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Abstract The limitations of native collagen, such as thermal stability and solubility in physiological environments, can be improved by applying bioconjugation and synthetic chemistry techniques. However, the exquisite control of the modification site of collagen remains a challenge. In this work, pH-responsive poly(acrylic acid) (PAA) with different chain lengths was attached to the N-terminal α-amino groups of succinylated collagen using a site-specific modification strategy. Additionally, the structure, thermal stability, and pH sensitivity of succinylated collagen were explored. The modification rate of amino groups in the succinylated collagen-PAA bioconjugate (SPSC-PAA) was evaluated by the 2,4,6-trinitrobenzene sulfonic acid assay. The impact of N-terminal modification of PAA and its chain length on the thermal stability of collagen was explored by CD and DSC. These techniques revealed that the thermal stability of SPSC-Col is pH-responsive and closely related to the chain length of grafted PAA. The pH sensitivity of SPSC-PAA was further explored by rheology and turbidity. Subsquently, the critical pH and isoelectric point of SPSC-PAAs were also examined by turbidity and isoelectric point titration, respectively. This work provides a new insight into the N-terminal modification of collagen on its properties. Graphical abstract

This study explores the use of diffuse reflectance spectroscopy (DRS) and multiwavelength fluorescence spectroscopy for real-time kidney stone identification during laser lithotripsy. Traditional methods are not suitable for in situ analysis, so the research focuses on optical techniques that can be integrated with lithotripsy fibers. Experiments were conducted ex vivo, using DRS and multiwavelength fluorescence spectroscopy (emission–excitation matrix (EEM)) to distinguish between 48 urinary stones of three types: urate, oxalate and hydroxyapatite, with infrared spectroscopy as a reference. A classification model was developed based on EEM and DRS data. Initial classification relying solely on EEM data achieved an f1-score of 87%, which increased to 92% when DRS data were included. The findings suggest that optical spectroscopy can effectively determine stone composition during laser lithotripsy, potentially enhancing surgical outcomes via the real-time automatic optimization of laser radiation parameters.

Extraordinary properties of two-dimensional materials make them attractive for applications in different fields. One of the prospective niches is optical applications, where such types of materials demonstrate extremely sensitive performance and can be used for labeling. However, the optical properties of liquid-exfoliated 2D materials need to be analyzed. The purpose of this work is to study the absorption and luminescent properties of MoS2 exfoliated in the presence of sodium cholate, which is the most often used surfactant. Ultrasound bath and mixer-assisted exfoliation in water and dimethyl sulfoxide were used. The best quality of MoS2 nanosheets was achieved using shear-assisted liquid-phase exfoliation as a production method and sodium cholate (SC) as a surfactant. The photoluminescent properties of MoS2 nanosheets varied slightly when changing the surfactant concentrations in the range C(SC) = 0.5–2.5 mg/mL. This work is of high practical importance for further enhancement of MoS2 photoluminescent properties via chemical functionalization.

A reaction of acyl chlorides derived from 1,10-phenanthroline-2,9-dicarboxylic acids with piperazine allows the preparation of the corresponding 24-membered macrocycles in good yield. The structural and spectral properties of these new macrocyclic ligands were thoroughly investigated, revealing promising coordination properties towards f-elements (Am, Eu). It was shown that the prepared ligands can be used for selective extraction of Am(III) from alkaline–carbonate media in presence of Eu(III) with an SFAm/Eu up to 40. Their extraction efficiency is higher than calixarene-type extraction of the Am(III) and Eu(III) pair. Composition of macrocycle–metal complex with Eu(III) was investigated by luminescence and UV-vis spectroscopy. The possibility of such ligands to form complexes of L:Eu = 1:2 stoichiometry is revealed.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell’s lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

We aimed to assess the influence of professional dental prophylaxis on the translucency and color stability of a novel simplified shade nanohybrid composite material. Sixty composite disks (5 mm in diameter and 2 mm thick) of light (n = 30) and dark (n = 30) shades were prepared. The specimens were randomly divided into the following three groups (n = 10) according to the prophylaxis procedure used: ultrasonic scaling, air-powder polishing with sodium bicarbonate, and controls. The specimens were submitted to translucency and color analysis based on the CIELab system. Two measurements were performed before and after 48-h storage in coffee. Translucency values of untreated light and dark specimens were 9.15 ± 0.38 and 5.28 ± 1.10, respectively. Air-powder polishing decreased the translucency of the light composite specimens. Storage in coffee resulted in color changes (∆E) ranging between 2.69 and 12.05 and a mean translucency decrease ranging between −0.88 and −6.91. The samples in the light group tended to exhibit greater staining; the treatment method had no effect on ∆E. It can be concluded that light-shade composite restorations are more prone to translucency and color changes resulting from air-powder polishing and contact with staining media. However, further research using other composites and powders is required.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells’ metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.

