**2.3 Sonodynamic excitation of photosensitizers**

Illumination is undoubtedly the easiest and most effective way to activate PSs. However, its use is restricted, due to limited penetration of visible light into tissues. There is an ongoing search for alternative methods of PS excitation in the dark in order to overcome this problem. Ultrasonic activation seems to be attractive as an alternative to illumination. As with light activation, ultrasound can be selectively focused on a specific area, thus activating only PS molecules located in the affected area. Ultrasound can also easily penetrate into tissues, which opens prospects for its application in treatment of internal lesions and infections, without the need for invasive devices [33, 34]. Ultrasonic irradiation of PSs initiates the formation of highly active cytotoxic species—ROS and free radicals—which lead to the death of pathogenic cells. It was found that some well-known PSs also have sonosensitizing properties. Among

them are porphyrins [35], RB [36, 37], chlorin e6 derivative, photodithazine [36], and curcumin [38]. Several studies found sonodynamic therapy (SDT) to be the promising treatment in various forms of cancerous tumors [39–43]. Sonodynamic therapy is also offered as treatment for atherosclerosis [44]. The applicability of sonodynamic antimicrobial chemotherapy (SACT) for the treatment of infectious diseases has been confirmed by various research groups [33, 34]. We have previously demonstrated the effectiveness of RB activated by ultrasonication for eradication of Gram-positive *S. aureus* and Gram-negative *E. coli* [29, 45, 46]. The effectiveness of SACT in inactivation of *S. aureus* by two other sensitizers—curcumin [38] and hematoporphyrin monomethyl ether [35]—was also reported. Alves et al. have recently reported on effective destruction of *Candida albicans* by photodithazine and RB in the dark under the ultrasonic excitation. A significant synergistic effect of the combination between PDT and SACT for combatting *C. albicans* biofilms was also found [36].

**Figure 2** demonstrates the effect of ultrasonic activation that we showed on the antibacterial activity of two PSs—RB (**Figure 2a**) and MB (**Figure 2b**)—against *S. aureus* compared to photodynamic activation. **Figure 2a** shows that 15 min of sonication reduces the number of living cells by almost two orders of magnitude, from 2 × 108 to 4 × 106 CFU mL<sup>−</sup><sup>1</sup> . RB alone applied in the dark causes a two orders of magnitude decrease in the cell concentration. However, sonication in the presence of 5 μM RB exerts a much stronger effect, reducing the cell concentration by 5 orders of magnitude. It should be noted that RB at the same concentration under illumination by visible light of 1.6 mW cm<sup>−</sup><sup>2</sup> fluence causes complete eradication of *S. aureus* cells, whereas light alone does not cause any significant harm to these cells. However, MB applied under sonication at the concentration causing complete destruction of *S. aureus* cells in the light did not eradicate microbial cells more than sonication alone (**Figure 2**).

#### **2.4 Activation of photosensitizers by radio waves**

Another possible way for activating PSs in the dark is by using nonionizing radiofrequency electromagnetic waves. The ability of radiofrequency waves to heat human tissue has been known for a long time and has already been applied for local destruction of cancerous tumors [47, 48]. The effectiveness of this method can be significantly improved by using suitable sensitizers, which can be targeted to the affected area and activated by means of radiofrequency radiation for selective destruction of cells. Tamarov et al. proposed the use of crystalline silicon-based nanoparticles as sensitizers induced by 27 MHz radiofrequency waves for effective treatment of Lewis lung carcinoma *in vivo* [48]. Another approach involved using gold nanoparticles, which were heated by an electric field using 13.56 MHz radiofrequency, and effectively destroyed human pancreatic cancer cells *in vitro* [49]. The same frequency was used in other studies to activate fullerene [50] and transferrin [51] and to eradicate cancer tumors *in vitro* and *in vivo*. A possible mechanism of radiosensitization, according to Tamarov et al. [48] and Chung et al. [51], may be thermal activation of sensitizers by hyperthermia, caused by dissipation of electromagnetic energy, which leads to thermal damage of cancer cells.

In our studies, we tested the possibility of using radiofrequency radiation to sensitize PSs in order to destroy microorganisms [29]. For this purpose, we irradiated *S. aureus* cells in physiological saline alone and in the presence of RB with radio waves at different frequencies—from 1 to 20 GHz. *S. aureus* cells in physiological saline in the dark (without RB and without radiation), *S. aureus* cells treated with radio waves (in the absence of RB), and *S. aureus* cells in the presence of RB, but not exposed to radio waves, were used as controls. Radiofrequency radiation alone did not significantly affect the survival of *S. aureus*. RB in the dark applied at the same

