Project description:Arabidopsis thaliana cell suspension cultures (ACSC) were subjected to 30-min, mild chemical treatments with three different singlet oxygen elicitors at low-medium light conditions (150 µE m–2 s–1) with the aim of getting a better understanding of singlet oxygen-mediated defence responses in plants. The three elicitors Indigo Carmine (IC), Methylene Violet (MV) and Rose Bengal (RB) at a concentration of 0.5 µM were chosen because they exhibited different abilities to permeate the plasma membrane and to accumulate in the cell soma or organelles such as chloroplasts. In addition, ACSC were treated with 500 µM H2O2 for comparison. Confocal image analysis of Arabidopsis cells revealed that IC was not retained in cells, whereas MV and RB permeated the plasma membrane and accumulated in the chloroplast envelope and inside chloroplasts, respectively. As a consequence of their different cellular location, the physiological, transcriptional and photosynthetic responses of Arabidopsis cells to singlet oxygen production varied from each other and the activation of programmed cell death (PCD) was observed in ACSC treated with 0.5 µM RB, but not with the other elicitor nor with 500 µM H2O2. The role of chloroplasts in the activation of PCD was further investigated when this physiological response was analyzed in dark-grown cell cultures containing undifferentiated plastids. Interestingly, PCD was only activated in light-grown, but not in dark-grown, Arabidopsis cell cultures, suggesting that singlet oxygen-mediated defence responses were initiated inside chloroplasts. Genome-wide transcriptional profile analyses were performed as well and the results proved that there were only statistically significant changes in the transcript expression of light-grown ACSC treated with 0.5 µM RB and 500 µM H2O2, but not with IC nor with MV. Functional enrichment analyses revealed that GO/Biological process terms associated with defence responses were common in the treatments with 0.5 µM RB and 500 µM H2O2; however, resistance response to pathogen and PCD terms were only significantly over-represented in the RB treatment. Moreover, the analysis of the up-regulated transcripts in ACSC treated with 0.5 µM RB brought out that both specific markers for singlet oxygen from the conditional fluorescence (flu) mutant of Arabidopsis and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present, although there was no evidence for the up-regulation of EDS1 encoding the ENHANCED DISEASE SUSCEPTIBILITY PROTEIN 1. Finally, a co-regulation analysis proved that ACSC treated with 0.5 µM RB exhibited higher correlation with the flu family mutants than with other singlet oxygen producer mutants of Arabidopsis or wild-type plants of Arabidopsis subjected to high light treatments, where singlet oxygen was produced in photosystem II and an acclimatory response was activated instead of PCD. 18 Samples total. Six Samples are controls, 3 Samples each treated with Rose Bengal, Hydrogen Peroxide, Methyl Violet, and Indigo Carmine.

Project description:Cutaneous melanoma is one of the most aggressive and metastatic forms of skin cancer. However, current therapeutic options present several limitations, and the annual death rate due to melanoma increases every year. Dermal delivery of nanomedicines can effectively eradicate primary melanoma lesions, avoid the metastatic process, and improve survival. Rose Bengal (RB) is a sono-photosensitizer drug with intrinsic cytotoxicity toward melanoma without external stimuli but the biopharmaceutical profile limits its clinical use. Here, we propose deformable lipid nanovesicles, also known as transfersomes (TF), for the targeted dermal delivery of RB to melanoma lesions to eradicate them in the absence of external stimuli. Considering RB's poor ability to cross the stratum corneum and its photosensitizer nature, transfersomal carriers were selected simultaneously to enhance RB penetration to the deepest skin layers and protect RB from undesired photodegradation. RB-loaded TF dispersion (RB-TF), prepared by a modified reverse-phase evaporation method, were nanosized with a ζ-potential value below -30 mV. The spectrophotometric and fluorimetric analysis revealed that RB efficiently interacted with the lipid phase. The morphological investigations (transmission electron microscopy and small-angle X-ray scattering) proved that RB intercalated within the phospholipid bilayer of TF originating unilamellar and deformable vesicles, in contrast to the rigid multilamellar unloaded ones. Such outcomes agree with the results of the in vitro permeation study, where the lack of a burst RB permeation peak for RB-TF, observed instead for the free drug, suggests that a significant amount of RB interacted with lipid nanovesicles. Also, RB-TF proved to protect RB from undesired photodegradation over 24 h of direct light exposure. The ex vivo epidermis permeation study proved that RB-TF significantly increased RB's amount permeating the epidermis compared to the free drug (78.31 vs 38.31%). Finally, the antiproliferative assays on melanoma cells suggested that RB-TF effectively reduced cell growth compared to free RB at the concentrations tested (25 and 50 μM). RB-TF could potentially increase selectivity toward cancer cells. Considering the outcomes of the characterization and cytotoxicity studies performed on RB-TF, we conclude that RB-TF represents a valid potential alternative tool to fight against primary melanoma lesions via dermal delivery in the absence of light.

Project description:Arabidopsis thaliana cell suspension cultures (ACSC) were subjected to 30-min, mild chemical treatments with three different singlet oxygen elicitors at low-medium light conditions (150 µE m–2 s–1) with the aim of getting a better understanding of singlet oxygen-mediated defence responses in plants. The three elicitors Indigo Carmine (IC), Methylene Violet (MV) and Rose Bengal (RB) at a concentration of 0.5 µM were chosen because they exhibited different abilities to permeate the plasma membrane and to accumulate in the cell soma or organelles such as chloroplasts. In addition, ACSC were treated with 500 µM H2O2 for comparison. Confocal image analysis of Arabidopsis cells revealed that IC was not retained in cells, whereas MV and RB permeated the plasma membrane and accumulated in the chloroplast envelope and inside chloroplasts, respectively. As a consequence of their different cellular location, the physiological, transcriptional and photosynthetic responses of Arabidopsis cells to singlet oxygen production varied from each other and the activation of programmed cell death (PCD) was observed in ACSC treated with 0.5 µM RB, but not with the other elicitor nor with 500 µM H2O2. The role of chloroplasts in the activation of PCD was further investigated when this physiological response was analyzed in dark-grown cell cultures containing undifferentiated plastids. Interestingly, PCD was only activated in light-grown, but not in dark-grown, Arabidopsis cell cultures, suggesting that singlet oxygen-mediated defence responses were initiated inside chloroplasts. Genome-wide transcriptional profile analyses were performed as well and the results proved that there were only statistically significant changes in the transcript expression of light-grown ACSC treated with 0.5 µM RB and 500 µM H2O2, but not with IC nor with MV. Functional enrichment analyses revealed that GO/Biological process terms associated with defence responses were common in the treatments with 0.5 µM RB and 500 µM H2O2; however, resistance response to pathogen and PCD terms were only significantly over-represented in the RB treatment. Moreover, the analysis of the up-regulated transcripts in ACSC treated with 0.5 µM RB brought out that both specific markers for singlet oxygen from the conditional fluorescence (flu) mutant of Arabidopsis and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present, although there was no evidence for the up-regulation of EDS1 encoding the ENHANCED DISEASE SUSCEPTIBILITY PROTEIN 1. Finally, a co-regulation analysis proved that ACSC treated with 0.5 µM RB exhibited higher correlation with the flu family mutants than with other singlet oxygen producer mutants of Arabidopsis or wild-type plants of Arabidopsis subjected to high light treatments, where singlet oxygen was produced in photosystem II and an acclimatory response was activated instead of PCD.

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Immune Modulation in Psoriasis Lesions by Topical PH-10 (Rose Bengal Disodium)

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