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Evaluation of physicochemical properties and bacterial photoinactivation of phenothiazine photosensitizers

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Abstract

We report herein the physicochemical properties and antimicrobial activity of a new monobrominated derivative of Azure B and its parent compound. These dyes are used as photosensitizers for photodynamic therapy and photodynamic antimicrobial chemotherapy. Relevant pharmaceutical properties (pKa, chemical and photochemical stability, and in vitro antimicrobial activity) were determined. A UV-visible spectro-photometry method was developed and validated according to the International Conference on Harmonization (ICH) guidelines for use in stability indicating studies and determination of the acid dissociation constant of Azure B and its monobrominated derivative. The results showed that both dyes were chemically stable. In addition, bromination of the phenothiazine dye decreased its photochemical stability and pKa value without affecting the ionization rate at physiological pH. The analytical parameters for validation of the method were linearity (r2 > 0.9981), limit of detection (LOD) (0.2-0.9 μM), limit of quantification (LOQ) (0.6-2.7 pM), and intra-day precision (0.76-1.40%) expressed as relative standard deviation (RSD). Recoveries ranging from 99.5 to 100.9% were obtained for the two dyes. Thus, this method provides a simple, sensitive, accurate, and precise assay for the determination of all compounds. The effect of photosensitizer concentration and visible irradiation time on lethal photosensitization against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was investigated. Both photosensitizers were active against the evaluated bacteria. However, the new monobrominated derivative was more effective than its predecessor and managed to eradicate these microorganisms by using different doses of the dye and light. In other words, a lower concentration of AzBBr and irradiation time were required to cause bacterial death equal to or greater than its precursor. The photodynamic efficacy of the two photosensitizers presented the following order: S. aureus > E. coli > P. aeruginosa. These studies indicated that the tested dyes satisfy the conditions of potential photosensitizers in terms of physicochemical and antimicrobial properties.

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References

  1. Y. Gao, B. Mai, A. Wang, M. Li, X. Wang, K. Zhang, Q. Liu, S. Wei and P. Wang, Antimicrobial properties of a new type of photosensitizer derived from phthalocyanine against planktonic and biofilm forms of Staphylococcus aureus, Photodiagn. Photodyn. Ther., 2018, 21, 316–326.

    Article  CAS  Google Scholar 

  2. C. S. A. Caires, C. R. B. Leal, C. A. N. Ramos, D. Bogo, A. R. Lima, E. J. Arruda, S. L. Oliveira, A. R. L. Caires and V. A. Nascimento, Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against Staphylococcus aureus and Escherichia coli, Lasers Med. Sci., 2017, 32, 1081–1088.

    Article  PubMed  Google Scholar 

  3. Y. Fang, T. Liu, Q. Zou, Y. Zhao and F. Wu, Water-soluble benzylidene cyclopentanone based photosensitizers for in vitro and in vivo antimicrobial photodynamic therapy, Sci. Rep., 2016, 6, 1–9.

    Article  CAS  Google Scholar 

  4. T. Felicetti, R. Cannalire, M. S. Burali, S. Massari, G. Manfroni, M. L. Barreca, O. Tabarrini, B. D. Schindler, S. Sabatini, G. W. Kaatz and V. Cecchetti, Searching for Novel Inhibitors of the S. aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-benzothiazine Analogues, ChemMedChem, 2017, 12, 1293–1302.

    Article  CAS  PubMed  Google Scholar 

  5. J. S. Monteiro, S. C. de Oliveira, G. M. Pires Santos, F. J. Pires Sampaio, L. G. Pinheiro Soares and A. L. Pinheiro, Effectiveness of antimicrobial photodynamic therapy (AmPDT) on Staphylococcus aureus using phenothiazine compound with red laser, Lasers Med. Sci., 2017, 32, 29–34.

    Article  PubMed  Google Scholar 

  6. G. M. P. Santos, S. C. P. S. de Oliveira, J. C. S. Monteiro, S. R. Fagnani, F. P. Sampaio, P. J. L. Crugeira, P. F. Almeida and A. L. B. Pinheiro, Effects of PACT using Phenothiazinederived drugs and red light on the macrophage x S. aureus interface, Photodiagn. Photodyn. Ther., 2018, 22, 96–100.

    Article  CAS  Google Scholar 

  7. A. F. Barbosa, B. B. Sangiorgi, S. L. Galdino, M. Barral-Netto, I. R. Pitta and A. L. Pinheiro, Photodynamic antimicrobial chemotherapy (PACT) using phenothiazine derivatives as photosensitizers against Leishmania brazi-liensis, Lasers Surg. Med., 2012, 44, 850–855.

