**3.1 Properties of RPy-bonded P-porphyrins**

**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 **Table 1**. In addition, **Table 1** lists the absorption coefficient (*ε*) of Soret band around 431 nm and Q-band at 562 nm in MeOH. These porphyrins could absorb strongly visible





**127**

*a*

*b*

*d*

*mL<sup>−</sup><sup>1</sup>*

**Table 2.**

*Irradiation time (t) in min.*

*Under dark conditions.*

*Photodynamic Inactivation of Escherichia coli with Cationic Porphyrin Sensitizers*

O2 efficiently, since the quantum yields for

O2 were found to be 0.88 for (HexPy3)2P(Tpp)3+ and 0.87 for

**Amount of bacteria ([***B***])/CFU mL<sup>−</sup><sup>1</sup> <sup>a</sup>** *t* **= 0/min c 20 40 60 80 100 120**

Results of PDI of *E. coli* are summarized in **Table 2**. As seen from **Table 2**, *Meso*-RPy-substituted P-porphyrins ((R'*m*)2P(RPyTpp)2+) have cytotoxicity, since *E. coli*

Based on **Table 2**, the survival ratios were calculated as 100*B*/*B*0 where *B*<sup>0</sup> is the initial amount of bacteria. From the time-course plots of survival ratios (100*B*/*B*0), the half-life (*T*1/2 in min), i.e., the time required to reduce *B* from *B*0 to 0.5*B*0, was measured. A typical example of time-course plots is the case of PDI of *E. coli* by (HexPy3)2P(Tpp)3+ as shown in **Figure 3**. In this case, the *T*1/2 value of

(MePy3)2P(tpp) 2.0 512 ± 22 450 ± 14 383 ± 13 344 ± 20 198 ± 13 103 ± 4.5 27 ± 1.2 (BuPy3)2P(tpp) 2.0 377 ± 56 216 ± 10 105 ± 9.9 39 ± 5.3 18 ± 3.2 6.0 ± 2.7 2.3 ± 0.6 (PentPy3)2P(tpp) 0.5 105 ± 12 65 ± 12 36 ± 4.6 19 ± 3.8 14 ± 4.0 11 ± 3.1 7.0 ± 2.0 (HexPy3)2P(tpp) 0.5 243 ± 23 156 ± 5.2 125 ± 5.8 86 ± 3.1 77 ± 7.5 60 ± 1.2 17 ± 6.0 (HeptPy3)2P(tpp) 0.4 203 ± 16 117 ± 9.1 53 ± 3.8 39 ± 3.1 15 ± 1.2 4.7 ± 2.1 3.0 ± 0 (OctPy3)2P(tpp) 0.5 294 ± 14 215 ± 15 194 ± 12 136 ± 16 103 ± 9.9 76 ± 10 44 ± 8.0 (HexPy3)2Sb(tpp) 1.0 152 ± 7.1 110 ± 4.7 76 ± 17 49 ± 4.2 36 ± 15 21 ± 4.5 45 ± 8.7 (MePy3)Sb(tpp) 1.0 170 ± 13 167 ± 17 134 ± 8.0 126 ± 6.8 102 ± 17 108 ± 26 113 ± 13 (HexPy3)Sb(tpp) 1.0 131 ± 28 120 ± 14 75 ± 11 55 ± 16 36 ± 11 23 ± 3.5 13 ± 1.7 (MePy5)2P(tpp) 1.0 29 ± 6.4 16 ± 4.2 12 ± 5.6 10 ± 1.0 13 ± 2.3 6.7 ± 2.1 6.7 ± 1.5 (EtPy5)2P(tpp) 0.25 167 ± 14 141 ± 18 59 ± 9.0 5.7 ± 0.6 1.7 ± 1.5 0.3 ± 0.6 0 (BuPy5)2P(tpp) 0.25 145 ± 11 123 ± 7.6 92 ± 7.5 63 ± 4.6 33 ± 8.4 6.7 ± 4.9 4.7 ± 0.6 (HexPy5)2P(tpp) 0.25 213 ± 10 213 ± 9.5 176 ± 16 166 ± 6.8 140 ± 8.2 132 ± 12 97 ± 4.4 (4-EtPy5)2P(tpp) 0.5 139 ± 14 85 ± 13 88 ± 16 62 ± 6.0 42 ± 8.7 32 ± 7.0 33 ± 1.5 (Me)2P(PyHex) 2.0 90 ± 13 88 ± 17 49 ± 7.8 27 ± 6.2 17 ± 5.1 13 ± 1.5 15 ± 3.1 (Me*1*)2P(PyHex) 0.5 89 ± 2.7 57 ± 2.9 42 ± 7.2 18 ± 3.5 16 ± 2.9 8.3 ± 4.0 5.7 ± 1.2 (Me*1*)2P(PyHex) d 0.5 109 ± 26 99 ± 13 59 ± 12 64 ± 10 65 ± 165 59 ± 42 41 ± 9.6 (Bu*1*)2P(PyMe) 0.5 24 ± 3.6 20 ± 4.5 13 ± 3.0 12 ± 1.2 7.3 ± 2.9 3.7 ± 2.1 4.7 ± 1.2 (Bu*1*)2P(PyMe) d 0.5 34 ± 5.0 25 ± 3.5 28 ± 6.1 31 ± 3.5 25 ± 1.5 20 ± 2.7 19 ± 2.1 (Bu*2*)2P(PyMe) 2.0 126 ± 14 56 ± 3.8 21 ± 4.9 8.7 ± 2.1 3.3 ± 3.5 1.7 ± 0.6 2.3 ± 2.1 (Bu*2*)2P(PyMe) d 2.0 150 ± 13 141 ± 5.5 129 ± 8.3 124 ± 11 116 ± 13 84 ± 14 94 ± 12 (Hex*2*)2P(PyMe) 1.0 63 ± 5.9 50 ± 7.5 56 ± 2.1 45 ± 8.1 39 ± 9.1 35 ± 6.1 33 ± 12

