Fluorescent properties in solid-state and solution of novel tricyclic derivatives of chloro/bromophenylchromanones and 2-methylpyrazoline.

In this work, we reported the synthesis and spectroscopic characterization of seven novel tricyclic compounds resulting from the reaction of 3-benzylidenechromanone with Cl or Br substituent in different positions and without halogen with methylhydrazine. The structural characterization of compounds was done through different techniques i.e., FTIR,1HNMR,a single and powder X-Ray diffraction. Moreover, fluorescence quantum yield and lifetime assessed their fluorescent properties in the solid state and various solvents. Derivatives with Cl or Br substituent in positions 2 and 4 are isostructural. 4-Cl, 4-Br and 3-Cl compounds exhibit fluorescence with moderate efficiency (quantum yield 0.11-0.26) in solid state due to specific arrangements, so-called π-stack brick stone with head-to-tail self-assembly. Other crystalline compounds (2-Cl, 2-Br and 3-Br) that exhibit negligible fluorescence quantum yield have crossed V-type arrangement. In the solution, the nonhalogenated compound shows the best fluorescence efficiency. In turn, the presence of halogen atoms results in fluorescence decreasing. TD-DFT study revealed that unsubstituted compound higher emissive in solution has a different electron density distribution at HOMO and LUMO levels than less emissive substituted compounds (A3 and A3).

Time-Resolved Fluorescence Spectroscopy of Blood, Plasma and Albumin as a Potential Diagnostic Tool for Acute Inflammation in COVID-19 Pneumonia Patients.

Fluorescence lifetime measurements of blood or plasma offer valuable insights into the microenvironment and molecular interactions of fluorophores, particularly concerning albumin. Neutrophil- and hypoxia-induced oxidative stress in COVID-19 pneumonia patients leads to hyperinflammation, various oxidative modifications of blood proteins, and potential alterations in the fluorescence lifetime of tryptophan-containing proteins, especially albumin. The objective of this study was to investigate the efficacy of time-resolved fluorescence spectroscopy of blood and plasma as a prompt diagnostic tool for the early diagnosis and severity assessment of COVID-19-associated pneumonia. This study examined a cohort of sixty COVID-19 patients with respiratory symptoms. To investigate whether oxidative stress is the underlying cause of the change in fluorescence lifetime, human serum albumin was treated with chloramine T. The time-resolved spectrometer Life Spec II (Edinburgh Instruments Ltd., Livingston, UK), equipped with a sub-nanosecond pulsed 280 nm diode, was used to measure the fluorescence lifetime of blood and plasma. The findings revealed a significant reduction in the fluorescence lifetime of blood (diluted 200 times) and plasma (diluted 20 times) at 360 nm in COVID-19 pneumonia patients compared with their respective values recorded six months post-infection and those of healthy individuals. Significant negative correlations were observed between the mean fluorescence lifetime of blood and plasma at 360 nm and several severity biomarkers and advanced oxidation protein products, while a positive correlation was found with albumin and the albumin-globulin ratio. The time-resolved fluorescence spectroscopy method demonstrates the potential to be used as a preliminary screening technique for identifying patients who are at risk of developing severe complications. Furthermore, the small amount of blood required for the measurements has the potential to enable a rapid fingerstick blood test.

Relationship between red blood cell aggregation and dextran molecular mass.

The aim of this study was to investigate the aggregation of red blood cells (RBCs) suspended in dextran solution at various levels of molecular mass. Dextran solutions at molecular mass 40, 70, 100 and 500 kDa at concentration from 2 to 5 g/dL were used to suspend the RBCs. The radius and velocity of sedimenting RBC aggregates were investigated using image analysis. The radius and sedimentation velocity of aggregates increased initially, then decreased after achieving maxima. The maximal velocity of RBC aggregates showed a bell-shaped dependence on dextran molecular mass and concentration, whereas maximal radius showed monotonic increase with both factors. Difference between aggregate and solution density was estimated using aggregate radius and sedimentation velocity and dextran solution viscosity, and was consistent across most molecular mass and concentration levels. This allowed to calculate the porosity of aggregates and to show that it monotonically decreased with the increase in the solution density, caused by the increase in the dextran concentration. The results provide insight into the RBC aggregation process in solutions of proteins of different size, reflecting various pathological conditions. The currently reported data can be potentially applied to specific pathophysiological conditions giving an interpretation that is not yet fully discussed in the literature.

The Mortality Risk and Pulmonary Fibrosis Investigated by Time-Resolved Fluorescence Spectroscopy from Plasma in COVID-19 Patients.

