Photodynamic Inactivation of Escherichia coli with Cationic Porphyrin Sensitizers

The activity of singlet-oxygen sensitizers for photodynamic inactivation (PDI) of microorganisms and photodynamic therapy of tumor cells has been evaluated using Escherichia coli , Saccharomyces cerevisiae , and human cancer cell lines. In this chapter, drug resistance of E. coli was examined based on the PDI activity of a variety of RPy-P-porphyrin sensitizers with different number of ionic valence and different hydrophobic characters. The PDI activities toward E. coli were evaluated using the minimum effective concentrations ([ P ]) of the porphyrin sensitizers. It was found that the [ P ] value for E. coli was larger than that for S. cerevisiae . E. coli has drug-resistance toward hydrophobic and mono-cationic porphyrins. However, E. coli has weak drug-resistance toward the porphyrins with both polycationic character and hydrophobicity. Since the outer membrane mainly consists of lipopolysaccharides and phospholipids that are negatively charged, cationic porphyrins are able to adsorb to the outer leaflet. Then the cationic porphyrins with hydrophobic character can interact with not only the outer leaflet but also inner leaflet of the outer membrane and the plasma membrane. Thus, porphyrins may be incorporated inside E. coli cells via the self-promoted uptake pathway. Moreover, polycationic porphyrins can interact with DNA and proteins by strong binding affinities.


Introduction
Singlet-oxygen ( 1 O 2 ) sensitizers for photodynamic inactivation (PDI) of microorganisms and photodynamic therapy of tumor cells have been developed using Escherichia coli, Saccharomyces cerevisiae, and human cancer cell lines (e.g., HeLa cell) as model cells [1][2][3][4]. As E. coli is a Gram-negative bacterium, the cell wall consists of an inner membrane, cytoplasmic membrane, a periplasmic space with a peptidoglycan layer, and an outer membrane [5]. Since the E. coli cell wall has a low permeability, there are only a few 1 O 2 -sensitizers that can permeate the cell wall and inactivate E. coli efficiently at low concentrations.
PDI refers to the use of a visible-light source, oxidizing agents (e.g., O 2 ), and photosensitizers. Photosensitizers absorb light energy that causes an energy transfer to O 2 , which leads to the formation of reactive oxygen such as 1 O 2 , thereby inactivating cells and bacteria. Preliminary studies on the photodynamic action for biological systems started in the 1930s by PDI of phages using methylene blue [6,7]. PDI of bacteria has received considerable attention as a methodology leading to the medical application of infection therapy beyond antimicrobial resistance. Among the large variety of photosensitizers developed for PDI over the last 60 years, porphyrins and metalloporphyrins became attractive sensitizers owing to their strong absorption band in the visible-light region [8][9][10][11].
In the case of porphyrin sensitizers, their solubilities in water are an important characteristic for handling them as aqueous solutions, since porphyrin derivatives, in general, are poorly soluble in water. The most popular method to improve the solubility in water is the introduction of ionic groups to the porphyrin ring. Especially, the introduction of an alkylpyridinium (RPy) group into porphyrins is a useful method to make porphyrins water-soluble [12,13]. A typical RPy-bonded porphyrin is represented by meso-tetra [4-(1-methyl-pyridinium)] porphyrin (TMP). The first application of TMP to PDI was reported by Ben Amor et al. in 1998 [14]. For the last two decades, a variety of RPy-bonded porphyrins have been prepared and studied for PDI [15][16][17][18][19][20][21].
We have interested in axially RPy-bonded tricationic P-porphyrins and their PDI activity [22][23][24][25][26]. It is advantageous that the water solubilization is easily achieved through the modification of the axial ligands of P-porphyrins. It is expected that polycationic porphyrins have strong binding affinities to DNA [27][28][29][30][31][32]. In this chapter, drug resistance of E. coli was discussed based on PDI activity of a variety of P-and Sb-porphyrin sensitizers with different number of ionic valence and different hydrophobic character. The typical structure of the porphyrin sensitizer is shown in Figure 1, and they are named P-type porphyrin.

