**3. Encapsulation of photosensitizers in liposomes**

Since PSs are usually inactive in the absence of excitation, focusing the beam of light, ultrasound or radio wave radiation on the affected area is the easiest way to achieve selective action of a PS. However, surrounding healthy tissues may also be affected by the PS, even under such focused processing. It is therefore very important to target the treatment directly to the infected site. Highly biocompatible and low immunogenic liposomes can serve as carriers for targeted delivery of PSs encapsulated into liposomes to the infected site [61–63].

Liposomes are spherical multi- or unilamellar vesicles consisting of phospholipids (e.g., phosphatidylcholines) with an internal hydrophilic cavity. They vary in composition, size, charge, and number of layers and can encapsulate and deliver both hydrophilic and hydrophobic compounds, which can be retained in the water core of liposomes or be encapsulated in the phospholipid bilayer, respectively.

**139**

**Figure 5.**

*external phospholipid bilayer.*

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

functional liposomes, and so on [62–66].

A variety of methods have been developed for the production of liposomes with a controlled size and special properties. The most widely used method for producing liposomes is hydration of thin lipid films. In this case, lipids with or without active substances are dissolved in an organic solvent, which is evaporated on a rotary evaporator, producing a thin film on a flask wall. The lipid film is then rehydrated by an aqueous phase. Membrane extrusion and sonication methods are most commonly used for control of liposome size [64]. Advanced strategies for liposome preparation include charging the liposomes, attaching the ligands such as antibodies or lectins to their surface, or altering the physiological conditions such as increasing the temperature or changing the pH in the target tissues to produce heat-sensitive or pH-sensitive liposomes [65]. The works of Ghosh, Li, Bulbake, Abu Lila, and Alavi summarize the latest developments in the field of liposome design and optimization, including passive and active targeting, extended circulation, building multi-

There exist several methods for PS encapsulation into liposomes (**Figure 5**). Hydrophilic PSs (e.g., MB, RB, or photofrin) are dissolved in aqueous buffer and are included into the internal cavity of liposomes. Hydrophobic compounds (such as temoporfin and bacteriochlorin a) are integrated in the phospholipid bilayer [62, 67]. Several groups have shown that encapsulation of PSs in liposomes improves their effectiveness against cancer *in vivo*. Back in 1983, Jori and colleagues reported that hematoporphyrin and its derivatives incorporated into liposomes on the basis of dipalmitoyl-phosphatidyl-choline are effective for systemic delivery of PSs to tumors in rats [68]. Enhancement of the photodynamic effects of photofrin encapsulated in a liposome carrier was later demonstrated on a human glioma implanted in rat brain [69]. A variety of PSs (temoporfin, zinc phthalocyanine, benzoporphyrin derivative monoacid, etc.) in various liposomal formulations, such

*Schematic representation of a liposome with PS entrapped in the internal aqueous phase and within the* 

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

*Microorganisms*

**Figure 4.**

exposed in the dark to MB only.

of internal organs.

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

*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.*

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

Since PSs are usually inactive in the absence of excitation, focusing the beam of light, ultrasound or radio wave radiation on the affected area is the easiest way to achieve selective action of a PS. However, surrounding healthy tissues may also be affected by the PS, even under such focused processing. It is therefore very important to target the treatment directly to the infected site. Highly biocompatible and low immunogenic liposomes can serve as carriers for targeted delivery of PSs

Liposomes are spherical multi- or unilamellar vesicles consisting of phospholipids (e.g., phosphatidylcholines) with an internal hydrophilic cavity. They vary in composition, size, charge, and number of layers and can encapsulate and deliver both hydrophilic and hydrophobic compounds, which can be retained in the water core of liposomes or be encapsulated in the phospholipid bilayer, respectively.

**3. Encapsulation of photosensitizers in liposomes**

encapsulated into liposomes to the infected site [61–63].