Rose Bengal (RB) is an anionic xanthene dye with multiple useful biological features, including photosensitization properties. RB was studied extensively as a photosensitizer, mostly for antibacterial and antitumor photodynamic therapy (PDT). The application of RB to virus inactivation is rather understudied, and no RB derivatives have been developed as antivirals. In this work, we used a synthetic approach based on a successful design of photosensitizing antivirals to produce RB derivatives for virus photoinactivation. A series of n-alkyl-substituted RB derivatives was synthesized and evaluated as antiviral photosensitizers. The compounds exhibited similar 1O2 generation rate and efficiency, but drastically different activities against SARS-CoV-2, CHIKV, and HIV; with comparable cytotoxicity for different cell lines. Submicromolar-to-subnanomolar activities and high selectivity indices were detected for compounds with C4-6 alkyl (SARS-CoV-2) and C6-8 alkyl (CHIKV) chains. Spectrophotometric assessment demonstrates low aqueous solubility for C8-10 congeners and a significant aggregation tendency for the C12 derivative, possibly influencing its antiviral efficacy. Initial evaluation of the synthesized compounds makes them promising for further study as viral inactivators for vaccine preparations.

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.

Photoacoustic and fluorescent methods are used intensely in biology and medicine. These approaches can also be used to investigate unicellular diatom algae that are extremely important for Earth’s ecology. They are enveloped within silica frustules (exoskeletons), which can be used in drug delivery systems. Here, we report for the first time the successful application of photoacoustic (PA) and fluorescent visualization of diatoms. Chlorophyll a and c and fucoxanthin were found likely to be responsible for the photoacoustic effect in diatoms. The PA signal was obtained from gel drops containing diatoms and was found to increase with the diatom concentration. The fluorescence lifetime of the diatom chromophores ranged from 0.5 to 2 ns. The dynamic light scattering, absorbance, and SEM characterization techniques were also applied. The results were considered in combination to elucidate the nature of the photoacoustic signal. Possible biotechnological applications are proposed for the remote photoacoustic monitoring of algae.

Cyanobacterial photoprotection is controlled by OCP and FRP proteins, but their dynamic interplay is not fully understood. Here, the authors combine protein engineering, disulfide trapping and structural analyses to provide mechanistic insights into the transient OCP-FRP interaction.

Diffuse reflectance spectroscopy (DRS) and imaging are increasingly being used in surgical guidance for tumor margin detection during endoscopic operations. However, the accuracy of the boundary detection with optical techniques may depend on the acquisition parameters, and its evaluation is in high demand. In this work, using optical phantoms with homogeneous and heterogeneous distribution of chromophores mimicking normal and pathological bladder tissues, the accuracy of tumor margin detection using single-fiber diffuse reflectance spectroscopy and spatial frequency domain imaging was evaluated. We also showed how the diffuse reflectance response obtained at different spatial frequencies with the spatial frequency domain imaging technique could be used not only to quantitatively map absorption and scattering coefficients of normal tissues and tumor-like heterogeneities but also to estimate the tumor depth localization. The demonstrated results could be helpful for proper analysis of the DRS data measured in vivo and for translation of optical techniques for tumor margin detection to clinics.

Abstract The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.

Signaling pathways of red blood cells’ (RBCs) micromechanics regulation, which are responsible for maintaining microcirculation, constitute an important property of RBC physiology. Selective control over these processes may serve as an indispensable tool for correction of hemorheological disorders, which accompany a number of systemic diseases (diabetes mellitus I&II, arterial hypertension, malaria, etc.). Activation of certain pathways involving adenylyl cyclase may provide fast adaptive regulation of RBC deformability (RBC-D). However the specific molecular conditions of intracellular signal transduction in mediating RBC microrheological properties at adenylyl cyclase stimulation remain unclear. In this paper, we present the results of the in vitro study of the effects of different signaling pathways in adenylyl cyclase stimulation on RBC-D. We studied (1) the direct stimulation of adenylyl cyclase with forskolin; (2) non-selective adrenoreceptor stimulation with epinephrine; (3) β2-adrenoreceptor agonist metaproterenol; (4) membrane-permeable analog of cAMP (dibutyryl-cAMP). Using laser ektacytometry, we observed a concentration-dependent increase in RBC-D for all studied effectors. The EC50 values for each substance were estimated to be in the range of 1–100 μM depending on the shear stress applied to the RBC suspension. The results can serve as an evidence of adenylyl cyclase signaling cascade involvement in the regulation of RBC micromechanical properties presenting a complex molecular pathway for fast increase of microcirculation efficiency in case of corresponding physiologic metabolic demands of the organism, e.g., during stress or physical activity. Further studies of this molecular system will reveal new knowledge which may improve the quality of medical treatment of hemorheological disorders.

