Anna Y. Gyurova

Plant extracts remain an important source of antiviral agents, while nanocarrier-based delivery systems have emerged as a promising strategy for improving their therapeutic safety and efficacy. Building on previous findings indicating that nanoliposomal encapsulation improved the anti-coronavirus activity of Aesculus hippocastanum seed extract against human coronavirus strain OC43 (HCoV-OC43), the present study evaluated whether encapsulation reduces the genotoxic effects of the extract and explored a potential molecular basis for the previously observed antiviral activity. Genotoxicity assessment using the alkaline comet assay in MRC-5 human lung fibroblasts demonstrated that at 1 mg/mL, liposomal encapsulation was associated with an approximately 86.0% reduction in %Tail DNA, from 21.301 ± 0.368% for the free extract to 2.984 ± 1.523% for the liposome-encapsulated extract. In parallel, chemical profiling by high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (HPLC–HRMS/MS) enabled the identification and tentative annotation of 46 metabolites, including triterpenoid saponins, flavonoids, indole-3-acetic acid glycosides, and organic acids. Based on this phytochemical profile, representative constituents were subjected to molecular docking against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural proteins Nsp5 and Nsp12 to investigate their potential contribution to the previously demonstrated antiviral activity of the extract. Among the evaluated compounds, hesperidin exhibited the most favorable predicted binding affinity toward both targets, while quercetin also showed favorable interactions, suggesting that flavonoid constituents may contribute to the antiviral activity of the extract. These findings indicate that liposomal encapsulation improves the biological safety profile of A. hippocastanum seed extract, while phytochemical characterization and molecular docking provide preliminary insights into the constituents that may contribute to its previously demonstrated anti-coronavirus activity.

A detailed investigation of the structure–property relationships of three-antennary oligoglycines in aqueous solutions is performed. Two representatives of these substances are investigated: CH₃C(-CH₂-NH-Gly₅)₃ and CH₃C(-CH₂-NH-Gly₇)₃. The aim is to clarify the effect of molecular peculiarities and the concentration of the oligoglycines on bulk-solution performance and on adsorption-layer properties at the solution–air interface. This study is focused on the clarification of the conditions for the onset of bulk and interfacial supramolecular species in the aqueous environment. The presence of oligoglycine antennae attached to a common carbon-atom center allows the formation of highly coordinated intra- and intermolecular ‘click-clack’ interactions and presumes the possibility for the development of extended H-bonded networks, e.g., in the form of Polyglycine II motifs. A combined study protocol, including dynamic light scattering, profile analysis tensiometry, and microscopic thin-liquid-film techniques, is applied. The results allow the drawing of essential conclusions about the possible coupling mechanism of bulk and interfacial phenomena. The outcomes give grounds to advance the following hypothesis: due to the synchronized action of noncovalent interactions, three types of tectomeric structures may appear—dimers, gel-like elements, and disk-like supramolecular entities. Options for fine-tuning of the tectomer formation in aqueous solutions are presented, and possible application routes are outlined.

Two-antennary oligoglycines are synthetic, biocompatible bola-amphiphiles composed of homologs with varying hydrophobic-to-hydrophilic ratios. These molecules exhibit a pronounced tendency for self-assembly in aqueous media, resulting in the formation of well-defined supramolecular structures known as tectomers. The present study is focused on one representative of the antennary olygoglycine and investigates its potential as a nanocarrier for hydrophobic drugs. The non-steroidal anti-inflammatory drug indomethacin is used as a model hydrophobic compound. To maximize the entrapment of the drug, we develop an experimental procedure, including the preliminary formation of tectomers at various pH values, followed by the addition of indomethacin. The obtained oligoglycine-indomethacin complexes are further characterized using UV-Vis spectroscopy, Dynamic Light Scattering, and Thin-Liquid-Film techniques. The results demonstrate that the two-antennary oligoglicine effectively captures indomethacin and forms stable complexes in aqueous media. We outline the optimal conditions for drug entrapment and propose hypotheses regarding the mechanisms behind the formation of the mixed complexes. This study advances the understanding of interactions between two-antennary oligoglycines and hydrophobic compounds and highlights their potential for biomedical applications.

