Research paperPhotophysical properties and photocytotoxicity of free and liposome-entrapped diazepinoporphyrazines on LNCaP cells under normoxic and hypoxic conditions
Graphical abstract
Introduction
For the last 20 years, macrocyclization reactions of 1,4-diazepine-2,3-dicarbonitrile derivatives have led to a plethora of various porphyrazines with annulated rings of diazepine [[1], [2], [3]], tetrahydrodiazepine [4,5], and styryldiazepine [[5], [6], [7], [8]]. Styryl-substituted diazepinoporphyrazines have been evaluated for their electronic properties, for the tendency for aggregation and photodegradation, singlet oxygen generation efficiency, and in vitro photodynamic activity at a nanomolar level against two oral squamous cell carcinoma cell lines [5,7,8]. 5,7-Diphenyl- and 5,7-di(4-tert-butylphenyl)-substituted diazepinoporphyrazines and tribenzodiazepinoporphyrazines have also been studied towards electronic [[1], [2], [3],9,10] and electrochemical properties [11,12].
Porphyrazine macrocycles seem to be suitable candidates for photodynamic therapy (PDT) for many reasons. They exhibit high generation quantum yields of singlet oxygen and have adequate light absorption spectra. Moreover, porphyrazines absorb light in the so-called therapeutic window in range 600–800 nm, where the tissues are the most permeable to such wavelengths. On the one hand, porphyrinoid photosensitizers are lipophilic and reveal high affinity to lipids, which seems beneficial, as cancer cells are often overexpressing LDL-receptors [13]. On the other hand, the high lipophilicity of porphyrinoids hampers their solubility in water and increases their tendency to form aggregates in polar solvents. This issue can be overcome by incorporation of photosensitizers into various pharmaceutical formulations, including liposomes proven as useful carriers [14]. Liposomes offer a huge advantage in lipophilic photosensitizer delivery, not only by enabling water-insoluble compound delivery but also in functionalization of the liposome membrane. The tunable charge of the carrier can also have a great effect on the effectiveness of the therapy. Photosensitizers like hydrophobic porphyrazines accumulate in liposomal membranes. In this regard, smaller porphyrazine macrocycles are preferred, as the thickness of membranes is limited thus making it impossible to incorporate bulky porphyrinoid structures.
To further address the issue of anticancer therapies, one of the characteristics of the cancer tissues such as hypoxia, must be taken into account. For many years until now, it is well known that tumor hypoxia has been a significant limitation of anticancer therapies, which depend on oxygen-mediated mechanisms such as radiation, photodynamic therapy as well as chemotherapy [15]. Although PDT has been found to be effective against the superficial tumor, the photodynamic efficacy for solid tumors, which are characterized by the hypoxic environment, is still challenging [16]. The rapid growth of the neoplasm cells and their increased metabolism are factors triggering hypoxia [17], which is a major problem in case of photodynamic therapy, as molecular oxygen is one of the three factors determining a successful treatment. The singlet oxygen (1O2) generated in type II photoreaction is still considered as the primary cytotoxic agent for PDT. Therefore, the low oxygen concentration may decrease the efficacy of photodynamic treatment to cancer cells [18,19]. To date, several strategies were designed to overcome tumor hypoxia in the context of PDT such as modification of the tumor microenvironment to ensure tissue re-oxygenation, the increase of intracellular oxygen concentration as well as the development of the photosensitizer, which may induce cytotoxic effects in environments of low molecular oxygen via type I photosensitization [15,16,20]. Thus, it is essential to develop photosensitizers which may exert therapeutic effects under both normoxia and hypoxia.
Currently, screening of photosensitizers during photodynamic therapy is mainly focused on normoxic condition. However, it should be noted that the median oxygenation in untreated tumors varies between approximately 0.3% and 4.2% oxygen and most tumors demonstrate median oxygen levels below 2% [21]. This range in tumor oxygenation depends on several factors, and hypoxic microregions are heterogeneously distributed within the tumor mass [22]. It is worth noting that many prostate and pancreatic tumors are strongly hypoxic [21]. Moreover, the hypoxia in prostate cancer is also related to advanced tumor stage, aggressive disease as well as increased resistance to androgen deprivation and radio- and chemotherapy [23]. Recently, we have reported the synthesis of diazepinoporphyrazine with G1-dendrimeric substituents and discussed its photophysical properties as well as singlet oxygen generation efficiency [12]. Herein, as an enhancement of our studies, we report the synthesis, physicochemical characterization, including absorption and emission properties, tendency to aggregation of magnesium(II) diazepinoporphyrazine and tribenzodiazepinoporhyrazine substituted in their C5 and C7 positions with 4-methoxyphenyl groups. Moreover, the anticancer potential of new porphyrazines was assessed in liposomal formulations in both normoxia and hypoxia conditions against prostate adenocarcinoma cells.
Section snippets
Synthesis and characterization
Novel magnesium diazepinoporphyrazine substituted at C5 and C7 positions with 4-methoxyphenyl groups (4) was synthesized in two steps (Scheme 1). Firstly, the condensation reaction of diaminomaleonitrile (1) and 1,3-bis(4-methoxyphenyl)-1,3-propanedione (2) was performed following the literature procedure [9], and led to the novel 5,7-disubstituted-1,4-diazepine-2,3-dicarbonitrile (3). Subsequent macrocyclization reaction of 3 using magnesium n-butanolate in n-butanol according to the Linstead
Materials and instruments
All reactions were conducted in oven-dried glassware under argon using Radleys Heat-On heating system. Solvents and all reagents were obtained from commercial suppliers and used without further purification. All solvents were removed by rotary evaporation at or below 50 °C. Dry flash column chromatography was carried out on Merck silica gel 60, particle size 40–63 μm, reverse phase Fluka C18 silica gel 90 and aluminum oxide 90 active neutral (activity stage I) for column chromatography
Conclusions
Novel 5,7-diaryl-substituted symmetrical diazepinoporphyrazine and tribenzodiazepinoporphyrazine were found to generate singlet oxygen in dimethylformamide and dimethyl sulfoxide in moderate yields up to ΦΔ = 0.307 in comparison to structurally similar G1-dendrimeric diazepinoporphyrazine. Absorbance and fluorescence measurements that were applied to study aggregation properties of novel macrocycles in the presence of tetramethylammonium fluoride as an antiaggregation agent proved that the
Acknowledgement
This study was supported by the National Science Centre, Poland under Grant No. 2012/05/E/NZ7/01204.
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