**137**

**Figure 3.**

*standard deviations.*

*Aspects of Photodynamic Inactivation of Bacteria DOI: http://dx.doi.org/10.5772/intechopen.89523*

concentration did not lead to any decrease in the bacterial concentration. However, exposure of *S. aureus* cells to radio waves in the presence of RB markedly reduced the number of live microorganisms. The rate of cell damage depended on the radio wave frequency. The most significant effect was observed in the frequency range of 9–12 GHz, where in the presence of RB, only 4.5% of the cells survived (**Figure 3**). For comparison, irradiation of cells treated by RB with radio waves in the frequency

 *in the dark. Error bars present* 

*Effect of RB at the 10 μM concentration under activation by radio waves at various frequency ranges on* 

*eradication of S. aureus at the initial cell concentration of 4.4 × 104 CFU mL<sup>−</sup><sup>1</sup>*

To the best of our knowledge, our work was the first attempt to sensitize a PS by radio waves for destruction of bacteria. This topic naturally necessitates a broader and deeper study to understand the mechanisms of excitation and the possibilities of applying this method. The most likely mechanism of RB excitation by radio waves is conversion of electromagnetic energy into heat, which causes activation of RB, followed by energy transfer to dissolved oxygen and the formation of ROS, affecting the cells. We assume that when PSs are exposed to radiofrequency radiation, they actually behave like thermosensitizers excited by heat instead of light [29].

range of 1–3 GHz caused only a 40% reduction in the number of live cells.

**2.5 Chemiluminescent and bioluminescent excitation of photosensitizers**

ous fields of medicine, pharmaceuticals, and bioanalytics [52, 53].

Another approach to overcoming the limitations of PACT in the treatment of deep infections is to replace the external light source by chemo- or bioluminescent light. Bioluminescence is a well-known phenomenon occurring in biological systems as a result of oxidation reactions of luciferins catalyzed by luciferases. This property is inherent in various microorganisms, worms, and insects, and the luciferins and luciferases of different organisms can be completely different. Bioluminescence is considered as a type of chemiluminescence, i.e., luminescence originating in the course of a chemical reaction. Bio- and chemiluminescence systems are used in vari-

One of the well-studied and most effective chemical reactions involving light emission is oxidation of luminol [52, 54, 55]. Most applications of this reaction are associated with treatment of cancers [55–57]. Use of chemiluminescence as a light source for PACT has not been studied as extensively. Ferraz and colleagues

*Aspects of Photodynamic Inactivation of Bacteria DOI: http://dx.doi.org/10.5772/intechopen.89523*

#### **Figure 3.**

*Microorganisms*

from 2 × 108

to 4 × 106

sonication alone (**Figure 2**).

illumination by visible light of 1.6 mW cm<sup>−</sup><sup>2</sup>

**2.4 Activation of photosensitizers by radio waves**

magnetic energy, which leads to thermal damage of cancer cells.

them are porphyrins [35], RB [36, 37], chlorin e6 derivative, photodithazine [36], and curcumin [38]. Several studies found sonodynamic therapy (SDT) to be the promising treatment in various forms of cancerous tumors [39–43]. Sonodynamic therapy is also offered as treatment for atherosclerosis [44]. The applicability of sonodynamic antimicrobial chemotherapy (SACT) for the treatment of infectious diseases has been confirmed by various research groups [33, 34]. We have previously demonstrated the effectiveness of RB activated by ultrasonication for eradication of Gram-positive *S. aureus* and Gram-negative *E. coli* [29, 45, 46]. The effectiveness of SACT in inactivation of *S. aureus* by two other sensitizers—curcumin [38] and hematoporphyrin monomethyl ether [35]—was also reported. Alves et al. have recently reported on effective destruction of *Candida albicans* by photodithazine and RB in the dark under the ultrasonic excitation. A significant synergistic effect of the combination between

**Figure 2** demonstrates the effect of ultrasonic activation that we showed on the antibacterial activity of two PSs—RB (**Figure 2a**) and MB (**Figure 2b**)—against *S. aureus* compared to photodynamic activation. **Figure 2a** shows that 15 min of sonication reduces the number of living cells by almost two orders of magnitude,

of magnitude decrease in the cell concentration. However, sonication in the presence of 5 μM RB exerts a much stronger effect, reducing the cell concentration by 5 orders of magnitude. It should be noted that RB at the same concentration under

of *S. aureus* cells, whereas light alone does not cause any significant harm to these cells. However, MB applied under sonication at the concentration causing complete destruction of *S. aureus* cells in the light did not eradicate microbial cells more than

Another possible way for activating PSs in the dark is by using nonionizing radiofrequency electromagnetic waves. The ability of radiofrequency waves to heat human tissue has been known for a long time and has already been applied for local destruction of cancerous tumors [47, 48]. The effectiveness of this method can be significantly improved by using suitable sensitizers, which can be targeted to the affected area and activated by means of radiofrequency radiation for selective destruction of cells. Tamarov et al. proposed the use of crystalline silicon-based nanoparticles as sensitizers induced by 27 MHz radiofrequency waves for effective treatment of Lewis lung carcinoma *in vivo* [48]. Another approach involved using gold nanoparticles, which were heated by an electric field using 13.56 MHz radiofrequency, and effectively destroyed human pancreatic cancer cells *in vitro* [49]. The same frequency was used in other studies to activate fullerene [50] and transferrin [51] and to eradicate cancer tumors *in vitro* and *in vivo*. A possible mechanism of radiosensitization, according to Tamarov et al. [48] and Chung et al. [51], may be thermal activation of sensitizers by hyperthermia, caused by dissipation of electro-

