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Oxidative stress on cysteine (Cys)-containing proteins has been associated with physiological disorders, as suggested for the human cofilin-1 (CFL-1) protein, in which the oxidized residues are likely implicated in the aggregation process of α-synuclein, leading to severe neuronal injuries. Considering the relevance of the oxidation state of cysteine, quantification of thiols may offer a guide for the development of effective therapies. This work presents, for the very first time, thiol quantification within CFL-1 in solution and on the surface following classic and adapted versions of Ellman's assay. The 1:1 stoichiometric Ellman's reaction occurs between 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol of the cysteine residue, producing two 2-nitro-5-thiobenzoate (TNB 2- ) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB 2- , on the surface, the mass of the attached TNB 2- ion to the protein allowed the quantification by means of the multiparametric surface plasmon resonance (MP-SPR) technique. The SPR angle change after the interaction of DTNB with immobilized CFL-1 gave a surface coverage of 26.5 pmol cm -2 for the TNB 2- ions (Γ TNB2- ). The ratio of this value to the surface coverage of CFL-1, Γ CFL-1 = 6.5 ± 0.6 pmol cm -2 (also determined by MP-SPR), gave 4.1 as expected for this protein, i.e., CFL-1 contains four Cys residues in its native form (reduced state). A control experiment with adsorbed oxidized protein showed no SPR angle change, thus proving the reliability of adapting Ellman's assay to the surface using the MP-SPR technique. The results presented in this work provide evidence of the heterogenization of Ellman's assay, offering a novel perspective for studying thiol-containing species within proteins. This may be particularly useful to ensure further studies on drug-like molecules that can be carried out with validated oxidized or reduced CFL-1 or other analogous systems.

Metallocompounds have emerged as promising new anticancer agents, which can also exhibit properties to be used in photodynamic therapy. Here, we prepared two ruthenium-based compounds with a 2,2'-bipyridine ligand conjugated to an anthracenyl moiety. These compounds coded GRBA and GRPA contain 2,2'-bipyridine or 1,10-phenathroline as auxiliary ligands, respectively, which provide quite a distinct behavior. Notably, compound GRPA exhibited remarkably high photoproduction of singlet oxygen even in water (ϕ Δ = 0.96), almost twice that of GRBA (ϕ Δ = 0.52). On the other hand, this latter produced twice more superoxide and hydroxyl radical species than GRPA , which may be due to the modulation of their excited state. Interestingly, GRPA exhibited a modest binding to DNA ( K b = 4.51 × 10 4 ), while GRBA did not show a measurable interaction only noticed by circular dichroism measurements. Studies with bacteria showed a great antimicrobial effect, including a synergistic effect in combination with commercial antibiotics. Besides that, GRBA showed very low or no cytotoxicity against four mammalian cells, including a hard-to-treat MDA-MB-231, triple-negative human breast cancer. Potent activities were measured for GRBA upon blue light irradiation, where IC 50 of 43 and 13 nmol L -1 were seen against hard-to-treat triple-negative human breast cancer (MDA-MB-231) and ovarian cancer cells (A2780), respectively. These promising results are an interesting case of a simple modification with expressive enhancement of biological activity that deserves further biological studies.

For more than 70 years, sodium nitroprusside (SNP) has been used to treat severe hypertension in hospital emergency settings. During this time, a few other clinical uses have also emerged such as in the treatment of acute heart failure as well as improving mitral incompetence and in the intra- and perioperative management during heart surgery. This drug functions by releasing nitric oxide (NO), which modulates several biological processes with many potential therapeutic applications. However, this small molecule has a short lifetime, and it has been administered through the use of NO donor molecules such as SNP. On the other hand, SNP also has some setbacks such as the release of cyanide ions, high water solubility, and very fast NO release kinetics. Currently, there are many drug delivery strategies that can be applied to overcome many of these limitations, providing novel opportunities for the use of old drugs, including SNP. This Perspective describes some nitroprusside properties and highlights new potential therapeutic uses arising from the use of drug delivery systems, mainly silica-based nanoparticles. There is a series of great opportunities to further explore SNP in many medical issues as reviewed, which deserves a closer look by the scientific community.

Silica-based nanoparticles have been developed as powerful platforms for drug delivery and might also prevent undesired side effects of drugs. Here, a fast method to synthesize positively charged mesoporous silica nanoparticles (ζ = 20 ± 0.5 mV, surface area = 678 m 2 g -1 , and 2.3 nm of porous size) was reported. This nanomaterial was employed to anchor sodium nitroprusside (SNP), a vasodilator drug with undesired cyanide release. A remarkable incorporation of 323.9 ± 7.55 μmol of SNP per gram of nanoparticle was achieved, and a series of studies of NO release were conducted, showing efficient release of NO along with major cyanide retention (ca. 64% bound to nanoparticle). Biological assays with mammalian cells showed only a slight drop in cell viability (13%) at the highest concentration (1000 μM), while SNP exhibited an LC 50 of 228 μM. Moreover, pharmacological studies demonstrated similar efficacy for vasodilation and sGC-PKG-VASP pathway activation when compared to SNP alone. Altogether, this new SNP silica nanoparticle has great potential as an alternative for wider and safer use of SNP in medicine with lower cyanide toxicity.

Ruthenium polypyridine complexes have shown promise as agents for photodynamic therapy (PDT) and tools for molecular biology (chromophore-assisted light inactivation). To accomplish these tasks, it is important to have at least target selectivity and great reactive oxygen species (ROS) photogeneration: two properties that are not easily found in the same molecule. To prepare such new agents, we synthesized two new ruthenium complexes that combine an efficient DNA binding moiety (dppz ligand) together with naphthyl-modified (1) and anthracenyl-modified (2) bipyridine as a strong ROS generator bound to a ruthenium complex. The compounds were fully characterized and their photophysical and photochemical properties investigated. Compound 2 showed one of the highest quantum yields for singlet oxygen production ever reported (Φ Δ = 0.96), along with very high DNA binding (log K b = 6.78). Such photochemical behavior could be ascribed to the lower triplet state involving the anthracenyl-modified bipyridine, which is associated with easier oxygen quenching. In addition, the compounds exhibited moderate selectivity toward G-quadruplex DNA and binding to the minor groove of DNA, most likely driven by the pendant ligands. Interestingly, they also showed DNA photocleavage activity even upon exposure to a yellow light-emitting diode (LED). Regarding their biological activity, the compounds exhibited an exciting antibacterial action, particularly against Gram-positive bacteria, which was enhanced upon blue LED irradiation. Altogether, these results showed that our strategy succeeded in producing light-triggered DNA binding agents with pharmacological and biotechnological potential.