**2. Olive oil-containing liposomes**

Liposomes (nano or micro-scale vesicles) can be obtained using phospholipids' property of self-assembly in the presence of an aqueous phase. Phospholipids spontaneously form a closed spherical phospholipid bilayer such that phosphate groups are in contact with the aqueous phase on the internal and external surfaces, and lipid chains are hidden within the membrane. Such a phospholipid assembly results in large multilamellar liposomes, which are constructed from alternating concentric lipid and aqueous layers. Treatment of multilamellar liposomes by ultrasound, membrane extrusion or other methods leads to the formation of unilamellar liposomes which consist of a single lipid bilayer (Chrai et al., 2001). Liposomes are convenient carriers of both hydrophilic and hydrophobic molecules, where the former can be incorporated into aqueous layers of multilamellar liposomes or

antioxidant (Cathcart, 1985). The scavenging ability in the triple mixtures after mechanochemical treatment was as good as that of the double mixtures of ascorbic acid with the supports. Mechanochemical inclusion of ascorbic acid into composites of olive oil with talc or olive oil with titanium dioxide successfully combined hydrophobic and hydrophilic components and provided high antioxidant properties to the entire system despite the

New olive oil-based composite materials exhibit pronounced bactericidal properties. The antimicrobial activity of the mechanochemically treated triple mixtures which were pressed into pellets was examined against the Gram-positive *S. aureus* and the Gram-negative *E. coli* bacteria. Samples containing ascorbic acid on a titanium dioxide support were more effective against both bacteria than a talc support, probably because of weaker bonding of ascorbic acid to titanium dioxide than to talc, which contributed to better diffusion of the ascorbic acid out of the pellets. Gram-positive *S. aureus* was more sensitive to all the ascorbic acid-containing samples than the Gram-negative *E. coli*, but *E. coli* responded to addition of olive oil into both talc-ascorbic acid and titanium dioxide-olive oil mixtures. In the latter case, the inhibitory activity of the triple composites was higher than that of double ascorbic acid-support composites. The antimicrobial activity of all the ascorbic acid-containing samples depended on the ascorbic acid content in the pellets. Olive oil, olive fruit and olive leaf extracts are known to exhibit a broad antimicrobial, antimycoplasmal and antifungal spectrum due to the presence of long chain unsaturated aldehydes, phenolic glycoside oleuropein and several other phenol compounds (Fleming et al. ,1973; Kubo et al., 1995; Bisignano et al., 2001; Furneri et al., 2002; Medina et al., 2007; Covas et al., 2009; Kampa et al., 2009). Mechanochemical combination of natural antimicrobial agents from olive oil with ascorbic acid, which is a strong bacterial suppressor, enabled the production of highly active

Hydrophilic and hydrophobic components can also be combined by encapsulating hydrophilic constituents in lipid vesicles called liposomes. Such lipid-based formulations are actually possible carriers for both hydrophobic and hydrophilic active components and can be applied as drug delivery systems. Liposome formulations possess enhanced abilities to penetrate the skin, thus improving the delivery process. Lipid-based drug administration can increase treatment efficiency in cases of skin infections and inflammations caused by

Liposomes (nano or micro-scale vesicles) can be obtained using phospholipids' property of self-assembly in the presence of an aqueous phase. Phospholipids spontaneously form a closed spherical phospholipid bilayer such that phosphate groups are in contact with the aqueous phase on the internal and external surfaces, and lipid chains are hidden within the membrane. Such a phospholipid assembly results in large multilamellar liposomes, which are constructed from alternating concentric lipid and aqueous layers. Treatment of multilamellar liposomes by ultrasound, membrane extrusion or other methods leads to the formation of unilamellar liposomes which consist of a single lipid bilayer (Chrai et al., 2001). Liposomes are convenient carriers of both hydrophilic and hydrophobic molecules, where the former can be incorporated into aqueous layers of multilamellar liposomes or

covalent bonding between the components (Nisnevitch et al, 2011).

solid-phase antibacterial composites.

**2. Olive oil-containing liposomes** 

bacterial invasion.

encapsulated in the inner space of unilamellar ones, and hydrophobic compounds can be incorporated into the lipid bilayers (Chrai et al., 2002).

Fig. 1. Structure of a triacylglycerol. R – various residues of fatty acids.

Table 1. Main virgin olive oil, egg phosphatidylcholine and dipalmitoyl phosphatidylcholine fatty acids. (Nichols & Sanderson, 2002; oliveoilsource.com)

Liposomes can be exploited as carriers for controlled drug delivery and targeting to cells. Liposome formulations of drugs have several advantages over the use of drugs in their free form: liposomes guarantee delivery of a highly concentrated drug, liposomes protect the drugs from degradation during the delivery process, liposomes are applicable for polar as well as for nonpolar drugs, and ingredients of the liposomes themselves are nontoxic and biodegradable (Chrai et al., 2002). Liposome components participate in drug delivery, but not in drug function, such that liposomes actually play the role of excipients (Chen, 2008). Additional ingredients can be incorporated into the phospholipid bilayer in order to impart needed properties to liposomes, as indicated by the following examples: negatively charged phosphatidylinositol or positively charged stearylamine can be incorporated into the phospholipid bilayer in order to obtain charged liposomes (Robinson et al., 2001); addition of cholesterol provides rigidity to the liposome structure (New, 1994). The latter example is

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication 475

The major ingredients of olive oil are triacylglycerols (Fig. 1) of unsaturated and saturated fatty acids (Table 1), mainly of oleic acid, but it also contains mixed triacylglycerols of palmitic-oleic-oleic, linoleic-oleic-oleic, palmitic-oleic-linoleic, stearic-oleic-oleic, linolenic-

Olive oil also contains a small amount of free fatty acids and several minor constituents necessary for health – tyrosol, hydroxytyrosol and their derivatives such as oleuropein, oleuropein aglycone, dialdehydic form of oleuropein aglycone, decarboxymethyl form of oleuropein aglycone and ligstroside aglycone; phenolic acids, for example, 4 hydroxybenzoic acid, protocatechuic acid, syringic acid and 4-hydroxy-phenylacetic acid; flavonoids and lignads, for instance, apigenin, luteolin, pinoresinol and acetopinoresinol; squalene, -tocopherol, vitamins E and K, pigments chlorophyll, pheophytin, carotenoids and other compounds (Boskou et al., 2006a,b; Boskou 2009a,b). In addition, olive oil includes phospholipids at a concentration range of 11-157 mg/kg in virgin olive oil (Hatzakis et al.,

oleic-oleic and other acids (Nichols & Sanderson, 2002; oliveoilsource.com ).

2008) and 21-124 mg/kg in cloudy (veiled) virgin olive oil (Koidis & Boscou, 2006).