*PDI of E. coli was performed in a phosphate buffer solution (10 mL, pH 7.6) containing E. coli (ca. 2 × 104*

*[P] was adjusted to attain the value of T1/2 between 20 and 120 min. <sup>c</sup>*

*PDI of E. coli with cationic porphyrins under visible light irradiation.*

*) and porphyrin sensitizers under the irradiation of a fluorescent lamp. CFU = colony formation unit.*

 *cell* 

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

light. Moreover, they could generate <sup>1</sup>

was inactivated under dark conditions.

**μM b**

the formation of <sup>1</sup>

**Sensitizers** *[P]***/**

(Bu2)2P(MePyTpp)2+ [23].

**3.2 Results of PDI of** *E. coli*

**Figure 2.** *Polycationic P- and Sb-porphyrins bonded to alkylpyridinium (RPy).*

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

light. Moreover, they could generate <sup>1</sup> O2 efficiently, since the quantum yields for the formation of <sup>1</sup> O2 were found to be 0.88 for (HexPy3)2P(Tpp)3+ and 0.87 for (Bu2)2P(MePyTpp)2+ [23].

### **3.2 Results of PDI of** *E. coli*

*The Universe of Escherichia coli*

**3.1 Properties of RPy-bonded P-porphyrins**

**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 **Table 1**. In addition, **Table 1** lists the absorption coefficient (*ε*) of Soret band around 431 nm and Q-band at 562 nm in MeOH. These porphyrins could absorb strongly visible

**3. Results**

**126**

**Figure 2.**

*Polycationic P- and Sb-porphyrins bonded to alkylpyridinium (RPy).*

Results of PDI of *E. coli* are summarized in **Table 2**. As seen from **Table 2**, *Meso*-RPy-substituted P-porphyrins ((R'*m*)2P(RPyTpp)2+) have cytotoxicity, since *E. coli* was inactivated under dark conditions.

