Tomasz Wybranowski

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.

Study of SERS efficiency of metallic colloidal systems

The aim of this work was to determine the influence of colloidal silver and gold nanoparticles on the intensity of Raman scattering observed for analytes dissolved in the water. The testing analytes were rhodamine B and rhodamine 6G. A special emphasis was placed on the choice of optimal method of preparation of colloids and also on the determination of the influence of the aggregation environment on surface-enhanced Raman scattering (SERS) efficiency and stability of colloids. Potassium chloride, sodium chloride and hydrochloric acid are used as aggregating agents. Simply Raman spectrometer and UV-Vis spectrophotometer were used for measurements. The total intensity of inelastic scattering on molecules exhibited up to 106-108fold increase in the presence of metallic nanoparticles with a detection limit of analyles being shifted up to the nanomolar concentration. It was observed that SERS enhancement factor varies strongly depending on kind and concentration of used aggregating agent (especially on the concentration of Cl−ions). These changes in SERS efficiency of colloid were reflected through changes on the absorption spectra.

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

Abstract. 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.

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.

Binding of drugs to serum albumin determined by changes in surface plasmon resonance signal of gold nanoparticles

Surface plasmon resonance of metal nanoparticles shifts if these nanoparticles are coated with proteins (e.g. BSA or HSA). It is related to the changes of refractive index of medium around nanoparticles. A value of this shift depends on the kind of nanoparticles, kind of proteins and pH of solution in which proteins are solved and nanoparticles suspended. The further shift in surface plasmon resonance occurs if molecules of drugs are bound to these protein layer coating nanoparticles. This additional shift is called as biosensor response. It depends on the number of drug molecules bound to protein layer. By measurement of biosensor response the affinity constant for the binding of drug to proteins is determined in this paper. The surface plasmon resonance was used also to monitor the structural changes of albumin at different pH values. The interaction between gold nanoparticles and albumin was investigated by red-shift of extinction maximum of gold nanoparticles.

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.

Spectroscopic study of surface-enhanced Raman scattering of rhodamine 6G on silver colloid

The aim of this work is to determine the influence of colloidal silver on the intensity of Raman scattering observed for rhodamine 6G (Rh6G) dissolved in the water. A special emphasis is placed on the exploration of the role of chloride ions and the influence of pH on the surface-enhanced Raman scattering (SERS) efficiency and stability of colloid. Rh6G is an extremely SERS active compound so in even relatively simple experiments up to 106 108 fold increase of Raman scattering is achieved. In the colloidal systems chloride ions are mainly responsible for an aggregation of nanoparticles what is the most important factor in the SERS enhancement. Moreover, Rh6G as a cationic dye co-adsorbing with chloride ions exhibits a strong direct interactions with the metallic surface. It results in additional enhancement of the intensity of Raman scattered light and probably is responsible for observed dimerization of Rh6G molecules. The reduction of pH by addition of nitric acid also leads to a decrease of surface potential of colloid and causes in its aggregation. Simply Raman spectrometer and UV-Vis spectrophotometer are used for spectroscopic measurements. The dynamic light scattering technique is used to estimate the size of particles and the degree of their aggregation. It is observed that the magnitude of SERS enhancement strongly depends on the kind and concentration of the used aggregating agent. The differences of SERS efficiency were reflected in the absorption spectra as well as in particles/aggregates size distribution.