Photodynamic inactivation of Enterococcus faecalis by conjugates of zinc(II) phthalocyanines with thymol and carvacrol loaded into lipid vesicles

Terpene-macrocycle conjugates, consisting of thymol or carvacrol and phthalocyanine were synthesized, characterized, and subjected to detailed optical and biological studies. The macrocyclization reactions of terpenesubstituted o-phthalonitrile derivatives were performed in a microwave reactor to give thymol- and carvacrolphthalocyanine conjugates, which were carefully purified by column chromatography and subsequently analyzed using HPLC and characterized using UV-Vis, NMR and MS. Both isolated phthalocyanine derivatives represent isomers of C-2v symmetry, which was confirmed by NMR study. The absorption UV-Vis spectra of studied thymol- and carvacrol-phthalocyanine conjugates with the Soret and the Q band possesses the same profile. Both molecules reveal emission in the red region of the spectrum with fluorescence quantum yields about two-fold lower in DMF and significantly lower in DMSO than that observed for zinc(II) phthalocyanine. The analyzed phthalocyanine derivatives generate singlet oxygen upon excitation with light at ca. three-fold lower level then that noticed for zinc(II) phthalocyanine. In comparison to zinc(II) phthalocyanine, both molecules also reveal two-fold lower photostability in DMF and similar stability in DMSO. Thymol- and carvacrol-phthalocyanine conjugates, zinc(II) phthalocyanine, thymol, and carvacrol were loaded into modified liposomes and subjected to biological activity study. Thymol-phthalocyanine conjugate at both 100 and 10 mu M revealed high, ca. 5 log photoinactivation potential on the growth of Enterococcus faecalis, similarly to reference zinc(II) phthalocyanine. For carvacrol-phthalocyanine conjugate, an increase of photoinactivation activity was observed at only 100 mu M in comparison to zinc(II) phthalocyanine, whereas a decrease at the concentration of 10 mu M was noticed. It is crucial that both studied phthalocyanines cross the border of 3 log photoinactivation potential, which indicates their bactericidal potential. In the Microtox (R) bioassay both conjugates revealed significantly decreased dark toxicity in comparison to thymol and carvacrol only. Interestingly, the carvacrol-phthalocyanine conjugate was found to be more toxic in comparison to thymol-phthalocyanine conjugate and zinc(II) phthalocyanine.