Preparation of E. coli suspension
E. coli K-12 (IFO 3301) was cultured aerobically at 30°C for 8 h in a LB medium (pH 6.5) consisting of bactotryptone (10 g L −1 ), yeast extract (5 g L −1 ), and NaCl (10 g L −1 ). After centrifugation of the cultured broth at 12,000 rpm for 10 min, the harvested cells were washed with physiological saline (NaCl, 7 g L −1 ) and then suspended in physiological saline, resulting in a cell suspension of E. coli. The cell concentrations were determined using a calibration curve and turbidity quantified by the absorbance measured at 600 nm on an UV-Vis spectrometer [24].

PDI of E. coli
PDI of E. coli was performed as follows. A phosphate buffer (0.1 M, pH 7.6) was prepared by dissolving Na 2 HPO 4 (2.469 g) and NaH 2 PO 4 (0.312 g) in 100 mL of water. The suspension of E. coli cells (1 × 10 5 cells mL −1 , 1.0 mL), an aqueous solution of the studied sensitizers (25-100 μM, 0.1 mL), and the phosphate buffer (0.1 M, pH 7.6, 8.9 mL) were introduced into L-type glass tubes, resulting in a buffer solution (10 mL) containing E. coli (1 × 10 4 cells mL −1 ) and the studied sensitizers (0.25-1.0 μM). Under dark conditions, the L-type glass tubes were set on a reciprocal shaker and shaken at 160 rpm at room temperature for 2 h [24]. And then the L-type glass tubes were irradiated using a fluorescent lamp (Panasonic FL-15ECW, Japan; wave length = 400-723 nm; the maximum intensity: 545 nm; 10.5 W cm −2 ) on a reciprocal shaker at room temperature. A portion of the reaction mixture (0.1 mL) was taken up to 2 h at 20-min intervals and plated on LB plates. The LB plates were incubated for 30 h at 30°C.
The amount of the living cells (B) was defined as the average number of E. coli colonies that appeared after an incubation period of 30 h in three replicate plates. The B values for the PDI sensitizers were recorded at each irradiation time.

Fluorescence imaging
Incorporation of porphyrin sensitizers inside cells can be examined by fluorescence microscopy images of E. coli on a confocal laser scanning microscope (CLSM) under laser excitation at 543 nm. The aqueous solution containing the porphyrin sensitizers and E. coli was incubated for 3 h at 25°C. The concentrated solution was sandwiched between a cover slip and an agar pad on a bottom cover slip to maintain its position within the same focal plane [36].  Figure 2 shows the structures of the prepared porphyrins, which were water soluble due to cationic complexes. The water solubility (C W ) is listed in light. Moreover, they could generate 1 O 2 efficiently, since the quantum yields for the formation of 1 O 2 were found to be 0.88 for (HexPy3) 2 P(Tpp) 3+ and 0.87 for (Bu2) 2 P(MePyTpp) 2+ [23].

Results of PDI of E. coli
Results of PDI of E. coli are summarized in Table 2. As seen from Table 2, Meso-RPy-substituted P-porphyrins ((R'm) 2 P(RPyTpp) 2+ ) have cytotoxicity, since E. coli was inactivated under dark conditions. Based on Table 2, the survival ratios were calculated as 100B/B 0 where B 0 is the initial amount of bacteria. From the time-course plots of survival ratios (100B/B 0 ), the half-life (T 1/2 in min), i.e., the time required to reduce B from B 0 to 0.5B 0 , was measured. A typical example of time-course plots is the case of PDI of E. coli by (HexPy3) 2 P(Tpp) 3+ as shown in Figure 3. In this case, the T 1/2 value of

