Photosensitization of human skin fibroblasts by vemurafenib promotes pleiotropic effects on membrane-enclosed organelles and apoptosis

© 2022 Elsevier B.V. All rights reserved.Vemurafenib (VB), a BRAF inhibitor and a first-line treatment for unresectable or metastatic melanoma, is strongly phototoxic towards normal skin cells. Herein, we show that in cultured HS 68 human diploid dermal fibroblasts, low concentrations of VB suffice to promote photosensitization to low doses of UVA (∼ 5 J/cm2), as evidenced by a significant decrease in cell viability. In contrast to data obtained in chemico our results support a role for ROS (reactive oxygen species). Indeed, peroxidation of cellular lipids was observed which could be alleviated by the lipophilic antioxidant BHT (2,6-di-tert-butyl-4-methylphenol). Using in vivo confocal laser scanning microscopy and vital fluorescent probes it was shown at the single cell level that the plasma membrane and lipid-rich organelles, namely mitochondria, endoplasmic reticulum, and lysosomes, as well as actin filaments, were severely damaged by the UVA-induced VB-photosensitization. Finally, we showed that mitochondrial impairment was concurrent with caspase 3/7 activation and cell death by apoptosis.This work was supported by AbbVie, Lda., donation FPJ937, and Fundação para a Ciência e Tecnologia/FCT, Portugal (FCT PTDC/MEC-DER/30198/2017 FARM ID).info:eu-repo/semantics/publishedVersio

Enhanced fluorescence of a dye on DNA-assembled gold nanodimers discriminated by lifetime correlation spectroscopy

The surface plasmon modes of metal nanoparticles provide a way to efficiently enhance the excitation and emission from a fluorescent dye. We have employed DNA-directed assembly to prepare dimers of gold nanoparticles and used their longitudinally coupled plasmon mode to enhance the fluorescence emission of an organic red-emitting dye, Atto-655. The plasmon-enhanced fluorescence of this dye using dimers of 80 nm particles was measured at the single-molecule detection level. The top enhancement factors were above 1000-fold in 71% of the dimers within a total of 32 dimers measured, and in some cases, they reached almost 4000-fold, in good agreement with model simulations. Additionally, fluorescence lifetime correlation analysis enabled the separation of enhanced from nonenhanced emission simultaneously collected in our confocal detection volume. This approach allowed us to recover a short relaxation component exclusive to enhanced emission that is attributed to the interaction of the dye with DNA in the interparticle gaps. Indeed, the frequency of enhancement events is larger than that expected from the volume occupancy of the gap region, thus suggesting that the interaction of the dye with DNA linkers favors the observation of emission enhancement in our dimer particles

Fluorescence Spectroscopy of Porphyrins and Phthalocyanines: Some Insights into Supramolecular Self-Assembly, Microencapsulation, and Imaging Microscopy

The molecular interactions of anionic tetrasulfonate phenyl porphyrin (TPPS) with poly(amido amine) (PAMAM) dendrimers of generation 2.0 and 4.0 (G2 and G4, respectively) forming H- or J-aggregates, as well as with human and bovine serum albumin proteins (HSA and BSA), were reviewed in the context of self-assembly molecular complementarity. The spectroscopic studies were extended to the association of aluminum phthtalocyanine (AlPCS4) detected with a PAMAM G4 dendrimer with fluorescence studies in both steady state and dynamic state, as well as due to the fluorescence quenching associated to electron-transfer with a distribution of lifetimes. The functionalization of TPPS with peripheral substituents enables the assignment of spontaneous pH-induced aggregates with different and well-defined morphologies. Other work reported in the literature, in particular with soft self-assembly materials, fall in the same area with particular interest for the environment. The microencapsulation of TPPS studies into polyelectrolyte capsules was developed quite recently and aroused much interest, which is well supported and complemented by the extensive data reported on the Imaging Microscopy section of the Luminescence of Porphyrins and Phthalocyanines included in the present review

Enhanced-Fluorescence of a Dye on DNA- assembled Gold Nano-Dimers Discriminated by Lifetime Correlation Spectroscopy

We have employed DNA-directed assembly to prepare dimers of gold nanoparticles and used their longitudinally coupled plasmon mode to enhance the fluorescence emission of an organic red-emitting dye, Atto-655. The plasmon- enhanced fluorescence of this dye using dimers of 80 nm particles was measured at single molecule detection level. The top enhancement factors were above 1000-fold in 71% of the dimers within a total of 32 dimers measured, and, in some cases, they reached almost 4000-fold, in good agreement with model simulations. Additionally, fluorescence lifetime correlation analysis enabled the separation of enhanced from non-enhanced emission simultaneously collected in our confocal detection volume. This approach allowed us to recover a short relaxation component exclusive to enhanced emission that is attributed to the interaction of the dye with DNA in the interparticle gaps. </div