Dipalmitoyl phosphatidylcholine 62 - Egg phosphatidylcholine - 64 Olive oil 30 28 Cholesterol 8 8

The liposomes used in this work were composed of dipalmitoyl phosphatidylcholine (DPPC, Northern Lipids Inc., Canada) or egg yolk phosphatidylcholine (EPC, Sigma, USA) also named L-α-lecithin. These phospholipids are constructed based on the phosphatidylcholine. However, DPPC has a homogeneous composition and contains only saturated palmitic acid residues, contrary to the heterogeneous composition of EPC, which includes several saturated and unsaturated fatty acid residues (Table 1). EPC liposomes are composed of two different fatty acid residues, where one residue is usually saturated and the other is unsaturated, as demonstrated in Table 2 (Kent & Carman, 1999). The most common fatty acids incorporated into the EPC structure are presented in Table 1. Phosphatidylcholine, the major membrane phospholipid in eukaryotic cells, is the source of the bioactive lipids lysophosphatidylcholine, phosphatidic acid, diacylglycerol, lysophosphatidylcholine, platelet activating factor and arachidonic acid. It also plays a role

We incorporated virgin olive oil (Yad Mordechai, Israel) into the lipid bilayer in order to enhance the biocompatibility of liposomes and enrich them with natural salubrious components. For this purpose, organic solutions of DPPC or EPC together with olive oil were prepared, and the organic solvent was evaporated in a round-bottom flask to dryness in a vacuum rotary evaporator to obtain a thin lipid film which was vigorously agitated

% (w/w)

EPC liposomes % (w/w)

Component DPPC liposomes,

Table 3. Weight compositions of olive oil based liposomes.

as a reservoir for several lipid messengers (Kent & Carman, 1999).

with buffer solutions with or without water-soluble active agents.

explained by an increase in the gel-to-liquid crystalline phase transition temperature (*T*c) of the lipid liposome layer upon the addition of cholesterol (Beaulac et al., 1998).

\*Alternative fatty acids residues are listed in the Table 1.

Table 2. Compounds used as a basis for liposome preparations.

explained by an increase in the gel-to-liquid crystalline phase transition temperature (*T*c) of

the lipid liposome layer upon the addition of cholesterol (Beaulac et al., 1998).

Dipalmitoyl phosphatidylcholine (DPPC)

Egg phosphatidylcholine (EPC)\*

Cholesterol

\*Alternative fatty acids residues are listed in the Table 1.

Table 2. Compounds used as a basis for liposome preparations.

Component Structure

The major ingredients of olive oil are triacylglycerols (Fig. 1) of unsaturated and saturated fatty acids (Table 1), mainly of oleic acid, but it also contains mixed triacylglycerols of palmitic-oleic-oleic, linoleic-oleic-oleic, palmitic-oleic-linoleic, stearic-oleic-oleic, linolenicoleic-oleic and other acids (Nichols & Sanderson, 2002; oliveoilsource.com ).

Olive oil also contains a small amount of free fatty acids and several minor constituents necessary for health – tyrosol, hydroxytyrosol and their derivatives such as oleuropein, oleuropein aglycone, dialdehydic form of oleuropein aglycone, decarboxymethyl form of oleuropein aglycone and ligstroside aglycone; phenolic acids, for example, 4 hydroxybenzoic acid, protocatechuic acid, syringic acid and 4-hydroxy-phenylacetic acid; flavonoids and lignads, for instance, apigenin, luteolin, pinoresinol and acetopinoresinol; squalene, -tocopherol, vitamins E and K, pigments chlorophyll, pheophytin, carotenoids and other compounds (Boskou et al., 2006a,b; Boskou 2009a,b). In addition, olive oil includes phospholipids at a concentration range of 11-157 mg/kg in virgin olive oil (Hatzakis et al., 2008) and 21-124 mg/kg in cloudy (veiled) virgin olive oil (Koidis & Boscou, 2006).


Table 3. Weight compositions of olive oil based liposomes.

The liposomes used in this work were composed of dipalmitoyl phosphatidylcholine (DPPC, Northern Lipids Inc., Canada) or egg yolk phosphatidylcholine (EPC, Sigma, USA) also named L-α-lecithin. These phospholipids are constructed based on the phosphatidylcholine. However, DPPC has a homogeneous composition and contains only saturated palmitic acid residues, contrary to the heterogeneous composition of EPC, which includes several saturated and unsaturated fatty acid residues (Table 1). EPC liposomes are composed of two different fatty acid residues, where one residue is usually saturated and the other is unsaturated, as demonstrated in Table 2 (Kent & Carman, 1999). The most common fatty acids incorporated into the EPC structure are presented in Table 1. Phosphatidylcholine, the major membrane phospholipid in eukaryotic cells, is the source of the bioactive lipids lysophosphatidylcholine, phosphatidic acid, diacylglycerol, lysophosphatidylcholine, platelet activating factor and arachidonic acid. It also plays a role as a reservoir for several lipid messengers (Kent & Carman, 1999).

We incorporated virgin olive oil (Yad Mordechai, Israel) into the lipid bilayer in order to enhance the biocompatibility of liposomes and enrich them with natural salubrious components. For this purpose, organic solutions of DPPC or EPC together with olive oil were prepared, and the organic solvent was evaporated in a round-bottom flask to dryness in a vacuum rotary evaporator to obtain a thin lipid film which was vigorously agitated with buffer solutions with or without water-soluble active agents.

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication 477

log *<sup>o</sup> <sup>n</sup> <sup>I</sup> <sup>K</sup> I*

– wavelength, and the liposome average size evaluation with a calibration curve representing *"n*"-values' dependence on vesicle sizes (Trofimov & Nisnevich, 1990; Nisnevitch et al, 2010). Higher "*n*"-values correspond to smaller vesicle sizes. Turbidity spectra of DPPC and EPC liposomes with and without addition of cholesterol and olive oil were measured (Fig. 3), and corresponding type (1) equations were found in each case. As can be seen from Table 4, "*n*" values in these equations and correspondingly, vesicle sizes, are different for DPPC and EPC liposomes, and vary when cholesterol and olive oil are

As can be seen from Table 4, the DPPC-based liposomes are smaller than the EPC ones obtained using the same treatment conditions. This phenomenon can be explained by two factors – by the lipid structure and by the lipid phase state of the liposomes. The homogeneous composition of DPPC, which contains only saturated palmitic acid residues, enables dense lipid packing in the liposome bilayers, in contradistinction to the heterogeneous composition of EPC, which includes several saturated and unsaturated fatty acid residues (Table 1). Such a denser package leads to the formation of unilamellar DPPC liposomes with a smaller diameter. At the temperatures of our experiments (from room temperature to 37oC), DPPC exists in a gel phase state (*T*c of DPPC is 41°C (avantilipids.com)), whereas EPC is found in a liquid crystal state (*T*c of EPC is –10oC (Kahl et al., 1989)). Acyl chains of phospholipids are more disordered and bulky in a fluid state, thus causing an increase in surface area per phospholipid molecule which results in bigger

Liposome composition DPPC-based liposomes EPC-based liposomes

Phospholipid alone 2.44 200 1.57 > 400 Phospholipid and cholesterol 2.50 190 2.00 > 400

olive oil 2.03 > 400 1.36 > 400

Addition of cholesterol to phospholipids resulted in an increase in the "*n*"-value, which means that the vesicle size decreased upon the addition of cholesterol. The liposome size increased again after olive oil was incorporated into the membrane structure (Table 4). These facts can be explained by taking the correlation between liposome rigidity and size into account. Addition of cholesterol caused the liposome vesicles to become more rigid and respectively smaller, and further addition of olive oil led to disturbance of the lipid layer

Table 4. Turbidity spectra parameters and vesicle size for liposomes of various

Vesicle size, nm

"*n*"-value in equation (1)

Vesicle size, nm

"*n*"-value in equation (1)

where log *oI*

incorporated into the phospholipid layers.

liposomes in the case of EPC liposomes (New, 1994).

