**4. Immobilization**

New prospects of using PSs are opened by the immobilization of PSs onto a solid phase. This approach may allow repeated or continuous use of PSs. PSs can be immobilized by adsorption and covalent bonding onto solid supports and by ionic bonding to ion-exchange resins or incorporation into polymer films. The photodynamic properties of immobilized PSs are reported to be retained for a long time [79–83]. PSs studied in the immobilized form include RB, MB, and TBO; the porphyrin derivatives 5,10,15,20-tetrakis (p-hydroxy phenyl) porphyrin, 5,10,15,20-tetrakis (p-aminophenyl) porphyrin, and zinc (II) phthalocyanine tetrasulfonic acid; and the ruthenium salts tris (4,4′-diphenyl-2,2′-bipyridine) ruthenium (II), tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II), tris (1,10-phenanthrolinyl-4,7-bis (benzenesulfonate) ruthenate (II), and tris (4,40-dinonyl-1,10-phenan throline) ruthenium (II). Solid supports applied for immobilization of PSs include polyethylene, polypropylene, polystyrene, polycarbonate, polymethyl methacrylate, polyester isophthalic resin, silicone, cationic nylon, porous silicones, poly (vinylidene difluoride), cellulose membranes, and chitosan [82–88]. Immobilized PSs demonstrated antibacterial properties against Gram-negative and Gram-positive bacteria in batch and continuous regimes and under reuse. Immobilized PSs were found more stable and resistant to photobleaching than in a free form [82, 86, 88].

Our group immobilized PSs in polymers using several techniques. The first method included mixing solutions of PSs in chloroform with solutions of polymers in the same solvent, followed by evaporation of the solvent, which yielded thin polymeric films with homogeneously incorporated PSs. This technique was applied to RB and MB immobilized onto polystyrene, polycarbonate, and polymethyl methacrylate [88–90]. In all cases, the obtained polymer films showed high antibacterial activity against Gram-positive and Gram-negative bacteria when exposed to an external source of white light. However, since this method involves using an organic solvent, it cannot be considered environmentally friendly. The second method is based on dissolution of PSs in a melted polymer under extrusion and does not require any additional chemical reagents [91]. The photosensitizers RB, Rose Bengal lactone, MB, and hematoporphyrin were immobilized in polyethylene and polypropylene using this method. The antibacterial efficiency of immobilized

**143**

disinfection.

**5. Conclusions**

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

complete eradication [91].

*Error bars present standard deviations.*

**Figure 8.**

of magnitude after 10 min of treatment.

PSs obtained as polymeric strips and beads was tested against *S. aureus* and *E. coli* in batch and continuous regimes under white fluorescent light. All immobilized PSs significantly reduced the concentration of the tested microorganisms, up to their

*Antibacterial activity of silicon-immobilized RB (5% w/w) under ultrasonic treatment in the dark. Control—S. aureus cells treated by ultrasound only. After the treatment, bacteria were tested by viable count.* 

Further development of immobilization methods and different PSs and polymers may expand the possibilities of this approach and yield the applications in various fields, such as the production of antibacterial surfaces and water

Numerous studies show that photodynamic antibacterial chemotherapy is a powerful tool for killing microorganisms. Since this method requires external illumination, it can be successfully applied only to the treatment of local superficial skin and oral cavity infections. Development of new modes of PS excitation by ultrasound, radio waves, chemiluminescent, and bioluminescent light opens new prospects for their use in treating internal infections. Encapsulation of PSs in liposomes may solve the problem of using hydrophobic PSs with poor solubility in the aqueous phase. It can also provide delivery of a concentrated PS directly to the target site, thus increasing efficiency and reducing side effects of the treatment. Immobilization of PSs in a solid phase enables using them repeatedly or in

Another immobilization technique was based on polymerization of silicon in the presence of RB as the photosensitizer. Silicon tablets produced by this method contained evenly distributed RB that was not bound to the support by covalent bonds [29]. The antibacterial activity of the immobilized RB was tested under illumination and using ultrasonic activation in the dark (**Figure 8**). **Figure 8** demonstrates the effect of immobilized RB on *S. aureus* cells when subjected to ultrasound in the dark. Silicone alone did not affect the microorganisms with and without sonication. However, the number of alive cells in samples subjected to immobilized RB under sonication decreased with sonication time and decreased by more than three orders

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

#### **Figure 8.**

*Microorganisms*

out in the dark.

**4. Immobilization**

use of encapsulated PSs [74].

