IC50 concentration (M) required for 50% inhibition of cell growth for MT-3 breast cancer

Table 2. Composition of OPP liposomes, relative portion of micelles obtained by EPR and

For a better understanding of the interaction between liposomal OPP formulations and tumor cells, a deeper understanding of liposomal bilayer organization is necessary. Therefore, EPR with spin labels was used to study the influence of cholesterol, charge and sterical stabilization by PEG2000 DSPE on physical and chemical characteristics of liposomal OPP formulations. For this purpose liposomes with the composition presented in Table 2 were spin labeled with 5 doxylpalmitoyl methyl ester (MeFASL(10,3), Fig. 3) and EPR spectra were measured. By computer simulation of the EPR spectra line-shape, information about membrane fluidity and membrane domain structure was obtained, taking into account that the membrane is heterogeneous, composed of regions with different fluidity characteristics (Koklic et al., 2002;

It was found that in general the experimental spectra are composed of at least three spectral components. Each spectral component corresponds to a mode of motion of a portion of spin probes partitioned in different parts of the membrane with the same physical properties and characterizes a certain type of lateral membrane domains with different fluidity characteristics. EPR parameters (order parameter S, rotational correlation time τc , polarity correction factor pA), which describe the motional modes of nitroxide in a certain domain

membranes and their interaction with cells.

N and P denote charge of the formulation (- or +, respectively)

(dimethylioctadecylammonium bromide) for P formulation)

phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000])

**Code Molar ratio** 

\* mol% of total lipids

cells, (Zeisig et al., 1998)

\*\* reference (Koklic et al., 2010)

their hemolytic and cytotoxic activity.

Fig. 5. A) EPR spectra of lipophilic spin-probe methyl ester of 5-doxylpalmitate (MeFASL(10,3)) in the membrane of OPP liposomes with different concentrations of cholesterol (the amount of cholesterol is indicated in mol%) in PBS buffer at 39 °C. The arrow points to a peak, which vanishes at around 50 mol% of cholesterol in the liposome membrane. B ) - E) Dependence of EPR spectral parameters of spectral components on cholesterol concentration ([cholesterol]). Spectral parameters of EPR spectra of spin-probe MeFASL(10,3) in membranes of liposomal OPP formulations were derived by fitting of the calculated to the experimental spectra. B) Relative proportions of spectral components with: the lowest order parameter – domain type 1 (●); middle order parameter – domain type 2 (◊); and the highest order parameter – domain type 3 (□). Solid black line is a linear fit to the relative proportion of domain type 1, C) Order parameter, D) rotational correlation time, and E) polarity correction factor of the less ordered domain type (domain type 1). (republished with permission from (Koklic et al., 2008)).

type and reflect the fluidity characteristics of the domains as well as the proportion of spin probes in each domain type were determined. They were found to depend mainly on the amount of cholesterol, and only to a minor part on charge and sterical stabilization (Koklic

Interaction of Alkylphospholipid Formulations with Breast Cancer

flasks (Batista et al., 2010).

**A) N5 (29% CH) + MCF7**

**B) N15 (56% CH) + MCF7**

as indicated at the bottom of each image.

Cells in the Context of Anticancer Drug Development 373

For liposomal OPP formulation with low cholesterol content N5 (circles) a fast decrease of the EPR signal was observed in first 10 minutes after mixing liposomes with cells (Fig. 6), indicating that about 30% of spin-probes were released fast from the liposome interior into the cell cytoplasm. On the other hand, for liposomal OPP formulation with high cholesterol content N15 (Fig. 6 sguares), only a very small amount of liposome entrapped ASL was released into the cells, since the intensity decrease was less than 10% at room and physiological temperature. This indicates that the liposomes remained intact either in the extracellular space or entered the cells by endocythosis, but remained intact at least for the time of measurement. It is important to note that at room temperature both cell lines behave similarly, while at physiological temperature significantly higher amount of liposomes with low CH (N5) interact with alkylphospholipid sensitive, estrogen receptor negative, MT-3 cells (open circles in Fig. 6B) than with alkylphospholipid resistant, estrogen receptor positive, MCF7 cells (Podlipec et al., manuscript in preparation). These results, obtained on trypsinated cells, which are presented here, agree well with the results published by Koklic et al. (Koklic et al., 2008), which were obtained on scraped MT-3 cells, although small differences could originate from different procedures of removal of cells from the culture

In order to investigate interaction of OPP liposomes with breast cancer cells in more detail, we have added 0.5 mol% of a phospholipid fluorescent probe C6-NBD-PC, where 7 nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) is attached to the phosphatidylcholine phospholipid (16:0-06:0 NBD PC, Avanti polar lipids, Alabaster, AL, USA) to OPP liposomes as described previously (Arsov et al., 2011). Results with the lipophilic fluorescence probe C6-NBD-PC (Fig. 7) confirm the EPR measurements, indicating that liposomes with high amount of

