Photoresponsive Azobenzene Nanocluster-Modified Liposomes: Mechanism Analysis Combining Experiments and Simulations.

Stimuli-responsive behaviors and controlled release in liposomes are pivotal in nanomedicine. To this end, we present an approach using a photoresponsive azobenzene nanocluster (AzDmpNC), prepared from azobenzene compounds through melting and aggregation. When integrated with liposomes, they form photoresponsive vesicles. The morphology and association with liposomes were investigated by using transmission electron microscopy. Liposomes loaded with calcein exhibited a 9.58% increased release after UV exposure. To gain insights into the underlying processes and elucidate the mechanisms involved. The molecular dynamic simulations based on the reactive force field and all-atom force field were employed to analyze the aggregation of isomers into nanoclusters and their impacts on phospholipid membranes, respectively. The results indicate that the nanoclusters primarily aggregate through π-π and T-stacking forces. The force density inside the cis-isomer of AzDmpNC formed after photoisomerization is lower, leading to its easier dispersion, rapid diffusion, and penetration into the membrane, disrupting the densification.

Photoswitchable Pseudoirreversible Butyrylcholinesterase Inhibitors Allow Optical Control of Inhibition in Vitro and Enable Restoration of Cognition in an Alzheimer's Disease Mouse Model upon Irradiation.

To develop tools to investigate the biological functions of butyrylcholinesterase (BChE) and the mechanisms by which BChE affects Alzheimer's disease (AD), we synthesized several selective, nanomolar active, pseudoirreversible photoswitchable BChE inhibitors. The compounds were able to specifically influence different kinetic parameters of the inhibition process by light. For one compound, a 10-fold difference in the IC50-values (44.6 nM cis, 424 nM trans) in vitro was translated to an "all or nothing" response with complete recovery in a murine cognition-deficit AD model at dosages as low as 0.3 mg/kg.

Photosensitive Polymeric Janus Micromotor for Enzymatic Activity Protection and Enhanced Substrate Degradation.

Immobilizing enzymes into microcarriers is a strategy to improve their long-term stability and reusability, hindered by (UV) light irradiation. However, in such approaches, enzyme-substrate interaction is mediated by diffusion, often at slow kinetics. In contrast, enzyme-linked self-propelled motors can accelerate this interaction, frequently mediated by the convection mechanism. This work reports on a new photosensitive polymeric Janus micromotor (JM) for UV-light protection of enzymatic activity and efficient degradation of substrates accelerated by the JMs. The JMs were assembled with UV-photosensitive modified chitosan, co-encapsulating fluorescent-labeled proteins and enzymes as models and magnetite and platinum nanoparticles for magnetic and catalytic motion. The JMs absorbed UV light, protecting the enzymatic activity and accelerating the enzyme-substrate degradation by magnetic/catalytic motion. Immobilizing proteins in photosensitive JMs is a promising strategy to improve the enzyme's stability and hasten the kinetics of substrate degradation, thereby enhancing the enzymatic process's efficiency.

Atomically Resolved Characterization of Optically Driven Ligand Reconfiguration on Nanoparticle Catalyst Surfaces.

Dynamic ligand layers on nanoparticle surfaces could prove to be critically important to enhance the functionality of individual materials. Such capabilities could complement the properties of the inorganic component to provide multifunctionality or the ability to be remotely actuated. Peptide-based ligands have demonstrated the ability to be remotely responsive to structural changes when adsorbed to nanoparticle surfaces via incorporation of photoswitches into their molecular structure. In this contribution, direct spectroscopic evidence of the remote actuation of a photoswitchable peptide adsorbed onto Au nanoparticles is demonstrated using X-ray absorption fine structure spectroscopic methods. From this analysis, Au-X (X = C or N) coordination numbers confirm the changes before and after photoswitching in the surface ligand conformation, which was correlated directly to variations in the catalytic application of the materials for nitrophenol reduction processes. In addition, the catalytic application of the materials was demonstrated to be significantly sensitive to the structure of the nitrophenol substrate used in the reaction, suggesting that changes in the reactivity are likely based upon the peptide conformation and substrate structure. Such results confirm that surface ligands can be remotely reconfigured on nanoparticle surfaces, providing pathways to apply such capabilities to a variety of applications beyond catalysis ranging from drug delivery to sensing.

