The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCP^O state to the red active signaling state, OCP^R, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCP^O suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCP^R. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCP^R to OCP^O. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

Because of their high biocompatibility, biological barrier negotiation, and functionalization properties, biological nanoparticles have been actively investigated for many medical applications. Biological nanoparticles, including natural extracellular vesicles (EVs) and synthetic extracellular vesicle–mimetic nanovesicles (EMNVs), represent novel drug delivery vehicles that can accommodate different payloads. In this study, we investigated the physical, biological, and delivery properties of EVs and EMNVs and analyzed their ability to deliver the chemotherapeutic drug doxorubicin. EMNVs and EVs exhibit similar properties, but EMNVs are more effectively internalized, while EVs show higher intracellular doxorubicin release activity. In addition, these nanotherapeutics were investigated in combination with the FDA-approved drug hydroxychloroquine (HCQ). We demonstrate that HCQ-induced lysosome destabilization and could significantly increase nanoparticle internalization, doxorubicin release, and cytotoxicity. Altogether, these data demonstrate that, from the delivery standpoint in vitro, the internalization of EMNVs and EVs and their payload release were slightly different and both nanotherapeutics had comparable cytotoxic performance. However, the synthesis of EMNVs was significantly faster and cost-effective. In addition, we highlight the benefits of combining biological nanoparticles with the lysosome-destabilizing agent HCQ that increased both the internalization and the cytotoxic properties of the particles.

Objectives. It was recently discovered that water samples with modified physicochemical properties can be obtained by successive vibration treatment of intact water together with a solution of a substance located in separate closely spaced vials. We refer to such samples as iterations. By adding the vibrational iterations into the initial substance, the physicochemical properties of the latter are changed, i.e., they demonstrate post-vibration activity. In addition, it has been shown that vibrational iterations can be obtained using water treated with a magnetic field as the initial substance. On this basis, we may hypothesize that the phenomenon of post-vibration activity is universal. To confirm this hypothesis, water treated with an electric signal having various parameters (electrochemically activated water) was used as the initial substance for the preparation of vibrational iterations.Methods. The physicochemical properties of vibrational iterations, which were obtained from electrochemically activated water, were studied by conductometry, terahertz spectroscopy, and radiometry. The effect of the initial substance or its vibrational iterations on intact water (a neutral carrier) was evaluated by dynamic light scattering. For this purpose, the intensity of light scattering by the sample and the hydrodynamic diameter of optical heterogeneities were measured. The attenuation coefficient of an additional electric signal applied to the samples was determined.Results. The obtained vibrational iterations differ from intact water and their mixtures with intact water in terms of specific electrical conductivity, power flux density of microwave radiation, as well as in the contribution of the main (Debye) relaxation process to the overall dielectric response. Mixtures of vibrational iterations with water also differ from intact water in terms of the size of optical heterogeneities. By analogy with the vibrational iterations for which solutions of high- and low-molecular-weight substances were used as the initial substance, vibrational iterations obtained using electrochemically activated water can be classified into different groups (fractions) according to their physicochemical characteristics. Different degrees of changes in the physicochemical characteristics are observed depending on the parameters of the electric signal used to obtain the initial substance. The efficiency of electrical signal propagation in these mixtures, as estimated by the signal strength attenuation coefficient, is additionally changed. The addition of the initial substance (electrochemically activated water) to intact water also leads to changes in the physicochemical properties of the resulting mixture compared to the control. Depending on the parameters of the electric signal used to obtain the initial substance, the magnitude of changes in the physicochemical characteristics of these mixtures similarly varies.Conclusions. The fundamental possibility of obtaining vibrational iterations from electrochemically activated water similarly to vibrational iterations prepared in other studies, was demonstrated. This confirms the universality of the phenomenon of post-vibration.

This paper investigates the effects of europium acetate and intensive stirring on the intermolecular properties of water in solutions. To do this, we studied aqueous solutions of europium acetate in a wide range of concentrations, which were prepared by serial dilution using a microfluidic unit. Water and similarly prepared water dilutions were used as controls. Raman spectroscopy and infrared (IR) spectroscopy were applied to assess the features of hydrogen bonds formed in the studied solutions. Using Raman spectroscopy, it was shown that intermolecular binding is stronger in solutions of europium acetate of 10^–1 M and 10^–3 M than in water controls. On the contrary, solutions of europium acetate at a concentration of 10^–10 М and some lower concentrations demonstrate weaker hydrogen bonding than in the respective water dilutions, which was shown by both methods. Such differences were observed even in solutions with a calculated concentration of europium acetate below 10^–24 M. When comparing water with control dilutions of water, it was established that intermolecular binding is different (stronger or weaker) in high dilutions of water than in water not subjected to the dilution procedure. This indicates that the dilution process itself significantly influences the properties of water in solutions. Additionally, the paper discusses the energy state of water molecules in the studied solutions.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to acidic environment observed in endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline of their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid endolysosomal compartment whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge associated with scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs, and indicate that EMNVs are more efficient in escaping or avoiding endolysosomal compartment.