Rational design of an arene ruthenium chlorin conjugate for in vivo anticancer activity
Graphical abstract
A tetranuclear p-cymene ruthenium 5,10,15,20-tetra(3-pyridyl)chlorin complex has been prepared and evaluated in vivo as dual photosensitizer and chemotherapeutic agent on mice bearing an ectopic human oral carcinoma xenograft. An in vitro study has showed that the ruthenium–chlorin complex accumulates preferentially in the endoplasmic reticulum. A mode of action in vivo dominated by a cytotoxic effect of the complex and not by photodynamic efficiency of the photosensitizer was suggested.
Introduction
In conventional chemotherapies, therapeutic windows are purely dose dependent [1]. On the other hand, in photodynamic therapy (PDT), several factors have to be optimised before being able to determine a therapeutic window for a new photosensitizer. Four adjustable factors are generally considered in PDT, the dose, the drug–light interval (DLI), the fluence and the fluence rate [2]. However, the influence of these factors can vary during the period of the treatment and also within the tumours, and the presence of molecular oxygen in the tumour tissue is crucial for an efficient photo-activation [3]. Therefore, finding the best treatment modality for a photodynamic agent remains a difficult task.
Mathematical tools are now available to facilitate the optimisation of photodynamic treatments [4]. These statistical models allow a better evaluation of the therapeutic effect of each factor, and give an estimation of their relative impact on the treatment. In addition, these models can highlight the existence of additive and synergistic effect between factors, still with a limited number of experiments, thus reducing to a minimum the number of animals needed for an in vivo study. Recently, we have used such a statistical method to evaluate the in vivo activity of two ruthenium–porphyrin photosensitizers (Fig. 1) [5].
The mononuclear and tetranuclear arene ruthenium complexes, [(p-cymene)RuCl2(ptp)] (ptp = 5-(3-pyridyl)-10,15,20-triphenylporphyrin) and [{(p-cymene)RuCl2}4(tpp)] (tpp = 5,10,15,20-tetra(3-pyridyl)porphyrin) (Fig. 1), were evaluated on mice bearing an ectopic human oral carcinoma xenograft. This study has showed that the tetranuclear derivative was the most active complex and that an additive effect was taking place between the p-cymene ruthenium units and the porphyrin core [5]. In that initial study, according to the absorption spectra of the ruthenium–porphyrin complexes, and to their photophysical properties, the irradiation of the tumour tissues was carried out at 514 nm, which in term of light penetration through tissues is not optimal. Wavelengths from 400 to 600 nm are naturally absorbed by biological compounds such as melanin, haemoglobin, oxy-haemoglobin or bilirubin [6]. Longer wavelengths can avoid being intercepted by biological chromophores, thus increasing significantly the amount of light available in tumours for photo-activation of the photosensitizers. Therefore, to obtain a better light penetration through the tissues and to potentially improve the efficiency of the tetranuclear p-cymene ruthenium–photosensitizer conjugate, we have now prepared the chlorin analogue.
Chlorins, the reduced form of porphyrins, are known to possess a strong Q band I above 650 nm, thus allowing photo-activation in the red [7]. The in vivo photodynamic study on mice bearing an ectopic human oral carcinoma xenograft was performed using a statistical model and an irradiation at 652 nm for better skin penetration. The intracellular localization was also evaluated in vitro on KB cells (human carcinoma cells of the nasopharynx).
Section snippets
Results and discussion
Two equivalents of the dinuclear arene ruthenium complex [(p-cymene)RuCl2]2 react in dichloromethane with 5,10,15,20-tetra(3-pyridyl)chlorin (tpc) to give, in quantitative yield, the corresponding tetranuclear complex [{(p-cymene)RuCl2}4(tpc)] (1), see Scheme 1. This synthetic strategy was based on the synthesis of the porphyrin-parent compound [{(p-cymene)RuCl2}4(tpp)] [8]. The tetranuclear ruthenium–chlorin complex has been fully characterised by 1H and 13C NMR spectroscopies, infrared,
Conclusions
In the search of combining photo- and cytotoxicity in a dual ruthenium–photosensitizer agent [18], a ruthenium–chlorin conjugate has been prepared. It was shown that the ruthenium–chlorin complex accumulates in tumours and, as observed with the ruthenium–porphyrin analogue [5], the presence of arene ruthenium units at the periphery of the photosensitizer facilitates the uptake in vivo. The accumulation of the ruthenium–chlorin complex in the endoplasmic reticulum was found to be undesirable in
General remarks
The starting material [(p-cymene)RuCl2]2 was prepared according to published methods [11], and 5,10,15,20-tetra(3-pyridyl)chlorin (tpc) was purchased from Inochem Ltd (Carnforth, Lancashire UK; Inochem Ltd being the European representative of Frontier Scientific, Logan USA). The 1H and 13C{1H} NMR spectra were recorded with a Bruker Avance II 400 MHz spectrometer using the residual protonated solvent. Infrared spectra were recorded as KBr pellets with a Perkin-Elmer FTIR 1720 X spectrometer. The
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