Rational Remodeling of Atypical Scaffolds for the Design of Photoswitchable Cannabinoid Receptor Tools.

Azobenzene-embedded photoswitchable ligands are the widely used chemical tools in photopharmacological studies. Current approaches to azobenzene introduction rely mainly on the isosteric replacement of typical azologable groups. However, atypical scaffolds may offer more opportunities for photoswitch remodeling, which are chemically in an overwhelming majority. Herein, we investigate the rational remodeling of atypical scaffolds for azobenzene introduction, as exemplified in the development of photoswitchable ligands for the cannabinoid receptor 2 (CB2). Based on the analysis of residue-type clusters surrounding the binding pocket, we conclude that among the three representative atypical arms of the CB2 antagonist, AM10257, the adamantyl arm is the most appropriate for azobenzene remodeling. The optimizing spacer length and attachment position revealed AzoLig 9 with excellent thermal bistability, decent photopharmacological switchability between its two configurations, and high subtype selectivity. This structure-guided approach gave new impetus in the extension of new chemical spaces for tool customization for increasingly diversified photo-pharmacological studies and beyond.

Photoswitches for controllable RNA binding: a future approach in the RNA-targeting therapy.

RNA is an emerging drug target that opens new perspectives in the treatment of viral and bacterial infections, cancer and a range of so far incurable genetic diseases. Among the various strategies towards the design and development of selective and efficient ligands for targeting and detection of therapeutically relevant RNA, photoswitchable RNA binders represent a very promising approach due to the possibility to control the ligand-RNA and protein-RNA interactions by light with high spatiotemporal resolution. However, the field of photoswitchable RNA binders still remains underexplored due to challenging design of lead structures that should combine high RNA binding selectivity with efficient photochemical performance. The aim of this highlight article is to describe the development of photoswitchable noncovalent RNA binders and to outline the current situation and perspectives of this emerging interdisciplinary field.

Azo modified hyaluronic acid based nanocapsules: CD44 targeted, UV-responsive decomposition and drug release in liver cancer cells.

Herein, we demonstrate a novel UV-induced decomposable nanocapsule of natural polysaccharide (HA-azo/PDADMAC). The nanocapsules are fabricated based on layer-by-layer co-assembly of anionic azobenzene functionalized hyaluronic acid (HA-azo) and cationic poly diallyl dimethylammonium chloride (PDADMAC). When the nanocapsules are exposed to 365 nm light, ultraviolet photons can trigger the photo-isomerization of azobenzene groups in the framework. The nanocapsules could decompose from large-sized nanocapsules to small fragments. Due to their optimized original size (~180 nm), the nanocapsules can effectively avoid biological barriers, provide a long blood circulation and achieve high tumor accumulation. It can fast eliminate nanocapsules from tumor and release the loaded drugs for chemotherapy after UV-induced dissociation. Besides, HA is an endogenous polysaccharide that shows intrinsic targetability to CD44 receptors on surface of cancer cells. The intracellular experiment shows that the HA-azo/PDADMAC nanocapsules with CD44 targeting ability and UV-controlled intracellular drug release are promising for cancer chemotherapy.

Sequence of Events during Peptide Unbinding from RNase S: A Complete Experimental Description.

The phototriggered unbinding of the intrinsically disordered S-peptide from the RNase S complex is studied with the help of transient IR spectroscopy, covering a wide range of time scales from 100 ps to 10 ms. To that end, an azobenzene moiety has been linked to the S-peptide in a way that its helicity is disrupted by light, thereby initiating its complete unbinding. The full sequence of events is observed, starting from unfolding of the helical structure of the S-peptide on a 20 ns time scale while still being in the binding pocket of the S-protein, S-peptide unbinding after 300 µs, and the structural response of the S-protein after 3 ms. With regard to the S-peptide dynamics, the binding mechanism can be classified as an induced fit, while the structural response of the S-protein is better described as conformational selection.

The Speed of Allosteric Signaling Within a Single-Domain Protein.