Fluorescent dye nano-assemblies by thiol attachment directed to the tips of gold nanorods for effective emission enhancement

The conjugation of dye-labelled DNA oligonucleotides with gold nanorods has been widely explored for the development of multifunctional fluorescent nanoprobes. Here, we show that the functionalization route is crucial to achieve enhanced emission in dye nano-assemblies based on gold nanorods. By using a tip-selective approach for thiol attachment of dye molecules onto gold nanorods, it was possible to effectively increase the emission by more than 10-fold relatively to that of a free dye. On the other hand, a non-selective approach revealed that indiscriminate surface functionalization has a detrimental effect on the enhancement. Simulations of discrete dipole approximation gave further insight into the surface distribution of plasmon-enhanced emission by confirming that tip regions afford an effective enhancement, while side regions exhibit a negligible effect or even emission quenching. The contrast between dye nano-assemblies obtained from tip- and non-selective functionalization was further characterized by single-particle fluorescence emission. These studies showed that tip-functionalized gold nanorods with an average of only 30 dye molecules have a comparable to or even stronger emission than non-selectively functionalized particles with approximately 10 times more dye molecules. The results herein reported could significantly improve the performance of dye nano-assemblies for imaging or sensing applications.UID/BIO/04565/2019, UID/QUI/00100/2019, UID/Multi/04326/2019, PD/BD/113630/2015, SFRH/BPD/111906/2015info:eu-repo/semantics/publishedVersio

Enhanced-Fluorescence of a Dye on DNA- assembled Gold Nano-Dimers Discriminated by Lifetime Correlation Spectroscopy

<div> <div> <div> <p>We have employed DNA-directed assembly to prepare dimers of gold nanoparticles and used their longitudinally coupled plasmon mode to enhance the fluorescence emission of an organic red-emitting dye, Atto-655. The plasmon- enhanced fluorescence of this dye using dimers of 80 nm particles was measured at single molecule detection level. The top enhancement factors were above 1000-fold in 71% of the dimers within a total of 32 dimers measured, and, in some cases, they reached almost 4000-fold, in good agreement with model simulations. Additionally, fluorescence lifetime correlation analysis enabled the separation of enhanced from non-enhanced emission simultaneously collected in our confocal detection volume. This approach allowed us to recover a short relaxation component exclusive to enhanced emission that is attributed to the interaction of the dye with DNA in the interparticle gaps. </p> </div> </div> </div

Merging Porphyrins with Gold Nanorods: Self Assembly Construct to High Fluorescent Polyelectrolyte Microcapsules

Dual probe porphyrin-gold nanorod polyelectrolyte microcapsules were developed to explore the enhancing effects of a plasmonic interface of self-assembled gold nanoparticles in the fluorescence emission from porphyrins loaded into the capsules&rsquo; core. An analysis of fluorescence lifetime imaging microscopy (FLIM) data reports a notable 105&ndash;106-fold increase in the maximum detected photon rates from diffraction-limited spots and an overall six-fold increase in fluorescence as averaged over the whole microcapsule area. Large emission enhancements were correlated with decreases in fluorescence lifetimes. The microcapsule&rsquo;s design proved effective in achieving high fluorescent hybrids and may shed light on new possibilities for advanced materials imaging applications

Enhanced Fluorescence of a Dye on DNA-Assembled Gold Nanodimers Discriminated by Lifetime Correlation Spectroscopy

The surface plasmon modes of metal nanoparticles provide a way to efficiently enhance the excitation and emission from a fluorescent dye. We have employed DNA-directed assembly to prepare dimers of gold nanoparticles and used their longitudinally coupled plasmon mode to enhance the fluorescence emission of an organic red-emitting dye, Atto-655. The plasmon-enhanced fluorescence of this dye using dimers of 80 nm particles was measured at the single-molecule detection level. The top enhancement factors were above 1000-fold in 71% of the dimers within a total of 32 dimers measured, and in some cases, they reached almost 4000-fold, in good agreement with model simulations. Additionally, fluorescence lifetime correlation analysis enabled the separation of enhanced from nonenhanced emission simultaneously collected in our confocal detection volume. This approach allowed us to recover a short relaxation component exclusive to enhanced emission that is attributed to the interaction of the dye with DNA in the interparticle gaps. Indeed, the frequency of enhancement events is larger than that expected from the volume occupancy of the gap region, thus suggesting that the interaction of the dye with DNA linkers favors the observation of emission enhancement in our dimer particles