A method of rapidly pointing out the risk of developing persistent pulmonary fibrosis from a sample of blood is extraordinarily needed for diagnosis, prediction of death, and post-infection prognosis assessment. Collagen scar formation has been found to play an important role in the lung remodeling following SARS-CoV-2 infection. For this reason, the concentration of collagen degradation products in plasma may reflect the process of lung remodeling and determine the extent of fibrosis. According to our previously published results of an in vitro study, an increase in the concentration of type III collagen degradation products in plasma resulted in a decrease in the fluorescence lifetime of plasma at a wavelength of 450 nm. The aim of this study was to use time-resolved fluorescence spectroscopy to assess pulmonary fibrosis, and to find out if the lifetime of plasma fluorescence is shortened in patients with COVID-19. The presented study is thus far the only one to explore the fluorescence lifetime of plasma in patients with COVID-19 and pulmonary fibrosis. The time-resolved spectrometer Life Spec II with the sub-nanosecond pulsed 360 nm EPLED® diode was used in order to measure the fluorescence lifetime of plasma. The survival analysis showed that COVID-19 mortality was associated with a decreased mean fluorescence lifetime of plasma. The AUC of mean fluorescence lifetime in predicting death was 0.853 (95% CI 0.735−0.972, p < 0.001) with a cut-off value of 7 ns, and with 62% sensitivity and 100% specificity. We observed a significant decrease in the mean fluorescence lifetime in COVID-19 non-survivors (p < 0.001), in bacterial pneumonia patients without COVID-19 (p < 0.001), and in patients diagnosed with idiopathic pulmonary fibrosis (p < 0.001), relative to healthy subjects. Furthermore, these results suggest that the development of pulmonary fibrosis may be a real and serious problem in former COVID-19 patients in the future. A reduction in the mean fluorescence lifetime of plasma was observed in many patients 6 months after discharge. On the basis of these data, it can be concluded that a decrease in the mean fluorescence lifetime of plasma at 450 nm may be a risk factor for mortality, and probably also for pulmonary fibrosis in hospitalized COVID-19 patients.

Study of Albumin Oxidation in COVID-19 Pneumonia Patients: Possible Mechanisms and Consequences.

Oxidative stress induced by neutrophils and hypoxia in COVID-19 pneumonia leads to albumin modification. This may result in elevated levels of advanced oxidation protein products (AOPPs) and advanced lipoxidation end-products (ALEs) that trigger oxidative bursts of neutrophils and thus participate in cytokine storms, accelerating endothelial lung cell injury, leading to respiratory distress. In this study, sixty-six hospitalized COVID-19 patients with respiratory symptoms were studied. AOPPs-HSA was produced in vitro by treating human serum albumin (HSA) with chloramine T. The interaction of malondialdehyde with HSA was studied using time-resolved fluorescence spectroscopy. The findings revealed a significantly elevated level of AOPPs in COVID-19 pneumonia patients on admission to the hospital and one week later as long as they were in the acute phase of infection when compared with values recorded for the same patients 6- and 12-months post-infection. Significant negative correlations of albumin and positive correlations of AOPPs with, e.g., procalcitonin, D-dimers, lactate dehydrogenase, aspartate transaminase, and radiological scores of computed tomography (HRCT), were observed. The AOPPs/albumin ratio was found to be strongly correlated with D-dimers. We suggest that oxidized albumin could be involved in COVID-19 pathophysiology. Some possible clinical consequences of the modification of albumin are also discussed.

A study of the oxidative processes in human plasma by time-resolved fluorescence spectroscopy.

The aim of this study was to examine the usefulness of time-resolved fluorescence spectroscopy in the evaluation of the oxidative processes in human plasma. To investigate the impact of oxidative stress on the fluorescence of plasma, five studied markers (thiobarbituric acid-reactive substances, ischemia modified albumin, carbonyl groups, hydrogen peroxide, advanced oxidation protein products) were chosen as oxidative damage approved markers. Our method presents several advantages over traditional methods as it is a direct, non-time-consuming, repeatable, and non-invasive technique that requires only simple pre-treatment of samples without additional reagents and the sample size needed for analysis is small. In principle, each modification of the protein in plasma can be expected to modify its fluorescence properties and hence its lifetime or intensity. The study involved 59 blood donors with no evidence of disease. The research was conducted at excitation wavelengths of 280 nm and 360 nm, and emission was measured at wavelengths of 350 nm and 440 nm, respectively. Our results, although preliminary, suggest that the application of fluorescence measurements can be considered as an effective marker of oxidative stress. Regression analyses showed that a notable growth in fluorescence intensity at 440 nm and a simultaneous decrease in fluorescence intensity and mean fluorescence lifetime at 350 nm are associated with higher levels of oxidative stress.

