No abstract

Characterizing how platinum metallocomplexes bind to human serum albumin (HSA) is essential in evaluating anticancer drug candidates. Using cisplatin as a reference complex, the application of capillary electrophoresis (CE) to reliably assess drug/HSA interactions was validated. Since this complex is small compared to the size of the protein, the binding response could only be recognized when applying CE coupled to a (platinum) metal-specific mode of detection, namely inductively coupled plasma-mass spectrometry (ICP-MS). This coupling allowed for confirmation of a specific affinity of cisplatin and novel Pt complexes to HSA, measurement of the kinetics of binding reactions, and determination of the number of drug molecules attached to the protein. As the cisplatin/HSA molar ratio increased, the reaction rate became faster with a maximum on the kinetic curve appearing at about 50 h of incubation at 20 times excess of cisplatin. The reaction was characterized as a pseudo-first order reaction with the rate constant k = 0.003 min(-1) at 37 degrees C. When incubated with a 20-fold excess of cisplatin, HSA bound up to 10 mol of Pt per mol of the protein. This is indicative for a strong metal-protein coordination occurring at several HSA sites other than the only protein cysteine residue. Structural analogs of cisplatin, bearing aminoalcohol ligands, showed comparable protein binding reactivity and stoichiometry but a common equilibrium was not reached even after one week of incubation. Also apparent was a two-step mechanism of the binding reaction. Results demonstrated the suitability of CE-ICP-MS as a rapid assay for high-throughput studying of drug/HSA interactions.

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A CE method has been developed to evidence and quantitatively characterize the interaction between platinum-based antitumor drugs and human serum proteins. This method is a variant of affinity CE modified regarding both experimental setup and data treatment so as to measure the peaks (or vacancies) that correspond to the bound drug when it slowly binds to the protein. Using the formalism of the Hummel-Dreyer method and cisplatin and oxaliplatin as test compounds, a protocol for determining albumin and transferrin binding constants and stoichiometries, including (and distinguished by) 48 hours of incubation of the reaction mixture, was elaborated. Relative affinities of drugs toward different proteins in aqueous solution at physiological pH, chloride concentration, and temperature were compared in terms of overall binding constants and numbers of drug molecules attached to the protein. The results indicate that both platinum drugs bind to albumin more strongly than to transferrin, supporting the concept that the albumin fraction is a major drug supply route for chemotherapeutical needs. From a comparison with the binding parameters measured previously for cisplatin by other methods, conclusions were drawn about the validity of CE as a simple and convenient method for assaying protein-drug reactions with slow kinetics.

Metal-based nanoscale particles possess unique optoelectronic or magnetic properties that make them highly promising as imaging agents in cancer therapy research. The fate of nanoparticles in vivo and particularly, the delivery to tumours are closely related to their interactions with plasma proteins. Furthermore, proteins can be used to modify the nanoparticle surface in order to facilitate active targeting to tumours. Therefore, there is an ongoing need for new and more capable analytical methodologies to characterize the protein-nanoparticle binding. Due to the small-sample volume requirement, high degree of resolution and, most importantly, mild, species-friendly separation conditions, capillary electrophoresis (CE) is gaining increasing popularity in the analysis of protein-nanoparticle interaction. This perspective article highlights the potential of CE in studying reactions associated with protein-mediated transformations of nanoparticles, with the focus on quantum dots, gold and iron oxide nanoparticles. Different ways by which CE can be applied to such monitoring are summarized and critically assessed using a representative coverage of recent publications.

The feasibility of capillary electrophoresis for distinguishing between the rhodium(III) species occurring in different acidic environments has been demonstrated. The separation was optimum under acidic electrolyte conditions in which the complexed Rh species were at their most stable and the electroosmotic flow approached zero, thereby aiding resolution. Identification of the forms of Rh and estimation of their relative equilibrium content were accomplished by use of a diode-array detector. The distribution of the metal complexes was highly dependent on the nature and concentration of the acid and the age of the rhodium stock solutions. On dilution Rh(III) tends to be readily hydrolyzed, giving rise to a wider variety (and a varied distribution) of complexed forms. In 0.1 mol L(-1) HCl, four differently charged chloro complexes--RhCl4(H2O)2-, RhCl3(OH)(H2O)2-, RhCl3(H2O)3, and RhCl2(H2O)4+--were separated and identified. When a stock solution in 11 mol L(-1) HCl was run, Rh produced a major peak ascribed to RhCl6(3-) and two slowly migrating peaks from ions in which one or two of the chloride ligands were probably replaced by water and hydroxyl ion, as a result of hydrolysis. The aquatic cationic species were found to be predominant in HClO4 and HNO3 solutions, whereas only negatively charged forms of Rh(III) occurred in sulfuric acid. This speciation information opens also new possibilities of assessing the catalytic activity of Rh in kinetic reactions.

A method based on combining inductively coupled plasma mass spectrometry (ICP-MS) with capillary electrophoresis (CE) or an ultrafiltration step was developed to study the speciation of the serum-protein adducts of a ruthenium anticancer drug under in vitro intracellular conditions. The formation of a reactive Ru species in the cell, following the metal release from the protein, is thought to play an important role in the drug's mode of action. Glutathione and ascorbic acid at their cancer cytosol concentrations were shown to be capable of altering the metal speciation in the drug adduct with holo-transferrin but not that with albumin. The appearance of the additional peaks in ICP-MS electropherograms (by recording both Ru- and Fe-specific signals) was found to be dependent on time which allowed for kinetic assessment of the evolution of novel metal species. On the contrary, after the addition of citric acid the ruthenium ion (within the appropriately complexed scaffold) remained sequestered in the adduct. This was inferred as a proof of the speciation changes taking place by a virtue of a redox mechanism rather than due to ligand-exchange transformations. The protein-bound metallodrug was further characterized by direct ICP-MS assaying so as to confirm a partial release of ruthenium induced by glutathione.

As metallic nanoparticles are growing in importance as analytes in CE, increases an interest in appropriate detection methods for their quantification in various samples. For gold nanoparticles (AuNPs), the most common UV detection poses intricacy of inadequate sensitivity that hinders the applicability of CE. With the objective of resolving this challenge, UV detection was compared with C(4) D and ICP-MS as alternative modes of detection for AuNPs. A C(4) D detector, applied under pressure-driven conditions, exhibited better sensitivity than a UV detector. However, C(4) D turned to be unsatisfactory to differentiate the signal of AuNPs at common CE conditions despite varying the nature of BGE and detection conditions. Due to intrinsic sensitivity and low background levels typical to Au, ICP-MS greatly surpasses UV detection. After optimization trials, CE-ICP-MS gained the LOD of AuNPs as low as 2 × 10(-15) M, as well as an excellent performance in terms of signal stability and linearity. Also importantly, the optimized BGE appears to be well matched to explore the behavior of AuNPs in biologically relevant systems. This was demonstrated by probing the interaction between AuNPs and the main blood-transporting protein, HSA.