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Lysaker, Norway

Wang J.T.-W.,University College London | Wang J.T.-W.,Kings College London | Berg K.,University of Oslo | Hogset A.,PCI Biotech AS | And 2 more authors.
Photochemical and Photobiological Sciences

This study investigated the photophysical and photobiological properties of a new amphiphilic chlorin photosensitiser, disulfonated tetraphenylchlorin (TPCS2a), for photochemical internalisation (PCI). The absorption and fluorescence spectra of TPCS2a were examined in a range of solvents together with fluorescence lifetime measurements. The fluorescence lifetime of TPCS2a was found to be 8.5 ns in methanol, whereas non-exponential decays were observed in distilled water due to sensitiser dimerisation. The singlet oxygen quantum yield of TPCS2a was determined as 0.62 in deuterated methanol by direct observation of singlet oxygen phosphorescence. In a human oral squamous carcinoma (HN5) cell line, intracellular co-localisation of TPCS2a and Alexa488-labelled saporin, a macromolecular toxin, was observed corresponding predominantly to a lysosomal distribution. Intracellular fluorescence redistribution of TPCS2a and Alexa488-saporin was observed after 405 nm irradiation. Using two-photon confocal microscopy at 840 nm, and fluorescence lifetime imaging (FLIM), the lifetime was measured as 6 ns in HN5 cells. PCI using TPCS2a was shown to be very effective, and a synergistic increase in saporin toxicity was achieved in HN5 cells where viability was significantly reduced after light exposure compared to saporin (25 nM) treatment alone. The results demonstrate the favourable photophysical and photobiological properties of TPCS2a for PCI, which induces the relocalisation of a macromolecular anti-cancer toxin inside cells and significantly enhances cell death. © 2013 The Royal Society of Chemistry and Owner Societies. Source

Bostad M.,University of Oslo | Bostad M.,Innovation Norway | Olsen C.E.,University of Oslo | Olsen C.E.,Innovation Norway | And 6 more authors.
Journal of Controlled Release

The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin (PCIAC133-saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133-saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133-saporin. PCI of picomolar levels of AC133-saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133-saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133-saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133-saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133-saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133-saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133 + cells in localized primary tumors or metastasis. © 2015 Elsevier B.V. Source

Berg K.,University of Oslo | Nordstrand S.,Synthetica AS | Selbo P.K.,University of Oslo | Tran D.T.T.,University of Oslo | And 2 more authors.
Photochemical and Photobiological Sciences

Photochemical internalisation (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers that locate in endocytic vesicles, and that upon activation by light induce a release of macromolecules from the endocytic vesicles. PCI has been shown to stimulate delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane. The preclinical evaluation of PCI has been performed with aluminum phthalocyanine disulfonate (AlPcS 2a) as photosensitizer. AlPcS 2a, due to its large number of isomers potentially with batch-to-batch ratio variations, is not an optimal photosenstizer for clinical use. Disulfonated tetraphenyl chlorin (TPCS 2a) has therefore been developed by di-imide reduction of disulfonated tetraphenyl porphine (TPPS 2a). The synthesized TPCS 2a contains 3 isomers as shown by HPLC with low (<4%) inter-batch variation with respect to isomer formation, less than 0.5% (w/w) of the starting material TPPS 2a and absorbs light at 652 nm. As prerequisites for a PCI photosensitizer TPCS 2a was found to localize in intracellular granules assumed to be endocytic vesicles. In cells in culture TPCS 2a-PCI induced activation of gelonin as seen by enhanced cytotoxicity, increased transfection efficacy by an enhanced green fluorescence protein (EGFP)-encoding plasmid, induced gene silencing by siRNA towards EGFP and induced in a synergistic manner tumor growth delay by TPCS 2a-mediated PCI of bleomycin in CT26.CL25 carcinomas growing subcutaneously in athymic mice. TPCS 2a-PCI of bleomycin was found superior to meso-tetraphenyl chlorin-based photodynamic therapy (mTHPC-PDT) with respect to inhibition of tumor growth. The tumor growth delay by PCI of bleomycin was independent of the time of bleomycin administration between 3 h prior to light to immediately after light, while bleomycin administered 24 h prior to or 24 h after the light exposure induced suboptimal or only additive effects on tumor growth delay respectively. TPCS 2a-PDT and -PCI induced indistinguishably strong edema the first 3-4 days after TPCS 2a-administration and only weak erythema the first day after TPCS 2a administration. In contrast, mTHPC-PDT induced moderate edema the first 7 days after mTHPC administration, but strong erythema resulting in open wounds and escar formation the first 2-3 days after mTHPC administration. The pharmacokinetic properties of TPCS 2a were evaluated in athymic mice. The plasma pharmacokinetics was best fit to a 2-compartment model with half-lives of 0.78 and 36 hrs. TPCS 2a was found to be a clinically suitable PCI photosensitizer for photochemical activation of molecules that do not readily penetrate the cellular plasma membrane. © The Royal Society of Chemistry and Owner Societies 2011. Source

Hakerud M.,University of Zurich | Hakerud M.,Institute for Cancer Research | Selbo P.K.,Institute for Cancer Research | Waeckerle-Men Y.,University of Zurich | And 5 more authors.
Journal of Controlled Release

Cancer vaccines aim to induce CD8 T cells infiltrating the tumour. For protein-based vaccines, the main biological barrier to overcome is the default MHC class-II-pathway, with activation of CD4 T cells rather than CD8 T cells. The latter requires antigens to access the cytosol and MHC class I antigen presentation. We applied photosensitiser and light to trigger disruption of antigen-containing endosomes and thereby MHC class I cross-presentation of a model cancer vaccine. This "photochemical internalisation" resulted in activation, proliferation, and IFN-γ production of cytotoxic CD8 T cells, which suppressed tumour growth by infiltrating CD8 T cells and caspase-3-dependent apoptosis. The process was independent of MHC class II, MyD88, and TLR4 signalling, but dependent on trypsin- and caspase-like proteasome activity and partly also on chloroquine. This novel method of vaccination may find applications in cancer immunotherapy where the activation of CD8 T cells is important. © 2014 Elsevier B.V. All rights reserved. Source

The present invention relates to a method for introducing an siRNA molecule into the cytosol of a cell, said method comprising i) contacting said cell with an siRNA molecule, a carrier and a photosensitising agent, and ii) irradiating the cell with light of a wavelength effective to activate the photosensitising agent, wherein said carrier comprises a cationic polyamine such as a lipopolyamine in a non-liposomal formulation, polyethyleneimine (PEI), a betacyclodextrin amine polymer, an amine group containing dendrimer, and a cationic peptide. Cells or a population of cells obtainable by the method, a composition containing an siRNA molecule and the carrier molecule, kits and therapeutic uses of the above are also provided.

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