Based on **Table 2**, the survival ratios were calculated as 100*B*/*B*0 where *B*<sup>0</sup> is the initial amount of bacteria. From the time-course plots of survival ratios (100*B*/*B*0), the half-life (*T*1/2 in min), i.e., the time required to reduce *B* from *B*0 to 0.5*B*0, was measured. A typical example of time-course plots is the case of PDI of *E. coli* by (HexPy3)2P(Tpp)3+ as shown in **Figure 3**. In this case, the *T*1/2 value of


*a PDI of E. coli was performed in a phosphate buffer solution (10 mL, pH 7.6) containing E. coli (ca. 2 × 104 cell mL<sup>−</sup><sup>1</sup> ) and porphyrin sensitizers under the irradiation of a fluorescent lamp. CFU = colony formation unit. b*

*[P] was adjusted to attain the value of T1/2 between 20 and 120 min. <sup>c</sup> Irradiation time (t) in min.*

*d Under dark conditions.*

#### **Table 2.**

*PDI of E. coli with cationic porphyrins under visible light irradiation.*

#### **Figure 3.**

*Typical example of time-course plots of survival ratio (100B/B0) in the PDT of E. coli with (HexPy3)2P(Tpp)3+ (0.5 μM) under visible light irradiation (•) and under dark conditions (*⃟*). The T1/2 was determined to be 31 min from the plots.*

(HexPy3)2P(Tpp)3+ was determined to be 31 min. The minimum concentrations of the sensitizer [*P*] were adjusted such that *T*1/2 attained values between 20 and 120 min. Thus, the bactericidal activity (*A*F in μM<sup>−</sup><sup>1</sup> h<sup>−</sup><sup>1</sup> ) was evaluated using the following equation: *A*F = 60/([*P*] × *T*1/2). **Table 3** summarizes [*P*] and *A*F values in the PDI of *E. coli.*

#### **3.3 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)2M(Tpp)3+ (M = P, Sb), RPy3Sb(Tpp)2+, and (RPy5)2P(Tpp)3+. **Figure 4A** shows the dependence of the *A*F values on *n* in the case of a series of (RPy3)2M(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)2P(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)2P(Tpp)3+).

**Figure 5** shows the fluorescence images of *E. coli* in the presence of depicting the emission from (MePy3)2P(Tpp)3+ and (HexPy3)2P(Tpp)3+ inside *E. coli*. The images show that (HexPy3)2P(Tpp)3+ was accumulated inside *E. coli*, whereas (MePy3)2P(Tpp)3+ was not. (HexPy3)2P(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*.

**129**

*a*

*b*

*c*

*d*

**Table 3.**

*under dark conditions*

*T1/2 = half-life in min. <sup>f</sup> AF = PDI activity in μM<sup>−</sup><sup>1</sup>*

*Z = charge of the complex.*

*Photodynamic Inactivation of Escherichia coli with Cationic Porphyrin Sensitizers*

**Sensitizera** *Z***<sup>b</sup> Metal** *n* **<sup>c</sup> [***P***]/μM d** *T***1/2 /min e** *A***F /μM<sup>−</sup><sup>1</sup>**

(MePy3)2P(tpp) +3 P 1 2.0 66 0.5 (BuPy3)2P(tpp) +3 P 4 2.0 27 1.1 (PentPy3)2P(tpp) +3 P 5 0.5 29 4.1 (HexPy3)2P(tpp) +3 P 6 0.5 31 3.8 (HeptPy3)2P(tpp) +3 P 7 0.4 24 6.3 (OctPy3)2P(tpp) +3 P 8 0.5 63 1.9 (HexPy3)2Sb(tpp) +3 Sb 6 1.0 36 1.7 (MePy3)Sb(tpp) +2 Sb 1 1.0 106 0.6 (HexPy3)Sb(tpp) +2 Sb 6 1.0 68 0.9 (MePy5)2P(tpp) +3 P 1 1.0 40 1.5 (EtPy5)2P(tpp) +3 P 2 0.25 32 7.5 (ButPy5)2P(tpp) +3 P 4 0.25 53 4.5 (HexPy5)2P(tpp) +3 P 6 0.25 120 2.0 (4EtPy5)2P(tpp) +3 P 2 0.5 50 2.4 (Me)2P(PyHex) +2 P 6 2.0 45 0.7 (Me*1*)2P(PyHex) +2 P 6 0.5 37 3.2 (Bu*1*)2P(PyMe) +2 P 1 0.5 55 2.2 (Bu*2*)2P(PyMe) +2 P 1 2.0 23 1.3 (Hex*2*)2P(PyMe) +2 P 1 1.0 116 0.5