PDI activity of the porphyrin sensitizers toward E. coli
As shown in Table 3, the A F values were dependent on the number of carbon atoms (n) in the alkyl group on the RPy group in (RPy3) 2 M(Tpp) 3+ (M = P, Sb), RPy3Sb(Tpp) 2+ , and (RPy5) 2 P(Tpp) 3+ . Figure 4A shows the dependence of the A F values on n in the case of a series of (RPy3) 2 M(Tpp) 3+ (M = P, Sb) and RPy3Sb(Tpp) 2+ . The maximum value of A F appeared at n = 7 whose [P] value was 0.40 μM. Moderately long alkyl chain made the sensitizer more active toward E. coli [24]. In the case of a series of (RPy5) 2 P(Tpp) 3+ (Figure 4B), the maximum value of A F appeared at n = 2 whose [P] value for E. coli was 0.25 μM [25]. Therefore, the A F and [P] values of 3-ethyl analog were compared with those of 4-ethyl isomer. It was found that the A F value of 4-ethyl isomer was lower than that of 3-ethyl isomer. In the case of the 4-ethyl analog, broadening of Soret and Q bands occurred due to aggregation of porphyrin chromophores. It is suggested that aggregation caused to lower the A F value of 4-ethyl isomer (4EtPy5) 2 P(Tpp) 3+ ). Figure 5 shows the fluorescence images of E. coli in the presence of depicting the emission from (MePy3) 2 P(Tpp) 3+ and (HexPy3) 2 P(Tpp) 3+ inside E. coli. The images show that (HexPy3) 2 P(Tpp) 3+ was accumulated inside E. coli, whereas (MePy3) 2 P(Tpp) 3+ was not. (HexPy3) 2 P(Tpp) 3+ , which had a large affinity to E. coli, had the high PDI activity. The RPy group with a long alkyl chain made the sensitizer reactive toward E. coli.

Comparison of the PDI activity in E. coli with the PDI activity in Saccharomyces cerevisiae
For comparison of the PDI activity in E. coli and other microorganisms, PDI of S. cerevisiae was performed using (RPy3) 2 P(Tpp) 3+ . It could photoinactivate S. cerevisiae in lower concentration compared with the case of E. coli [23]. For example, the [P] values of (MePy3) 2 P(Tpp) 3+ for S. cerevisiae were 0.05 μM, while that for E. coli was 2.0 μM. Moreover, PDI of S. cerevisiae was performed using other porphyrins (Type E, Figure 6), which were monocationic and highly hydrophobic. The PDI of S. cerevisiae occurred efficiently by Type E porphyrins [37]. The [P] values for the PDI of S. cerevisiae were optimized to be 0.005 μM. Thus, S. cerevisiae has low drug resistance for hydrophobic sensitizers rather than polycationic sensitizers, since the [P] value of tricationic porphyrins was larger than that of monocationic porphyrins (Type E). On the contrary, no PDI of E. coli by Type E porphyrins occurred at all. This result shows that a more positive character is required for an efficient PDI of E. coli.

Discussion
The mechanism behind the PDI activity in E. coli is still not completely understood. However, it is known that the first contact of porphyrin photosensitizers occurs at the outer membrane. The outer leaflet of the outer membrane mainly consists of lipopolysaccharides and phospholipids, which are negatively charged and are stabilized with divalent cations such as Ca 2+ and Mg 2+ [38]. Therefore, electrostatic interaction between cationic photosensitizers and the outer leaflet instead of these divalent cations promotes destabilization of the outer membrane [39]. In the case of the cationic porphyrins with hydrophobic character, or the amphiphilic one, they can also interact with not only the outer leaflet but also the inner leaflet of the outer membrane and the plasma membrane (Figure 7). Thus, the amphiphilic porphyrins may be incorporated inside E. coli cells via the self-promoted uptake pathway [37]. The porphyrin sensitizers passed through the cell wall may reach biogenic proteins, lipids, and DNA. Under irradiation, reactive oxygen such as 1 O 2 was generated near to these molecules to induce cell death. Although E-type porphyrins generate 1 O 2 efficiently under visible light irradiation, the lifetime of 1 O 2 in aqueous medium is very short (~3 μs) [40]. Thus, for efficient PDI, 1 O 2 should be generated as close as possible to the target molecules. The P type porphyrins with amphiphilic characters, which can be incorporated inside E. coli, will be advantageous to PDI via 1 O 2 generation.

Conclusion
PDI of E. coli K-12 (IFO 3301) was examined using 19 kinds of cationic porphyrin sensitizers. In conclusion, (1) E. coli has high drug-resistance toward the hydrophobic and monocationic porphyrins such as Type E. (2) However, E. coli has low drug-resistance toward polycationic porphyrins such as Type P. (3) Especially, E. coli has low drug-resistance toward polycationic porphyrins with moderately long alkyl chain, for example, (HeptPy3) 2 P(Tpp) 3+ and (EtPy5) 2 P(Tpp) 3+ . Alkyl chains might result in moderate hydrophobicity to take advantage of interaction between hydrophobic parts of cell membranes. (4) Polycationic porphyrins can interact with the anionic outer membrane at the first step and DNA and proteins inside the cells with strong binding affinities.
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