While much is known about different allosteric regulation mechanisms, the nature of the allosteric signal and the time scale on which it propagates remains elusive. The PDZ3 domain from postsynaptic density-95 protein is a small protein domain with a terminal third α-helix, i.e., the α3-helix, which is known to be allosterically active. By cross-linking the allosteric helix with an azobenzene moiety, we obtained a photocontrollable PDZ3 variant. Photoswitching triggers its allosteric transition, resulting in a change in binding affinity of a peptide to the remote binding pocket. Using time-resolved infrared and UV/vis spectroscopy, we follow the allosteric signal transduction and reconstruct the timeline in which the allosteric signal propagates through the protein within 200 ns.

Facile production of three-dimensional chitosan fiber embedded with zinc oxide as recoverable photocatalyst for organic dye degradation.

Herein we report on a facile and green strategy for continuous production of chitosan-zinc oxide fibers and then compare their photodegradation performance against three organic dyes (i.e., methylene blue (MB), methyl orange (MO) and Rhodamine B, respectively) under different lights. Chitosan-zinc hydrogel fibers (CS/Zn) with different zinc loadings are obtained by direct mixing of chitosan and zinc acetate solutions using a double-syringe injection device. The as-prepared CS/Zn fibers are then immersed into glutaraldehyde (GA) and sodium hydroxide solutions, respectively, and dried at T = 50 °C. The resultant CS/ZnO/GA fibers of ca. 617 µm in diameter are characterized using X-ray diffraction (XRD), thermogravimetric analysis and field emission scanning electron microscope (FE-SEM). XRD and FE-SEM data confirm that the CS/ZnO/GA fibers consist of a large amount of hexagonal wurtzite ZnO nanorods up to 550 nm in length, and exhibit three-dimensional interconnected macroporous architecture. Photodegradation results clearly show that the CS/ZnO/GA fibers are effective for the removal of organic dyes upon UV irradiation and can be easily recovered and reused for at least 6 consecutive cycles. Unlike most reported CS/ZnO nanocomposites, the current CS/ZnO/GA fiber shows a higher adsorption of cationic MB rather than anionic MO, the mechanism of which is proposed.

Response Surface Methodology: Photocatalytic Degradation Kinetics of Basic Blue 41 Dye Using Activated Carbon with TiO2.

Water decontamination still remains a major challenge to some developing countries not having centralized wastewater systems. Therefore, this study presents the optimization of photocatalytic degradation of Basic Blue 41 dye in an aqueous medium by an activated carbon (AC)-TiO2 photocatalyst under UV irradiation. The mesoporous AC-TiO2 synthesized by a sonication method was characterized by X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectroscopy for crystal phase identification and molecular bond structures, respectively. The efficiency of the AC-TiO2 was evaluated as a function of three input variables viz. catalyst load (2-4 g), reaction time (15-45 min) and pH (6-9) by using Box-Behnken design (BBD) adapted from response surface methodology. Using color and turbidity removal as responses, a 17 run experiment matrix was generated by the BBD to investigate the interaction effects of the three aforementioned input factors. From the results, a reduced quadratic model was generated, which showed good predictability of results agreeable to the experimental data. The analysis of variance (ANOVA), signposted the selected models for color and turbidity, was highly significant (p < 0.05) with coefficients of determination (R2) values of 0.972 and 0.988, respectively. The catalyst load was found as the most significant factor with a high antagonistic impact on the process, whereas the interactive effect of reaction time and pH affected the process positively. At optimal conditions of catalyst load (2.6 g), reaction time (45 min), and pH (6); the desirability of 96% was obtained by a numerical optimization approach representing turbidity removal of 93% and color of 96%.

Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythms.

Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of the cis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity together with the accompanying modulation of cellular circadian rhythms in which CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.

Structural Aspects of Photopharmacology: Insight into the Binding of Photoswitchable and Photocaged Inhibitors to the Glutamate Transporter Homologue.

Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation of inhibitory potency in general is still missing, which poses a major design challenge for this emerging field of research. Here we solved crystal structures of the glutamate transporter homologue GltTk in complex with photoresponsive transport inhibitors-azobenzene derivative of TBOA (both in trans and cis configuration) and with the photocaged compound ONB-hydroxyaspartate. The essential role of glutamate transporters in the functioning of the central nervous system renders them potential therapeutic targets in the treatment of neurodegenerative diseases. The obtained structures provide a clear structural insight into the origins of photocontrol in photopharmacology and lay the foundation for application of photocontrolled ligands to study the transporter dynamics by using time-resolved X-ray crystallography.