**138**

A variety of methods have been developed for the production of liposomes with a controlled size and special properties. The most widely used method for producing liposomes is hydration of thin lipid films. In this case, lipids with or without active substances are dissolved in an organic solvent, which is evaporated on a rotary evaporator, producing a thin film on a flask wall. The lipid film is then rehydrated by an aqueous phase. Membrane extrusion and sonication methods are most commonly used for control of liposome size [64]. Advanced strategies for liposome preparation include charging the liposomes, attaching the ligands such as antibodies or lectins to their surface, or altering the physiological conditions such as increasing the temperature or changing the pH in the target tissues to produce heat-sensitive or pH-sensitive liposomes [65]. The works of Ghosh, Li, Bulbake, Abu Lila, and Alavi summarize the latest developments in the field of liposome design and optimization, including passive and active targeting, extended circulation, building multifunctional liposomes, and so on [62–66].

There exist several methods for PS encapsulation into liposomes (**Figure 5**). Hydrophilic PSs (e.g., MB, RB, or photofrin) are dissolved in aqueous buffer and are included into the internal cavity of liposomes. Hydrophobic compounds (such as temoporfin and bacteriochlorin a) are integrated in the phospholipid bilayer [62, 67]. Several groups have shown that encapsulation of PSs in liposomes improves their effectiveness against cancer *in vivo*. Back in 1983, Jori and colleagues reported that hematoporphyrin and its derivatives incorporated into liposomes on the basis of dipalmitoyl-phosphatidyl-choline are effective for systemic delivery of PSs to tumors in rats [68]. Enhancement of the photodynamic effects of photofrin encapsulated in a liposome carrier was later demonstrated on a human glioma implanted in rat brain [69]. A variety of PSs (temoporfin, zinc phthalocyanine, benzoporphyrin derivative monoacid, etc.) in various liposomal formulations, such

#### **Figure 5.**

*Schematic representation of a liposome with PS entrapped in the internal aqueous phase and within the external phospholipid bilayer.*

as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylcholine, and others, were found to be effective on HT29 and Meth A tumor models *in vivo* [62]. However, the only clinically approved liposomal PS drug to date is Visudyne, developed by QLT in Vancouver, and produced by Novartis AG, Switzerland. This formulation is produced from a derivative of benzoporphyrin monoacid encapsulated in unilamellar dimyristoylphosphatidylcholine/egg phosphatidylglycerol liposomes. The liposomes in this drug not only dissolve the

#### **Figure 6.**

*MIC values of free and liposome encapsulated MB and NR determined against (a) S. aureus and (b) E. coli. Liposomes were prepared from dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol at 15 mg/ mL total lipid concentration by sonication for 10 sec. Bacteria at 3 × 104 CFU mL<sup>−</sup><sup>1</sup> concentration were treated by MB and NR at doubled dilutions, illuminated at room temperature by white light of 1.6 mW cm<sup>−</sup><sup>2</sup> intensity for 1 h, and incubated overnight in the dark by shaking at 37°C. Error bars present standard deviations.*

**141**

**Figure 7.**

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

and for infected burn wounds *in vivo* [78].

*aureus* (**Figure 6a**) and *E. coli* (**Figure 6b**).

absorption in tumor tissues [62, 64].

lipophilic PS for intravenous administration but also contribute to its enhanced

Liposomal PS preparations are suitable for antibacterial applications. This approach ensures the delivery of the compound at a higher concentration, thus increasing the cytotoxicity of the drug. In addition, the local use of liposomal preparations provides a slow release of active components, which helps prolong their effect in infected tissues. In Gram-negative bacteria, fusion between liposomes and the outer cell membranes leads to the delivery of concentrated liposome contents directly into the cytoplasm [70–72]. In Gram-positive bacteria, the PS is probably released when liposomes interact with the external peptidoglycan and diffuse through the cell wall [72–74]. Various researchers have demonstrated the effectiveness of liposomal formulations of various PSs against Gram-positive and Gram-negative microorganisms and also against fungal infections *in vitro* and *in vivo*. Ferro et al. showed high efficacy of porphyrin incorporated into cationic liposomes against *S. aureus*, compared to the free drug [75, 76]. Tsai also showed an increase in the bactericidal efficacy of hematoporphyrin against a number of Grampositive bacteria, including *S. aureus*, as a result of its incorporation into liposomes [77]. Yang proved the efficacy of chlorine e6 encapsulated in cationic liposomes against susceptible and drug-resistant clinical isolates of *C. albicans* both *in vitro*