    Article  PubMed  Google Scholar 

  8. M. Jeleň, E. I. Bavavea, M. Pappa, A. P. Kourounakis, B. Morak-Mlodawska and K. Pluta, Synthesis of quinoline/ naphthalene-containing azaphenothiazines and their potent in vitro antioxidant properties, Med. Chem. Res., 2015, 24, 1725–1732.

    Article  PubMed  CAS  Google Scholar 

  9. C. Gopi, V. G. Sastry and M. D. Dhanaraju, Synthesis, spectroscopic characterization, X-ray crystallography, structural activity relationship and antimicrobial activity of some novel 4-(5-(10-(3- N, N -dimethylamino)propyl)-10 H -pheno-thiazine-3-yl)-1, 3, 4-thiadiazole-2-yl) Azo dye/Schiff base de, Future J. Pharm. Sci., 2017, 3, 79–89.

    Article  Google Scholar 

  10. K. Pluta, B. Morak-Mlodawska and M. Jeleň, Recent progress in biological activities of synthesized phenothiazines, Eur. J. Med. Chem., 2011, 46, 3179–3189.

    Article  CAS  PubMed  Google Scholar 

  11. A. P. Pinto, I. B. Rosseti, M. L. Carvalho, B. G. M. da Silva, C. Alberto-Silva and M. S. Costa, Photodynamic Antimicrobial Chemotherapy (PACT), using Toluidine blue O inhibits the viability of biofilm produced by Candida albicans at different stages of development, Photodiagn. Photodyn. Ther., 2018, 21, 182–189.

    Article  CAS  Google Scholar 

  12. T. Huang, C. Chen, S. Ding and C. Chen, Photodiagnosis and Photodynamic Therapy Antimicrobial e ffi cacy of methylene blue-mediated photodynamic therapy on titanium alloy surfaces in vitro, Photodiagn. Photodyn. Ther., 2019, 25, 7–16.

    Article  CAS  Google Scholar 

  13. A. Henrique, C. Pereira, J. Guerra, M. Aparecida and A. Freitas, Photodiagnosis and Photodynamic Therapy Methylene blue internalization and photodynamic action against clinical and ATCC Pseudomonas aeruginosa and Staphyloccocus aureus strains, Photodiagn. Photodyn. Ther., 2018, 22, 43–50.

    Article  CAS  Google Scholar 

  14. V. G. Garcia, M. Longo, E. C. Gualberto Júnior, A. F. Bosco, M. J. H. Nagata, E. Ervolino and L. H. Theodoro, Effect of the concentration of phenothiazine photosensitizers in antimicrobial photodynamic therapy on bone loss and the immune inflammatory response of induced periodontitis in rats, J. Periodontal Res., 2014, 49, 584–594.

    Article  CAS  PubMed  Google Scholar 

  15. P. Calzavara-Pinton, M. T. Rossi, R. Sala and M. Venturini, Photodynamic antifungal chemotherapy, Photochem. Photobiol., 2012, 88, 512–522.

    Article  CAS  PubMed  Google Scholar 

  16. C. M. L. Francisco, M. L. A. Gonçalves, B. S. Brum, T. P. C. Santos, A. Lino-dos-santos-franco, D. F. T. Silva and C. Pavani, The photodynamic efficiency of phenothiazinium dyes is aggregation dependent, New J. Chem., 2017, 41, 14438–14443.

    Article  CAS  Google Scholar 

  17. D. A. Phoenix and F. Harris, Phenothiazinium-based photosensitizers: Antibacterials of the future?, Trends Mol. Med., 2003, 9, 283–285.

    Article  CAS  PubMed  Google Scholar 

  18. M. N. Montes De Oca, J. Vara, L. Milla, V. Rivarola and C. S. Ortiz, Physicochemical properties and photodynamic activity of novel derivatives of triarylmethane and thiazine, Arch. Pharm., 2013, 346, 255–265.

    Article  CAS  Google Scholar 

  19. W. Shao, H. Wang, S. He, L. Shi, K. Peng, Y. Lin, L. Zhang, L. Ji and H. Liu, Photophysical properties and singlet oxygen generation of three sets of halogenated corroles, J. Phys. Chem. B, 2012, 116, 14228–14234.

    Article  CAS  PubMed  Google Scholar 

  20. M. G. Alvarez, M. N. Montes de Oca, M. E. Milanesio, C. S. Ortiz and E. N. Durantini, Photodynamic properties and photoinactivation of Candida albicans mediated by brominated derivatives of triarylmethane and phenothiazinium dyes., Photodiagn. Photodyn. Ther., 2014, 11, 148–155.

