Research paper
Photophysical properties and photocytotoxicity of free and liposome-entrapped diazepinoporphyrazines on LNCaP cells under normoxic and hypoxic conditions

https://doi.org/10.1016/j.ejmech.2018.02.064Get rights and content

Highlights

  • Porphyrazine derivatives were synthesized and characterized.

  • Singlet oxygen generation and aggregation properties of porphyrazines were evaluated.

  • Photocytotoxicity of porphyrazines was examined in free form and in liposomes.

  • Photocytotoxicity of porphyrazines was studied under normoxic and hypoxic conditions.

Abstract

5,7-Diaryl-substituted symmetrical diazepinoporphyrazine and tribenzodiazepinoporphyrazine were synthesized and characterized using UV–Vis, MS MALDI, and various NMR techniques. The expected photosensitizing potentials of these porphyrazines were evaluated by measuring their abilities to generate singlet oxygen in organic solvents and by comparing them with that of the recently obtained dendrimeric G1-type diazepinoporhyrazine. Absorbance and fluorescence measurements were performed to study the aggregation properties of the novel macrocycles. The photocytotoxicity of tribenzodiazepinoporphyrazine towards LNCaP cells in its free form and after its incorporation into liposomes was examined using MTT assay under normoxic and hypoxic conditions. It is interesting that all tested liposome formulations maintained their phototoxic activity in hypoxia. Also, tribenzodiazepinoporphyrazine incorporated into liposomes revealed better photocytotoxic effect (IC50 values of 0.600 ± 0.357 μM and 0.378 ± 0.002 μM) than its free form (IC50 values of 3.135 ± 0.156 μM). Following the in vitro experiments, the most promising liposomal formulation containing l-α-phosphatidyl-DL-glycerol for tribenzodiazepinoporphyrazine was found. Moreover, tribenzodiazepinoporphyrazine incorporated into liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) revealed moderate phototoxicity at 5 × 10−5 μM for antibacterial photodynamic therapy. It was established that an irradiation of planktonic bacterial strains significantly reduced CFUs of Staphylococcus aureus ATCC 25923 in comparison to tribenzodiazepinoporphyrazine containing l-α-phosphatidyl-DL-glycerol liposomes.

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.

References (53)

  • T. Goslinski et al.

    Polyhedron

    (2011)
  • J. Piskorz et al.

    Inorg. Chem. Commun.

    (2012)
  • P.A. Tarakanov et al.

    Spectrochim. Acta A

    (2015)
  • E.N. Tarakanova et al.

    Dyes Pigments

    (2015)
  • E. Wieczorek et al.

    Tetrahedron Lett.

    (2017)
  • S. Anand et al.

    Canc. Lett.

    (2012)
  • C. Michiels et al.

    BBA - Rev. Cancer

    (2016)
  • N.J. Rupp et al.

    J. Pathol. Inf.

    (2016)
  • V. Chauke et al.

    J. Photochem. Photobiol., A

    (2007)
  • A.T. Bilgiçli et al.

    Polyhedron

    (2010)
  • M. Çamur et al.

    Polyhedron

    (2009)
  • A.P. Castano et al.

    Photodiagn. Photodyn. Ther.

    (2004)
  • I. Seotsanyana-Mokhosi et al.

    J. Photochem. Photobiol., A

    (2001)
  • J.M. Dąbrowski et al.

    Coord. Chem. Rev.

    (2016)
  • Z. Jiang et al.

    J. Pharmaceut. Biomed. Anal.

    (2014)
  • K. Lang et al.

    Coord. Chem. Rev.

    (2004)
  • S. Md et al.

    Drug Discov. Today

    (2017)
  • E.S. Nyman et al.

    J. Photochem. Photobiol., B

    (2004)
  • R. Weijer et al.

    J. Photochem. Photobiol., C

    (2015)
  • P. Skupin-Mrugalska et al.

    Drug Discov. Today

    (2013)
  • N. Düzgünes

    Methods Enzymol.

    (2003)
  • M. Kryjewski et al.

    Polyhedron

    (2015)
  • U. Bhardwaj et al.

    Int. J. Pharm.

    (2010)
  • H. Zhao et al.

    Photodiagn. Photodyn. Ther.

    (2014)
  • L.H. Li et al.

    Biochim. Biophys. Acta, Biomembr.

    (1997)
  • Y. Sadzuka et al.

    Canc. Lett.

    (2006)
  • Cited by (24)

    • Artificial tumor microenvironment regulated by first hemorrhage for enhanced tumor targeting and then occlusion for synergistic bioactivation of hypoxia-sensitive platesomes

      2022, Acta Pharmaceutica Sinica B
      Citation Excerpt :

      Upon laser irradiation, UV absorption of DPBF in L/DP&PPa and PML/DP&PPa was reduced rapidly, similar to a PPa DMSO solution, indicating the significant production of ROS. When PPa was entrapped in the liposome membrane to prevent aggregation, liposomal PPa was endowed with a high-efficiency PDT property38,39. Furthermore, the oxygen concentration before and after laser irradiation was also measured (Fig. 4B).

    • Synthesis of sulfanyl porphyrazines with bulky peripheral substituents – Evaluation of their photochemical properties and biological activity

      2021, Journal of Photochemistry and Photobiology A: Chemistry
      Citation Excerpt :

      To evaluate the anticancer activity of Pzs 4–7, they were incorporated into two types of liposomes composed of (Fig. 1): (i) L-a-phosphatidyl-DL-glycerol (chicken egg, PG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), with molar ratio 2:8 or (ii) 1,2-dioleoyl-3-trimethylammonium propane (chloride salt, DOTAP) and POPC with molar ratio 2:8. The thin-film hydration method was used according to the previously elaborated procedure [10,37]. All lipids were purchased from Avanti Polar Lipids Inc.

    • Tribenzoporphyrazines with dendrimeric peripheral substituents and their promising photocytotoxic activity against Staphylococcus aureus

      2020, Journal of Photochemistry and Photobiology B: Biology
      Citation Excerpt :

      The supernatant was removed, and PBS was added to the precipitate to obtain a suspension with a density of approximately 107 CFU/mL (S. aureus - 1.2 × 107–3.9 × 107 CFU/mL, E. coli - 1.0 × 107–2.0 × 107 CFU/mL, and C. albicans - 1.9 × 107–2.8 × 107 CFU/mL). The photodynamic activity was examined based on a previously developed procedure [22]. Solutions of tribenzoporphyrazine (with ≤1% addition of DMSO as a solubilizer) at the appropriate concentration were added to the microbial suspensions and then incubated in the dark for 20 min at room temperature.

    View all citing articles on Scopus
    View full text