Edema, i.e., fluid accumulation in the interstitial space, accompanies numerous pathological states of the human organism, including heart failure (HF), inflammatory response, and lymphedema. Nevertheless, techniques for quantitative assessment of the edema’s severity and dynamics are absent in clinical practice, and the analysis is mainly limited to physical examination. This fact stimulates the development of novel methods for fast and reliable diagnostics of fluid retention in tissues. In this work, we focused on the possibilities of two microscopic techniques, nailfold video capillaroscopy (NVC) and confocal laser scanning microscopy (CLSM), in the assessment of the short-term and long-term cutaneous edema. We showed that for the patients with HF, morphological parameters obtained by NVC—namely, the apical diameter of capillaries and the size of the perivascular zone—indicate long-term edema. On the other hand, for healthy volunteers, the application of two models of short-term edema, venous occlusion, and histamine treatment of the skin, did not reveal notable changes in the capillary parameters. However, a significant reduction of the NVC image sharpness was observed in this case, which was suggested to be due to water accumulation in the epidermis. To verify these findings, we made use of CLSM, which provides the skin structure with cellular resolution. It was observed that for the histamine-treated skin, the areas of the dermal papillae become hyporefractive, leading to the loss of contrast and the lower visibility of capillaries. Similar effect was observed for patients undergoing infusion therapy. Collectively, our results reveal the parameters can be used for pericapillary edema assessment using the NVC and CLSM, and paves the way for their application in a clinical set-up.

An elevated concentration of fibrinogen in blood is a significant risk factor during many pathological diseases, as it leads to an increase in red blood cells (RBC) aggregation, resulting in hemorheological disorders. Despite the biomedical importance, the mechanisms of fibrinogen-induced RBC aggregation are still debatable. One of the discussed models is the non-specific adsorption of fibrinogen macromolecules onto the RBC membrane, leading to the cells bridging in aggregates. However, recent works point to the specific character of the interaction between fibrinogen and the RBC membrane. Fibrinogen is the major physiological ligand of glycoproteins receptors IIbIIIa (GPIIbIIIa or αIIββ3 or CD41/CD61). Inhibitors of GPIIbIIIa are widely used in clinics for the treatment of various cardiovascular diseases as antiplatelets agents preventing the platelets’ aggregation. However, the effects of GPIIbIIIa inhibition on RBC aggregation are not sufficiently well studied. The objective of the present work was the complex multimodal in vitro study of the interaction between fibrinogen and the RBC membrane, revealing the role of GPIIbIIIa in the specificity of binding of fibrinogen by the RBC membrane and its involvement in the cells’ aggregation process. We demonstrate that GPIIbIIIa inhibition leads to a significant decrease in the adsorption of fibrinogen macromolecules onto the membrane, resulting in the reduction of RBC aggregation. We show that the mechanisms underlying these effects are governed by a decrease in the bridging components of RBC aggregation forces.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

Endogenous autofluorescence of biological tissues is an important source of information for biomedical diagnostics. Despite the molecular complexity of biological tissues, the list of commonly known fluorophores is strictly limited. Still, the question of molecular sources of the red and near-infrared excited autofluorescence remains open. In this work we demonstrated that the oxidation products of organic components (lipids, proteins, amino acids, etc.) can serve as the molecular source of such red and near-infrared excited autofluorescence. Using model solutions and cell systems (human keratinocytes) under oxidative stress induced by UV irradiation we demonstrated that oxidation products can contribute significantly to the autofluorescence signal of biological systems in the entire visible range of the spectrum, even at the emission and excitation wavelengths higher than 650 nm. The obtained results suggest the principal possibility to explain the red fluorescence excitation in a large class of biosystems—aggregates of proteins and peptides, cells and tissues—by the impact of oxidation products, since oxidation products are inevitably presented in the tissue. The observed fluorescence signal with broad excitation originated from oxidation products may also lead to the alteration of metabolic imaging results and has to be taken into account.

Heterogeneity of tumor metabolism is an important, but still poorly understood aspect of tumor biology. Present work is focused on the visualization and quantification of cellular metabolic heterogeneity of colorectal cancer using fluorescence lifetime imaging (FLIM) of redox cofactor NAD(P)H. FLIM-microscopy of NAD(P)H was performed in vitro in four cancer cell lines (HT29, HCT116, CaCo2 and CT26), in vivo in the four types of colorectal tumors in mice and ex vivo in patients’ tumor samples. The dispersion and bimodality of the decay parameters were evaluated to quantify the intercellular metabolic heterogeneity. Our results demonstrate that patients’ colorectal tumors have significantly higher heterogeneity of energy metabolism compared with cultured cells and tumor xenografts, which was displayed as a wider and frequently bimodal distribution of a contribution of a free (glycolytic) fraction of NAD(P)H within a sample. Among patients’ tumors, the dispersion was larger in the high-grade and early stage ones, without, however, any association with bimodality. These results indicate that cell-level metabolic heterogeneity assessed from NAD(P)H FLIM has a potential to become a clinical prognostic factor.