    Article  CAS  Google Scholar 

  21. J. Vara, M. S. Gualdesi, S. G. Bertolotti and C. S. Ortiz, Two phenothiazine dyes as photosensitizers for the production of singlet oxygen. Photophysics, photochemistry and effects of aggregation, J. Mol. Struct., 2019, 1181,1–7.

    Article  CAS  Google Scholar 

  22. B. R. Smith, C. M. Eastman and J. T. Njardarson, Beyond C, H, O, and N! Analysis of the Elemental Composition of U.S. FDA Approved Drug Architectures, J. Med. Chem., 2014, 57, 9764–9773.

    Article  CAS  PubMed  Google Scholar 

  23. M. Zhao, Y. Xu, M. Xie, L. Zou, Z. Wang and S. Liu, Halogenated Aza-BODIPY for Imaging-Guided Synergistic Photodynamic and Photothermal Tumor Therapy, Adv. Healthcare Mater., 2018, 7, 1800606.

    Article  CAS  Google Scholar 

  24. J. Zou, Z. Yin, K. Ding, Q. Tang and J. Li, Article BODIPY Derivatives for Photodynamic Therapy: Influence of Configuration versus Heavy Atom Effect BODIPY Derivatives for Photodynamic Therapy: Influence of Configuration versus Heavy Atom Effect, ACS Appl. Mater. Interfaces, 2017, 9, 32475–32481.

    Article  CAS  PubMed  Google Scholar 

  25. C. Garcia, A. Burgain, J. Chaillot, É. Pic, I. Khemiri and A. Sellam, A phenotypic small-molecule screen identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell invasion, Sci. Rep., 2018, 8, 11559.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. K. Plaetzer, B. Krammer, J. Berlanda, F. Berr and T. Kiesslich, Photophysics and photochemistry of photodynamic therapy: Fundamental aspects, Lasers Med. Sci., 2009, 24, 259–268.

    Article  CAS  PubMed  Google Scholar 

  27. D. Dohoda, K. Tsinman, O. Tsinman, H. Wang and K. Y. Tam, Spectrophotometric pKa determination of ionizable pharmaceuticals: Resolution of molecules with weak pH-dependent spectral shift, J. Pharm. Biomed. Anal., 2015, 114, 88–96.

    Article  CAS  PubMed  Google Scholar 

  28. M. M. Pandey, A. Jaipal, A. Kumar, R. Malik and S. Y. Charde, Determination of pKa of felodipine using UV-Visible spectroscopy, Spectrochim. Acta, Part A, 2013, 115, 887–890.

    Article  CAS  Google Scholar 

  29. M. Blessy, R. D. Patel, P. N. Prajapati and Y. K. Agrawal, Development of forced degradation and stability indicating studies of drugs - A review, J. Pharm. Anal., 2014, 4, 159–165.

    Article  CAS  PubMed  Google Scholar 

  30. S. Bajaj, N. Sakhuja, D. Singla and S. Bajaj Principal, Stability Testing of Pharmaceutical Products, J. Appl. Pharm. Sci., 2012, 02, 129–138.

    Google Scholar 

  31. J. Ferreira, P. F. C. Menezes, C. Kurachi, C. Sibata, R. R. Allison and V. S. Bagnato, Photostability of different chlorine photosensitizers, Laser Phys. Lett., 2008, 5, 156–161.

    Article  Google Scholar 

  32. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmon. Tripart. Guidel. Q2(R1). Valid. Anal. Proced. Text Methodol.

  33. J. Goicoechea, F. J. Arregui, J. M. Corres and I. R. Matias, Study and Optimization of Self-Assembled Polymeric Multilayer Structures with Neutral Red for pH Sensing Applications, J. Sens., 2008, 2008, 1–7.

    Article  Google Scholar 

  34. J. W. Wydra, N. B. Cramer, J. W. Stansbury and C. N. Bowman, The reciprocity law concerning light dose relationships applied to BisGMA/TEGDMA photopolymers: Theoretical analysis and experimental characterization, Dent. Mater., 2014, 30, 605–612.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing, 26th edn, 2016.

    Google Scholar 

  36. H. Naghili, H. Tajik, K. Mardani, S. M. Razavi Rouhani, A. Ehsani and P. Zare, Validation of drop plate technique for bacterial enumeration by parametric and nonparametric tests., Vet. Res. Forum, 2013, 4, 179–183.

