Photodynamic Inactivation of *Escherichia coli* with Cationic Porphyrin Sensitizers

*Jin Matsumoto, Tomoko Matsumoto, Kazuya Yasuda and Masahide Yasuda*

### **Abstract**

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.

**Keywords:** PDT sensitizer, singlet oxygen, porphyrins, PDI activity, *Escherichia coli*, *Saccharomyces cerevisiae*

### **1. Introduction**

Singlet-oxygen (1 O2) 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 [1–4]. 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 O2-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., O2), and photosensitizers. Photosensitizers absorb light energy that causes an energy transfer

#### **Figure 1.**

*Typical structure of porphyrin sensitizer (P type).*

to O2, which leads to the formation of reactive oxygen such as <sup>1</sup> O2, 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 [8–11].

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 [15–21].

We have interested in axially RPy-bonded tricationic P-porphyrins and their PDI activity [22–26]. 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 [27–32]. 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.

#### **2. Materials and methods**
