Eleni Alexandratou

Human fibroblast alterations induced by low power laser irradiation at the single cell level using confocal microscopy

Low power laser irradiation is regarded to have a significant role in triggering cellular proliferation and in treating diseases of diverse etiologies. The present work contributes to the understanding of the mechanisms of action by studying low power laser effects in human fibroblasts. Confocal laser scanning microscopy is used for irradiation and observation of the same area of interest allowing the imaging of laser effects at the single cell level and in real time. Coverslip cultures were placed in a small incubation chamber for in vivo microscopic observation. Laser stimulation of the cells was performed using the 647 nm line of the confocal laser through the objective lens of the microscope. Mitochondrial membrane potential (delta psi(m)), intracellular pH, calcium alterations and generation of reactive oxygen species (ROS) were monitored using specific fluorescent vital probes. The induced effects were quantified using digital image processing techniques. After laser irradiation, a gradual alkalinization of the cytosolic pH and an increase in mitochondrial membrane potential were observed. Recurrent spikes of intracellular calcium concentration were also triggered by laser. Reactive oxygen species were generated as a result of biostimulation. No such effects were monitored in microscopic fields other than the irradiated ones.

Topical photodynamic therapy of murine non-melanoma skin carcinomas with aluminum phthalocyanine chloride and a diode laser: pharmacokinetics, tumor response and cosmetic outcomes

Background/purpose: Topical photodynamic therapy (PDT) is potentially useful for the treatment of non‐melanoma skin cancer and other skin diseases. We investigated the therapeutic effects of PDT using topical application of aluminum phthalocyanine chloride (AlClPc) and a diode laser emitting at 670 nm in murine non‐melanoma skin carcinomas.

Comparative characterization of the cellular uptake and photodynamic efficiency of Foscan® and Fospeg in a human prostate cancer cell line

BACKGROUND m-THPC (Foscan(®)) is one of the most potent second generation photosensitizers used in photodynamic therapy, photoactivated at higher wavelengths (652 nm). However, its strongly hydrophobic nature causes aggregation of the molecules and prevents its unbiased bioavailability in the biological media, resulting in lower accumulation in the tumor cells. Several strategies have been adopted to improve the photodynamic characteristics of the photosensitizer. Among them, very promising seems to be the encapsulation of the molecule into liposomes, due to the superior properties of liposomes as drug carriers. METHODS In this paper the photodynamic characteristics of the PEGylated liposomal formulation of m-THPC, Fospeg, using the human prostate cancer cell line LNCaP, as an in vitro model, were investigated. In addition the spectral characteristics, cellular uptake and localization, dark and light induced cytotoxicity and photodynamic efficacy of Foscan(®) and Fospeg were compared. RESULTS Fospeg, compared with Foscan, showed higher intracellular uptake at any concentration and incubation time. Regarding PDT efficacy, Fospeg produced more severe cytotoxicity than Foscan(®) at any concentration and energy dose. Using Fospeg, the lowest concentration (0.22 μM) and energy dose (180 mJ/cm(2)) was adequate to result in the death of 50% of the cells 24h post PDT while an approximately 10 times higher Foscan(®) concentration (1.8 μM) was needed to result in the same cytotoxicity. CONCLUSIONS The use of the PEGylated liposomal formulation of m-THPC resulted in the improvement of its intracellular uptake and the enhancement of its photodynamic activity. Fospeg, compared to Foscan(®), proved to be a more advantageous photosensitizer for photodynamic therapy.

Fluorescence and absorption assessment of a lipid mTHPC formulation following topical application in a non-melanotic skin tumor model.

Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [+/- standard deviation (SD)] mTHPC concentration within lesions of 6.0(+/-3.1) ngmg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.

Investigation of the influence of UV irradiation on collagen thin films by AFM imaging

Collagen is the major fibrous extracellular matrix protein and due to its unique properties, it has been widely used as biomaterial, scaffold and cell-substrate. The aim of the paper was to use Atomic Force Microscopy (AFM) in order to investigate well-characterized collagen thin films after ultraviolet light (UV) irradiation. The films were also used as in vitro culturing substrates in order to investigate the UV-induced alterations to fibroblasts. A special attention was given in the alteration on collagen D-periodicity. For short irradiation times, spectroscopy (fluorescence/absorption) studies demonstrated that photodegradation took place and AFM imaging showed alterations in surface roughness. Also, it was highlighted that UV-irradiation had different effects when it was applied on collagen solution than on films. Concerning fibroblast culturing, it was shown that fibroblast behavior was affected after UV irradiation of both collagen solution and films. Furthermore, after a long irradiation time, collagen fibrils were deformed revealing that collagen fibrils are consisting of multiple shells and D-periodicity occurred on both outer and inner shells. The clarification of the effects of UV light on collagen and the induced modifications of cell behavior on UV-irradiated collagen-based surfaces will contribute to the better understanding of cell-matrix interactions in the nanoscale and will assist in the appropriate use of UV light for sterilizing and photo-cross-linking applications.

Nanotopography of collagen thin films in correlation with fibroblast response

Abstract. Collagen thin films consisting of randomly oriented and oriented collagen fibrils/fibers are fabricated by hydrodynamic flow and spin coating, and then they are characterized by atomic force microscopy (AFM). Fibroblasts are cultured on these films in order to correlate their morphology and alignment, which are assessed with fluorescence and AFM imaging with different film characteristics. The results showed that the formed films could be used as substrates for culturing cells. Furthermore, cells reacted to film nanocharacteristics and especially to the orientation of fibrils/fibers. The investigation of the influence that the substrate nanotopography has on cells will help to elucidate the mechanisms of cell–biomaterial interactions, and will enable the design of intelligent coatings for implants and tissue engineering purposes.