Selected Drug-Likeness Properties of 2-Arylidene-indan-1,3-dione Derivatives-Chemical Compounds with Potential Anti-Cancer Activity.

2-Arylidene-indan-1,3-done derivatives have very different properties, thanks to which they find various applications in science, medicine, and industry. Selected derivatives show antiviral, antibacterial, and anti-inflammatory activity. This paper presents a procedure for the synthesis of a series of indan-1,3-dione derivatives that present antiproliferative activity. The aim of the work was to develop a method of simple synthesis and purification, evaluate the fulfillment of the Lipinski's and Veber's rule, and determine the potential scope of application of the obtained series of compounds. The structure of the synthesized compounds was confirmed, and their lipophilicity was determined using experimental and computational methods. Their antiproliferative activity against selected cell lines was tested in accordance with the MTT protocol; the ability to bind to albumin was tested, and the parameters related to the toxicity of substances in silico were determined. The selected compounds which showed antiproliferative activity were strongly bound to albumin and, in most cases, met the Lipinski's and Veber's rule. Thus, the obtained results suggest that 2-arylidene-indan-1,3-done derivatives appear to be good candidates for drugs with a potential leading structure for further development.

The impact of oxidative stress on binding of drugs with plasma proteins studied by fluorescence anisotropy methods.

The aim of this study was to investigate the effect of oxidative stress on drug binding affinity, free and bound fraction. The fluorescence anisotropy method was used to examine the interactions of commercial human serum albumin (HSA) and human plasma proteins with ochratoxin A (OTA) or carboxylate form of camptothecin (CPT-C). In-vitro method revealed significant reduction in bound fraction of drugs to HSA oxidized by chloramine T. Oxidative stress was determined by measuring the plasma level of advanced oxidation protein products (AOPP). A significant positive correlation between the AOPP and the plasma free fraction of tested drugs in thirty healthy patients was also found. The oxidative stress monitoring by AOPP is very important for improvement of dosage of drugs highly bound to albumin and with narrow therapeutic index. As a result of severe oxidative stress, the drug pharmacokinetics and therapeutic effects are prone to change. This study highlights the issues of therapeutic drug monitoring and it can explain the behaviour of drugs in pathological situations.

INTERACTION OF CAMPTOTHECIN WITH HUMAN SERUM ALBUMIN DETERMINED BY FLUORESCENCE ANISOTROPY SPECTROSCOPY.

The study can be useful for understanding the interaction of camptothecin with human serum albu- min. There are two forms of camptothecin (the carboxylate form (CPT-C) and the lactone form (CPT-L)) but only the lactone one is pharmacologically active. It was reported earlier that in the presence of HSA, the active lactone form of camptothecin changes to inactive carboxylate form and it reduces the antitumor activities of camptothecin. However, those studies were performed at physiological pH (7.4) and with non-oxidized and non-glycosylated albumin. The aim of this study was to investigate the effect of oxidative stress, glycosylation, pH changes and competitor drugs on inactivation of lactone form of camptothecin in albumin solution using measurements of fluorescence anisotropy spectroscopy. It was tried to prove that in vivo camptothecin may be present in higher amount in lactone form than previously thought. Due to a reduction of pH value, a decreased rate of hydrolysis from CPT-L to CPT-C was observed. It was found in vitro a significant reduction in bound fraction of CPT-C to HSA oxidized by chloramine T or glycosylated by glucose. Moreover, as a result of block- ing binding of CPT-C to HSA by competitive compound (flurbiprofen), a decrease in the fluorescence anisotropy of the HSA-CPT complex was found. This study opens the way to review an application of CPT and its derivatives in therapy.

Properties of Ultraviolet Exposed Camptothecin Studied by Using Optical Spectroscopy Methods.

Camptothecin (CPT) and its analogs as inhibitors of topoisomerase I are anticancer compounds. Their antitumor potency is seriously limited due to hydrolysis of lactone form of camptothecins in solutions at pH>5.5, which leads to the formation of inactive carboxylate form with open lactone ring. Furthermore, the clinical application of CPT is also restricted by strong affinity of its carboxylate form to human serum albumin which destabilizes the active lactone form. By UV irradiation of the CPT carboxylate authors of this paper received camptothecin compound which has biophysical properties similar to the lactone form. The specific objective of the project is to determine the properties using the methods of absorption and steady-state fluorescence spectra analysis, fluorescence lifetime measurements as well as steady-state fluorescence anisotropy. The results suggest that the UV exposed camptothecin carboxylate changes the chemical structure. The high-throughput assays based on the steady state fluorescence anisotropy measurements proved that the form obtained as a result of UV irradiation of CPT carboxylate exhibits weaker affinity to albumin than CPT carboxylate however stronger than CPT lactone. This property is very desirable from the point of view of clinical applications.