Phospholipid, cholesterol and

and to an increase in size (Table 4).

compositions.

*I* – a measured turbidity value, *I*o – initial light intensity, *I* – light intensity and

(1)

Fig. 2. A schematic representation of unilamellar olive oil including liposome. Phospholipid molecules are orange coloured, olive oil components are green, cholesterol molecules are grey coloured. Internal liposome volume containing water-soluble active components is pink coloured.

As can be seen from Table 1, both used by us phospholipids are built from the same fatty acids as olive oil triacylglycerols and olive oil phospholipids, although in different proportions. This fact points to high compatibility between the used phospholipids and olive oil. Various combinations of phospholipids and olive oil were attempted, and it was found that a homogeneous lipid film could not be obtained with any combination of olive oil and EPC and at a high olive oil content added to DPPC. A small amount of cholesterol (Table 2) was added to the lipid mixture solution in order to increase the lipid film's rigidity. Even and homogeneous films were attained after this addition, which resulted in stable liposomes. The multilamellar liposomes were transformed into unilamellar liposomes by sonication, as described previously (Nisnevitch et al., 2010; Nakonechny et al., 2010). Final compositions found to be appropriate for liposome preparation are presented in Table 3.

A schematic representation of the olive oil-based unilamellar liposomes is presented in Fig. 2. Triacylglycerol olive oil components are organically incorporated into the phospholipidbased structure, hydrophobic olive oil constituents such as polyphenols or vitamins are incorporated into the liposome bilayer and the aqueous solution is located in the inner liposome space. The prepared liposomes were used for encapsulation of active bactericidal factors as described in part 4.

The prepared olive oil-based liposomes were characterized by average size, evaluated by measuring the turbidity spectra. This method is based on the determination of an equation of the turbidity spectra curves, estimation of the power "*n*" in the equation (1):

Fig. 2. A schematic representation of unilamellar olive oil including liposome. Phospholipid molecules are orange coloured, olive oil components are green, cholesterol molecules are grey coloured. Internal liposome volume containing water-soluble active components is

As can be seen from Table 1, both used by us phospholipids are built from the same fatty acids as olive oil triacylglycerols and olive oil phospholipids, although in different proportions. This fact points to high compatibility between the used phospholipids and olive oil. Various combinations of phospholipids and olive oil were attempted, and it was found that a homogeneous lipid film could not be obtained with any combination of olive oil and EPC and at a high olive oil content added to DPPC. A small amount of cholesterol (Table 2) was added to the lipid mixture solution in order to increase the lipid film's rigidity. Even and homogeneous films were attained after this addition, which resulted in stable liposomes. The multilamellar liposomes were transformed into unilamellar liposomes by sonication, as described previously (Nisnevitch et al., 2010; Nakonechny et al., 2010). Final compositions found to be appropriate for liposome preparation are

A schematic representation of the olive oil-based unilamellar liposomes is presented in Fig. 2. Triacylglycerol olive oil components are organically incorporated into the phospholipidbased structure, hydrophobic olive oil constituents such as polyphenols or vitamins are incorporated into the liposome bilayer and the aqueous solution is located in the inner liposome space. The prepared liposomes were used for encapsulation of active bactericidal

The prepared olive oil-based liposomes were characterized by average size, evaluated by measuring the turbidity spectra. This method is based on the determination of an equation

of the turbidity spectra curves, estimation of the power "*n*" in the equation (1):

pink coloured.

presented in Table 3.

factors as described in part 4.

$$\log \frac{I\_o}{I} = K\mathcal{X}^{-n} \tag{1}$$

where log *oI I* – a measured turbidity value, *I*o – initial light intensity, *I* – light intensity and

– wavelength, and the liposome average size evaluation with a calibration curve representing *"n*"-values' dependence on vesicle sizes (Trofimov & Nisnevich, 1990; Nisnevitch et al, 2010). Higher "*n*"-values correspond to smaller vesicle sizes. Turbidity spectra of DPPC and EPC liposomes with and without addition of cholesterol and olive oil were measured (Fig. 3), and corresponding type (1) equations were found in each case. As can be seen from Table 4, "*n*" values in these equations and correspondingly, vesicle sizes, are different for DPPC and EPC liposomes, and vary when cholesterol and olive oil are incorporated into the phospholipid layers.

As can be seen from Table 4, the DPPC-based liposomes are smaller than the EPC ones obtained using the same treatment conditions. This phenomenon can be explained by two factors – by the lipid structure and by the lipid phase state of the liposomes. The homogeneous composition of DPPC, which contains only saturated palmitic acid residues, enables dense lipid packing in the liposome bilayers, in contradistinction to the heterogeneous composition of EPC, which includes several saturated and unsaturated fatty acid residues (Table 1). Such a denser package leads to the formation of unilamellar DPPC liposomes with a smaller diameter. At the temperatures of our experiments (from room temperature to 37oC), DPPC exists in a gel phase state (*T*c of DPPC is 41°C (avantilipids.com)), whereas EPC is found in a liquid crystal state (*T*c of EPC is –10oC (Kahl et al., 1989)). Acyl chains of phospholipids are more disordered and bulky in a fluid state, thus causing an increase in surface area per phospholipid molecule which results in bigger liposomes in the case of EPC liposomes (New, 1994).


Table 4. Turbidity spectra parameters and vesicle size for liposomes of various compositions.

Addition of cholesterol to phospholipids resulted in an increase in the "*n*"-value, which means that the vesicle size decreased upon the addition of cholesterol. The liposome size increased again after olive oil was incorporated into the membrane structure (Table 4). These facts can be explained by taking the correlation between liposome rigidity and size into account. Addition of cholesterol caused the liposome vesicles to become more rigid and respectively smaller, and further addition of olive oil led to disturbance of the lipid layer and to an increase in size (Table 4).

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication 479

Fig. 4. A scheme of photosensitizer (PS) activation upon illumination which visible light and

Photosensitizes refer to several chemical groups - porphyrins, phenothiazinium, phthalocyanines, xanthenes, chlorin derivatives and others. However, a feature common to all of these groups is the presence of conjugated double bonds, which allow effective absorbance of light energy. The history, mechanism of action and biomedical applications of PACT have been reviewed extensively (Nitzan & Pechatnikov, 2011; Malik et al., 2010; Reddy et al., 2009; Randie et al., 2011; Daia et al., 2009). Two photosensitizers, Rose Bengal and Methylene Blue, were used in this work. Rose Bengal relates to a xanthene (halogenated xanthenes) group of photosensitizers, and is negatively charged under physiological conditions. Methylene Blue represents a phenothiaziniums group and exists in cationic

Fig. 5. Structures of photosensitizers Methylene Blue (upper) and Bengal Rose (lower).