In our studies, we tested the effect of different PSs in different liposome formulations on Gram-positive and Gram-negative bacteria. **Figure 6** presents a comparison between the MICs of free and dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol liposome-encapsulated MB and NR against *S.* 

*Chemiluminescent photodynamic antimicrobial treatment effect on the viability of S. aureus and E. coli. Cells were incubated with 25 μM MB liposome (lip) encapsulated together with 0.7 mM luminol (LM). After the* 

*treatment, the bacteria were tested by viable count. Error bars present standard deviations.*

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

*Microorganisms*

as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylcholine, and others, were found to be effective on HT29 and Meth A tumor models *in vivo* [62]. However, the only clinically approved liposomal PS drug to date is Visudyne, developed by QLT in Vancouver, and produced by Novartis AG, Switzerland. This formulation is produced from a derivative of benzoporphyrin monoacid encapsulated in unilamellar dimyristoylphosphatidylcholine/egg phosphatidylglycerol liposomes. The liposomes in this drug not only dissolve the

*MIC values of free and liposome encapsulated MB and NR determined against (a) S. aureus and (b) E. coli. Liposomes were prepared from dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol at 15 mg/*

*by MB and NR at doubled dilutions, illuminated at room temperature by white light of 1.6 mW cm<sup>−</sup><sup>2</sup>*

*for 1 h, and incubated overnight in the dark by shaking at 37°C. Error bars present standard deviations.*

 *concentration were treated* 

 *intensity* 

*mL total lipid concentration by sonication for 10 sec. Bacteria at 3 × 104 CFU mL<sup>−</sup><sup>1</sup>*

**140**

**Figure 6.**

lipophilic PS for intravenous administration but also contribute to its enhanced absorption in tumor tissues [62, 64].

Liposomal PS preparations are suitable for antibacterial applications. This approach ensures the delivery of the compound at a higher concentration, thus increasing the cytotoxicity of the drug. In addition, the local use of liposomal preparations provides a slow release of active components, which helps prolong their effect in infected tissues. In Gram-negative bacteria, fusion between liposomes and the outer cell membranes leads to the delivery of concentrated liposome contents directly into the cytoplasm [70–72]. In Gram-positive bacteria, the PS is probably released when liposomes interact with the external peptidoglycan and diffuse through the cell wall [72–74]. Various researchers have demonstrated the effectiveness of liposomal formulations of various PSs against Gram-positive and Gram-negative microorganisms and also against fungal infections *in vitro* and *in vivo*. Ferro et al. showed high efficacy of porphyrin incorporated into cationic liposomes against *S. aureus*, compared to the free drug [75, 76]. Tsai also showed an increase in the bactericidal efficacy of hematoporphyrin against a number of Grampositive bacteria, including *S. aureus*, as a result of its incorporation into liposomes [77]. Yang proved the efficacy of chlorine e6 encapsulated in cationic liposomes against susceptible and drug-resistant clinical isolates of *C. albicans* both *in vitro* and for infected burn wounds *in vivo* [78].

In our studies, we tested the effect of different PSs in different liposome formulations on Gram-positive and Gram-negative bacteria. **Figure 6** presents a comparison between the MICs of free and dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol liposome-encapsulated MB and NR against *S. aureus* (**Figure 6a**) and *E. coli* (**Figure 6b**).

#### **Figure 7.**

*Chemiluminescent photodynamic antimicrobial treatment effect on the viability of S. aureus and E. coli. Cells were incubated with 25 μM MB liposome (lip) encapsulated together with 0.7 mM luminol (LM). After the treatment, the bacteria were tested by viable count. Error bars present standard deviations.*

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 use of encapsulated PSs [74].

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 out in the dark.

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 increase in the efficiency and specificity of this technique.
