent form of human immunotoxicity. It is caused by reactive low molecular weight chemicals, that is, haptens, coming in contact with the skin where hapten-peptide complexes are formed, activating the immune system. By using sensitizing fluorescent thiol-reactive haptens, that is, bromobimanes, we show how keratinocytes respond to hapten exposure in vitro and reveal, for the first time in a living system, an exact site of haptenation. Rapid internalization and reaction of haptens with keratin filaments were visualized. Subsequently, keratinocytes respond in vitro to hapten exposure by release of membrane blebs, which contain haptenated keratins 5 and 14. Particularly, cysteine 54 of K5 was found to be a specific target. A mechanism is proposed where neoepitopes, otherwise hidden from the immune system, are released after hapten exposure via keratinocyte blebbing. The observed expulsion of modified keratins by keratinocytes in vitro might play a role during hapten sensitization in vivo and should be subject to further investigations.
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| 2. |
- Borglin, Johan, 1986, et al.
(author)
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Improving multiphoton microscopy using annular beam shaping, focusing on imaging of human skin
- 2014
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In: Multiphoton Microscopy in the Biomedical Sciences XIV: 2-4 February 2014, San Francisco, California, United States. Progress in Biomedical Optics and Imaging - Proceedings of SPIE. - : SPIE. - 1605-7422. ; 8948
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Conference paper (peer-reviewed)abstract
- Multiphoton fluorescence microscopy (MPM) is a method for high resolution, non-invasive investigations of biological tissue. The aim of introducing an annular shaped laser beam is to reduce the ouf-of-focus generated background signal improving imaging of light scattering tissue such as human skin. Simulations show that 50% of the beam radius can be blocked, while preserving the shape of the point spread function. Initial experiments performed on a phantom consisting of fluorescein and fluorescent beads embedded in agar by using a custom built MPM-set up show that by introducing a simple beam blocker to create an annular beam, the background signal is reduced with approximately 5%. Future work will include optimizing the set up, and creating phantoms with more light scattering properties. © 2014 SPIE.
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| 3. |
- Evenbratt, Hanne, 1980, et al.
(author)
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Cubic and Sponge Phases in Ether Lipid-Solvent-Water Ternary Systems: Phase Behavior and NMR Characterization
- 2013
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In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 29:42, s. 13058-13065
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Journal article (peer-reviewed)abstract
- The phase behavior of 1-glyceryl monoleyl ether (GME) in mixtures of water and the solvents 1,5-pentanediol (POL) or N-methyl-2-pyrrolidone (NMP) was investigated by ocular inspection, polarization microscopy, and small-angle X-ray diffraction (SAXD). Phase diagrams were constructed based on analyses of more than 200 samples prepared using the two different solvents at 20 degrees C. The inverse hexagonal phase formed by GME in excess of water was transformed into the cubic and sponge phase with the increasing amount of each solvent. Particularly POL allowed for the formation of an extended sponge phase area in the phase diagram, comprising up to 70% POL water mixture. The phase behavior using NMP was found to be similar to the earlier investigated solvent propylene glycol. The extended sponge phase for the POL system was attributed to POLs strong surface/interfacial activity with the potential to stabilize the polar/apolar interface of the sponge phase. The cubic and sponge phases formed using POL were further studied by NMR in order to measure the partitioning of POL between the lipid and aqueous domains of the phases. The domain partition coefficient K (lipid domain/aqueous domain) for POL in cubic and sponge phases was found to be 0.78 +/- 0.14 and constant for the two phases.
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| 4. |
- Evenbratt, Hanne, 1980, et al.
(author)
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In vivo study of an instantly formed lipid-water cubic phase formulation for efficient topical delivery of aminolevulinic acid and methyl-aminolevulinate
- 2013
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In: International Journal of Pharmaceutics. - : Elsevier BV. - 0378-5173 .- 1873-3476. ; 452:1-2, s. 270-275
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Journal article (peer-reviewed)abstract
- We demonstrate a rapidly formed cubic liquid crystalline phase, i.e. typically 1 g cubic phase in less than 1 min confirmed by X-ray diffraction, consisting of an ether lipid, 1-glyceryl monooleyl ether (GME), an aprotic solvent (propylene glycol or pentane-1,5-diol) and water. The efficacy of the cubic formulation was tested in vivo by administrating formulations containing 3% (w/w) of the HCl salts of delta-aminolevulinic acid (ALA) or methylaminolevulinate (MAL) to hairless mice. The endogenous formation of protoporphyrin IX (PpIX) was monitored spectrophotometrically as a marker for cellular uptake of active compound. As reference, a commercial product containing 16% (w/w) MAL in an oil-in-water emulsion (Metvix (R)), and a cubic phase based on an ester lipid (glyceryl monooleate, GMO), previously shown to facilitate topical delivery of both ALA and MAL, were applied. It was found that in general the cubic phases gave rise to higher fluorescence levels than the mice exposed to the commercial product. The instantly formed cubic formulations based on GME demonstrated the same efficiency as the GMO based formulations. The results imply that instantly formed cubic formulations opens up new opportunities, particularly for transdermal drug delivery of substances subject to stability problems in, e. g. aqueous environments.
