The effect of low-dose photodynamic therapy using the photosensitizer chloroaluminum phthalocyanine on a scratch wound model in skin fibroblasts.

Different approaches on wound healing have been developed over the years but they suffer from high costs and adverse effects for the patients. The current paper was designed to study low dose PDT, a novel healing approach, in an in vitro fibroblasts wound healing model. Chloroaluminum phthalocyanine (AlClPc) was used as photosensitizer and was activated by a red diode laser at 661 nm. After PDT optimization, wound closure rate and reactive oxygen species were quantified by image processing and analysis. Our results revealed that wound healing rates were significantly higher in PDT treated groups than in the control. Additionally, the study revealed that a prolonged ROS increase did not promote wound closure, while a small increase acted as a trigger, resulting in faster wound closure. Concluding, low dose PDT using AlClPc enhances wound healing in vitro in a ROS dependent manner, allowing the assumption of similar positive effects in vivo.

Photobiomodulation and Wound Healing: Low-Level Laser Therapy at 661 nm in a Scratch Assay Keratinocyte Model.

This study aims to investigate the effectiveness of low power red light (661 nm) in accelerating the wound healing process of an in vitro scratch assay model of keratinocytes. Furthermore, the study aims to clarify the role of light irradiation parameters, optimize them and gain additional insight into the mechanisms of wound closure as a result of photobiomodulation. Wound healing was studied using scratch assay model of NCTC 2544 keratinocytes. Cells were irradiated with a laser at various power densities and times. Images were acquired at 0, 24, 48 and 72 h following the laser treatment. Cellular proliferation was studied by MTT. ROS were studied at 0 and 24 h by fluorescence microscopy. Image analysis was used to determine the wound closure rates and quantify ROS. The energy range of 0.18-7.2 J/cm2 was not phototoxic, increased cell viability and promoted wound healing. Power and irradiation time proved to be more important than energy. The results indicated the existence of two thresholds in both power and irradiation time that need to be overcome to improve wound healing. An increase in ROS production was observed at 0 h only in the group with the lowest healing rate. This early response seemed to block proliferation and finally wound healing. Low level laser light at 661 nm enhanced both proliferation and migration in keratinocytes, providing evidence that it could possibly stimulate wound healing in vivo. The observed results are dependent on irradiance and irradiation time rather than energy dose in total.

Preparation and characterization of solid lipid nanoparticles incorporating bioactive coumarin analogues as photosensitizing agents.

Daphnetin (7,8-dihydroxy-coumarin, DAPH) is a naturally occurring coumarin presenting a wide array of biological activities. In the present study, daphnetin and its novel synthetic analogue 7,8-dihydroxy-4-methyl-3-(4-hydroxyphenyl)-coumarin (DHC) were encapsulated in solid lipid nanoparticles (SLNs) with Encapsulation Efficiency values of 80% and 40%, respectively. Nanoparticles of an average hydrodynamic diameter of approximately 250 nm were formed, showing a good stability in aqueous dispersion (polydispersity index 0.3-0.4), as determined by Dynamic Light Scattering (DLS). The SLNs were also characterized using Fourier Transform-Infrared (FT-IR) spectroscopy and Thermogravimetric Analysis (TGA). TEM images of the blank-SLNs indicated a spherical morphology and a size of 20-50 nm. The release studies of the coumarin analogues indicated a non-Fickian diffusion mechanism, while the release profiles better fitted on the Higuchi kinetic model. Moreover, the coumarin analogues and their SLNs were examined for their antioxidant activity using DPPH and anti-lipid peroxidation assays, exhibiting stronger antioxidant activity when encapsulated than in their free form. The coumarin derivatives and their SLNs were examined for their photodynamic therapy (PDT) efficacy against the human squamous carcinoma A431 cell line, with DHC coumarin both in its free and encapsulated form exhibiting significant PDT activity, reducing the cell viability to 11% after irradiation with a fluence rate of 2.16 J/cm2. Finally, the intracellular localization studies indicated the enhanced cellular uptake of the coumarin analogues when loaded in the SLNs.

