Rosmarinic acid turned α-syn oligomers into non-toxic species preserving microtubules in Raw 264.7 cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide, and the therapeutic is focused on several approaches including the inhibition of fibril formation by small compounds, avoiding the formation of cytotoxic oligomers. Thus, we decided to explore the capacity of compounds carrying catechol moieties to inhibit the progression of α-synuclein. Overall, the compounds rosmarinic acid (1), carnosic acid (2), carnosol (3), epiisorosmanol (4), and rosmanol (5) avoid the progression of fibril formation assessed by Thiofavine T (ThT), and atomic force microscopy images showed that morphology is influenced for the actions of compounds over fibrillization. Moreover, ITC experiments showed a Kd varying from 28 to 51 µM, the ΔG showed that the reaction between compounds and α-syn is spontaneous, and ΔH is associated with an exothermic reaction, suggesting the interactions of hydrogen bonds among compounds and α-syn. Docking experiments reinforce this idea showing the intermolecular interactions are mostly hydrogen bonding within the sites 2, 9, and 3/13 of α-synuclein, and compounds 1 and 5. Thus, compound 1, rosmarinic acid, interestingly interacts better with site 9 through catechol and Lysines. In cultured Raw 264. 7 cells, the presence of compounds showed that most of them can promote cell differentiation, especially rosmarinic acid, and rosmanol, both preserving tubulin cytoskeleton. However, once we evaluated whether or not the aggregates pre-treated with compounds could prevent the disruption of microtubules of Raw 264.7 cells, only pre-treated aggregates with rosmarinic acid prevented the disruption of the cytoskeleton. Altogether, we showed that especially rosmarinic acid not only inhibits α-syn but stabilizes the remaining aggregates turning them into not-toxic to Raw 264.7 cells suggesting a main role in cell survival and antigen processing in response to external α-syn aggregates.

Direct Oral FXa Inhibitors Binding to Human Serum Albumin: Spectroscopic, Calorimetric, and Computational Studies.

Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban, edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies. The interaction of active compounds with human serum albumin (HSA), the most abundant protein in blood plasma, is a key research area and provides crucial information about drugs' pharmacokinetics and pharmacodynamic properties. This research focuses on the study of the interactions between HSA and four commercially available direct oral FXa inhibitors, applying methodologies including steady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and molecular dynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and the complex formation in the ground states affects the fluorescence of HSA, with a moderate binding constant of 104 M-1. However, the ITC studies reported significantly different binding constants (103 M-1) compared with the results obtained through spectrophotometric methods. The suspected binding mode is supported by molecular dynamics simulations, where the predominant interactions were hydrogen bonds and hydrophobic interactions (mainly π-π stacking interactions between the phenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications of the obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.

Supramolecular co-encapsulation of a photosensitizer and chemotherapeutic drug in cucurbit[8]uril for potential chemophototherapy.

Supramolecular strategies as well as combinatorial approaches have been proposed to improve cancer therapeutics. In this work, we investigated the encapsulation of the photosensitizer acridine orange (AO) and the chemotherapeutic drug oxaliplatin (OxPt) in cucurbit[8]uril (CB[8]), and tested their effect both separate and combined on tumoral cells cultivated in vitro. Binding constants and enthalpies of reaction for the AO@CB[8], (AO)2@CB[8] and OxPt@CB[8] complexes were determined by isothermal titration calorimetry. In the case of AO, a negative cooperativity for the binding of the second AO molecule was found, in agreement with previous fluorescence titration data. We show herein that the AO@CB[8] complex was effectively incorporated within the cells and showed important phototoxicity, while the OxPt@CB[8] complex was cytotoxic only at long incubation times (24 h). Pre-treatment of the cells with the OxPt@CB[8] complex for 24 h inhibited any photodynamic action by the later treatment with the AO@CB[8] complex. However, when both complexes were co-incubated for 90 min, the combined cytotoxicity/phototoxicity was superior to any of the treatments individually. A cooperative effect was identified that added up to an extra 30% cytotoxicity/phototoxicity. The results point to an interesting system where a photosensitizer and chemotherapeutic drug are co-encapsulated in a macrocycle to develop chemophototherapy applications.