Both photosensitizes absorb visible light, and their absorption spectra are presented in Fig. 6.

form. The structures of these compounds are shown in Fig.5.

its cytotoxic action.

Fig. 3. Turbidity spectra of liposomes with and without additions of cholesterol (Chol) and olive oil (O-O). a – EPC-based liposomes, b – DPPC- based liposomes.

#### **3. Photosensitizers encapsulated in olive oil-containing liposomes**

Bacterial resistance to antibiotics has become a serious problem worldwide, causing an urgent need to develop new approaches and ways to overcome the evolution and spread of drug-resistant strains (Patterson, 2006; Maragakis et al., 2008; Moellering et al., 2007). One alternative to treatment of infections by antibiotics is photodynamic antimicrobial chemotherapy (PACT), which is based on the use of non-toxic compounds – photosensitizers, which can be activated by visible light. Excited photosensitizer molecules return to a ground level by transfering their energy to dissolved molecular oxygen with production of reactive oxygen species, which leads to direct damage of cellular components (Macdonald & Dougherty, 2001; Wainwright, 1998). This process is explained in Fig. 4.

**EPC EPC+Chol EPC+Chol+O-O** **a** 

**b** 

**400 450 500 550 600 650 700**

**Wavelength, nm**

**DPPC DPPC+Chol DPPC+Chol+O-O**

Fig. 3. Turbidity spectra of liposomes with and without additions of cholesterol (Chol) and

**400 450 500 550 600 650 700**

**Wavelength, nm**

Bacterial resistance to antibiotics has become a serious problem worldwide, causing an urgent need to develop new approaches and ways to overcome the evolution and spread of drug-resistant strains (Patterson, 2006; Maragakis et al., 2008; Moellering et al., 2007). One alternative to treatment of infections by antibiotics is photodynamic antimicrobial chemotherapy (PACT), which is based on the use of non-toxic compounds – photosensitizers, which can be activated by visible light. Excited photosensitizer molecules return to a ground level by transfering their energy to dissolved molecular oxygen with production of reactive oxygen species, which leads to direct damage of cellular components (Macdonald & Dougherty, 2001; Wainwright, 1998). This process is explained in Fig. 4.

olive oil (O-O). a – EPC-based liposomes, b – DPPC- based liposomes.

**0 0.2 0.4 0.6 0.8 1 1.2 1.4**

> **0 0.2 0.4 0.6 0.8 1 1.2 1.4**

**log(Io/I)**

**log(Io/I)**

**3. Photosensitizers encapsulated in olive oil-containing liposomes** 

Fig. 4. A scheme of photosensitizer (PS) activation upon illumination which visible light and its cytotoxic action.

Photosensitizes refer to several chemical groups - porphyrins, phenothiazinium, phthalocyanines, xanthenes, chlorin derivatives and others. However, a feature common to all of these groups is the presence of conjugated double bonds, which allow effective absorbance of light energy. The history, mechanism of action and biomedical applications of PACT have been reviewed extensively (Nitzan & Pechatnikov, 2011; Malik et al., 2010; Reddy et al., 2009; Randie et al., 2011; Daia et al., 2009). Two photosensitizers, Rose Bengal and Methylene Blue, were used in this work. Rose Bengal relates to a xanthene (halogenated xanthenes) group of photosensitizers, and is negatively charged under physiological conditions. Methylene Blue represents a phenothiaziniums group and exists in cationic form. The structures of these compounds are shown in Fig.5.

Fig. 5. Structures of photosensitizers Methylene Blue (upper) and Bengal Rose (lower). Both photosensitizes absorb visible light, and their absorption spectra are presented in Fig. 6.

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication 481

enhanced cytotoxicity. A mechanism of drug delivery by liposomes was examined for Gram-negative and Gram-positive bacteria. Gram-negative and Gram-positive bacteria differ in their cell wall structure. Gram-negative cells possess an outer membrane which contains phospholipids, lipoproteins, lipopolysaccharides and proteins, peptidoglycan and cytoplasmic membrane. Gram-positive bacteria do not have an outer membrane, and their cell wall consists of peptidoglycan and an inner cytoplasmic membrane (Baron, 1996).

In Gram-negative bacteria, fusion between drug-containing liposomes and the bacterial outer membranes occurs, which results in the delivery of the liposomal contents into the cytoplasm. This mechanism was verified by scanning electron microscopy (Mugabe et al.,

Fig. 7. A schematic representation of liposome-encapsulated drug delivery to (a) Gram-

provides sustained release of active components (Storm & Crommelin, 1998).

In Gram-positive bacteria, liposomes are assumed to release their content after interaction with the external peptidoglycan barrier, enabling passive diffusion through the cell wall (Furneri et al., 2000). This drug delivery mechanism is demonstrated in Fig. 7b. Application of liposomal forms of drugs leads to prolongation of their action in infected tissues and

Gram-positive and Gram-negative bacteria respond differently to PACT, with the former being more susceptible to the treatment. Gram-negative bacteria do not bind anionic photosensitizers (Minnock et al., 2000), unless additional manipulations facilitating membrane transport are used (Nitzan et al., 1992), due to the more complex molecular and physico-chemical structure of their cell wall. PACT is considered to have good perspectives in the control of oral and otherwise localized infections (Meisel & Kocher, 2005; O'Riordan et al., 2005). Local application of liposome-entrapped drugs can prolong their action in infected tissues and provide sustained release of active components (Storm & Crommelin, 1998). It should be mentioned that bacterial resistance to phosphosensitizers has not been

Liposome formulations of photosensitizers showed high efficiency in eradication of both Gram-negative and Gram-positive bacteria. Liposome or micelle-entrapped hematoporphyrin and chlorin *e6* were found to be effective against several Gram-positive bacteria, including

negative and (b) Gram-positive bacteria cells.

methicillin-resistant *S. aureus* (Tsai et al., 2009).

reported to date.

**b**

**a**

2006; Sachetelli et al., 2000), and it is schematically shown on the Fig. 7a.

Fig. 6. Absorption spectra of (a) Methyle Blue and (b) Bengal Rose.

The described photosensitizers were encapsulated into DPPC and EPC liposomes with and without addition of olive oil as previously described by us (Nisnevitch et al., 2010). Liposomes with encapsulated photosensitizers were separated from free photosensitizers by centrifugation, and absorption of free photosensitizers was measured at the appropriate wavelengths (665 nm for Methylene Blue and 550 nm for Rose Bengal, Fig. 6).

$$\frac{A\_o \cdot V\_o - A \cdot V}{A\_o \cdot V\_o} \cdot 100\% \tag{2}$$

where - *A0* - absorbance of the initial photosensitizer in the volume *Vo* and *A*- absorbance of the free photosensitizer in the volume *V*. The encapsulation rate reached 50±5% in all cases.

