The binding properties of some novel ruthenium (III) complexes with human serum transferrin

Aim. The transferrin cycle gained increased interest in recent years and it holds promise as an attractive system for strategies of drug targeting to tumors. Neoplasic cells exhibit a large demand of iron and therefore express highly transferrin receptors. As a consequence, transferrin conjugates can preferentially interact with cancer cells. This strategy is exploited nowadays for targeting novel anti-cancer drugs. Recent data showed that ruthenium (III) compounds possess antitumor and antimetastatic effects, due to their affinity for crucial biomolecules (like transferrin). Methods. The paper presents the transferrin-binding properties of some novel ruthenium (III) complexes with general formula RuL2(DMSO) mCl3·nH2O ((Ru-nf) L: norfloxacin (nf), m = 1, n = 1; (Ru-cpx) L: ciprofloxacin (cpx), m = 2, n = 2; (Ru-oflo) L: ofloxacin (oflo), m = 1, n = 1; (Ru-levo) L: levofloxacin (Levo), m = 2, n = 8; (Ru-pip) L: pipemidic acid (pip), m = 1, n = 2, DMSO: dimethylsulfoxide). We investigated, in vitro, the interactions of these ligands with human transferrin through spectroscopic techniques, with the ultimate goal of preparing adducts with good selectivity for cancer cells. Results. All studied complexes interact with human serum transferrin; the molar ratio [complex]/[transferrin] strongly influences the binding affinity. Conclusions. The best interaction between the complexes studied and transferrin is achieved for a molar ratio of 8; the best interaction was registered for Ru-pip, followed by Ru-nf.


