Investigation of Ruthenium-Based Complexes for Anti-Cancer Therapy

Investigation of Ruthenium-Based Complexes for Anti-Cancer Therapy

L.Paduano

, M. Vaccaro

, G.Mangiapia

, A.Radulescu

, H.Frielinghaus

1University of Naples “Federico II”, Department of Chemistry – CSGI Consorzio per lo Sviluppo dei Sistemi a Grande Interfase

2Procter and Gamble, Cobalt 3, Newcastle upon Tyne, UK

3JCNS: Jülich Centre for Neutron Science

A series of amphiphilic ruthenium-based molecules with potential anti-cancer activity has been synthesized and characterized. The SANS investigations at KWS-2 have revealed a rich variety of aggregates, spanning from vesicles to cubic bicontinuous phases, with a good antiproliferative activity.

Despite of many advances achieved by the medicine, numerous efforts are nowadays addressed by biologists, chemists and physicians for finding new solutions able to treat efficiently tumoral pathologies.

This is not surprising since the huge complexity of the topic.

Currently, platinum-based drugs, such as cisplatin, carboplatin and oxaliplatin are successfully used in the treatment of the most common cancer types. It has been estimated that 50-70% of all cancer patients in the world are treated with cisplatin. However, there are problems associated with the use of cisplatin, which highly toxic leading to side effects that limit the dose that can be administrated. On the other hand, a possible improvement of the antitumoral drug properties is represented by the efficacy toward the formation and growth of metastases. In fact, many tumors can develop metastases often very extended at the diagnosis time, making scarcely effective the surgical treatment.

FIG. 1: Molecular structure of the molecules synthesized:

ToThyRu (A), HoThyRu (B) and DoHuRu (C), and DOPURu (D).

In this perspective, ruthenium complexes have attracted much interest as a promising alternative to platinum, showing a remarkable anti-tumor and anti- metastatic activity, together with lower toxicity.

Ruthenium complexes exhibit a higher affinity with respect to platinum compounds toward serum proteins, such as transferrin and albumins. This allows delivery of the cytotoxic Ru agents to tumor

cells. Although a rather large number of studies on Ru-complexes have been published in recent years, no example of nanovectors carrying ruthenium complexes for anti-cancer teraphy is reported. Lipid- based nanovectors, such as vesicles, are the most promising carriers for Ru complexes due to their tunable physic-chemical features and good pharmacological properties. The use of amphiphilic nanovectors allows delivery of a controlled amount of ruthenium with respect to the use of single ruthenium complex.

FIG. 2: Cryo-TEM images of cubosome-like particles (A) and multilamellar vesicles (B) present in the (DOPC/DOPE)/DOPURu in physiological condition (pH = 7.4), at 60/40 and 50/50 phospho-lipids/Ru complex molar ratios.

Scale bars are 100 nm. The spherical particles indicated by arrows are frost; the star indicates the lacey carbon support.

Inspired by the results of NAMI-A, one of the most promising ruthenium-based drug experimentally used in oncology therapy [1], we have synthesized and characterized a series of amphiphilic complexes able to form supramolecular aggregates: the chemical structures of these complexes, baptized DOPURu, ToThyRu, HoThyRu and DoHuRu, are shown in Figure 1. All of them, differently from NAMI-A that is not able to aggregate, are composed by a pyrimidinic or a purinic nucleoside that acts as a poly-functional main scaffold. One or two oleic chains have been inserted onto it, as well as a poly-(ethylene oxide) chain able to make the aggregates resistant toward the enzymatic degradation. Finally, a chelating moiety able to complex Ru(III) ions has been linked.

Physico-chemical characterization, along with the anti-proliferation activity of such molecules, has been performed for the synthesized molecules, in the presence of POPC, DOPC and DOPE phospholipids, in order to modulate the amount of ruthenium administered and for increasing the biocompatibility.

FIG. 3: Scattering cross sections obtained for multilamellar vesicles found in systems containing POPC and the ToThyRu (blue), HoThyRu (green), and DoHuRu (red) Ru-complexes in physiological condition (pH=7.4). Lines correspond to the fits according to structural models [2,3].

Small-angle neutron scattering (SANS), dynamic light scattering, cryo-TEM microscopy, electron paramagnetic resonance spectroscopy are the techniques intensively used for acquiring information on the aggregation properties of the systems

investigated. In particular, through SANS investigations performed at the KWS-2 high-intensity / wide Q-range SANS diffractometer of JCNS located at the research neutron source Heinz Maier-Leibnitz (FRM II) in Garching-München, it has been possible to discover that the synthesized molecules are able to form a rich variety of bilayer aggregates (liposomes, cubosomes, small micelles, see Figures 2 and 3), whose size and density parameters have been obtained by modeling the experimental data with appropriate structural models [2-4]. Finally, it has been observed a very good antiproliferative activity of the synthesized molecules, among the most promising for ruthenium complexes presented in literature, as shown for some cell lines in Figure 4.

The evaluation of the “cell survival index” arising from the combination of cell viability evaluation with cell counting, for human MCF-7 and WiDr cancer cells and rat C6 glioma cells, suggested that these nanocarriers produce a cytotoxic effect substantially similar to that of the organometallic Ru-complex AziRu, but at a ruthenium concentration of about 6 times smaller.

The present results open new scenarios for the application of lipid based supramolecular systems in ruthenium anti-cancer therapy.

FIG. 4: Concentration/effect curves and cell survival index. Cell survival index, evaluated by MTT assay and total cell count, in MCF-7 (A), WiDr (C) and C6 (E) cell lines incubated for 48 h with POPC (open squares), with different Ru-containing formulations (red, green, violet) and with AziRu (full squares). In panels B, D and F, for MCF-7, WiDr and C6 cells, are reported the corresponding concentrations-effect curves obtained by normalizing for the actual amount of Ru contained within DoHuRu/POPC, HoThyRu/POPC and ToThyRu/POPC liposomes. Data are expressed as percentage of untreated control cells and reported as mean of three independent experiments ± SEM.

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[3] Mangiapia, G., Vaccaro, M., D'Errico, G., Frielinghaus,

H., Radulescu, A., Pipich, V., Carnerup, A.M., Paduano, L., Soft Matter 2011, 7, 10577-10580.

[4] G. Mangiapia, G. D’Errico, L. Simeone, C. Irace, A.

Radulescu, A. Di Pascale, A. Colonna, D. Montesarcio, and L. Paduano, Biomaterials, 33, 3770 (2012).