The extent of the photosensitizers encapsulation in liposomes was estimated by formula (2) as the ratio of the encapsulated photosensitizer amount, taken as the difference between initial and free photosensitizer amount, and the initial amount.

#### **4. Bactericidal properties of photosensitizers encapsulated in olive oil-based liposomes**

Application of liposomal forms of various drugs is widely used in cases of cancer and bacterial infection treatment. Treatment of tumours by liposomal forms of doxorubicin led to a manifold accumulation of the drug in the malignant cells (Drummond et al., 1999). Entrapment of photosensitizers into liposomes was also successfully applied for eradication of cancer cells (Derycke & de Witte, 2004). Liposome-encapsulated tobramycin, unlike its free form, was demonstrated to be highly effective against chronic pulmonary *P. aeruginosa* infection in rats (Beaulac et al., 1996). Drug administration using liposomes provided a delivery of active components in a more concentrated form and contributed to their

The described photosensitizers were encapsulated into DPPC and EPC liposomes with and without addition of olive oil as previously described by us (Nisnevitch et al., 2010). Liposomes with encapsulated photosensitizers were separated from free photosensitizers by centrifugation, and absorption of free photosensitizers was measured at the appropriate

> *o o* 100% *o o A V AV A V*

where - *A0* - absorbance of the initial photosensitizer in the volume *Vo* and *A*- absorbance of the free photosensitizer in the volume *V*. The encapsulation rate reached 50±5% in all cases. The extent of the photosensitizers encapsulation in liposomes was estimated by formula (2) as the ratio of the encapsulated photosensitizer amount, taken as the difference between

**4. Bactericidal properties of photosensitizers encapsulated in olive oil-based** 

Application of liposomal forms of various drugs is widely used in cases of cancer and bacterial infection treatment. Treatment of tumours by liposomal forms of doxorubicin led to a manifold accumulation of the drug in the malignant cells (Drummond et al., 1999). Entrapment of photosensitizers into liposomes was also successfully applied for eradication of cancer cells (Derycke & de Witte, 2004). Liposome-encapsulated tobramycin, unlike its free form, was demonstrated to be highly effective against chronic pulmonary *P. aeruginosa* infection in rats (Beaulac et al., 1996). Drug administration using liposomes provided a delivery of active components in a more concentrated form and contributed to their

(2)

**a** 

**b** 

Fig. 6. Absorption spectra of (a) Methyle Blue and (b) Bengal Rose.

initial and free photosensitizer amount, and the initial amount.

**liposomes** 

wavelengths (665 nm for Methylene Blue and 550 nm for Rose Bengal, Fig. 6).

enhanced cytotoxicity. A mechanism of drug delivery by liposomes was examined for Gram-negative and Gram-positive bacteria. Gram-negative and Gram-positive bacteria differ in their cell wall structure. Gram-negative cells possess an outer membrane which contains phospholipids, lipoproteins, lipopolysaccharides and proteins, peptidoglycan and cytoplasmic membrane. Gram-positive bacteria do not have an outer membrane, and their cell wall consists of peptidoglycan and an inner cytoplasmic membrane (Baron, 1996).

In Gram-negative bacteria, fusion between drug-containing liposomes and the bacterial outer membranes occurs, which results in the delivery of the liposomal contents into the cytoplasm. This mechanism was verified by scanning electron microscopy (Mugabe et al., 2006; Sachetelli et al., 2000), and it is schematically shown on the Fig. 7a.

Fig. 7. A schematic representation of liposome-encapsulated drug delivery to (a) Gramnegative and (b) Gram-positive bacteria cells.

In Gram-positive bacteria, liposomes are assumed to release their content after interaction with the external peptidoglycan barrier, enabling passive diffusion through the cell wall (Furneri et al., 2000). This drug delivery mechanism is demonstrated in Fig. 7b. Application of liposomal forms of drugs leads to prolongation of their action in infected tissues and provides sustained release of active components (Storm & Crommelin, 1998).

Gram-positive and Gram-negative bacteria respond differently to PACT, with the former being more susceptible to the treatment. Gram-negative bacteria do not bind anionic photosensitizers (Minnock et al., 2000), unless additional manipulations facilitating membrane transport are used (Nitzan et al., 1992), due to the more complex molecular and physico-chemical structure of their cell wall. PACT is considered to have good perspectives in the control of oral and otherwise localized infections (Meisel & Kocher, 2005; O'Riordan et al., 2005). Local application of liposome-entrapped drugs can prolong their action in infected tissues and provide sustained release of active components (Storm & Crommelin, 1998). It should be mentioned that bacterial resistance to phosphosensitizers has not been reported to date.

Liposome formulations of photosensitizers showed high efficiency in eradication of both Gram-negative and Gram-positive bacteria. Liposome or micelle-entrapped hematoporphyrin and chlorin *e6* were found to be effective against several Gram-positive bacteria, including methicillin-resistant *S. aureus* (Tsai et al., 2009).

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication 483

We have previously shown that Methylene Blue encapsulated in liposomes composed of DPPC or EPC effectively deactivated several Gram-positive and Gram-negative bacteria, including *S. lutea*, *E. coli*, *S. flexneri*, *S. aureus* and MRSA, and that liposomal Rose Bengal

Olive oil-containing liposomes loaded with photosensitizers were tested for their antimicrobial activity under white light illumination against two Gram-positive bacteria of the genus *Staphylococcus* – *S. aureus* and *S. epidermidis*. Although *S. epidermidis* is part of the normal skin flora, it can provoke skin diseases such as folliculitis, and may cause infections of wounded skin, in particular around surgical implants. *S. aureus* is defined as a human opportunistic pathogen and is a causative agent in up to 75% of primary pyodermas, including carbuncle,

*S.epidermidis*

Fig. 9. Eradication of *S. epidermidis* by various concentrations of Rose Bengal (RB) in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at initial

106 cells/mL.

**0 0.01 0.02 0.03 0.04 0.05**

**Rose Bengal concentration, M**

**0 0.1 0.2 0.3 0.4 0.5 0.6**

free RB

free RB

RB in liposomes

RB in liposomes

**a** 

**b** 

**Rose Bengal concentration, M**

*S.epidermidis*

108 cells/mL and (b) 3.

bacteria concentration of (a) 3.

**0**

**0**

**500**

**1000**

**1500**

**CFU/mL**

**2000**

**2500**

**5000**

**10000**

**15000**

**CFU/mL**

**20000**

**25000**

also eradicated *P. aeruginosa* (Nisnevitch et al., 2010; Nakonechny et al., 2010; 2011).

ecthyma, folliculitis, furunculosis, impetigo and others (Maisch et al., 2004).

Fig. 8. Eradication of *S. aureus* by various concentrations of Rose Bengal (RB) in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at initial bacteria concentration of (a) 3. 109 cells/mL and (b) 3. 107 cells/mL.