Use of time-resolved fluorescence spectroscopy to evaluate diagnostic value of collagen degradation products.

The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs' characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The in vitro model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial.

A new approach to determine camptothecin and its analogues affinity to human serum albumin.

A novel and fast method for the determination of the binding kinetic data of ligand to protein has been developed. A new tool including human serum albumin-coated magnetic beads (HSA-MB) was used to determine the affinity of camptothecin (CPT) and its analogues to HSA. From the biological activity point of view, these compounds have potential anticancer activity. However, the numerous studies indicate that some of these analogues have a strong affinity to plasma proteins stopping their effective therapy. Thus, the problem of plasma protein binding behavior of CPT's analogues was the subject of this study.

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin.

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.

The comparison of biophysical properties of DB-67 and its ester DB-67-4ABTFA determined by fluorescence spectroscopy methods.

Fluorescence spectroscopy methods are applied to the study of camptothecin analogue DB-67 and its ester DB-67-4ABTFA (trifluoroacetic acid salt of 20(S)-aminobutyrate substituted DB-67). Camptothecin and many of its analogues exhibit anticancer properties. They are fluorescent compounds, so using the method of fluorescence anisotropy measurements and fluorescence spectra recording many biophysical properties can be determined including affinity to proteins and membranes. One can also observe the process of conversion of the ester into DB-67. Active lactone form of camptothecin in fluids at pH 7.4 hydrolyses and converts into inactive carboxylate. Process of camptothecin deactivation is accelerated in plasma and after about 2h the total conversion to carboxylate form occurs. It is caused by fast and irreversible binding of carboxylate form to the human serum albumin (HSA). Camptothecin carboxylate bound to HSA does not lactonise. On the other hand, camptothecin lactone binding to membranes is reversible, but as long as lactone form bound to membranes does not hydrolyse. Knowledge of binding properties to proteins and membranes permits to select among many camptothecin analogues the ones exhibiting desirable behavior in physiological conditions: high affinity of lactone form to membranes and low affinity of carboxylate form to albumin. The studied DB-67 and DB-67-4ABTFA fulfill these requirements.

Hydroxycamptothecin deactivation rates and binding to model membranes and HSA determined by fluorescence spectra analysis.

Camptothecins (CPTs) are fluorescent compounds exhibiting anticancer activity. They can exist in two forms, a lactone and a carboxylate. In neutral and base solution, lactone forms hydrolyse and convert into carboxylates. Only the lactone forms of CPTs are biologically active. Because of strong affinity of the carboxylate form of the parent drug camptothecin to human serum albumin (HSA), this protein promotes the deactivation of this compound. On the other hand, the lactone forms of camptothecins do not hydrolyse and are stabilized when bound to membranes. The following three hydroxycamptothecins, 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxy-camptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67) were studied. Factor analysis of a set of fluorescence excitation spectra recorded during lactone hydrolysis facilitated the high-throughput determination of the deactivation rates of camptothecin and each hydroxycamptothecin in phosphate buffered saline. The fluorescence spectra of hydroxycamptothecins diluted in HSA solution or suspended in DMPC liposomes were recorded, and the association constants of these drugs to membranes and plasma proteins were calculated. Among the analysed agents, DB-67 exhibited the most desirable properties including low affinity of the carboxylate form for albumin and high affinity of its lactone form for model membranes.

Determination of hydroxycamptothecin affinities to albumin and membranes by steady-state fluorescence anisotropy measurements.

Camptothecin (CPT) and its hydroxycamptothecin analogs are fluorescent compounds exhibiting strong anticancer properties. They exist in two forms: active lactone and inactive carboxylate. The deactivation proceeds via hydrolysis in neutral and base solutions. A serious limitation to the clinical application of CPT is the strong affinity of its carboxylate form to human serum albumin (HSA) which destabilizes its active lactone form. However, binding to membranes in blood improves the stability of the lactone form of CPT and its analogs. A high-throughput screening assay based on the steady-state fluorescence anisotropy method was used to determine the protein- and membrane-binding properties of 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67). The relative affinities of hydroxycamptothecins to HSA and model membranes in the form of DMPC liposomes were determined, and DB-67 exhibited the most desirable properties including the highest affinity to membranes in its lactone form and low affinity to HSA in its carboxylate form.