Hydrogen Bond Acceptors and Additional Cationic Charges in Methylene Blue Derivatives: Photophysics and Antimicrobial Efficiency

Abstract

Photodynamic inactivation of bacteria (PIB) by efficient singlet oxygen photosensitizers might be a beneficial alternative to antibiotics in the struggle against multiresistant bacteria. Phenothiazinium dyes belong to the most prominent classes of such sensitizers due to their intense absorption in the red-light region (λ abs, max ca. 600–680 nm, ε > 50000 L mol−1 cm−1), their low toxicity, and their attachment/penetration abilities. Except simple substituents like alkyl or hydroxyalkyl residues, nearly no modifications of the phenothiaziniums have been pursued at the auxochromic sites. By this, the properties of methylene blue derivatives and their fields of application are limited; it remains unclear if their potential antimicrobial efficacy may be enhanced, also to compete with porphyrins. We prepared a set of six mainly novel methylene blue derivatives with the ability of additional hydrogen bonding and/or additional cationic charges to study the substituents' effect on their activity/toxicity profiles and photophysical properties. Direct detection of singlet oxygen was performed at 1270 nm and the singlet oxygen quantum yields were determined. In suspensions with both, Gram-positive and Gram-negative bacteria, some derivatives were highly active upon illumination to inactivate S. aureus and E. coli up to 7 log10 steps (99.99999%) without inherent toxicities in the nonirradiated state.

Antibacterial photodynamic therapy: overview of a promising approach to fight antibiotic-resistant bacterial infections

“…Methylene blue derivatives (structures are summarized in Table 2) such as new methylene blue, dimethyl methylene blue, and methylene green were developed with different functional substituents and shown to have improved effectiveness of killing bacteria. As indicated previously, increasing the positive charge on methylene blue correlated positively with increased APDT efficacy than native methylene blue [19,20], mainly because substitution with cationic functional groups improved the binding and uptake of the PS by bacteria relative to the parent structure [19][20][21]. For example, Felgentrager et al revealed that derivatization of methylene blue with tertiary ammonium substituents increased the uptake by microbial cells due to the fact that these substituents imposed a greater cationic charge in a pH-equilibrated aqueous solution, and therefore more avid bacterial cell binding, compared to primary or secondary substituents [19].…”

“…As indicated previously, increasing the positive charge on methylene blue correlated positively with increased APDT efficacy than native methylene blue [19,20], mainly because substitution with cationic functional groups improved the binding and uptake of the PS by bacteria relative to the parent structure [19][20][21]. For example, Felgentrager et al revealed that derivatization of methylene blue with tertiary ammonium substituents increased the uptake by microbial cells due to the fact that these substituents imposed a greater cationic charge in a pH-equilibrated aqueous solution, and therefore more avid bacterial cell binding, compared to primary or secondary substituents [19]. Furthermore, the tertiary ammonium-substituted methylene blue derivative possesses lower pK a values than the lower-order substituents, which results in faster deprotonation when bound to the negatively charged bacterial cell envelope structure and better uptake.…”

“…The PSs that have been tested in vivo include phenothiaziniums (section 2.1) such as methylene blue [19,209], Rose Bengal [210], and EtNBS [188] (Table 1), ZnPc derivative RLP068/Cl [211], (Table 1), and C70 fullerene [60,64,68] (Table 1), among others. Animal models have been developed that mimic clinical infections, including a mouse model of skin abrasion caused by MRSA [212] and a guinea pig model of burn infections with S. aureus [213].…”

Photodynamic inactivation of Escherichia coli by methylene blue and malachite green under red LED light

“…Vilela et al (2012) did not observe any cytotoxic effect using 3000 M and 300 M of MG and MB, respectively, in the absence of light. Ergaieg and Seux (2009) used 3.65 M of MB and observed no inhibitory effect on cell viability in the dark, while Felgenträger et al (2013) reached the same conclusion using MB 100 M. Although all of the studies cited used the same standard E. coli strain (ATCC 25922), the results are different since other factors must be considered as conditions for cultivating bacteria, growth stage, PS solutions (in de-ionized water, saline or culture medium), contact time with the bacteria in PS in the dark, among other Rev.…”

Strategies to optimize photosensitizers for photodynamic inactivation of bacteria

“…Therefore MB was substituted with methyl-groups (dimethyl methylene blue), ethyl-group (new methylene blue) or a nitro-group (methylene green) [38][39][40]. Felgentrager et al showed that additional cationic charges (primary, secondary and tertiary ammonium residues) in methylene blue derivatives (MB3 & MB4) lead to a better aPDI efficiency [41]. The aPDI efficacy of these MB derivatives was improved from primary to secondary to tertiary ammonium residues.…”