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Transport in the blood of an anti-tumor water-soluble ruthenium cyclopentadienyl complex: a Fluorescence study on albumin binding
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Abstract(s)
Ruthenium (Ru) complexes have captivated an increasing interest in recent years; they are a promising alternative to platinum-based complexes due to the fact that some of them have been repeatedly described for showing higher selectivity and lower cytotoxicity regarding their effects on cancer cells.
The interactions studied between the Ru-complexes and serum proteins such as fatty acid free Human Serum Albumin (fafHSA) are of very high importance considering that their bioavailability and effects depend on their binding to albumin to reach the targeted cells. In addition, the Food and Drug Administration (FDA) has set the report on plasma protein binding for prodrugs as a requirement in order to do the screening for potential therapeutic agents.
The interactions and several aspects related to the binding between the ruthenium (II) complex [RuII(Cp)(bipy)(CO)][PF6] (or RuC, also named pmc44) and HSA are addressed using fluorescence spectroscopy, steady-state and timeresolved measurements, and UV-Visible absorption in 10 mM HEPES buffered medium pH 7,4. Additionally, in order to obtain a deeper insight into the HSA binding properties of the complex at the specific drug binding sites, displacement experiments with the site markers Warfarin and Dansylglycine (for which the binding site is well known) were performed.
The results show that the complex strongly quenches the intrinsic fluorescence of albumin and that the interaction is quite fast, indicating that the complex successfully binds the protein. The outcome from the Stern-volmer linearizations (both time-resolved and steady-state measurements) displayed more than one type of quenching mechanism; albumin fluorescence is quenched by a combination of dynamic and static quenching mechanisms. The most representative values obtained for KD and KS from the averages were (4,8 ± 0,3)x104 M-1 and (2,3 ± 0,7)x104 M-1 respectively, calculated with the SternVolmer linearization at em=338 nm by a Minimum Square Fit procedure with variance analysis (ANOVA) with a 95% confidence level.
A speciation model with two protein-RuC adducts is proposed with global conditional binding constants of log β’1= (4,78 ± 0,01) (for the 1:1 adduct {HSARuC}) and log β’2= (9,52 ± 0,01) (for the 1:2 adduct {HSA-(RuC)2} with a fitting parameter of 1,16x105 using the computer program PSEQUAD.
The conclusions achieved with the site markers´ experiments revealed that the complex binds to both sites I and II of the protein.
Altogether, the results from this work indicated that the complex studied can bind human serum albumin in a moderate way and thus it be efficiently transported in the blood by HSA.