9 research outputs found
Conjugation to the Cell-Penetrating Peptide TAT Potentiates the Photodynamic Effect of Carboxytetramethylrhodamine
Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents
Apoptosis- and necrosis-induced changes in light attenuation measured by optical coherence tomography
Optical coherence tomography (OCT) was used to determine optical properties of pelleted human fibroblasts in which necrosis or apoptosis had been induced. We analysed the OCT data, including both the scattering properties of the medium and the axial point spread function of the OCT system. The optical attenuation coefficient in necrotic cells decreased from 2.2βΒ±β0.3Β mmβ1 to 1.3βΒ±β0.6Β mmβ1, whereas, in the apoptotic cells, an increase to 6.4βΒ±β1.7Β mmβ1 was observed. The results from cultured cells, as presented in this study, indicate the ability of OCT to detect and differentiate between viable, apoptotic, and necrotic cells, based on their attenuation coefficient. This functional supplement to high-resolution OCT imaging can be of great clinical benefit, enabling on-line monitoring of tissues, e.g. for feedback in cancer treatment
Photosensitizers as Model Systems to Study Reactive Oxygen Effects in Biological Preparations
In vitro evaluation of the oxidation efficacy of transgingival photodynamic therapy
OBJECTIVE: To evaluate the capability of soft laser light to penetrate blood, serum, gingival connective tissue and pure collagen type I.
MATERIALS AND METHODS: A 1:1 mixture of methylene blue (MB) and diphenylisobenzofuran (DPBF) was irradiated for 60 s with a diode laser (670 nm, 0.3 W) through blood, serum, gingival connective tissue and collagen type I (2 mm transillumination thickness). The oxidation of DPBF by MB was determined spectrophotometrically by measuring the optical density (oD) at 410 nm. The absorption spectra of DPBF/MB irradiated through MB (1%) and strawberry red solution (3%) served as control.
RESULTS: The mean oD of non-irradiated DPBF/MB was 1.98 Β± 0.04. Irradiation through MB showed no oxidation of DPBF (1.98 Β± 0.02; p > 0.05), while interposition of strawberry red and serum resulted in almost complete oxidation of DPBF (0.13 Β± 0.09, 0.06 Β± 0.03; p β€ 0.0001). Irradiation through gingiva and collagen reduced the oxidation of DPBF significantly (1.0 Β± 0.04, 0.7 Β± 0.04; p β€ 0.0001), accounting for 50% to 35% of the non-irradiated DPBF/MB solution.
CONCLUSION: Red light from a diode laser can penetrate blood and gingival tissues. However, light absorption for collagen and connective tissue can hamper the oxidation process