Disease’s severity, mortality rates, and common failures to achieve clinical improvement during the unprecedented COVID-19 pandemic exposed the emergency need for new antiviral therapeutics with higher efficacy and fewer adverse effects. This study explores the potential to encapsulate multi-component plant extracts in liposomes as optimized delivery systems and to verify if they exert inhibitory effects against human seasonal betacoronavirus OC43 (HCoV-OC43) in vitro. The selection of Sambucus nigra, Potentilla reptans, Allium sativum, Aesculus hippocastanum, and Glycyrrhiza glabra L. plant extracts was based on their established pharmacological and antiviral properties. The physicochemical characterization of extract-loaded liposomes was conducted by DLS and electrokinetics. Encapsulated amounts of the extract were evaluated based on the total flavonoid content (TFC) and total polyphenol content (TPC) by colorimetric methods. The BALB 3T3 neutral red uptake (NRU) phototoxicity/cytotoxicity assay was used to estimate compounds’ safety. Photo irritation factors (PIFs) of the liposomes containing extracts were A. hippocastanum extract showed 16 times higher inhibitory properties reaching a selectivity index (SI) of 58.96. Virucidal and virus-adsorption effects were investigated using the endpoint dilution method and ∆lgs comparison with infected and untreated controls. The results confirmed that nanoparticles do not directly affect the viral surface or cell membrane, but only serve as carriers of the active substances and the observed protection is due solely to the intracellular action of the extracts.

The difficulties faced by the society in its goal of reducing mortality and improving the health status during the period of the COVID-19 pandemic showed that it is necessary to develop new approaches in the therapy of infectious diseases, leading to higher treatment efficiency and few side effects. The subject of the present study is the formation and characterization of the proper-ties of stable liposomes loaded with extracts from Bulgarian medicinal plants and monitoring their antiviral action against the human strain of coronavirus OC43 (HCoV-OC43) in vitro. The hydrodynamic diameter and -potential of the liposomes containing each type of payload were determined with DLS and electrokinetics. The encapsulated amount of extracts was evaluated against quercetin and gallic acid by using colourimetric methods. The BALB 3T3 Neutral Red Uptake (NRU) phototoxicity/cytotoxicity assay was used to estimate the safety of the compounds. For assessment of the antiviral activity against HCoV-OC43 of liposomes (Lip) containing medicinal plant extracts, the cytopathic effect inhibition test was used. Measurements of the virucidal activity and the effect on the virus adsorption stage was carried out using the endpoint dilution method, and ∆lgs were calculated by comparison with untreated controls. The estimated photo irritation factors (PIF) for the Lip containing extracts were < 2 which assigns them to the category of non-phototoxic substances. When the extracts were introduced into the liposomes as their carriers, the antiviral activity of the vesicles against the replication of HCoV-OC43 in vitro in-creased by several times compared to their individual activity described in our previous study. The determination of the effect of structures loaded with extracts on the extracellular virions and the stage of their adsorption to sensitive HCT-8 cells proved that the liposomes do not affect directly the viral surface or cell membrane, but only perform a function of carriers of the active substances and the observed result is due solely to the intracellular action of the extracts.

