Photodynamic therapy (PDT) is a promising treatment method for various diseases, driven by the need for targeted approaches that minimize off-target effects. It employs photosensitizers that generate reactive oxygen species (ROS) upon light irradiation to induce localized cell death. The ability to bind with DNA, primary molecular target for numerous therapeutic agents, provides a significant advantage over non-targeted photosensitizers, as it promotes to the localized ROS generation in close proximity to the target. Here, we present a novel bifunctional conjugate Qn-St combining a photocytotoxic quinolizinium derivative Qn with a fluorescent styryl dye St for targeted delivery and imaging. Using optical methods and 1H NMR spectroscopy, simultaneous binding of both conjugate fragments to DNA was demonstrated, similar to the parent compounds. The quinolizinium derivative intercalates between CG base pairs, while the styryl dye is located along the AT-rich minor groove of DNA. Agarose gel electrophoresis shows that irradiation of Qn and Qn-St leads to efficient photoinduced plasmid damage with the formation of the linear form. Confocal microscopy demonstrated mitochondrial localization of St and Qn-St in HCT116 colorectal carcinoma cells, proving the dye's vector function. Furthermore, both Qn and Qn-St exhibited comparable ROS production and reduced cell survival after irradiation, suggesting that combinations of quinolizinum derivatives and styryl dyes represent a promising platform for development of PDT theranostics.

Few genetically encoded systems based on aptamer-dye pairs meet all the requirements of bioimaging in terms of brightness, contrast, etc., thus necessitating their optimization. Here, we report a new biotin-free fluorogenic ligand that demonstrates increased selectivity for Mango II over other DNA/RNA structures in tube and brightness in complex with genetically encoded aptamer-labeled RNA in live cells compared to the known dye.

In this communication, the results of theoretical investigations of a 1,3,5,7-tetramethyl-BODIPY derivative (TMB) and a dimeric molecule composed of two TMB fluorophores linked by a flexible siloxane spacer (di-TMB) in various solvents are presented. The existence of two energetically close configurations for the di-TMB molecule is revealed. The analysis of the patterns of frontier molecular orbitals indicates that the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are predominantly localized on the chromophores. Furthermore, in the di-TMB molecule, a nearly complete transfer of electron density occurs between the chromophores.

In the present work, two molecular rotors based on styryl dyes were synthesized and investigated. Both dyes showed significant sensitivity to the viscosity of the medium. Due to their significant positive charge, they efficiently accumulated in mitochondria of living cells. Binding to mitochondria resulted in increased fluorescence intensity and fluorescence lifetime of the dyes, allowing them to be visualized without washing off the unbound dye. We have also shown that the fluorescence lifetime of the studied molecular rotors is determined not only by the viscosity of the medium, but also by interactions with cell components such as proteins and nucleic acids. The present work clearly shows that these interactions do not allow a reliable estimation of viscosity in cell organelles using the synthesized dyes. This compromises the results of previous viscosity studies using molecular rotors at least of the styryl type. Nevertheless, induction of cell apoptosis by benzylviologen led to an increase in brightness and fluorescence lifetime of the dyes, which can be caused by both changes in viscosity and changes in the expression profile of proteins localized in mitochondria and interacting with the dyes. Thus, the obtained styryl dyes, like previously published homologous compounds, cannot be used for viscosity measurements, but can be used for identification of pathological states of the cell.

In this paper, we provide a thorough electrochemical study of redox-active nanosized cationic gels which are promising materials for redox flow battery electrolytes. We use two-step synthesis under mild aqueous conditions: precipitation polymerization of nanogels based on poly-(N-isporopylacrylamide-co-N-(3-aminopropyl) methacrylamide hydrochloride) (PNIPAM-co-APMA), and grafting of redox-active 4-(3-carboxypropanamido)TEMPO units to the nanogels. We demonstrate stable reduction-oxidation behavior of such nanogels and suggest a universal approach to evaluate the "effective" concentration and diffusion coefficient of redox-active groups grafted to nanogel particles. For the TEMPO-grafted PNIPAM-co-APMA nanogels we find the "effective" concentration of TEMPO-groups to be approximately 50 % of their total concentration and demonstrate an increase of the "effective" concentration upon electrode rotation. Also, we investigate electron transfer kinetics of redoxactive nanogels and provide an evidence that the adsorbed layer of nanogels facilitates electron transfer.