Binding of toluidine blue-myristic acid derivative to cucurbit[7]uril and human serum albumin: computational and biophysical insights towards a biosupramolecular assembly.

A new toluidine blue-myristic acid photosensitizer derivate (TBOMyr) was investigated as a design molecule to bind simultaneously to cucurbit[7]uril (CB[7]) and human serum albumin (HSA) with the aim of constructing a biosupramolecular assembly. Molecular docking and dynamics calculations revealed the main supramolecular and bio-molecular interactions of TBOMyr with the macrocycle or the protein, respectively. The addition of the negatively charged myristic acid-like tail resulted in a unique conformation of the CB[7] complex where the phenothiazine core was included in the cavity of CB[7], leaving the fatty acid portion free to interact with the protein. A favorable ternary interaction between TBOMyr, CB[7] and HSA was suggested by the calculations, and an experimental binding affinity in the order of 105 M-1 was determined for the TBOMyr@CB[7] complex with HSA. The new TBOMyr derivative could find applications in photodynamic therapy benefiting from the biosupramolecular interactions as a transport system.

New Benzotriazole and Benzodithiophene-Based Conjugated Terpolymer Bearing a Fluorescein Derivative as Side-Group: In-Ternal Förster Resonance Energy Transfer to Improve Organic Solar Cells.

A new benzodithiophene and benzotriazole-based terpolymer bearing a fluorescein derivative as a side group was synthesized and studied for organic solar cell (OSC) applications. This side group was covalently bounded to the backbone through an n-hexyl chain to induce the intramolecular Förster Resonance Energy Transfer (FRET) process and thus improve the photovoltaic performance of the polymeric material. The polymer exhibited good solubility in common organic chlorinated solvents as well as thermal stability (TDT10% > 360 °C). Photophysical measurements demonstrated the occurrence of the FRET phenomenon between the lateral group and the terpolymer. The terpolymer exhibited an absorption band centered at 501 nm, an optical bandgap of 2.02 eV, and HOMO and LUMO energy levels of −5.30 eV and −3.28 eV, respectively. A preliminary study on terpolymer-based OSC devices showed a low power-conversion efficiency (PCE) but a higher performance than devices based on an analogous polymer without the fluorescein derivative. These results mean that the design presented here is a promising strategy to improve the performance of polymers used in OSCs.

Mechanism of Visible-Light Photooxidative Demethylation of Toluidine Blue O.

Experiments and theoretical calculations by density functional theory (DFT) have been carried out to examine a self-sensitized type I photooxidation of toluidine blue O (TBO+). This study attempts to build a connection between visible-light photolysis and demethylation processes of methylamine compounds, such as TBO+. We show that controlled photoinduced mono- and double-demethylation of TBO+ can be achieved. The kinetics for the appearance rate of the mono-demethylated TBO+ and the double-demethylated TBO+ were found to fit pseudo-first-order kinetics. DFT calculations have been used to examine the demethylation of TBO+ and included N, N-dimethylaniline as a model compound for TBO+. The results show an oxygen-dependent demethylation process. The mechanism for the sequential methyl loss is proposed to be due to H• or e-/H+ transfer to 3TBO+* followed by a reaction of TBO+• with O2, yielding a C-peroxyTBO+• intermediate. Instead of aminyl radical peroxyl formation, i.e., N-peroxyTBO+•, the C-centered peroxyTBO+• is favored, that upon dimerization (Russell mechanism) leads to dissociation of formaldehyde from the methylamine site.

Fluorescence properties of aurone derivatives: an experimental and theoretical study with some preliminary biological applications.