Photoregulated "Breathing" Vesicle with Inversed Supramolecular Chirality.

Though phospholipids possess chiral centers, their chiral aggregation within bilayer cell membranes has seldom been referred and recognized. Insight into the chirality at higher levels in artificial molecular bilayer assemblies such as vesicles or liposomes is important to better understand biomembrane functions. In this work, we illustrate the fabrication of chiral vesicles with photoresponsive supramolecular chirality and structural transformation property. Cholesterol was conjugated to azobenzene via different spacers, of which molecular chirality underwent transfer to supramolecular level upon aggregation in water. The resultant building block self-assembled into unilamellar vesicles that could respond to light irradiation by showing reversible extension/contraction behavior. Such "breathing" behavior was accompanied with supramolecular chirality inversion from M- to P-handedness, confirmed by the solid-state crystal structure and electronic circular dichroism spectra based on density functional theory. The vesicle membrane behaves as a matrix to accommodate guest molecules via aromatic interactions, which significantly elevated the UV light resistance with respect to the structural and supramolecular chirality transformation. This work offers an unprecedented rational control over supramolecular chirality using photoresponsiveness in vesicular membranes.

Theoretical insights into the effect of size and substitution patterns of azobenzene derivatives on the DNA G-quadruplex.

Introducing photoswitches into the DNA G-quadruplex provides excellent opportunities to control folding and unfolding of these assemblies, demonstrating their potential in the development of novel nanodevices with medical and nanotechnology applications. Using a quantum mechanics/molecular mechanics (QM/MM) scheme, we carried out a series of simulations to identify the effect of the size and substitution patterns of three azobenzene derivatives (AZ1, AZ2 and AZ3) on the excitation energies of the two lowest excited states of the smallest photoswitchable G-quadruplex reported to date. We demonstrated that the size and the substitution pattern do not affect the ultrafast cis-trans photoiomerization mechanism of the azobenzene derivatives significantly, in agreement with the experiment. However, molecular dynamics simulations revealed that while AZ2 and AZ3 G-quadruplexes are structurally stable during the simulations, the AZ1 G-quadruplex undergoes larger structural changes and shows two ground state populations that differ in the azobenzene backbone adopting two different conformations. AZ1, with para-para substitution pattern, provides more flexibility to the whole G-quadruplex structure compared to AZ2 and AZ3, and can thus facilitate the photoisomerization reaction between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after trans-cis isomerization occurring in a longer time dynamics, in agreement with the experimental findings. The QM/MM simulations of the absorption spectra indicated that the thermal fluctuation plays a more crucial role in the main absorption band of the azobenzene derivatives than the inclusion of the G-quadruplex, implying that the influence of the G-quadruplex environment is minimal. We propose that the latter is attributed to the position of the azobenzene linkers in the G-quadruplexes, i.e. the edgewise loops containing the azobenzene moieties that are located above the G-quartets, not being fully embedded inside or involved in the stacked structure. Our theoretical findings provide support to a recent study of the photoresponsive formation of photoswitchable G-quadruplex motifs.

Controlling Supramolecular Structures of Drugs by Light.

Controlling physicochemical properties of light-unresponsive drugs, by light, prima facie, a paradox approach. We expanded light control by ion pairing light-unresponsive salicylate or ibuprofen to photoswitchable azobenzene counterions, thereby reversibly controlling supramolecular structures, hence the drugs' physicochemical and kinetic properties. The resulting ion pairs photoliquefied into room-temperature ionic liquids under ultraviolet light. Aqueous solutions showed trans-cis-dependent supramolecular structures under a light with wormlike aggregates decomposing into small micelles and vice versa. Light control allowed for permeation through membranes of cis-ibuprofen ion pairs within 12 h in contrast to the trans ion pairs requiring 72 h. In conclusion, azobenzene ion-pairing expands light control of physicochemical and kinetic properties to otherwise light-unresponsive drugs.

ROS-initiated chemiluminescence-driven payload release from macrocycle-based Azo-containing polymer nanocapsules.