Encapsulation of photosensitizers into liposomes does not always result in enhancement compared to the free-form cytotoxic activity. The activity of m-tetrahydroxyphenylchlorin in liposomal form was comparable to the free form activity of PACT inactivation of a methicillin-resistant *S. aureus* strain (Bombelli et al., 2008). When tested against methicillinresistant *S. aureus,* chlorophyll *a* was reported to be more efficient in free form than in a liposomal formulation, whereas hematoporphyrin as well as a positively charged PS 5-[4-(1 dodecanoylpyridinium)]-10,15,20-triphenyl-porphyrin were less effective in free form than upon encapsulation in liposomes. These results were explained by differences in photosensitizer chemistry which may influence their association with liposomal components, lipid fluidity and localization in liposome vesicles (Ferro et al., 2006; 2007).

*S.aureus*

**012345**

*S.aureus*

**Rose Bengal concentration, M**

free RB

RB in liposomes

free RB

RB in liposomes

**a** 

**b** 

Fig. 8. Eradication of *S. aureus* by various concentrations of Rose Bengal (RB) in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at initial

**0 0.2 0.4 0.6 0.8 1 1.2**

**Rose Bengal concentration, M**

Encapsulation of photosensitizers into liposomes does not always result in enhancement compared to the free-form cytotoxic activity. The activity of m-tetrahydroxyphenylchlorin in liposomal form was comparable to the free form activity of PACT inactivation of a methicillin-resistant *S. aureus* strain (Bombelli et al., 2008). When tested against methicillinresistant *S. aureus,* chlorophyll *a* was reported to be more efficient in free form than in a liposomal formulation, whereas hematoporphyrin as well as a positively charged PS 5-[4-(1 dodecanoylpyridinium)]-10,15,20-triphenyl-porphyrin were less effective in free form than upon encapsulation in liposomes. These results were explained by differences in photosensitizer chemistry which may influence their association with liposomal components, lipid fluidity and localization in liposome vesicles (Ferro et al., 2006; 2007).

107 cells/mL.

109 cells/mL and (b) 3.

bacteria concentration of (a) 3.

**0**

**50**

**100**

**CFU/mL**

**150**

**CFU/mL**

We have previously shown that Methylene Blue encapsulated in liposomes composed of DPPC or EPC effectively deactivated several Gram-positive and Gram-negative bacteria, including *S. lutea*, *E. coli*, *S. flexneri*, *S. aureus* and MRSA, and that liposomal Rose Bengal also eradicated *P. aeruginosa* (Nisnevitch et al., 2010; Nakonechny et al., 2010; 2011).

Olive oil-containing liposomes loaded with photosensitizers were tested for their antimicrobial activity under white light illumination against two Gram-positive bacteria of the genus *Staphylococcus* – *S. aureus* and *S. epidermidis*. Although *S. epidermidis* is part of the normal skin flora, it can provoke skin diseases such as folliculitis, and may cause infections of wounded skin, in particular around surgical implants. *S. aureus* is defined as a human opportunistic pathogen and is a causative agent in up to 75% of primary pyodermas, including carbuncle, ecthyma, folliculitis, furunculosis, impetigo and others (Maisch et al., 2004).

**Rose Bengal concentration, M**

Fig. 9. Eradication of *S. epidermidis* by various concentrations of Rose Bengal (RB) in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at initial bacteria concentration of (a) 3. 108 cells/mL and (b) 3. 106 cells/mL.

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication 485

*S.aureus*

RB in liposomes

free RB

**6 7 8 9 10**

**log10 CFU(initial)/mL**

**b** 

**a** 

*S.epidermidis*

free RB

RB in liposomes

Fig. 10. Eradication of (a) *S. aureus* by 0.25 M and (b) *S. epidermidis* by 0.01 M Rose Bengal (RB) in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at various initial bacteria concentrations presented in a logarithmic form.

**6789**

**log10 CFU(initial)/mL**

Olive oil-containing liposomes with encapsulated Methylene Blue were tested against *S. epidermidis*. Bacterial sensitivity to this photosensitizer was much lower than to Rose Bengal in both free and liposomal forms. Thus, at the same initial bacterial concentration of 3.

cells/mL, total eradication of *S. epidermidis* by liposomal Rose Bengal was achieved at 0.02 M (Fig. 9b), and by liposomal Methylene Blue only at a concentration of 62.5 M (Fig. 12). As to the general effect of free and liposomal Methylene Blue, it can be said that this photosensitizer exhibits the same trends as Rose Bengal. A liposome-encapsulated form was twice to three times more effective than the free form at all Methylene Blue concentrations

(Fig. 12).

**0**

**5000**

**10000**

**15000**

**CFU/mL**

**20000**

**25000**

**CFU/mL**

106

The water-soluble photosensitizers Rose Bengal and Methylene Blue were encapsulated in the above-described unilamellar liposomes at various concentrations and were examined under white light illumination against various cell concentrations by a viable count method as described previously (Nakonechny et al., 2010) and the number of bacterial colony forming units (CFU) was determined. This number characterized the concentration of bacterial cells which survived after a treatment.

The antimicrobial effect of liposomes incorporated with olive oil and loaded with Rose Bengal was strongly dependent on its concentration (Fig. 8 and 9). As can be seen from Fig. 8a, treatment of *S. aureus* with EPC-based liposomes caused a million-fold suppression of the bacterial cells at 0.25 M of Rose Bengal and total eradication at a concentration of 2 M when tested at an initial cell concentration of 3. 109 cells/mL. Total eradication of *S. aureus* at an initial concentration of 3. 107 cells/mL occurred already at a liposome-encapsulated Rose Bengal concentration of 0.5 M (Fig 8b).

A principal similar trend was observed for *S. epidermidis*. It was necessary to apply liposome-encapsulated Rose Bengal at a concentration of 0.25 M for total eradication of bacteria at an initial concentration of 3. 108 cells/mL (Fig. 9a), and it was enough to apply 0.02 M encapsulated photosensitizer for killing bacteria at 3. 106 cells/mL (Fig. 9b). *S. epidermidis* exhibited a higher sensitivity than *S. aureus* for the liposome formulation of Rose Bengal compared with its free form. For *S. aureus*, liposomal Rose Bengal was only twice as effective as its free form – at each Rose Bengal concentration its liposomal form caused twofold higher suppression of the bacteria. In contradistinction, *S. epidermidis* was suppressed three to twelve times more effectively by Rose Bengal encapsulated in liposomes than by the free photosensitizer.