Ιnclusion Complexes of Magnesium Phthalocyanine with Cyclodextrins as Potential Photosensitizing Agents.

In this work, the preparation of inclusion complexes, (ICs) using magnesium phthalocyanine (MgPc) and various cyclodextrins (ß-CD, γ-CD, HP-ß-CD, Me-ß-CD), using the kneading method is presented. Dynamic light scattering (DLS) indicated that the particles in dispersion possessed mean size values between 564 to 748 nm. The structural characterization of the ICs by infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy provides evidence of the formation of the ICs. The release study of the MgPc from the different complexes was conducted at pH 7.4 and 37 °C, and indicated that a rapid release ("burst effect") of ~70% of the phthalocyanine occurred in the first 20 min. The kinetic model that best describes the release profile is the Korsmeyer-Peppas. The photodynamic therapy studies against the squamous carcinoma A431 cell line indicated a potent photosensitizing activity of MgPc (33% cell viability after irradiation for 3 min with 18 mW/cm2), while the ICs also presented significant activity. Among the different ICs, the γ-CD-MgPc IC exhibited the highest photokilling capacity under the same conditions (cell viability 26%). Finally, intracellular localization studies indicated the enhanced cellular uptake of MgPc after incubation of the cells with the γ-CD-MgPc complex for 4 h compared to MgPc in its free form.

The effects of low power laser light at 661 nm on wound healing in a scratch assay fibroblast model.

Wound treatment, especially for chronic and infected wounds, has been a permanent socio-economical challenge. This study aimed to investigate the ability of red light at 661 nm to accelerate wound healing an in vitro wound model using 3T3 fibroblasts. The purpose is further specified in clarifying the mechanisms of wound closure by means of intracellular ROS production, proliferation and migration of cells, and cellular orientation. Illumination effects of red light from a diode laser (661 nm) at different doses on 3T3 cell viability was assessed via MTT assay and tested in a scratch wound model. Wound closure rates were calculated by image analysis at 0, 24, and 48 h after laser treatment. ROS production was monitored and quantified immediately and 24 h after the treatment by fluorescence microscopy. Cellular orientation was quantified by image analysis. No phototoxic energy doses used and increased cell viability in most of the groups. Scratch assay revealed an energy interval of 3 - 4.5 J/cm2 that promote higher wound healing rate 24 h post treatment. An increase in ROS production was also observed 24 h post irradiation higher in the group with the highest wound healing rate. Also, cellular orientation toward the margin of the wound was observed and quantified after irradiation. Low power laser light at 661 nm activated both the migration and proliferation in the in vitro model used, providing evidence that it could also accelerate wound healing in vivo. Also, ROS production and cellular orientation seem to play an important role in wound healing process.

Assessment of natural antioxidants' effect on PDT cytotoxicity through fluorescence microscopy image analysis.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) is a cancer treatment modality mediated by reactive oxygen species (ROS). However, the intracellular antioxidant defense system antagonizes PDT-generated ROS, impeding PDT efficacy. This study aimed to evaluate the enhancement of PDT cytotoxicity by its combination with natural antioxidants in pro-oxidant concentrations. METHODS: A rich natural antioxidant mixture originating from Pinus halepensis bark extract was studied for its potential to enhance the efficacy of m-tetrahydroxyphenylchlorin (m-THPC)-PDT on LNCaP prostate cancer cells, in vitro. Various P. halepensis concentrations, at two different incubation times, were used in combination with m-THPC-PDT. Assessment of cellular viability and intracellular ROS levels evaluated the treatments' outcome. A novel method was developed for the assessment of the intracellular ROS levels, based on image analysis and data extraction from fluorescence microscopy images. RESULTS: P. halepensis bark extract increased the intracellular ROS levels in a concentration-dependent but not in an incubation-dependent manner. The higher concentrations used (≥50 µg/ml) reduced cellular viability even by 50%. One hour pretreatment with 30 µg/ml P. halepensis before m-THPC-PDT exceeded the levels of cellular death by approximately 15%. CONCLUSIONS: The results provided evidence of the cytotoxic effect of P. halepensis bark extract on LNCaP cells, showing the potential of P. halepensis to be used as an anticancer agent in prostate cancer treatment. The results also provided evidence of enhancement of m-THPC-PDT by P. halepensis bark extract showed the potential to be used as a supplementary agent to improve prostate cancer PDT treatment.