An object of the present study was the development of liposomes loaded with the medicine Veklury^® (remdesivir) stabilized by electrostatic adsorption of polysaccharide film formed from chitosans with different physicochemical characteristics and hyaluronic acid. The functionalization of the structures was achieved through the inclusion of an aptamer (oligonucleotide sequence) with specific affinity to the spike protein of the human coronavirus HCoV-OC43. The hydrodynamic size, electrokinetic potential and stability of the structures were evaluated at each step in the procedure. The encapsulation efficiency and loaded amount of remdesivir (99% and 299 µg/mL) were estimated by UV–vis spectroscopy. Our investigations showed manifestation of promising tendencies for prolonged periods of the drug release and increased effectiveness of its antiviral action. Among all studied versions of the delivery system, the most distinguished and suitable in a model coronavirus therapy are the liposomes formed from chitosan oligosaccharides. The cytotoxicity of the liposomes was determined against the HCT-8 cell line. A cytopathic effect inhibition test was used for the assessment of the antiviral activity of the compounds. The virucidal activity and the effect on the viral adsorption of the samples were reported by the end-point dilution method, and the alteration in viral titer was determined as Δlgs compared to untreated controls. The redox-modulating properties of the nanoparticles were studied in vitro in certain/several/a few chemical model systems. Our investigations showed a manifestation of promising tendencies for a prolonged effect of the drug release and increased effectiveness of its antiviral action.

Specific self-assembly is registered in aqueous solution formulations based on four-antennary oligoglycines (T4), namely a spontaneous onset of highly ordered nanostructures—tectomers. This phenomenon is initiated by the action of hydrogen-bonding interactions that promote molecular recognition propensities involving Polyglycine-II-type non-canonical architecture. The result is the formation of positively charged supramolecular entities. These have high potential to capture bacterial endotoxins, like lipopolysaccharides (LPSs). By now, it has been established that the overall properties of these systems can be precisely regulated and gradually changed through fine-tuning the parameters in the aqueous environment (composition, pH, etc.). One unexplored option is to clarify the impact of temperature variations. The aim of the present study is to implement systematic investigations on how changes in temperature influence the various options for the removal of trace LPS quantities, captured by the T4 tectomers. The additional goal is to verify the possibility to develop consecutive paths of recovering the extra T4 quantities that have not participated in the formation of T4+LPS complexes. Some prospects for further applications, e.g., in medicine and pharmaceutics, are also generally outlined.

Bacterial lipopolysaccharides (LPS) are designated as endotoxins, because they cause fever and a wide range of pathologies in humans. It is important to develop effective methodologies to detect trace quantities of LPS in aqueous systems. The present study develops a fine-tuning procedure for the entrapment of trace quantities of LPS from E. coli EH100. The capture agents are self-assemblies (tectomers) formed by synthetic four-antennary oligoglycine (C-(CH₂-NH-Gly₇)₄, T4). Based on previously performed investigations of bulk and adsorption-layer properties of aqueous solutions containing T4 and LPS, the optimal conditions for the entrapment interactions are further fine-tuned by the pH regulation of aqueous systems. A combined investigation protocol is developed, including dynamic light scattering, profile analysis tensiometry, microscopic thin-liquid-film techniques, and transmission electron microscopy. The key results are: (1) two types of complexes between T4 and LPS are generated—amphiphilic species and “sandwich-like” hydrophilic entities; the complexes are smaller at lower pH, and larger at higher pH; (2) an optimum range of pH values is established within which the whole quantity of the LPS is entrapped by the tectomers, namely pH = 5.04–6.30. The obtained data substantiate the notion that T4 may be used for an effective capture and the removal of traces of endotoxins in aqueous systems.

The electric properties of bacteria determine their non-specific interactions with the environment, in particular their pathogenic activity. The electric polarizability of Escherichia coli HB101 (K-12 strain) was studied while inactivation with ethanol (20–40 vol.%). The current investigation might be regarded as a continuation of previous research on the polarizability of E. coli at lower ethanol concentration (≤ 20 vol.%) and higher frequencies (≥ 20 kHz). The bacteria polarizability at low frequencies (4 Hz) shows anomalies (unexpected increase in the polarizability at certain ethanol concentrations), while the parameter decreases with an increase in the ethanol concentration at higher frequencies. We investigated for the possible reasons causing the anomalies — in our case reduced to the medium dielectric permittivity, the average cell length and the surface electric charge density distribution, related to bacterial lipopolysaccharides. We suggest a hypothesis for the molecular mechanism of changing the surface charge of E. coli, carried by lipopolysaccharides, induced by the non-ionic ethanol.