Claudia Scalfi-Happ

Evaluation of photodynamic treatment using aluminum phthalocyanine tetrasulfonate chloride as a photosensitizer: new approach

Photodynamic therapy (PDT) has been the subject of several clinical studies. Evidence to date suggests that direct cell death may involve apoptosis. T(24) cells (bladder cancer cells, ATCC-Nr. HTB-4) were subjected to PDT with aluminum phthalocyanine tetrasulfonate chloride (AlS(4)Pc-Cl) and red laser light at 670 nm. Morphological changes after PDT were visualized under confocal microscopy. Raman microspectroscopy is considered as one of the newly established methods used for the detection of cytochrome c as an apoptotic marker. Results showed that PDT treated T(24) cells seem to undergo apoptosis after irradiation with 3 J cm(-2). Cytochrome c could not be detected from cells incubated with AlS(4)Pc-Cl using Raman spectroscopy whereas AlS(4)Pc-Cl seems to interfere with the Raman spectrum of cytochrome c.

FLIM and SLIM for molecular imaging in PDT

Various problems arising during molecular imaging of different fluoroprobes and metabolites used in photodynamic therapy could be circumvented by focusing on time-resolved detection. For this, an interesting new method seems to be time-correlated single photon counting, where a time-to-amplitude converter determines the temporal position and a scanning interface connected to the scanning unit of a laser microscope determines the spatial location of a signal. In combination with spectral resolved detection (spectral lifetime imaging) the set-up achieves the features of highly sophisticated lifetime imaging systems. The photoactive substance on which 5-ALA PDT is based, is protoporphyrine IX which is synthesized in mitochondria. Alternatively, other metabolites from 5-ALA could be involved. Subcellular differentiation of those metabolites without extensive extraction procedures is not trivial, because of highly overlapping spectral properties. Measuring the fluorescence lifetime on a subcellular level could be a successful alternative. To record lifetime images (τ-mapping) a setup consisting on a laser scanning microscope equipped with detection units for time-correlated single photon counting and ps diode lasers for short-pulsed excitation was implemented. The time-resolved fluorescence characteristics of 5-ALA metabolites were investigated in solution and in cell culture. The lifetimes were best fitted by a biexponential fitting routine. Different lifetimes could be found in different cell compartments. During illumination, the lifetimes decreased significantly. Different metabolites of 5-ALA could be correlated with different fluorescence lifetimes. In addition cells were coincubated with the nuclear staining dye DAPI, in order to investigate the cell cycle. Using appropriate filtering or alternatively spectral lifetime imaging the time-resolved fluorescence of DAPI could be very well distinguished from 5-ALA-metabolites. In contrast to ALA, the lifetime of DAPI, which was best fitted monoexponentially did not change during photobleaching, making this dye a perfect internal standard.

Confocal Raman microscopy for investigation of the level of differentiation in living neuroblastoma tumor cells

The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods. In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumor in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor neuroblastoma cell differentiation. Neuroblastoma cells at different degrees of differentiation were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation. Integration time per spectrum was 80-100 ms. A lateral resolution in submicrometer range was achieved by using a 60x water immersion lens with a numerical aperture of 1,0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands, by basis analysis using reference spectra or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images providing insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for differentiation. The results of this work may have applications in the monitoring of molecular changes and distribution of biomolecules and in particular of low molecular weight markers as they occur during the differentiation of neuroblastoma cells.

Chlorin Nanoparticles for Tissue Diagnostics and Photodynamic Therapy

BACKGROUND Organic crystalline nanoparticles (NPs) are not fluorescent due to the crystalline structure of the flat molecules organized in layers. In earlier experiments with Aluminum Phthalocyanine (AlPc)-derived NPs, the preferential uptake and dissolution by macrophages was demonstrated [3]. Therefore, inflamed tissue or cancer tissue with accumulated macrophages may exhibit specific fluorescence in contrast to healthy tissue which does not fluoresce. The present study addresses the photobiological effects of NP generated from Temoporfin (mTHPC), a clinically utilized photosensitizer belonging to the chlorin family. METHODS In-vitro investigations addressing uptake, dissolution and phototoxicity of mTHPC NP vs. the liposomal mTHPC formulation Foslip were performed using J774A.1 macrophages and L929 fibroblasts. For total NP uptake analysis, the cells were lysed, the nanoparticles dissolved and the fluorescence quantified. The intracellular molecular dissolution was measured by flow cytometry. Fluorescence microscopy served for controlling intracellular localization of the dissolved fluorescing molecules. Reaction mechanisms after PDT (mitochondrial activity, apoptosis) were analyzed using fluorescent markers in cell-based assays and flow cytometry. RESULTS Organic crystalline NP of different size were produced from mTHPC raw material. NP were internalized more efficiently in J774A.1 macrophages when compared to L929 fibroblasts, whereas uptake and fluorescence of Foslip was similar between the cell lines. NP dissolution correlated with internalization levels for larger particles in the range of 200-500 nm. Smaller particles (45 nm in diameter) were taken up at high levels in macrophages, but were not dissolved efficiently, resulting in comparatively low intracellular fluorescence. Whereas Foslip was predominantly localized in membranes, NP-mediated fluorescence also co-localized with acidic vesicles, suggesting endocytosis/phagocytosis as a major uptake mechanism. In macrophages, phototoxicity of NPs was stronger than in fibroblasts, even exceeding Foslip when administered in identical amounts. In both cell lines, phototoxicity correlated with mitochondrial depolarization and enhanced activation of caspase 3. CONCLUSIONS Due to their preferential uptake/dissolution in macrophages, mTHPC NP may have potential for the diagnosis and photodynamic treatment of macrophage-associated disorders such as inflammation and cancer.