Atomic Force Microscopy on Biological Materials Related to Pathological Conditions.

Atomic force microscopy (AFM) is an easy-to-use, powerful, high-resolution microscope that allows the user to image any surface and under any aqueous condition. AFM has been used in the investigation of the structural and mechanical properties of a wide range of biological matters including biomolecules, biomaterials, cells, and tissues. It provides the capacity to acquire high-resolution images of biosamples at the nanoscale and allows at readily carrying out mechanical characterization. The capacity of AFM to image and interact with surfaces, under physiologically relevant conditions, is of great importance for realistic and accurate medical and pharmaceutical applications. The aim of this paper is to review recent trends of the use of AFM on biological materials related to health and sickness. First, we present AFM components and its different imaging modes and we continue with combined imaging and coupled AFM systems. Then, we discuss the use of AFM to nanocharacterize collagen, the major fibrous protein of the human body, which has been correlated with many pathological conditions. In the next section, AFM nanolevel surface characterization as a tool to detect possible pathological conditions such as osteoarthritis and cancer is presented. Finally, we demonstrate the use of AFM for studying other pathological conditions, such as Alzheimer's disease and human immunodeficiency virus (HIV), through the investigation of amyloid fibrils and viruses, respectively. Consequently, AFM stands out as the ideal research instrument for exploring the detection of pathological conditions even at very early stages, making it very attractive in the area of bio- and nanomedicine.

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.

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.

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.

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. METHODS: AlClPc solution (0.7% w/v) was applied to tumors in mice for 1-6 h. The penetration depth and the optimum drug-light interval were assessed using pharmacokinetic studies. Then, PDT was performed on a murine model of non-melanoma skin cancer using seven different combinations of therapeutic parameters (fluence rate and energy dose). RESULTS: Pharmacokinetic studies revealed that AlClPc was absorbed 40 times more and penetrated 19 times deeper in tumors than normal skin. PDT using AlClPc (0.7% w/v) and a diode laser (75 mW/cm(2), 150 J/cm(2)) resulted in complete tumor remission in 60% of the mice, excellent cosmetic outcomes and growth retardation of tumors with partial remission. CONCLUSIONS: The results indicate that AlClPc-PDT is an effective treatment for non-melanoma skin carcinomas in experimental mouse models.

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.

A confocal microscopy study of the very early cellular response to oxidative stress induced by zinc phthalocyanine sensitization.

Oxidative stress has been involved in several biological and pathological processes. Reactive oxygen species have been shown to play both beneficial and deleterious roles. The present work contributes to the understanding of the very early events of cellular response to oxidative stress. Oxidative stress was produced intracellularly by light activation of zinc phthalocyanine (ZnPc) at a light dose that did not lead to apoptosis or necrosis. Phthalocyanine was photoactivated using the 647-nm laser line of a confocal microscope through the objective lens causing oxidative stress and allowing observation of the evoked phenomena at the single cell level and in real time. Mitochondria membrane potential (DeltaPsi(m)), intracellular pH, calcium concentration, and generation of reactive oxygen species (ROS) were recorded using specific vital fluorescent probes and quantified by image processing and analysis. Subcellular localization of ZnPc was also studied in order to determine the primary and intermediate ROS target.

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.