In our studies, we tested the possibility of using radiofrequency radiation to sensitize PSs in order to destroy microorganisms [29]. For this purpose, we irradiated *S. aureus* cells in physiological saline alone and in the presence of RB with radio waves at different frequencies—from 1 to 20 GHz. *S. aureus* cells in physiological saline in the dark (without RB and without radiation), *S. aureus* cells treated with radio waves (in the absence of RB), and *S. aureus* cells in the presence of RB, but not exposed to radio waves, were used as controls. Radiofrequency radiation alone did not significantly affect the survival of *S. aureus*. RB in the dark applied at the same

. RB alone applied in the dark causes a two orders

fluence causes complete eradication

PDT and SACT for combatting *C. albicans* biofilms was also found [36].

CFU mL<sup>−</sup><sup>1</sup>

**136**

*Effect of RB at the 10 μM concentration under activation by radio waves at various frequency ranges on eradication of S. aureus at the initial cell concentration of 4.4 × 104 CFU mL<sup>−</sup><sup>1</sup> in the dark. Error bars present standard deviations.*

concentration did not lead to any decrease in the bacterial concentration. However, exposure of *S. aureus* cells to radio waves in the presence of RB markedly reduced the number of live microorganisms. The rate of cell damage depended on the radio wave frequency. The most significant effect was observed in the frequency range of 9–12 GHz, where in the presence of RB, only 4.5% of the cells survived (**Figure 3**). For comparison, irradiation of cells treated by RB with radio waves in the frequency range of 1–3 GHz caused only a 40% reduction in the number of live cells.

To the best of our knowledge, our work was the first attempt to sensitize a PS by radio waves for destruction of bacteria. This topic naturally necessitates a broader and deeper study to understand the mechanisms of excitation and the possibilities of applying this method. The most likely mechanism of RB excitation by radio waves is conversion of electromagnetic energy into heat, which causes activation of RB, followed by energy transfer to dissolved oxygen and the formation of ROS, affecting the cells. We assume that when PSs are exposed to radiofrequency radiation, they actually behave like thermosensitizers excited by heat instead of light [29].

#### **2.5 Chemiluminescent and bioluminescent excitation of photosensitizers**

Another approach to overcoming the limitations of PACT in the treatment of deep infections is to replace the external light source by chemo- or bioluminescent light. Bioluminescence is a well-known phenomenon occurring in biological systems as a result of oxidation reactions of luciferins catalyzed by luciferases. This property is inherent in various microorganisms, worms, and insects, and the luciferins and luciferases of different organisms can be completely different. Bioluminescence is considered as a type of chemiluminescence, i.e., luminescence originating in the course of a chemical reaction. Bio- and chemiluminescence systems are used in various fields of medicine, pharmaceuticals, and bioanalytics [52, 53].

One of the well-studied and most effective chemical reactions involving light emission is oxidation of luminol [52, 54, 55]. Most applications of this reaction are associated with treatment of cancers [55–57]. Use of chemiluminescence as a light source for PACT has not been studied as extensively. Ferraz and colleagues

#### **Figure 4.**

*Effect of chemiluminescent photodynamic antimicrobial treatment (CPAT) on the viability of S. aureus and E. coli. Cells were incubated with MB at 25 μM concentration in the presence of 0.7 mM luminol. After the treatment, bacteria were tested by viable count. Error bars present standard deviations.*

evaluated the potential of chemiluminescent-excited photogem in killing *S. aureus* cells [58]. Our group demonstrated the effectiveness of chemiluminescent photodynamic antimicrobial therapy (CPAT) for destruction of *S. aureus* and *E. coli* by exposing these bacteria to the photosensitizer MB in the presence of luminol [46, 59, 60]. The results presented in **Figure 4** show that the rate of growth inhibition by MB increased in the presence of luminol compared to untreated cells or to cells exposed in the dark to MB only.

The dark effect of MB discussed in the above "Dark Activity" section can be seen in **Figure 4**, where the exposure of *S. aureus* and *E. coli* to 25 μM MB in the dark reduced the number of live cells by about 10-fold. Luminol alone had no toxic effect on the tested microorganisms. However, when combined with MB, it reduced the number of surviving bacteria by two additional orders of magnitude for *S. aureus* and 1.5 orders of magnitude in the case of *E. coli*. Thus, the use of chemiluminescence may expand the capabilities of PDT, allowing the use of PSs for the treatment of internal organs.