In this paper, we explored the fluorescence properties of eight aurone derivatives bearing methoxy groups and bromine atoms as substituents in the benzene rings. All derivatives showed strong solvatochromic absorption and emission properties in solvents of different polarities. Some of them showed high fluorescence quantum yields, which make them potential compounds for sensing applications. The position of the methoxy groups in the benzofuranone moiety and the presence of bromine atoms in the benzene ring had a strong influence on the fluorescence behaviour of the aurones. DFT calculations allowed us to explain the emission properties of aurones and their solvatochromism, which was related to an excited state with strong charge-transfer character. Aurone 4 has the most promising characteristics showing a large difference in the quantum yields and large Stokes shifts depending on the solvent polarities. These results prompted us to explore some preliminary biological applications for aurone 4 such as the sensing of hydrophobic pockets of a protein and its thermotropic behaviour in liposomes.

Photochemical behavior of biosupramolecular assemblies of photosensitizers, cucurbit[n]urils and albumins.

Biosupramolecular assemblies combining cucurbit[n]urils (CB[n]s) and proteins for the targeted delivery of drugs have the potential to improve the photoactivity of photosensitizers used in the photodynamic therapy of cancer. Understanding the complexity of these systems and how it affects the properties of photosensitizers is the focus of this work. We used acridine orange (AO+) as a model photosensitizer and compared it with methylene blue (MB+) and a cationic porphyrin (TMPyP4+). Encapsulation of the photosensitizers into CB[n]s (n = 7, 8) modified their photoactivity. In particular, for AO+, the photo-oxidation of HSA was enhanced in the presence of CB[7]; meanwhile it was decreased when included into CB[8]. Accordingly, peroxide generation and protein fragmentation were also increased when AO+ was encapsulated into CB[7]. The triplet excited state lifetimes of all the photosensitizers were lengthened by their encapsulation into CB[n]s, while the singlet oxygen quantum yield was enhanced only for AO+ and TMPyP4+, but it decreased for MB+. The results obtained in this work prompt the necessity of further investigating these kinds of hybrid assemblies as drug delivery systems because of their possible applications in biomedicine.

Electrostatically promoted dynamic hybridization of glucans with cationic polythiophene.

Hybridizing natural macromolecules with synthetic polymers is an efficient general method for constructing sophisticated supramolecular architectures. To comprehensively elucidate the controversial hybridization mechanism of glucans with synthetic polymers, the hybridization behaviors of triple-stranded curdlan (Cur) and schizophyllan (SPG) with cationic polythiophene (PyPT) were investigated in aqueous DMSO solutions by using UV-vis, circular dichroism (CD), fluorescence, fluorescence excitation, and NMR spectroscopy methods, as well as theoretical calculations, dynamic light scattering, and zeta potential measurements. Upon mixing with glucan, a hetero-triplex formed, which was dynamic and greatly accelerated by heating and by adding a base or a salt. The hetero-triplex disassembled into a hetero-duplex in highly basic solutions. Thus, polycationic polymers, such as PyPT, are expected to serve as a versatile tool for unzipping glucan homo-triplexes and promoting subsequent hybridization in aqueous solution, while the detailed mechanism elucidated in the present study contributes to the rational design of hybridization partners.

A study of the Fenton-mediated oxidation of methylene blue-cucurbit[n]uril complexes.

Cucurbit[n]urils efficiently decreased the Fenton-mediated oxidation of encapsulated dyes, providing a mechanism for some control and selectivity over the degradation. The encapsulation of methylene blue into cucurbit[7]uril made it highly refractory against Fenton oxidation in the dark or under UVA light irradiation. However, the oxidation of the encapsulated dye was significantly enhanced under visible light irradiation. This behavior was selective for the cucurbit[7]uril complex and not for the cucurbit[8]uril complex, which achieved the same degree of protection irrespective of the irradiation conditions. This different reactivity of the complexes was further discussed in terms of their excited state properties. The main mechanism for protection was the seclusion of the dye into cucurbit[n]urils as shown by the fact that the non-encapsulated dye safranin was protected much less than methylene blue. Additionally, cucurbit[n]urils efficiently trapped hydroxyl radicals, which contributed significantly to the protection of the dyes from Fenton-mediated oxidation.

Time-resolved fluorescence anisotropy as a tool to study guest-cucurbit[n]uril-protein ternary supramolecular interactions.