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| 5. |
- Guldbrand, Stina, 1970, et al.
(author)
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Measuring the diffusion of fluorophores in human skin by two-photon fluorescence correlation spectroscopy combined with measurements of point spread function
- 2011
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In: MULTIPHOTON MICROSCOPY scigloo.IN THE BIOMEDICAL SCIENCES XI Book Series: Proceedings of SPIE. ; 7903, s. 7903291-7903296
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Journal article (peer-reviewed)abstract
- Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been used in combination with measurements of the point spread function (PSF), for quantitative analysis of fluorophores in excised human skin. Measurements have been performed at depths between 0 and 40 μm. The PSF, measured as full width at half maximum, was found not to depend on the depth. Measurements revealed difference in diffusion coefficient depending on extra- or intracellular location of fluorophore. The number of molecules was accumulating close to the surface and then decreased by the depth. The results from our study show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
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| 6. |
- Guldbrand, Stina, 1970, et al.
(author)
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Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin
- 2010
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In: Optics Express. - 1094-4087. ; 18:15, s. 15289-15302
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Journal article (peer-reviewed)abstract
- Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 ± 0.05 μm in the lateral direction, and 1.2 ± 0.4 μm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 × 10 −8 cm2/s. The diffusion coefficient is not dependent on depths down to 40 μm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
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| 7. |
- Guldbrand, Stina, 1970, et al.
(author)
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Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin
- 2013
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In: European Journal of Pharmaceutics and Biopharmaceutics. - : Elsevier BV. - 0939-6411. ; 84:2, s. 430-436
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Journal article (peer-reviewed)abstract
- There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity. The measurements were performed at tissue depths in the range 0 and 20 pm, and the diffusion coefficients at skin depths 5 and 10 mu m were found to be significantly different (P < 0.05). Overall median values for the diffusion coefficients were found to be 4.0 x 10(-13) m(2)/s and 2.0 x 10(-13) m(2)/s for RB and RBITC, respectively. These values correspond to the diffusion of a hard sphere with a volume eight times larger for RBITC compared to RB. This indicates that the RBITC have bound to biomolecules in the skin, and the measured signal is obtained from the RBITC-biomolecule complexes, demonstrating the potential of the TPM-FCS method to track molecular interactions in an intricate biological matrix such as human skin.
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| 8. |
- Kandoth, N., et al.
(author)
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Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles
- 2014
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In: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 15:5, s. 1768-1776
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Journal article (peer-reviewed)abstract
- We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a β-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen (1O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells. The combination of steady-state and time-resolved spectroscopic and photochemical techniques shows that the two photoresponsive guests do not interfere with each other while being enclosed in their supramolecular container and can thus be operated in parallel under control of light stimuli. Specifically, two-photon fluorescence microscopy allows mapping of the nanoassembly, here applied to epidermal cancer cells. By detecting the red emission from the phthalocyanine fluorophore it was also possible to investigate the tissue distribution after topical delivery onto human skin ex vivo. Irradiation of the nanoassembly with visible light triggers the simultaneous delivery of cytotoxic 1O 2 and NO, resulting in an amplified cell photomortality due to a combinatory effect of the two cytotoxic agents. The potential of dual therapeutic photodynamic action and two-photon fluorescence imaging capability in a single nanostructure make this system an appealing candidate for further studies in biomedical research. © 2014 American Chemical Society.
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| 9. |
- Kantere, Despoina, et al.
(author)
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Anti-Stokes fluorescence from endogenously formed protoporphyrin IX - Implications for clinical multiphoton diagnostics.
- 2013
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In: Journal of biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 6:5, s. 409-415
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Journal article (peer-reviewed)abstract
- Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two-photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one-photon anti-Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
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| 10. |
- Kirejev, Vladimir, 1984, et al.
(author)
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A polymer-based nanodevice for the photoregulated release of NO with two-photon fluorescence reporting in skin carcinoma cells
- 2014
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In: Journal of Materials Chemistry B. - : Royal Society of Chemistry (RSC). - 2050-750X .- 2050-7518. ; 2:9, s. 1190-1195
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Journal article (peer-reviewed)abstract
- We have developed a multifunctional biocompatible nanoconstruct based on polymeric nanoparticles encapsulating a molecular conjugate, able to photorelease nitric oxide (NO) with a fluorescent reporting function. We demonstrate that two-photon excitation (TPE) using biofriendly NIR 700 nm laser light can be applied for monitoring as well as triggering the release of NO, wherein the uncaging of a strongly fluorescent co-product acts in turn as a TPE fluorescent reporter for the simultaneous NO release from the nanoassembly. This supramolecular nanodevice internalizes in skin carcinoma cells, induces significant cell death upon light excitation and preserves its TPE properties, allowing the nearly instantaneous quantification of the NO photoreleased in cancer cells by two-photon NIR fluorescence microscopy. © 2014 The Royal Society of Chemistry.
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