Bacterial eradicating ability of the encapsulated as well as of the free Rose Bengal was demonstrated to depend on the initial concentration of the bacteria. When tested at the same Rose Bengal concentration, a suppression of both bacteria varied from partial to total. As can be seen from Fig. 10a, a 0.25 M concentration of Rose Bengal encapsulated in EPC-olive oil liposomes caused a decrease of up to 6. 102 cells/mL in the *S. aureus* concentration when taken at an initial concentration of 3. 109 cells/mL (corresponding to 6.7 log10 CFU/mL) and up to zero cell concentration when taken at 3. 107 or 3. 106 cells/mL. In the case of *S. epidermidis,* 0.01M encapsulated Rose Bengal induced bacterial reduction of up to 1.5. 104 cells/mL from the initial concentration of 108 cells/mL, and to the zero concentration at an initial concentration of 3. 106 cells/mL (Fig. 10b).

DPPC-based liposomes were also examined, in addition to EPC-based olive oil-containing liposomes. The results showed high antimicrobial efficiency of the olive oil-containing liposomes in both bases, which was not less than that of the liposomes without olive oil supplements. Fig. 11 relates to the antimicrobial activity of Rose Bengal, applied against *S. epidermidis*, in free form or encapsulated in olive oil-containing ECP- and DPPC-liposomes, as well as to EPC-liposomes without olive oil. The data presented in Fig. 11 indicate that at each initial concentration, all liposomal forms of Rose Bengal eradicated bacteria more effectively than its free form (P-value 0.015), but there was no statistically significant difference in the photosensitizer activity when encapsulated in various types of liposomes (P-value 0.86).

The water-soluble photosensitizers Rose Bengal and Methylene Blue were encapsulated in the above-described unilamellar liposomes at various concentrations and were examined under white light illumination against various cell concentrations by a viable count method as described previously (Nakonechny et al., 2010) and the number of bacterial colony forming units (CFU) was determined. This number characterized the concentration of

The antimicrobial effect of liposomes incorporated with olive oil and loaded with Rose Bengal was strongly dependent on its concentration (Fig. 8 and 9). As can be seen from Fig. 8a, treatment of *S. aureus* with EPC-based liposomes caused a million-fold suppression of the bacterial cells at 0.25 M of Rose Bengal and total eradication at a concentration of 2 M

A principal similar trend was observed for *S. epidermidis*. It was necessary to apply liposome-encapsulated Rose Bengal at a concentration of 0.25 M for total eradication of

*epidermidis* exhibited a higher sensitivity than *S. aureus* for the liposome formulation of Rose Bengal compared with its free form. For *S. aureus*, liposomal Rose Bengal was only twice as effective as its free form – at each Rose Bengal concentration its liposomal form caused twofold higher suppression of the bacteria. In contradistinction, *S. epidermidis* was suppressed three to twelve times more effectively by Rose Bengal encapsulated in liposomes than by the

Bacterial eradicating ability of the encapsulated as well as of the free Rose Bengal was demonstrated to depend on the initial concentration of the bacteria. When tested at the same Rose Bengal concentration, a suppression of both bacteria varied from partial to total. As can be seen from Fig. 10a, a 0.25 M concentration of Rose Bengal encapsulated in EPC-olive oil

*epidermidis,* 0.01M encapsulated Rose Bengal induced bacterial reduction of up to 1.5.

106 cells/mL (Fig. 10b).

cells/mL from the initial concentration of 108 cells/mL, and to the zero concentration at an

DPPC-based liposomes were also examined, in addition to EPC-based olive oil-containing liposomes. The results showed high antimicrobial efficiency of the olive oil-containing liposomes in both bases, which was not less than that of the liposomes without olive oil supplements. Fig. 11 relates to the antimicrobial activity of Rose Bengal, applied against *S. epidermidis*, in free form or encapsulated in olive oil-containing ECP- and DPPC-liposomes, as well as to EPC-liposomes without olive oil. The data presented in Fig. 11 indicate that at each initial concentration, all liposomal forms of Rose Bengal eradicated bacteria more effectively than its free form (P-value 0.015), but there was no statistically significant difference in the photosensitizer activity when encapsulated in various types of liposomes

109 cells/mL. Total eradication of *S. aureus* at

108 cells/mL (Fig. 9a), and it was enough to apply

102 cells/mL in the *S. aureus* concentration when

106 cells/mL. In the case of *S.* 

104

109 cells/mL (corresponding to 6.7 log10 CFU/mL) and

107 or 3.

106 cells/mL (Fig. 9b). *S.* 

107 cells/mL occurred already at a liposome-encapsulated Rose

bacterial cells which survived after a treatment.

when tested at an initial cell concentration of 3.

Bengal concentration of 0.5 M (Fig 8b).

bacteria at an initial concentration of 3.

liposomes caused a decrease of up to 6.

up to zero cell concentration when taken at 3.

taken at an initial concentration of 3.

0.02 M encapsulated photosensitizer for killing bacteria at 3.

an initial concentration of 3.

free photosensitizer.

initial concentration of 3.

(P-value 0.86).

Fig. 10. Eradication of (a) *S. aureus* by 0.25 M and (b) *S. epidermidis* by 0.01 M Rose Bengal (RB) in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at various initial bacteria concentrations presented in a logarithmic form.

Olive oil-containing liposomes with encapsulated Methylene Blue were tested against *S. epidermidis*. Bacterial sensitivity to this photosensitizer was much lower than to Rose Bengal in both free and liposomal forms. Thus, at the same initial bacterial concentration of 3. 106 cells/mL, total eradication of *S. epidermidis* by liposomal Rose Bengal was achieved at 0.02 M (Fig. 9b), and by liposomal Methylene Blue only at a concentration of 62.5 M (Fig. 12). As to the general effect of free and liposomal Methylene Blue, it can be said that this photosensitizer exhibits the same trends as Rose Bengal. A liposome-encapsulated form was twice to three times more effective than the free form at all Methylene Blue concentrations (Fig. 12).

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication 487

emulsion, composed of isotropic mixtures of oil triacylglycerols, surfactant and one or more hydrophilic solvents. The typical particle size of such systems is between 100 and 300 nm (Constantinides, 1995). Another system, called a lipidic self-microemulsifying drug delivery system, represents transparent microemulsions with a particle size of 50-100 nm (Constantinides, 1995; Holm et al., 2003). The described emulsions and microemulsions were based on structural triacylglycerols or sunflower oil. Such systems were proven to appropriately deliver lipophilic drugs such as cyclosporine A, saquinavir, ritonavir and halofantrine (Charman et al., 1992; Holm et al., 2002). A soybean lecithin-based nanoemulsion enriched with triacylglycerols was used for efficient delivery of Amphotericin B (Filippin et al., 2008). An additional example represents solid lipid nanoparticles which were shown to not only deliver glucocorticoids, but also to enhance drug penetration into the skin (Schlupp et al., 2011). Colloid dispersions of solid triacylglycerol 140 nm-sized nanoparticles stabilized with poly(vinyl alcohol) were applied for delivery of the drugs diazepam and ubidecarenone (Rosenblat & Bunje, 2009). Soybean and olive oils were suggested as drug delivery vehicles for the steroids progesterone, estradiol and testosterone (Land et al., 2005). All of the abovementioned examples illustrate successful use of lipid-based systems for delivery of hydrophobic drugs. However, they are all unsuitable for carrying hydrophilic components.