Raman and fluorescence microscopy to study the internalization and dissolution of photosensitizer nanoparticles into living cells

In this present study we applied Raman and fluorescence microscopy to investigate the internalisation, cellular distribution and effects on cell metabolism of photosensitizer nanoparticles for photodynamic therapy in fibroblasts and macrophages.

Label-Free Detection of Tumor Markers in a Colon Carcinoma Tumor Progression Model by Confocal Raman Microspectroscopy

Living colon carcinoma cells were investigated by confocal Raman microspectroscopy. An in vitro model of tumor progression was established. Evaluation of data sets by cluster analysis reveals that lipid bodies might be a valuable diagnostic parameter for early carcinogenesis.

Photodynamic activity of Temoporfin nanoparticles induces a shift to the M1-like phenotype in M2-polarized macrophages

The monocyte/macrophage cell lineage reveals an enormous plasticity, which is required for tissue homeostasis, but is also undermined in various disease states, leading to a functional involvement of macrophages in major human diseases such as atherosclerosis and cancer. We recently generated in vivo evidence that crystalline, nonfluorescent nanoparticles of the hydrophobic porphyrin-related photosensitizer Aluminum phthalocyanine are selectively dissolved and thus may be used for specific fluorescent labelling of rejected, but not of accepted xenotransplants. This led us to hypothesize that nanoparticles made of planar photosensitizers such as porphyrins and chlorins were preferentially taken up and dissolved by macrophages, which was verified by in vitro studies. Here, using an in vitro system for macrophage differentiation/polarization of the human monocyte THP-1 cell line, we demonstrate differential uptake/dissolution of Temoporfin-derived nanoparticles in polarized macrophages, which resulted in differential photosensitivity. More importantly, low dose photodynamic sensitization using Temoporfin nanoparticles can be used to trigger M1 re-polarization of THP-1 cells previously polarized to the M2 state. Thus, sublethal photodynamic treatment using Temoporfin nanoparticles might be applied to induce a phenotypic shift of tumor-associated macrophages for the correction of an immunosuppressive microenvironment in the treatment of cancer, which may synergize with immune checkpoint inhibition.

Improving fluorescence diagnosis of cancer by SLIM

Although during the last years, significant progress was made in cancer diagnosis, using either intrinsic or specially designed fluorophores, still problems exist, due to difficulties in spectral separation of highly overlapping probes or in lack of specificity. Many of the problems could be circumvented by focusing on time-resolved methods. In combination with spectral resolved detection (spectral fluorescence lifetime imaging, SLIM) highly sophisticated fluorescence lifetime imaging can be performed which might improve specificity of cell diagnosis. To record lifetime images (τ-mapping) with spectral resolution a setup was realized consisting of a laser scanning microscope equipped with a 16 channel array for time-correlated single photon counting (TCSPC) and a spectrograph in front of the array. A Ti:Saphir laser can be used for excitation or alternatively ps diode lasers. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in solution and in cell culture. As an example, not only the mitochondria staining dye rhodamine 123 could be easily distinguished from DAPI, which intercalates into nucleic acids, but also different binding sites of DAPI. This was proved by the appearance of different lifetime components within different spectral channels. Another example is Photofrin, a photosensitizer which is approved for bladder cancer and for palliative lung and esophageal cancer in 20 countries, including the United States, Canada and many European countries. Photofrin is a complex mixture of different monomeric and aggregated porphyrins. The phototoxic efficiency during photodynamic therapy (PDT) seems to be correlated with the relative amounts of monomers and aggregates. With SLIM different lifetimes could be attributed to various, spectrally highly overlapping compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during Photofrin-PDT.

Imaging time-resolved fluorescence characteristics of organelle specific fluorophores and photosensitizers using ps pulsed diode lasers and TCSPC techniques

A time-correlated single photon counting (TCSPC) module (SPC-730, Becker & Hickl, Germany) was connected to a laser scanning microscope (Zeiss, Germany) equipped with an ultrafast photomultiplier. Short pulse excitation was achieved with two laser diodes emitting at 398nm and 434nm with a pulse duration of 70ps and 60 ps (PicoQuant, Germany) to allow intracellular fluorescence lifetime imaging (FLIM). With this setup, fluorescence lifetime of the mitochondrial marker Rhodamine 123 could be studied in solution under the same instrumental conditions as used for fluorescence lifetime imaging of cell monolayers. With the same set of parameters, fluorescence lifetime of Rhodamine 123 was calculated with good reproducibility in mitochondria of living cells. We present here a comparison of different fitting routines, including a multiexponential fitting based on the method of Laplace transformation. Fluorescence lifetimes calculated with the multiexponential fitting routine proved to be particularly useful to study the distribution of 5-ALA metabolites in cell monolayers.

Thermal effects in IR-laser-irradiated living cells

Irradiation of cell-layers with focussed 2.8 μm ir-laser allows to control the cell temperature from room temperature up to 100°C. Temperatures were calculated for a cell culture model and verified experimentally by thermal mapping of the cell-surrounding medium by means of thermochromic liquid crystals (TLC). Irradiation power and time were varied and associated biological effects like necrosis and apoptosis were observed with respect to the irradiation dosis.