Photodynamic Studies and Photoinactivation of Escherichia coli Using meso-Substituted Cationic Porphyrin Derivatives with Asymmetric Charge Distribution¶

Abstract

The photodynamic activities of novel asymmetrically meso-substituted cationic porphyrins, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 1 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 2 and its metal complex with Pd(II) 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. The amphiphilic character of porphyrin 2 was increased by the presence of a high-lipophilic trifluoromethyl group and its photophysical properties changed by forming a complex with Pd(II). Absorption and fluorescence spectroscopic studies were compared in different media. Fluorescence quantum yields (phi(F)) of 0.16 for 1 in tetrahydrofuran and 0.08 for 2 in N, N-dimethylformamide (DMF) were calculated, whereas no significant emission was detected for Pd(II) porphyrin 3. The singlet molecular oxygen, O(2)((1)Delta(g)), production was evaluated using 9,10-dimethylanthracene in DMF yielding relative values of 1, 0.55 and 0.47 for porphyrins 3, 2 and 1, respectively. A faster decomposition of l-tryptophan was obtained using Pd(II) porphyrin 3 as sensitizer with respect to the free-base porphyrins 1 and 2. In biological medium, the behavior of cationic porphyrins 1-3 were compared with that of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrin 4, which was used as a noncationic sensitizer. These porphyrins are rapidly bound to E. coli cells in 5 min and the amount of cell-bound sensitizer is not appreciably changed incubating the cultures for longer times. The recovered porphyrin 2 after one washing step reaches a value of approximately 2.9 nmol/10(6) cells and this amount remains high even after three washes, indicating that this sensitizer is tightly bound to cells. Photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. In both cases, a higher photoinactivation of cells was found for tricationic porphyrin 2 and 3, causing a approximately 5.5 log (99.999%) decrease of cell survival, when treated with 10 microM of sensitizer. Under these conditions, a lower effect was found for porphyrin 1 (approximately 4 log) whereas sensitizer 4 did not produce appreciable photodamage. The results were also confirmed by growth delay experiments. These studies show that the amphiphilic tricationic porphyrin 2 and 3 bearing a trifluoromethyl group can be a promising model for phototherapeutic agents with potential applications in inactivation of bacteria by photodynamic therapy.

Photodynamic inactivation of Escherichia coli with cationic meso-tetraarylporphyrins – The charge number and charge distribution effects

“…Although it has been shown that molecular charge is important in determining antibacterial activity [22,30,32,34], as cationic compounds are taken up through the so called self-promoted uptake pathway [21,52], the increase in the number of charges increases the PS affinity to the bacteria, which enhances retention [25,26]. In fact, cationic molecules have a 2 to 4 orders of magnitude higher affinity to binding sites on the surface of LPS molecules of Gram negative bacteria than the divalent cations Ca 2+ or Mg 2+ , and they competitively displace these cations.…”

“…an amphiphilic behaviour [56]. The increase in the amphiphilic character of the PS enhances its affinity for bacteria [26,57] increasing PDI efficiency [36]. The amphiphilic PS 4a is more efficient against E. coli than the hydrophilic PS 5a.…”

“…Positively charged PS, namely cationic porphyrins, are generally more efficient and can act at lower concentrations than neutral and anionic PS [21][22][23][24]. The charge number increases the amphiphilic character of the PS, and consequently its affinity for bacteria, helping its accumulation in the cells [25,26] which is usually accompanied by an increase in the photocytotoxic activity. Neutral and anionic PS can efficiently inactivate Gram-positive bacteria because these types of PS can easily cross the cell wall.…”

“…The number of charges [22,26,[35][36][37] and their distribution in the PS core have an important role on PDI efficiency [22,38]. For instance, Alves et al [22] found that the photodynamic efficiency of cationic porphyrins in the photosensitization of Escherichia coli and Enterococcus faecalis increases with the number of charges.…”

Synthesis and bactericidal properties of porphyrins immobilized in a polyacrylamide support: influence of metal complexation on photoactivity

“…Palladium was chosen to be inserted into compound 5 because Pd forms stable complexes with several porphyrins and it can be used to enhance the photodynamic action of the sensitiser 23 inducing the heavy atom effect which increases the singlet oxygen quantum yields by increasing the triplet state lifetime.…”