Fig. 7. Interaction of fluorescently labeled Perifosine (OPP) liposomal formulations with MCF7 breast cancer cells. Fluorescence microscopy was performed to localize C6-NBD-PC labeled liposomal formulation immediately after addition to cells, at room temperature. A) Negatively charged liposomal OPP formulation (N5) with 29 mol % of cholesterol (1 mM final total lipid concentration) were added to MCF7 cells attached to the bottom of a well and the distribution of the lipophilic fluorescent probe C6-NBD-PC was followed with time

B) Negatively charged liposomal OPP formulation (N15) with 56 mol % cholesterol were

added to MCF7 cells and measured under same conditions as in experiment A.

et al., 2002). Dependance of EPR parameters, reflecting the properties of the least ordered domain type on cholesterol concentration is presented in Fig. 5 B, C, and D. A sudden increase in order parameter and rotational correlation time was observed when cholesterol concentration increases from 45 mol% to 50 mol%, while at the same time the polarity correction factor decreased, indicating that the spin probes in the domain type with lowest order parameter are less accessible to water. Relative proportion of this domain type (Fig. 5B) decreases at higher cholesterol concentrations, whereas the relative proportion of the domain type with the highest order parameter increases. It seems that above 50 mol% cholesterol the least ordered domains are transformed into a new type of domains with higher order parameter (S = 0.15) and with proportion of 15 %.

#### **5.3 Release of liposome encapsulated material during the interaction of Perifosine (OPP) liposomal formulations with breast cancer cells**

In order to better understand the factors that determine the therapeutic activity of liposomal OPP formulations, the interaction of liposomal OPP formulations at different cholesterol/Perifosine (CH/OPP) ratios with MT-3 and MCF7 breast cancer cells was measured and correlated with the membrane domain structure of liposomal OPP formulations (Koklic et al., 2008). For this purpose, spin labeled tempocholine (ASL) (Fig. 3), which cannot penetrate an intact liposome membrane easily, was entrapped into the liposomes. Labeled liposomal formulations were mixed with the cells and the kinetics of ASL reduction in the presence of human breast cancer cells was measured by EPR. ASL gets reduced to EPR non-visible hydroxylamine when it is released from liposomes and exposed to the oxy-redoxy systems inside the cells (Chen et al., 1988; Swartz et al., 1986; Ueda et al., 2003), which is reflected in an EPR spectra intensity decrease. Therefore, from the kinetics of EPR spectra intensity decrease information about the interaction of liposomes with cells can be obtained. Results are presented in Fig. 6.

Fig. 6. EPR spectra intensity decrease after mixing of MCF7 cells (closed signs) or MT-3 cells (open signs) with Perifosine (OPP) liposomal formulations with two concentrations of cholesterol: 29 mol% (N5) (circles) and 56 mol% (N15) (squares) at A) room temperature and B) 37 oC. Symbols represent mean values of two to three measurements with error bars representing standard deviations.

et al., 2002). Dependance of EPR parameters, reflecting the properties of the least ordered domain type on cholesterol concentration is presented in Fig. 5 B, C, and D. A sudden increase in order parameter and rotational correlation time was observed when cholesterol concentration increases from 45 mol% to 50 mol%, while at the same time the polarity correction factor decreased, indicating that the spin probes in the domain type with lowest order parameter are less accessible to water. Relative proportion of this domain type (Fig. 5B) decreases at higher cholesterol concentrations, whereas the relative proportion of the domain type with the highest order parameter increases. It seems that above 50 mol% cholesterol the least ordered domains are transformed into a new type of domains with

**5.3 Release of liposome encapsulated material during the interaction of Perifosine** 

In order to better understand the factors that determine the therapeutic activity of liposomal OPP formulations, the interaction of liposomal OPP formulations at different cholesterol/Perifosine (CH/OPP) ratios with MT-3 and MCF7 breast cancer cells was measured and correlated with the membrane domain structure of liposomal OPP formulations (Koklic et al., 2008). For this purpose, spin labeled tempocholine (ASL) (Fig. 3), which cannot penetrate an intact liposome membrane easily, was entrapped into the liposomes. Labeled liposomal formulations were mixed with the cells and the kinetics of ASL reduction in the presence of human breast cancer cells was measured by EPR. ASL gets reduced to EPR non-visible hydroxylamine when it is released from liposomes and exposed to the oxy-redoxy systems inside the cells (Chen et al., 1988; Swartz et al., 1986; Ueda et al., 2003), which is reflected in an EPR spectra intensity decrease. Therefore, from the kinetics of EPR spectra intensity decrease information about the interaction of liposomes with cells can

higher order parameter (S = 0.15) and with proportion of 15 %.

**(OPP) liposomal formulations with breast cancer cells** 

be obtained. Results are presented in Fig. 6.

**● MCF7 + N5 ■ MCF7 + N15**

representing standard deviations.