Computer vision algorithms in DNA ploidy image analysis

The high incidence and mortality rates of prostate cancer have stimulated research for prevention, early diagnosis and appropriate treatment. DNA ploidy status of tumour cells is an important parameter with diagnostic and prognostic significance. In the current study, DNA ploidy analysis was performed using image cytometry technique and digital image processing and analysis. Tissue samples from prostate patients were stained using the Feulgen method. Images were acquired using a digital imaging microscopy system consisting of an Olympus BX-50 microscope equipped with a color CCD camera. Segmentation of such images is not a trivial problem because of the uneven background, intensity variations within the nuclei and cell clustering. In this study specific algorithms were developed in Matlab based on the most prominent image segmentation approaches that emanate from the field of Mathematical Morphology, focusing on region-based watershed segmentation. First biomedical images were simplified under non-linear filtering (alternate sequential filters, levelings), and next image features such as gradient information and markers were extracted so as to lead the segmentation process. The extracted markers are used as seeds; watershed transformation was performed to the gradient of the filtered image. Image flooding was performed isotropically from the markers using hierarchical queues based on Beucher and Meyer methodology. The developed algorithms have successfully segmented the cell from its background and from cells clusters as well. To characterize the nuclei, we attempt to derive a set of effective color features. By analyzing more than 50 color features, we have found that a set of color features, hue, saturation-weighted hue, I1=(R+G+B)/3, I2=(R-B),I3=(2G-R-B)/2, Karhunen-Loeve transformation and energy operator, are effective.

mTHPC pharmacokinetics following topical administration

Measurements of concentration of sensitizers for photodynamic therapy can provide important information in the dosimetry planning and can also give input to the optimal time for treatment. There has been skepticism towards fluorescence techniques for this purpose, as the signal depends on the fluorescence yield and optical properties of the tissue. Absorption based techniques, lack on the other hand, often the sensitivity required for many sensitizers with relative weak absorption in a wavelength region where hemoglobin absorption is dominant. A direct comparison between absorption and fluorescence techniques for measuring mTHPC concentration after topical application on hairless SKH-1 mice bearing skin carcinomas has been performed. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumor area and the concentration of mTHPC was measured at 4 and 6 hours after drug application by two methods: 1. A fluorescence imaging system capturing images at two wavelengths (500 and 650 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 650 to 500 was calculated. 2. A diffuse reflectance spectroscopy system with a fiber separation of 2 mm, providing the absorbance at 652 nm. Both systems provided consistent results related to the photosensitizer concentration. The methods show a remarkable difference in the concentration of photosensitizer in normal skin and tumor. No significant difference in mTHPC concentration in tumor could be observed between the 4 and 6h groups after drug application.

Low-power laser effects at the single-cell level: a confocal microscopy study

Confocal microscopy was used for irradiation and observation of the same area of interest, allowing the imaging of low power laser effects in subcellular components and functions, at the single cell level. Coverslips cultures of human fetal foreskin fibroblasts (HFFF2) were placed in a small incubation chamber for in vivo microscopic observation. Cells were stimulated by the 647 nm line of the Argon- Krypton laser of the confocal microscope (0.1 mW/cm2). Membrane permeability, mitochondrial membrane potential ((delta) Psim), intracellular pHi, calcium alterations and nuclear chromatin accessibility were monitored, at different times after irradiation, using specific fluorescent vital probes. Images were stored to the computer and quantitative evaluation was performed using image- processing software. After irradiation, influx and efflux of the appropriate dyes monitored changes in cell membrane permeability. Laser irradiation caused alkalizatoin of the cytosolic pHi and increase of the mitochondrial membrane potential ((delta) Psim). Temporary global Ca2+ responses were also observed. No such effects were noted in microscopic fields other than the irradiated ones. No toxic effects were observed, during time course of the experiment.

Combination of Fospeg-IPDT and a natural antioxidant compound prevents photosensitivity in a murine prostate cancer tumour model

BACKGROUND The aim of the present research was to investigate the potential use of a natural compound rich in antioxidant agents, derived from Pinus halepensis (P. halepensis), to prevent PDT induced photosensitivity. The present research progressed in two levels. The first one evolved the optimization of Fospeg-interstitial photodynamic therapy (IPDT) in a prostate cancer animal model. In the second one, P. halepensis bark extract, was evaluated for its potential use to prevent photosensitivity. METHODS Two sets of experiments were performed, IPDT only and IPDT in the presence of antioxidant. For both of them, Fospeg was administrated intravenously to SCID mice bearing prostate cancer, followed by IPDT after 6 h. For the IPDT+antioxidant experiments, P. halepensis was injected intratumourously 1 h prior the tumour illumination. Treatment outcome was monitored twice a week by an imaging system and by measuring tumour dimensions using a caliper. Photosensitivity was assessed by monitoring erythema of the tail using the imaging system. RESULTS IPDT with Fospeg and 15 J total light energy is a therapeutic scheme that can eliminate tumours in the murine model of prostate cancer. Two months after complete tumour remission no tumour recurrence was observed. Also, the cosmetic outcome of the research was excellent. The major drawback of this treatment scheme was that 90% of the animals developed photosensitivity. The addition of P. halepensis bark extract resulted in prevention of the photosensitivity, leaving PDT outcome unaffected. CONCLUSIONS The combined use of PDT and the used antioxidant agent could broaden the implementation of photodynamic therapy, by eliminating photosensitivity.