Reactive oxygen species (ROS) overproduction is involved in many pathological processes, particularly in inflammatory diseases. Therefore, ROS-responsive nanocarriers for specific drug release have been highly sought after. Herein we developed a ROS-responsive drug delivery system based on covalently self-assembled polymer nanocapsules (Azo-NCs) formed via crosslinking macrocyclic cucurbit[6]urils by a photo-sensitive azobenzene derivative (Azo). Luminol, a chemiluminescent molecule activatable by ROS, was co-loaded into Azo-NCs together with a therapeutic payload. When exposed to high ROS concentration that is typically encountered in inflammatory cells or tissues, the ROS-initiated blue chemiluminescence of luminol drives photoisomerization of the Azo groups within Azo-NCs, leading to Azo-NCs' surface transformation and distortion of the nanostructure, and subsequent payload release. As a proof-of-concept, ROS-responsive payload release from luminol-loaded Azo-NCs in inflammatory cells and zebrafish was demonstrated, showing promising anti-inflammatory effects in vitro and in vivo.

Spatiotemporal regulation of dynamic cell microenvironment signals based on an azobenzene photoswitch.

Dynamic biochemical and biophysical signals of cellular matrix define and regulate tissue-specific cell functions and fate. To recapitulate this complex environment in vitro, biomaterials based on structural- or degradation-tunable polymers have emerged as powerful platforms for regulating the "on-demand" cell-material dynamic interplay. As one of the most prevalent photoswitch molecules, the photoisomerization of azobenzene demonstrates a unique advantage in the construction of dynamic substrates. Moreover, the development of azobenzene-containing biomaterials is particularly helpful in elucidating cells that adapt to a dynamic microenvironment or integrate spatiotemporal variations of signals. Herein, this minireview, places emphasis on the research progress of azobenzene photoswitches in the dynamic regulation of matrix signals. Some techniques and material design methods have been discussed to provide some theoretical guidance for the rational and efficient design of azopolymer-based material platforms. In addition, considering that the UV-light response of traditional azobenzene photoswitches is not conducive to biological applications, we have summarized the recent approaches to red-shifting the light wavelength for azobenzene activation.

Reversible spatial and temporal control of lipid signaling.

Herein, we introduce versatile molecular tools that enable specific delivery and visualization of photoswitchable lipids at cellular membranes, namely at the plasma membrane and internal membranes. These molecules were prepared by tethering ortho-nitrobenzyl-based fluorescent cages with a signaling lipid bearing an azobenzene photoswitch. They permit two sequential photocontrolled reactions, which are uncaging of a lipid analogue and then its repeated activation and deactivation. We used these molecules to activate GPR40 receptor transiently expressed in HeLa cells and demonstrated downstream modulation of intracellular Ca2+ levels.

Enhancing the performance of pollution degradation through secondary self-assembled composite supramolecular heterojunction photocatalyst BiOCl/PDI under visible light irradiation.

A novel n-n type inorganic/organic heterojunction of flaky-like BiOCl/PDI photocatalyst was constructed by water bath heating method. Meanwhile, a simple method - secondary self-assembly was used to prepare the BiOCl/PDI with a special band structure. The photocatalytic activities were evaluated by degrading aqueous organic pollutants under visible light (λ > 420 nm). The removal rates of 5 mg L-1 phenol (non-ionic type), methyl orange (MO, anionic type), rhodamine B (RhB, cationic type) and 10 mg L-1 RhB by secondary self-assembly BiOCl/PDI (BiOCl/PDI-2) were 8.0%, 3.4%, 27.8% and 78.9% higher than self-assembly BiOCl/PDI (BiOCl/PDI-1) under visible light (λ > 420 nm). The better photocatalytic activity for BiOCl/PDI-2 was attributed to the optimization of energy-band structures, which arose from different exposed surfaces, narrower interplanar spacing and stronger visible light absorption performance. Under acidic condition, BiOCl/PDI-2 showed a good photocatalytic activity, which was not affected by neutral ionic intensity and had good recycling properties. Moreover, the photocatalytic mechanism was explored by free radical capture test and electron paramagnetic resonance (EPR), and contribution of active species was calculated. The main active species of BiOCl/PDI-2 were ·O2-, 1O2 and h+. Our work may provide a route to design efficient inorganic/organic heterojunctions for organic pollutants degradation.