Ternary supramolecular complexes involving cucurbit[n]urils and proteins are of potential interest for improving drug transport and delivery. We report here time-resolved fluorescence studies for acridine orange complexes with cucurbit[7]uril and cucurbit[8]uril in the presence of human serum albumin as a model system. A detailed characterization of the fluorescence lifetime and anisotropy properties of the different acridine orange complexes with cucurbit[n]urils and human serum albumin was performed. Of particular importance is the analysis of the stepwise binding for acridine orange-cucurbit[8]uril complexes and the assignment of the fluorescence and anisotropy properties to the 2 : 1 complex. Anisotropy decay measurements were essential to detect protein-bound species and to discriminate between different complexes. Based on the fluorescence evidence, ternary interactions with the protein are suggested for the acridine orange-cucurbit[7]uril complex but not for the cucurbit[8]uril complex. We highlight here the usability and sensitivity of the combined fluorescence analysis.

Gramicidin conformational changes during riboflavin photosensitized oxidation in solution and the effect of N-methylation of tryptophan residues.

In the present work, we evaluated the role of gramicidin conformation in its photosensitized oxidation in organic solvents when irradiated in the presence of riboflavin. Gramicidin conformation has been described as monomeric in trifluoroethanol and as an intertwined dimer in methanol. Gramicidin showed extensive photo-oxidation upon irradiation in the presence of riboflavin in both solvents, and tryptophan residues were identified to be involved. We synthesized a gramicidin derivative methylated at position 1 of the indole ring of tryptophan to assess its effect on gramicidin conformation and photo-oxidation. Methylated gramicidin showed very similar absorption and emission spectra to gramicidin, but different conformations were identified by circular dichroism spectra. Upon irradiation, N-methylated tryptophan residues in the gramicidin derivative were not easily photo-oxidized by riboflavin compared to gramicidin. Circular dichroism spectra for gramicidin in methanol changed significantly upon irradiation in the presence of riboflavin indicating a change in conformation, while in trifluoroethanol no such changes were observed. Time-resolved fluorescence and anisotropy studies showed that oxidized gramicidin in methanol had shorter fluorescence lifetimes and a shorter rotational correlation time compared to non-irradiated gramicidin. Additionally, SDS-PAGE analysis showed a marked change in the electrophoretic pattern, whereas the high-molecular-weight bands disappeared upon irradiation. We interpret all these results in terms of a riboflavin photosensitized shift in gramicidin conformation from intertwined to monomeric.

On the mechanism of visible-light sensitized photosulfoxidation of toluidine blue O.

We report on the formation of toluidine blue O (TBO) sulfoxide by a self-sensitized photooxidation of TBO. Here, the photosulfoxidation process was studied by mass spectrometry (MS) and discussed in the context of photodemethylation processes which both contribute to TBO consumption over time. Analysis of solvent effects with D2O, H2O, and CH3CN along with product yields and MS fragmentation patterns provided mechanistic insight into TBO sulfoxide's formation. The formation of TBO sulfoxide is minor and detectable up to 12% after irradiation of 3 h. The photosulfoxidation process is dependent on oxygen wherein instead of a type II (singlet oxygen, 1O2) reaction, a type I reaction involving TBO to reach the TBO sulfoxide is consistent with the results. Density functional theory results point to the formation of the TBO sulfoxide by the oxidation of TBO via transiently formed peroxyl radical or thiadioxirane intermediates. We discover that the TBO photosulfoxidation arises competitively with TBO photodemethylation with the latter leading to formaldehyde formation.

Effect of Chitosan-Riboflavin Bioconjugate on Green Mold Caused by Penicillium digitatum in Lemon Fruit.

