**3.4 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)2P(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)2P(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*.

#### **Figure 4.**

*Relationship between the AF values and number of carbon atoms (n) in the alkyl group on the alkylpyridinium (RPy) in PDI of E. coli using (A) P-porphyrins ((RPy3)2P(Tpp)3+,* ○*) and Sb-porphyrins ((RPy3)2Sb(Tpp)3+ and RPy3Sb(Tpp)2+,* ∆*) and (B) 3-alkyl-substituted P-porphyrins ((RPy5)2P(Tpp)3+,* ⃟*) and their 4-ethylanalog ((4EtPy5)2P(Tpp)3+,* ◆*).*

#### **Figure 5.**

*The incorporation of porphyrins inside bacteria through self-promoted mechanism. (i) Cationic porphyrin adsorbs to the anionic outer membrane; (ii) amphiphilic porphyrin interacts with hydrophobic parts of outer and inner membranes; (iii) porphyrin is incorporated inside the cell.*

**131**

**Figure 7.**

*P-porphyrins (Type E) substituted with alkylethyleneglycol ligands.*

**Figure 6.**

**4. Discussion**

*obtained by overlapping images in D and E.*

*Photodynamic Inactivation of Escherichia coli with Cationic Porphyrin Sensitizers*

*Fluorescence images of E. coli obtained with a CLSM under laser-excitation at 543 nm. Fluorescence coming from inside the cells was observed with the addition of (HexPy3)2P(Tpp)3+ (D), but not observed with the addition of (MePy3)2P(Tpp)3+ (A). Transmission images of E. coli containing (HexPy3)2P(Tpp)3+ (E) and (MePy3)2P(Tpp)3+ (B). The image of C is obtained by overlapping images in A and B, and the image in F is* 

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 Ca2+ and Mg2+ [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

*DOI: http://dx.doi.org/10.5772/intechopen.82645*

*Photodynamic Inactivation of Escherichia coli with Cationic Porphyrin Sensitizers DOI: http://dx.doi.org/10.5772/intechopen.82645*

#### **Figure 6.**

*The Universe of Escherichia coli*

**130**

**Figure 5.**

**Figure 4.**

*analog ((4EtPy5)2P(Tpp)3+,* ◆*).*

*Relationship between the AF values and number of carbon atoms (n) in the alkyl group on the alkylpyridinium (RPy) in PDI of E. coli using (A) P-porphyrins ((RPy3)2P(Tpp)3+,* ○*) and Sb-porphyrins ((RPy3)2Sb(Tpp)3+ and RPy3Sb(Tpp)2+,* ∆*) and (B) 3-alkyl-substituted P-porphyrins ((RPy5)2P(Tpp)3+,* ⃟*) and their 4-ethyl-*

*The incorporation of porphyrins inside bacteria through self-promoted mechanism. (i) Cationic porphyrin adsorbs to the anionic outer membrane; (ii) amphiphilic porphyrin interacts with hydrophobic parts of outer* 

*and inner membranes; (iii) porphyrin is incorporated inside the cell.*

*Fluorescence images of E. coli obtained with a CLSM under laser-excitation at 543 nm. Fluorescence coming from inside the cells was observed with the addition of (HexPy3)2P(Tpp)3+ (D), but not observed with the addition of (MePy3)2P(Tpp)3+ (A). Transmission images of E. coli containing (HexPy3)2P(Tpp)3+ (E) and (MePy3)2P(Tpp)3+ (B). The image of C is obtained by overlapping images in A and B, and the image in F is obtained by overlapping images in D and E.*

#### **4. 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 Ca2+ and Mg2+ [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

**Figure 7.** *P-porphyrins (Type E) substituted with alkylethyleneglycol ligands.* sensitizers passed through the cell wall may reach biogenic proteins, lipids, and DNA. Under irradiation, reactive oxygen such as <sup>1</sup> O2 was generated near to these molecules to induce cell death. Although E-type porphyrins generate <sup>1</sup> O2 efficiently under visible light irradiation, the lifetime of <sup>1</sup> O2 in aqueous medium is very short (~3 μs) [40]. Thus, for efficient PDI, <sup>1</sup> O2 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 <sup>1</sup> O2 generation.