Introduction.
A number of biological studies have highlighted in the last years a novel mechanism of action for anticancer drugs, regardless their covalent binding with the DNA.The literature presents experimental evidence for an important affinity between the chemotherapeutic medicines and other crucial biomolecules like plasma proteins.
In this regard, transferrin became an interesting and promising biomolecule over the last years for the development of new biochemical mechanisms of drug targeting to tumor tissues [1][2][3].Neoplasic cells demand high amounts of iron for their growth and therefore express the transferrin receptor at a high rate.As a consequence, transferrin conjugates can preferentially interact with cancer cells through transferrin receptors.This strategy is exploited nowadays for targeting novel anti-cancer drugs.
Ruthenium polyaminocarboxylate complexes, a class of ruthenium (III) complexes with polydentate mixed donor ligands from the H 4 EDTA family were prepared and tested by the group of Gonzalez-Vilchez [10].
The literature studies demonstrated the affinity of these complexes for transferrin and provide evidence for formation of stable adducts between them [11][12][13][14][15][16].Therefore transferrin is investigated presently as a natural carrier of the drugs to the tumor tissue, mainly because of the high affinity of this iron-protein and the large number of transferrin receptors on the surface of tumor cells.
This paper presents the transferrin-binding properties of some novel ruthenium (III) complexes with mixed ligands of the type RuL 2 (DMSO [17][18][19][20].All complexes display an octahedral stereochemistry with the quinolone ligand acting as monodentate coordinated through N4 atom of piperazinyl ring.The structures of the ligands and the general structure of complexes are presented in Fig. 1. Materials and methods.During the experiment the following reagents were used: Tris («Merck», Germany), serum transferrin («Merck»), working solutions of the studied ruthenium (III) complexes.The experimental procedure implied the following steps: The first step implied the preparation of Tris buffer pH 7 (5×10 -3 M Tris, 5×10 -2 M NaCl).
The preparation of each of the ruthenium complexes stock solutions 10 -5 M, using Tris buffer pH 7 as solvent.When using this stock solution it was diluted 1:10 with Tris buffer (final complex working solution 10 -6 M).
It was prepared a 10 -5 M stock solution of human serum transferrin in pH 7 Tris buffer: in a 10 ml volumetric flask 0.0077g of human seric transferrin were weighed and disolved in pH 7 Tris buffer.When using this stock solution it was diluted 1:10 with Tris buffer (final transferrin working solution 10 -6 M).
For each studied complex we used 6 test tubes containing the reagents, as described in Table 1.
The test tubes (performed in triplicate) were incubated for 24 hours at room temperature, in the dark.Afterwards, the UV spectra (l = 275 nm) were immediately recorded using an UV-Vis Cary 100 Bio spectrophotometer («Varian Inc.», USA).
Results and discussion.In this study we conducted an extensive in vitro characterization of adducts Fig. 1.Structures of the investigated quinolones and general structure of their Ru (III) complexes between some possible ruthenium (III) drugs (newly synthesized) and human serum transferrin and we gained insight into the intriguing chemistry of these complexes.It is to mention that we also possess unpu lished data regarding the interactions of our newly synthesized ruthenium (III) complexes with human serum albumin (HSA) and DNA, but their affinity and binding to both HSA and DNA is importantly different, compared with the interactions with transferrin (from with the interaction stoichiometry to the reactivity patterns).
The present study evoked that there exists chemical binding between the substances investigated and transferrin and also that this interaction (including the chemical stoichiometry) is complicated and needs proper investigations in respect to their solution behaviour (reflected in the organisms fluids behaviour) and their protein binding ability.Nevertheless, the accurate knowledge of this binding (interaction) is crucial at this stage of the development of a newly possible antitumor drug since it directly reflects in the IC 50 and toxicity.
The knowledge of the transferrin cycle has rapidly increased in recent years and it holds promise as an attractive system for strategies of drug targeting to tumor tissues.Indeed, tumor cells exhibit a large demand of iron for their growth and therefore express the transferrin receptor at a high rate.As a consequence, transferrin conjugates that retain a good affinity for the trans-ferrin receptor can preferentially interact with cancer cells.This strategy is exploited nowadays for targeting novel anti-cancer drugs.For example, Keppler's complexes were the first ruthenium (III) complexes whose interactions with human serum transferrin were investigated in solution by various techniques, including spectrophotometry, at different stoichiometry binding stages [8,9,14].Also, such a strategy was used to achieve targeting on adriamycin to neoplastic tissues [2].
In our case, the major behaviour of our ruthenium (III) complexes reflected in the transferrin spectroscopic absorbance.In this regard transferrin absorbance in UV was modified (has decreased) in the presence of the studied ruthenium complexes, as it can be seen in Table 2 and Fig. 2-6.
The studied ruthenium complexes interact in vitro with transferrin.This fact has been experimentally observed by the diminish of the absorbance value of transferrin in UV, consequent to the direct contact complextransferrin.
By analyzing the obtained results, it can be concluded that the complex Ru-nf produces the most important decrease in the UV transferrin absorbance, when used in the molar ratio [Ru-nf]/[transferrin] = 1/1.The decrease in the UV absorbance of this protein due to an interaction with the research complexes (molar ratio [complex]/[transferrin] = 1/1) has varied as follows: Ru-nf > Ru-pip > Ru-levo > Ru-oflo > Ru-cpx.When using the molar ratio [complex]/[transferrin] = 2/1 it was observed, as well, a decrease of the UV absorbance of human serum transferrin, in vitro.The procentual dynamics of this phenomenon is: Ru-pip > > Ru-nf > Ru-levo > Ru-oflo > Ru-cpx.
When using the molar ratio [complex]/[transferrin] = 4/1 it was observed a decrease of the UV absorbance of human serum transferrin, in vitro.The pro-centual variation of the absorbances was as follows: Ru-pip > Ru-nf > Ru-oflo > Ru-levo > Ru-cpx.
When using the molar ratio [complex]/[transferrin] = 8/1 it was observed a decrease of the UV absorbance of human serum transferrin, in vitro.The procentual dynamics of the absorbances was as follows: Ru-pip > Ru-nf > Ru-levo > Ru-oflo > Ru-cpx.
Our further studies will focus on other critical drug development issues, like testing of these complexes and on in vivo tumor models.
Conclusions.The experimental results regarding the interaction of the studied ruthenium complexes with human serum transferrin have revealed a series of important aspects: All the studied complexes interact in vitro with transferrin, fact proved by the decrease of transferrin UV absorbance due to the contact with these compounds, in all concentrations; The ruthenium complex with pipemidic acid intensively reduces the transferrin UV absorbance, followed by the ruthenium complex with norfloxacin; The weakest interaction complex-transferrin has been registered in the case of ruthenium-ciprofloxacin; The molar ratio [complex]/[transferrin] = 8/1 has presented the strongest UV detectable interactions, in the chosen experimental conditions, for all the evaluated complexes.

Table 2
Procentual decrease of the transferrin absorbance due to the interaction with the studied ruthenium complexes

Table 1
Working tehnique for studing the interactions of the studied complexes with transferrin