Penicillium digitatum is the causal agent of green mold, a primary postharvest disease of citrus fruits. This study evaluated the efficacy of a novel photoactive chitosan-riboflavin bioconjugate (CH-RF) to control green mold in vitro and in lemon fruit. The results showed total inhibition of P. digitatum growth on APDA supplemented with CH-RF at 0.5% (w/v) and a significant reduction of 84.8% at 0.25% (w/v). Lemons treated with CH-RF and kept under controlled conditions (20 °C and 90-95% relative humidity) exhibited a noteworthy reduction in green mold incidence four days post-inoculation. Notably, these effects persisted, with all treatments remaining significantly distinct from the control group until day 14. Furthermore, CH-RF showed high control of green mold in lemons after 20 days of cold storage (5 ± 1 °C). The disease incidence five days after cold storage indicated significant differences from the values observed in the control. Most CH-RF treatments showed enhanced control of green mold when riboflavin was activated by white-light exposure. These findings suggest that this novel fungicide could be a viable alternative to conventional synthetic fungicides, allowing more sustainable management of lemon fruit diseases.

Explaining the highly enantiomeric photocyclodimerization of 2-anthracenecarboxylate bound to human serum albumin using time-resolved anisotropy studies.

The mechanism for the high enantiomeric excess (ee) (80-90%) observed in the photocyclodimerization of 2-anthracenecarboxylate (AC) in the chiral binding sites of human serum albumin (HSA) was studied using fluorescence anisotropy. A long rotational correlation time of 36 ns was observed for the excited states of the ACs bound to the HSA site responsible for the high ee, suggesting that the ACs have restricted rotational mobility in this site. The ACs in this site have the same prochiral face protected by the protein, and this protection is responsible for the high ee observed. These insights provide a strategy for the rational design of supramolecular photochirogenic systems.

α-Synuclein Drives Tau's Cytotoxic Aggregates Formation through Hydrophobic Interactions.

Tau and α-synuclein are proteins involved in pathologies known as tauopathies and synucleinopathies, respectively. Moreover, evidence shows that there is a crosstalk between them as is seen in the brains of individuals with sporadic neurodegenerative disorders. Based on that, we present data showing that the hydrophobic α-peptide 71 VTGVTAVAQKTV82 induces the aggregation of the full-length tau fragment in the absence of heparin assessed by ThT. Moreover, AFM images reveal the presence of straight filaments and amorphous aggregates of full-length tau in the presence of the α-peptide. Additionally, ITC experiments showed the interaction of the α-peptide with tau full-length (441 amino acids),4R (amino acids from 244 to 369), and both hexapeptides 275 VQIINK280 and 306 VQIVYK311 through hydrophobic interactions. The Raman spectroscopy spectra showed conformational changes in the Amide region in the aggregates formed with full-length tau and α-syn peptide. Furthermore, the incubation of extracellular aggregates with N2a cells showed morphological differences in the cellular body and the nucleus suggesting cell death. Moreover,, the incubation of different types of aggregates in cell culture provokes the release of Lactate dehydrogenase (LDH). Altogether, we found that α-synuclein peptide can drive the aggregation of full-length tau-provoking morphological and structural changes evoking cytotoxic effects.

Recent Photosensitizer Developments, Delivery Strategies and Combination-based Approaches for Photodynamic Therapy†.

Photodynamic therapy of cancer (PDT) is a therapeutic technique, minimally invasive, which is currently used to treat cancerous lesions and tumors that have been in the spotlight for its potential over the recent decades. Nonetheless, PDT still needs further development to become a first-option treatment for patients. This review compiles recent progress in several aspects of the current research in the constantly growing area of PDT to overcome the main challenges as an attempt to serve as a guide and reference for newcomers into this research area. This review has been prepared to highlight the use of chemical modifications on photosensitizers to improve their solubility, photostability, selectivity and phototoxicity. Additionally, the use of liposomes and cavitands as drug delivery systems to aid in the biodistribution and bioaccumulation of photosensitizers is presented. Also, the combination of PDT with chemotherapy or immunotherapy as an option to boost and improve treatment outcomes is discussed. Finally, the inhibition of antioxidant enzymes as a strategy for a synergistic effect to ameliorate the performance of the photosensitizers in PDT is presented as an alternative for future researchers.

Extraordinary Control of Photosensitized Singlet Oxygen Generation by Acyclic Cucurbituril-like Containers.

