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.

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.

Synthesis, Bioactivity, Pharmacokinetic and Biomimetic Properties of Multi-Substituted Coumarin Derivatives.

A series of novel multi-substituted coumarin derivatives were synthesized, spectroscopically characterized, and evaluated for their antioxidant activity, soybean lipoxygenase (LOX) inhibitory ability, their influence on cell viability in immortalized human keratinocytes (HaCaT), and cytotoxicity in adenocarcinomic human alveolar basal epithelial cells (A549) and human melanoma (A375) cells, in vitro. Coumarin analogues 4a-4f, bearing a hydroxyl group at position 5 of the coumarin scaffold and halogen substituents at the 3-phenyl ring, were the most promising ABTS•+ scavengers. 6,8-Dibromo-3-(4-hydroxyphenyl)-4-methyl-chromen-2-one (4k) and 6-bromo-3-(4,5-diacetyloxyphenyl)-4-methyl-chromen-2-one (3m) exhibited significant lipid peroxidation inhibitory activity (IC50 36.9 and 37.1 µM). In the DCF-DA assay, the 4'-fluoro-substituted compound 3f (100%), and the 6-bromo substituted compounds 3i (80.9%) and 4i (100%) presented the highest activity. The 3'-fluoro-substituted coumarins 3e and 4e, along with 3-(4-acetyloxyphenyl)-6,8-dibromo-4-methyl-chromen-2-one (3k), were the most potent lipoxygenase (LOX) inhibitors (IC50 11.4, 4.1, and 8.7 µM, respectively) while displaying remarkable hydroxyl radical scavenging ability, 85.2%, 100%, and 92.9%, respectively. In silico docking studies of compounds 4e and 3k, revealed that they present allosteric interactions with the enzyme. The majority of the analogues (100 µΜ) did not affect the cell viability of HaCaT cells, though several compounds presented over 60% cytotoxicity in A549 or A375 cells. Finally, the human oral absorption (%HOA) and plasma protein binding (%PPB) properties of the synthesized coumarins were also estimated using biomimetic chromatography, and all compounds presented high %HOA (>99%) and %PPB (60-97%) values.

Novel 3-aryl-5-substituted-coumarin analogues: Synthesis and bioactivity profile.

Eighteen 3-aryl-5-substituted-coumarins-six 5-acetyloxy-derivatives, six 5-hydroxy-derivatives, and six 5-geranyloxy-derivatives-were synthesized, structurally characterized and their antioxidant activity, lipoxygenase inhibitory ability, as well as their cytotoxic activity against human neuroblastoma SK-N-SH and HeLa adenocarcinoma cell lines were evaluated. The 5-acetyloxy-compounds 3a-3f were found to be the best cytotoxic agents among all the compounds studied. The bromo-substituted coumarins 3a and 3b were remarkably active against HeLa cell line showing IC50 1.8 and 6.1 µM, respectively. Coumarin 5e possessing a geranyloxy-chain on position 5 of the coumarin scaffold presented dual bioactivity, while 5-geranyloxy-coumarin 5f was the most competent soybean lipoxygenase inhibitor of this series (IC50 10 µM). As shown by in silico docking studies, the studied molecules present allosteric interactions with soybean lipoxygenases.