 **h<sup>−</sup><sup>1</sup> <sup>f</sup>**

**3.4 Comparison of the PDI activity in** *E. coli* **with the PDI activity in** 

*: AF = 60/([P] × T1/2).*

*The [P], T1/2, and AF values in the PDI of E. coli by cationic porphyrins.*

shows that a more positive character is required for an efficient PDI of *E. coli*.

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

*The PDI did not occur under dark conditions except for meso-RPy-substituted P-porphyrins, which were cytotoxic* 

*[P] = minimum concentrations of the porphyrins adjusted to attain the value of T1/2 between 20 and 120 min. <sup>e</sup>*

*Saccharomyces cerevisiae*

*n = carbon number of the alkyl chain on the AP.*

 *h<sup>−</sup><sup>1</sup>*

*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*

*a The PDI did not occur under dark conditions except for meso-RPy-substituted P-porphyrins, which were cytotoxic under dark conditions*

*b Z = charge of the complex.*

*c n = carbon number of the alkyl chain on the AP.*

*d [P] = minimum concentrations of the porphyrins adjusted to attain the value of T1/2 between 20 and 120 min. <sup>e</sup>*

*T1/2 = half-life in min. <sup>f</sup>*

*AF = PDI activity in μM<sup>−</sup><sup>1</sup> h<sup>−</sup><sup>1</sup> : AF = 60/([P] × T1/2).*

#### **Table 3.**

*The Universe of Escherichia coli*

(HexPy3)2P(Tpp)3+ was determined to be 31 min. The minimum concentrations of the sensitizer [*P*] were adjusted such that *T*1/2 attained values between 20 and

*(HexPy3)2P(Tpp)3+ (0.5 μM) under visible light irradiation (•) and under dark conditions (*⃟*). The T1/2 was* 

*Typical example of time-course plots of survival ratio (100B/B0) in the PDT of E. coli with* 

following equation: *A*F = 60/([*P*] × *T*1/2). **Table 3** summarizes [*P*] and *A*F values in

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)2M(Tpp)3+ (M = P, Sb), RPy3Sb(Tpp)2+, and (RPy5)2P(Tpp)3+. **Figure 4A** shows the dependence of the *A*F values on *n* in the case of a series of (RPy3)2M(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)2P(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

**Figure 5** shows the fluorescence images of *E. coli* in the presence of depicting the emission from (MePy3)2P(Tpp)3+ and (HexPy3)2P(Tpp)3+ inside *E. coli*. The images show that (HexPy3)2P(Tpp)3+ was accumulated inside *E. coli*, whereas (MePy3)2P(Tpp)3+ was not. (HexPy3)2P(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

h<sup>−</sup><sup>1</sup>

) was evaluated using the

120 min. Thus, the bactericidal activity (*A*F in μM<sup>−</sup><sup>1</sup>

**3.3 PDI activity of the porphyrin sensitizers toward** *E. coli*

the PDI of *E. coli.*

*determined to be 31 min from the plots.*

**Figure 3.**

(4EtPy5)2P(Tpp)3+).

reactive toward *E. coli*.

**128**

*The [P], T1/2, and AF values in the PDI of E. coli by cationic porphyrins.*