Liposomes are devoid of this serious disadvantage and are applicable for delivery of both hydrophobic and hydrophilic agents. In case of dermal application, lipid-based drug formulations exhibit enhanced abilities to penetrate into skin, improving the delivery process of active agents, thus enabling an increase in treatment efficiency in cases of skin infections and inflammations caused by bacterial invasion. Liposomes were shown to carry the encapsulated hydrophilic agents into the human stratum corneum and possibly into the deeper layers of the skin (Verma et al., 2003). Packaging of drugs into liposomes enables a more concentrated delivery, enhanced cytotoxicity, improved pharmacokinetic qualities,

In this chapter we considered only one type of antimicrobial agents delivered by olive oilcontaining liposomes, but the list of active drugs can be continued and expanded. Incorporation of olive oil into the lipid bilayer increases the biocompatibility of liposomes and enriches them with a broad spectrum of natural bioactive compounds. Integration of olive oil into the liposome lipid bilayer enriches the liposome features by new properties. Such enriched liposomes can not only fulfill a passive role in drug delivery, but can also supply active components for post-treatment recovery of skin. It has been proven that daily treatment with olive oil lowered the risk of dermatitis (Kiechl-Kohlendorfer et al., 2008). Olive oil vitamins and antioxidants could help overcome skin damage caused by skin infection and by the active treatment itself. Olive oil-containing liposomes can thus be converted from passive excipients into active supporting means of drug delivery systems. Totally natural and biocompatible olive oil-containing liposomes carrying any of the antimicrobial agents can be administrated in ointments and creams for application on skin

Olive oil can be incorporated into the liposome phospholipid bilayer, composed of an egg phosphatidylcholine or a dipalmitoyl phosphatidylcholine bilayer. The photosensitizers Rose Bengal and Methylene Blue encapsulated in olive oil-containing liposomes showed

sustained release and prolonged action of active components.

areas contaminated with bacteria.

**6. Conclusions** 

Fig. 11. Eradication of *S. epidermidis* under white light illumination by 0.01M Rose Bengal (RB) in a free form and when encapsulated into liposomes with or without olive oil (O-O) and cholesterol (Chol) at various initial bacteria concentrations presented in a logarithmic form.

Fig. 12. Eradication of *S. epidermidis* by various concentrations of Methylene Blue in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at initial bacteria concentration of 3. 106 cells/mL.

It is important to mention that in no case did olive oil incorporation into the membrane of liposomes with encapsulated photosensitizers cause any decrease in their antimicrobial activity.

#### **5. Perspectives for application of olive oil-containing liposomes**

Several types of drug delivery systems containing lipids for oral, intravenous or dermal administration are described in the literature (Wasan, 2007). One of them is an oil-in-water

free RB

free MB

MB in liposomes

RB/EPC+Chol

RB/EPC+Chol+O-O RB/DPPC+Chol+O-O

Fig. 11. Eradication of *S. epidermidis* under white light illumination by 0.01M Rose Bengal (RB) in a free form and when encapsulated into liposomes with or without olive oil (O-O) and cholesterol (Chol) at various initial bacteria concentrations presented in a logarithmic form.

*S.epidermidis*

**678**

*S. epidermidis*

**log10 CFU(initial)/mL**

Fig. 12. Eradication of *S. epidermidis* by various concentrations of Methylene Blue in a free form and encapsulated into EPC–olive oil liposomes under white light illumination at initial

**0 50 100 150**

**Methylene Blue concentration, M**

It is important to mention that in no case did olive oil incorporation into the membrane of liposomes with encapsulated photosensitizers cause any decrease in their antimicrobial

Several types of drug delivery systems containing lipids for oral, intravenous or dermal administration are described in the literature (Wasan, 2007). One of them is an oil-in-water

106 cells/mL.

**5. Perspectives for application of olive oil-containing liposomes** 

bacteria concentration of 3.

**0**

**CFU/mL**

**1**

**2**

**logCFU/mL**

**3**

**4**

**5**

activity.

emulsion, composed of isotropic mixtures of oil triacylglycerols, surfactant and one or more hydrophilic solvents. The typical particle size of such systems is between 100 and 300 nm (Constantinides, 1995). Another system, called a lipidic self-microemulsifying drug delivery system, represents transparent microemulsions with a particle size of 50-100 nm (Constantinides, 1995; Holm et al., 2003). The described emulsions and microemulsions were based on structural triacylglycerols or sunflower oil. Such systems were proven to appropriately deliver lipophilic drugs such as cyclosporine A, saquinavir, ritonavir and halofantrine (Charman et al., 1992; Holm et al., 2002). A soybean lecithin-based nanoemulsion enriched with triacylglycerols was used for efficient delivery of Amphotericin B (Filippin et al., 2008). An additional example represents solid lipid nanoparticles which were shown to not only deliver glucocorticoids, but also to enhance drug penetration into the skin (Schlupp et al., 2011). Colloid dispersions of solid triacylglycerol 140 nm-sized nanoparticles stabilized with poly(vinyl alcohol) were applied for delivery of the drugs diazepam and ubidecarenone (Rosenblat & Bunje, 2009). Soybean and olive oils were suggested as drug delivery vehicles for the steroids progesterone, estradiol and testosterone (Land et al., 2005). All of the abovementioned examples illustrate successful use of lipid-based systems for delivery of hydrophobic drugs. However, they are all unsuitable for carrying hydrophilic components.

Liposomes are devoid of this serious disadvantage and are applicable for delivery of both hydrophobic and hydrophilic agents. In case of dermal application, lipid-based drug formulations exhibit enhanced abilities to penetrate into skin, improving the delivery process of active agents, thus enabling an increase in treatment efficiency in cases of skin infections and inflammations caused by bacterial invasion. Liposomes were shown to carry the encapsulated hydrophilic agents into the human stratum corneum and possibly into the deeper layers of the skin (Verma et al., 2003). Packaging of drugs into liposomes enables a more concentrated delivery, enhanced cytotoxicity, improved pharmacokinetic qualities, sustained release and prolonged action of active components.

In this chapter we considered only one type of antimicrobial agents delivered by olive oilcontaining liposomes, but the list of active drugs can be continued and expanded. Incorporation of olive oil into the lipid bilayer increases the biocompatibility of liposomes and enriches them with a broad spectrum of natural bioactive compounds. Integration of olive oil into the liposome lipid bilayer enriches the liposome features by new properties. Such enriched liposomes can not only fulfill a passive role in drug delivery, but can also supply active components for post-treatment recovery of skin. It has been proven that daily treatment with olive oil lowered the risk of dermatitis (Kiechl-Kohlendorfer et al., 2008). Olive oil vitamins and antioxidants could help overcome skin damage caused by skin infection and by the active treatment itself. Olive oil-containing liposomes can thus be converted from passive excipients into active supporting means of drug delivery systems. Totally natural and biocompatible olive oil-containing liposomes carrying any of the antimicrobial agents can be administrated in ointments and creams for application on skin areas contaminated with bacteria.