Supramolecular control of singlet oxygen generation is incredibly valuable for several fields with broad applications and thus still challenging. However, macrocyclic inclusion complexes inherently restrict the interaction of photosensitizers with surrounding oxygen in the media. To circumvent this issue, we turned our attention in this work to acyclic cucurbituril-like containers and uncover their properties as supramolecular hosts for photosensitizers with extraordinary control of their photophysics, including singlet oxygen generation. Thermodynamic and photophysical studies were carried out showing that these acyclic containers compare very favorably to benchmark macrocycles such as cucurbiturils and cyclodextrins in terms of their binding affinities and supramolecular control of singlet oxygen generation. Acyclic container with terminal naphthalene walls offers a similar cavity to cucurbit[7]uril and the same carbonyl-lined portals for a tight binding of phenothiazinium dye methylene blue and stabilizing its singlet and triplet excited states. Thus, generation of singlet oxygen for this container is higher than for other macrocycles and even higher than the free photosensitizer. While the acyclic container with smaller terminal benzene walls, stacks over the dye through sulfur-π and π-π interactions deactivating the singlet and triplet excited states, thus showing the lowest generation of singlet oxygen out of all of the studied systems. Due to the great water solubility and biocompatibility of these systems, they possess great potential for novel applications in photocatalysis, synthesis, and biomedical fields, among others.

Aggregation behavior of pegylated bile acid derivatives.

Bile acids are amphiphilic endogenous steroids that act as anionic surfactants in the digestive tract and aggregate in aqueous solutions. Nonionic surfactants were synthesized by grafting poly(ethylene glycol) chains of various lengths (pegylation) to three bile acids (lithocholic, deoxycholic, and cholic acid) using anionic polymerization. The aggregation properties of the derivatives were studied with viscosity measurements and light scattering as well as with steady-state and time-resolved fluorescence techniques, and the aggregates were visualized by transmission electron microscopy to elucidate the effect of pegylation on the aggregation process. The fluorescence results showed a good correlation with the capacity of the bile acid derivatives to solubilize a hydrophobic drug molecule. The solubilization of ibuprofen depends on the length and the number of grafted PEG chains, and the solubilization efficiency increases with fewer PEG chains on the bile acid. The results indicate their potential for use in the design of new bile acid-based drug-delivery systems.

Studies of the solvatochromic emission properties of N-aroylurea derivatives II: influence of hydrogen-bonding interactions.

The solvatochromic emission properties of five naphthoylurea derivatives with different substitution patterns at the naphthoylurea functionality were investigated, with a particular focus on the influence of inter- and intramolecular H-bonding interactions. The bathochromic shifts of the emission maxima correlate well with the acceptor number or Catalán's acidity of the solvent (Δλ = 47-86 nm), indicating an excited species with a pronounced negative charge that is stabilized by H-bond donating (HBD) solvents. In media with restricted free volume the formation of the charged species is not favored, because the required conformational change to establish an intramolecular charge transfer (ICT) between the fluorophore and the acylurea substituent is hindered, and the emission mainly originates from the locally excited state. This relationship between the alignment of the naphthoyl carbonyl functionality relative to the naphthyl ring and the spectroscopic shift was confirmed by the comparison of the ground state conformation and the emission spectra of the naphthoylurea derivatives in the solid state. Time-resolved experiments revealed different excited entities, whose lifetimes are significantly influenced by the HBD properties and the temperature of the environment. With few exceptions the naphthoylurea derivatives exhibit only two emissive species in the nanosecond range. All experimental data point to conformational relaxation and solvent reorganization leading to the cis and trans isomers of one preferential conformer with respect to the acylurea unit. The structure of the preferred conformation is mainly determined by the possible inter- or intramolecular H-bonds and is therefore also strongly influenced by the HBD and H-bond accepting (HBA) properties of the polar solvents. As the NH groups of the acylurea functionality contribute mainly to the entire inter- and intramolecular H-bond arrangement the variation of the substitution pattern of the urea unit, specifically the presence and position of the NH groups, leads to derivatives with significantly different steady-state and time-resolved emission properties.