This article is devoted to the construction and study of a model cell line expressing neuroglobin. The obtained data show the manifestation of anti-apoptotic properties by neuroglobin under condi-tions of apoptosis via the cytochrome C-dependent pathway

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4–6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70–85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70–85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Objective: Today, treatment of oncological diseases mostly relies on radiation, radioisotope, and hormone therapy, immunotherapy, and chemotherapy methods. These methods display poor specificity and have adverse side effects on the body. Nowadays, targeted cancer therapies are under development, in particular, those using targeting peptides that selectively bind to cancer cells. Methods: As shown in the prior studies cancer cells are characterized by a reduced pH in the extracellular space (pH 6.0–6.7). A polypeptide from bacteriorhodopsin (pHLIP) that spontaneously binds to the lipid bilayer at acidic pHs (below pH 6.8) was identified. Here, by using EGFP–pHLIP conjugates, the influence of the linker sequence, connecting pHLIP and EGFP, on the functional properties of such constructs was evaluated. Results and Discussion: In this study, we prepared EGFP–pHLIP constructs separated with the peptide linkers of different sequences. The influence of the linker sequence on the pH-dependent binding of these constructs to cancer cells and on the efficiency of the EGFP chromophore synthesis within the protein-peptide constructs was demonstrated. Conclusions: Using EGFP-pHLIP constructs with either of two pH-sensitive peptide variants, pHLIPwt and pHLIPvar3, the influence of the linker sequence on the functional properties of such constructs was demonstrated. The prepared constructs contained two types of linkers, IEGRCGS (EGFP-IEGRCGS-pHLIPwt and EGFP-IEGRCGS-pHLIPvar3) and GS (EGFP-GS-pHLIPwt and EGFP-GS-pHLIPvar3). It was found that the constructs containing the IEGRCGS linker pH-dependently bind to HeLa cells with higher affinity. The fusion of EGFP with the pHLIP peptides was shown to affect to some extent the maturation of the chromophore of EGFP. Also, it was demonstrated that the pHLIPwt-carrying protein-peptide constructs display a higher binding affinity to cell membranes at decreased pH values compared to the analogous constructs bearing pHLIPvar3.

Fluorescent dyes which exhibit emission/excitation in the second near-infrared (NIR-II, 1000-1350 nm) region are currently attracting significant attention in bioimaging and diagnostics applications. Furthermore, dyes with high two-photon absorption cross-section (TPA), such as BODIPY derivatives, are of a particular interest due to deeper signal penetration into biological tissues, better image contrast, reduced phototoxicity and photobleaching. Herein we report the synthesis and properties of new monomeric and dimeric di-styryl-BODIPY dyes, which have absorption maxima near 625 nm and emission in the range of 600-800 nm (NIR-I, 650-950 nm). For the first time, we used a femtosecond Cr:Forsterite laser with a wavelength of 1250 nm (NIR-II) for the excitation of NIR-I di-styryl-BODIPY dyes by TPA. A cooperative effect was observed for TPA for the dimeric di-styryl-BODIPY dyes. The results obtained may be of great interest due to their potential applications in bioimaging and photodynamic therapy.

Two new styryl-type dyes modified with the succinimide ester group were prepared for conjugation with antibodies. The optical characteristics of the obtained compounds in different solvents were studied, and the solvatochromic properties of these dyes were revealed. The applicability of the dyes as protein labels was demonstrated in cyto-fluorometry and fluorescence microscopy.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems.

It is generally accepted that the use of two different plasmids with the identical origins of replication in bacteria is not desirable due to their "incompatibility". The utilization of the same bacterial enzymatic apparatus for replication of different plasmids is thought to cause a significant redistribution in favor of one of them. In the present work, examining co-expression of two different fluorescent proteins in Escherichia coli, we have shown that the use of highly homologous plasmids with identical origins of replication and providing resistance to different antibiotics results in high representation of both plasmids in bacteria. Meanwhile, the level of gene expression and the amount of proteins produced may differ and is determined mostly by their sequence rather than by the "incompatibility" of the plasmids.

Creating new tools for the early diagnosis and treatment of cancer is one of the most important and intensively developing areas of modern medicine. Currently, photodynamic cancer therapy (PDT) is attracting increasing attention as a unique modality of minimally invasive treatment and due to the absence of acquired resistance. However, PDT is associated with undesirable activities, such as non-specific photodynamic effects of sunlight on healthy tissues. Therefore, an important fundamental task is the development of improved PDT agents that selectively act on the affected areas. Here, we report the development of a hybrid protein-peptide system for the selective pH-dependent binding and subsequent photodynamic cancer cells ablation. It is known that a distinctive feature of cancer cells is a decreased pH level in the extracellular space. In this study we exploited a peptide fragment (pHLIP) as a targeting module, which spontaneously binds and embeds into the cell membrane when pH decreases below neutral. A mutant of miniSOG protein fused to pHLIP was used as a photosensitizing constituent. We demonstrate that this protein-peptide photosensitizing system selectively binds to HeLa cells at pH below 6.8 and kills them when exposed to light. These findings demonstrate the feasibility of using genetically encoded MiniSOG fusions with pHLIP for the targeted delivery of PSs to cancer cells and subsequent highly precise photodynamic therapy.

The photophysical properties of functional distyryl derivatives of dipyrrometheneboron difluoride (BODIPY) in various solvents have been studied. It was shown that in all solvents, the studied compounds have similar absorption and fluorescence spectra, which are characteristic of the distyryl derivatives of BODIPY. Small bathochromic shifts of the spectra with increasing solvent polarity, as well as insignificant changes in the rate constants of radiative and nonradiative processes, provide evidence that the S1 states are of the (π,π*) type. These dyes can be used to visualize processes in living systems.

Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear. In the present work, we studied echinenone (ECN) delivery by cyanobacterial carotenoprotein AnaCTDH (C-terminal domain homolog of the Orange Carotenoid Protein from Anabaena), into liposome membranes labelled with BODIPY fluorescent probe. We observed that addition of AnaCTDH-ECN to liposomes led to the significant changes in the fast-kinetic component of the fluorescence decay curve, pointing on the dipole-dipole interactions between the probe and ECN within the membrane. It may serve as an indirect evidence of ECN delivery into membrane. To study the delivery in detail, we carried out molecular dynamics modeling of the localization of ECN within the lipid bilayer and calculate its orientation factor. Next, we exploited FRET to assess concentration of ECN delivered by AnaCTDH. Finally, we used time-resolved fluorescence anisotropy to assess changes in microviscosity of liposomal membranes. Incorporation of liposomes with β-carotene increased membrane microviscosity while the effect of astaxanthin and its mono- and diester forms was less pronounced. At temperatures below 30 °C addition of AnaCTDH-ECN increased membrane microviscosity in a concentration-dependent manner, supporting the protein-mediated carotenoid delivery mechanism. Combining all data, we propose FRET-based analysis and assessment of membrane microviscosity as potent approaches to characterize the efficiency of carotenoids delivery into membranes.

Novel BODIPY and DBMBF2dyads connectedviaa flexible trisiloxane linker were synthesized and their photophysical properties were investigated.

Here, we present a direct comparison of different dyes and assays for the determination of protein concentrations. We compared the classical Bradford assay with two modern assays based on the fluorogenic dyes QuDye and ProteOrange and showed that the Bradford reagent achieved excellent results in the determination of protein concentrations as compared with more modern rivals. We also showed that standard approaches for determining the limit of detection (LoD) and limit of quantification (LoQ) may not work correctly with the tested dyes. We proposed a new approach that extends the standard algorithm for LoD and LoQ determination. This approach works well with both classical colorimetric and fluorogenic dyes, as well as with nontrivial fluorescent probes.

Compounds sensitive to reactive oxygen species are widely used in the study of processes in living cells and in the development of therapeutic agents for photodynamic therapy. In the present work, we have synthesized a dyad in which the BODIPY dye is chemically bound to 9,10-diphenylanthracene (DPA). Here, DPA acts as a specific sensor of singlet oxygen and BODIPY as a reference dye. We studied the photophysical properties of the BODIPY-DPA dyad and showed that energy transfer occurs between the chromophores. As a result, the compound has excitation maxima in the absorption region of both DPA and BODIPY, but the fluorescence emission occurs mainly from BODIPY. In the presence of singlet oxygen, the excitation maximum of DPA decreases, while the intensity of the excitation maximum of BODIPY remains almost unchanged. This allows the BODIPY-DPA dyad to be used as a ratiometric sensor of singlet oxygen.

A series of multifluorophore compounds with two, four, six and eight BODIPY residues linked to a siloxane core via a flexible spacer was synthesized. An increasing number of BODIPY units caused an enhanced propensity to chromophore aggregation with characteristic photophysical effects evoked by the aggregation. The increase in the number of fluorophores additionally resulted in considerable increase in molar extinction coefficient. Meanwhile, a slight drop of the extinction coefficient as calculated per a single chromophore was detected. Multifluorophore conjugates showed a significant broadening of fluorescence emission bands in polar solvents and a substantial decrease in the fluorescence quantum yield due to the aggregation-caused quenching. Time resolved fluorescence measurements of multifluorophore compounds in different solvents showed that the fluorescence decays have a multiexponential character and strongly depend on the number of fluorophores in the conjugate, polarity/viscosity of the solvent and the detection wavelength.

The targeted delivery of nanodrugs to malignant neoplasm is one of the most pressing challenges in the development of modern medicine. It was reported earlier that a bacteriorhodopsin-derived pH low insertion peptide (pHLIP) targets acidic tumors and has the ability to translocate low molecular weight cargoes across the cancer cell membrane. Here, to better understand the potential of pHLIP-related technologies, we used genetically engineered fluorescent protein (EGFP) as a model protein cargo and examined targeting efficiencies of EGFP-pHLIP hybrid constructs in vitro with the HeLa cell line at different pHs. By two independent monitoring methods we observed an increased binding affinity of EGFP-pHLIP fusions to HeLa cells at pH below 6.8. Confocal images of EGFP-pHLIP-treated cells showed bright fluorescence associated with the cell membrane and fluorescent dots localized inside the cell, that became brighter with time. To elucidate the pHLIP-mediated EGFP cell entry mechanisms, we performed a series of experiments with specific inhibitors of endocytosis. Our results imply that EGFP-pHLIP internalization is realized by endocytosis of various types.