As can be seen from the results, incorporation into liposomes significantly increased the antibacterial activity of MB and NR. Following encapsulation, the MIC of MB decreased by approximately 2-fold and that of NR by about 1.4-fold for both tested microorganisms (**Figure 6**). We tested the effect of liposome composition on the delivery of these PSs to cells and determined the conditions for efficient

In addition, we tried to apply liposomal forms of PSs to CPAT by encapsulating not only PSs in liposomes but also luminol and introduced to activate PSs in sites inaccessible to external lighting [59]. We monitored the survival of the cells following their exposure to either liposomal MB or luminol, as well as to liposomes containing both compounds together (**Figure 7**) when the experiments were carried

It can be seen (**Figure 7**) that luminol itself did not lead to cell damage. MB in the liposomal form exhibited certain dark activity, similar to that in a free form discussed in the "Dark Activity" section. The addition of luminol to MB liposomes markedly increased its antibacterial activity toward *S. aureus* and *E. coli*. Liposomes were not targeted in this study. Targeting of liposomes can lead to an additional

New prospects of using PSs are opened by the immobilization of PSs onto a solid phase. This approach may allow repeated or continuous use of PSs. PSs can be immobilized by adsorption and covalent bonding onto solid supports and by ionic bonding to ion-exchange resins or incorporation into polymer films. The photodynamic properties of immobilized PSs are reported to be retained for a long time [79–83]. PSs studied in the immobilized form include RB, MB, and TBO; the porphyrin derivatives 5,10,15,20-tetrakis (p-hydroxy phenyl) porphyrin, 5,10,15,20-tetrakis (p-aminophenyl) porphyrin, and zinc (II) phthalocyanine tetrasulfonic acid; and the ruthenium salts tris (4,4′-diphenyl-2,2′-bipyridine) ruthenium (II), tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II), tris (1,10-phenanthrolinyl-4,7-bis (benzenesulfonate) ruthenate (II), and tris (4,40-dinonyl-1,10-phenan throline) ruthenium (II). Solid supports applied for immobilization of PSs include polyethylene, polypropylene, polystyrene, polycarbonate, polymethyl methacrylate, polyester isophthalic resin, silicone, cationic nylon, porous silicones, poly (vinylidene difluoride), cellulose membranes, and chitosan [82–88]. Immobilized PSs demonstrated antibacterial properties against Gram-negative and Gram-positive bacteria in batch and continuous regimes and under reuse. Immobilized PSs were found more stable and resistant to photobleaching than in a free form [82, 86, 88]. Our group immobilized PSs in polymers using several techniques. The first method included mixing solutions of PSs in chloroform with solutions of polymers in the same solvent, followed by evaporation of the solvent, which yielded thin polymeric films with homogeneously incorporated PSs. This technique was applied to RB and MB immobilized onto polystyrene, polycarbonate, and polymethyl methacrylate [88–90]. In all cases, the obtained polymer films showed high antibacterial activity against Gram-positive and Gram-negative bacteria when exposed to an external source of white light. However, since this method involves using an organic solvent, it cannot be considered environmentally friendly. The second method is based on dissolution of PSs in a melted polymer under extrusion and does not require any additional chemical reagents [91]. The photosensitizers RB, Rose Bengal lactone, MB, and hematoporphyrin were immobilized in polyethylene and polypropylene using this method. The antibacterial efficiency of immobilized

increase in the efficiency and specificity of this technique.

**142**

*Antibacterial activity of silicon-immobilized RB (5% w/w) under ultrasonic treatment in the dark. Control—S. aureus cells treated by ultrasound only. After the treatment, bacteria were tested by viable count. Error bars present standard deviations.*

PSs obtained as polymeric strips and beads was tested against *S. aureus* and *E. coli* in batch and continuous regimes under white fluorescent light. All immobilized PSs significantly reduced the concentration of the tested microorganisms, up to their complete eradication [91].

Another immobilization technique was based on polymerization of silicon in the presence of RB as the photosensitizer. Silicon tablets produced by this method contained evenly distributed RB that was not bound to the support by covalent bonds [29]. The antibacterial activity of the immobilized RB was tested under illumination and using ultrasonic activation in the dark (**Figure 8**). **Figure 8** demonstrates the effect of immobilized RB on *S. aureus* cells when subjected to ultrasound in the dark. Silicone alone did not affect the microorganisms with and without sonication. However, the number of alive cells in samples subjected to immobilized RB under sonication decreased with sonication time and decreased by more than three orders of magnitude after 10 min of treatment.

Further development of immobilization methods and different PSs and polymers may expand the possibilities of this approach and yield the applications in various fields, such as the production of antibacterial surfaces and water disinfection.

### **5. Conclusions**

Numerous studies show that photodynamic antibacterial chemotherapy is a powerful tool for killing microorganisms. Since this method requires external illumination, it can be successfully applied only to the treatment of local superficial skin and oral cavity infections. Development of new modes of PS excitation by ultrasound, radio waves, chemiluminescent, and bioluminescent light opens new prospects for their use in treating internal infections. Encapsulation of PSs in liposomes may solve the problem of using hydrophobic PSs with poor solubility in the aqueous phase. It can also provide delivery of a concentrated PS directly to the target site, thus increasing efficiency and reducing side effects of the treatment. Immobilization of PSs in a solid phase enables using them repeatedly or in

a continuous mode. It can be assumed that PSs have a good potential for various clinical and nonclinical applications.