    PubMed  PubMed Central  Google Scholar 

  37. B. Ghalami-Choobar and A. Ghiami-Shomami, Theoretical calculation of pKa values of the Nortryptiline and Amitryptiline drugs in aqueous and non-aqueous solvents, Comput. Theor. Chem., 2013, 1018, 66–70.

    Article  CAS  Google Scholar 

  38. T. H. Shayesteh, M. Radmehr, F. Khajavi and R. Mahjub, Application of chemometrics in determination of the acid dissociation constants (pKa) of several benzodiazepine derivatives as poorly soluble drugs in the presence of ionic surfactants, Eur. J. Pharm. Sci., 2015, 69, 44–50.

    Article  CAS  PubMed  Google Scholar 

  39. A. Mills, D. Hazafy, J. Parkinson, T. Tuttle and M. G. Hutchings, Effect of alkali on methylene blue (C.I. Basic Blue 9) and other thiazine dyes, Dyes Pigm., 2011, 88, 149–155.

    Article  CAS  Google Scholar 

  40. L. Cincotta, J. W. Foley and A. H. Cincotta, Novel red absorbing benzo[a]phenoxazinium and benzo[a]phenothiazinium photosensitizers: in vitro evaluation., Photochem. Photobiol., 1987, 46, 751–758.

    Article  CAS  PubMed  Google Scholar 

  41. J. Dandriyal, R. Singla, M. Kumar and V. Jaitak, Recent developments of C-4 substituted coumarin derivatives as anticancer agents, Eur. J. Med. Chem., 2016, 119, 141–168.

    Article  CAS  PubMed  Google Scholar 

  42. T. Kiesslich, A. Gollmer, T. Maisch, M. Berneburg and K. Plaetzer, A comprehensive tutorial on in vitro characterization of new photosensitizers for photodynamic antitumor therapy and photodynamic inactivation of microorganisms, BioMed Res. Int., 2013, 840417.

    Google Scholar 

  43. Y. A. Handoko, F. S. Rondonuwu and L. Limantara, The photosensitizer stabilities of Tookad® on aggregation, acidification, and day-light irradiation, Procedia Chem., 2015, 14, 474–483.

    Article  CAS  Google Scholar 

  44. S. S. Martínez and E. V. Uribe, Enhanced sonochemical degradation of azure B dye by the electroFenton process, Ultrason. Sonochem., 2012, 19, 174–178.

    Article  PubMed  CAS  Google Scholar 

  45. J. Luan, W. Zhao, J. Feng, H. Cai, Z. Zheng, B. Pan, X. Wu, Z. Zou and Y. Li, Structural, photophysical and photocatalytic properties of novel Bi2AlVO7, J. Hazard. Mater., 2009, 164, 781–789.

    Article  CAS  PubMed  Google Scholar 

  46. M. S. Gualdesi, C. I. Alvarez Igarzabal, J. Vara and C. S. Ortiz, Synthesis and physicochemical properties of polyacrylamide nanoparticles as photosensitizer carriers, Int. J. Pharm., 2016, 512, 213–218.

    Article  CAS  PubMed  Google Scholar 

  47. A. T. Zenuz, H. Eslami, H. S. Kafil, E. Safari, M. Ghanizadeh and A. Mohammadi, The application of antimicrobial photodynamic therapy on pseudomonas aeuroginosa and enterococcus fecalis using heperecin and methylene blue photosensitizers, Biomed. Pharmacol. J., 2016, 9, 443–450.

    Article  Google Scholar 

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Acknowledgements

This work was supported by grants from Secretaría de Ciencia y Técnica (SeCyT) 316/2016, 113/2017. JV gratefully acknowledges receipt of a fellowship from CONICET. MSG and VA are career members of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

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Authors and Affiliations

  1. Departamento de Ciencias Farmacéuticas, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina

    Jimena Vara, Maria S. Gualdesi, Virginia Aiassa & Cristina S. Ortiz

  2. UNITEFA-CONICET, Argentina

    Jimena Vara & Virginia Aiassa

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  1. Jimena Vara
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  2. Maria S. Gualdesi
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  3. Virginia Aiassa
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  4. Cristina S. Ortiz
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Correspondence to Cristina S. Ortiz.

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Vara, J., Gualdesi, M.S., Aiassa, V. et al. Evaluation of physicochemical properties and bacterial photoinactivation of phenothiazine photosensitizers. Photochem Photobiol Sci 18, 1576–1586 (2019). https://doi.org/10.1039/c8pp00584b

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