**A B**

**○ MT-3 + N5 □ MT-3 + N15**

Fig. 6. EPR spectra intensity decrease after mixing of MCF7 cells (closed signs) or MT-3 cells (open signs) with Perifosine (OPP) liposomal formulations with two concentrations of cholesterol: 29 mol% (N5) (circles) and 56 mol% (N15) (squares) at A) room temperature and B) 37 oC. Symbols represent mean values of two to three measurements with error bars

For liposomal OPP formulation with low cholesterol content N5 (circles) a fast decrease of the EPR signal was observed in first 10 minutes after mixing liposomes with cells (Fig. 6), indicating that about 30% of spin-probes were released fast from the liposome interior into the cell cytoplasm. On the other hand, for liposomal OPP formulation with high cholesterol content N15 (Fig. 6 sguares), only a very small amount of liposome entrapped ASL was released into the cells, since the intensity decrease was less than 10% at room and physiological temperature. This indicates that the liposomes remained intact either in the extracellular space or entered the cells by endocythosis, but remained intact at least for the time of measurement. It is important to note that at room temperature both cell lines behave similarly, while at physiological temperature significantly higher amount of liposomes with low CH (N5) interact with alkylphospholipid sensitive, estrogen receptor negative, MT-3 cells (open circles in Fig. 6B) than with alkylphospholipid resistant, estrogen receptor positive, MCF7 cells (Podlipec et al., manuscript in preparation). These results, obtained on trypsinated cells, which are presented here, agree well with the results published by Koklic et al. (Koklic et al., 2008), which were obtained on scraped MT-3 cells, although small differences could originate from different procedures of removal of cells from the culture flasks (Batista et al., 2010).

In order to investigate interaction of OPP liposomes with breast cancer cells in more detail, we have added 0.5 mol% of a phospholipid fluorescent probe C6-NBD-PC, where 7 nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) is attached to the phosphatidylcholine phospholipid (16:0-06:0 NBD PC, Avanti polar lipids, Alabaster, AL, USA) to OPP liposomes as described previously (Arsov et al., 2011). Results with the lipophilic fluorescence probe C6-NBD-PC (Fig. 7) confirm the EPR measurements, indicating that liposomes with high amount of

Fig. 7. Interaction of fluorescently labeled Perifosine (OPP) liposomal formulations with MCF7 breast cancer cells. Fluorescence microscopy was performed to localize C6-NBD-PC labeled liposomal formulation immediately after addition to cells, at room temperature. A) Negatively charged liposomal OPP formulation (N5) with 29 mol % of cholesterol (1 mM final total lipid concentration) were added to MCF7 cells attached to the bottom of a well and the distribution of the lipophilic fluorescent probe C6-NBD-PC was followed with time as indicated at the bottom of each image.

B) Negatively charged liposomal OPP formulation (N15) with 56 mol % cholesterol were added to MCF7 cells and measured under same conditions as in experiment A.

Interaction of Alkylphospholipid Formulations with Breast Cancer

**A B**

N5 (29%)

N15 (56%)

A) in the membrane of liposomal OPP formulation with different concentrations of cholesterol (the amount of cholesterol is indicated in mol%) in PBS buffer at room temperature. The arrows point to peaks corresponding to free 5P, which is neither incorporated in liposomes, neither in micelles. A broad spectrum corresponds to

B) in the membrane of MCF7 cells. Spectra were recorded 2 minutes after mixing of spin labeled OPP liposomes (1.5 µL) with pellet of MCF7 cells (1.5 µL with 5 -10 x 106 cells).

At first glance these results are in stark contrast with the experiments with fluorescently labeled liposomal OPP formulations (Fig. 7) and with OPP liposomes with the entrapped hydrophilic probe (Fig. 6), since those experiments suggest that OPP liposomes with high amount of cholesterol almost do not interact with breast cancer cells. Fast transfer of OPP from liposomes to other membranes would lead to destabilization of liposomes, which was not the case for N15 liposomes with high amount of CH. It seems that OPP differs from spin labeled OPP (5P), which is not surprising with respect to the doxyl group attached to the

**6. Summary of differences between MT-3 and MCF7 breast cancer cell lines** 

1. Plasma membrane fluidity is slightly larger for estrogen receptor negative (ER-) MT-3

Main differences between OPP sensitive MT-3 and OPP resistant MCF7 cells are:

1 mT 1 mT

Fig. 8. EPR spectra of lipophilic spin-probe – spin labeled OPP (5P)

alkyl chain, which probably prevents the condensing effect of cholesterol.

supramolecular structures of OPP (liposomes and micelles);

as for estrogen receptor positive (ER+) MCF7.

of liposomes seems to be similar.

Cells in the Context of Anticancer Drug Development 375

condensation is reached at a ratio of HePC/sterol around 50:50 (mol/mol) (Rakotomanga et al., 2004). This kind of behavior is generally known as the condensing effect of cholesterol towards phospholipids (Chapman et al., 1969; Ghosh & Tinoco, 1972). Micelles constituted a reservoir of monomers both for monomer insertion between condensed phospholipids and for insertion of groups of monomers between fluid phospholipids. Since biological membranes are composed of dynamically condensed domains surrounded by fluid domains, it has been suggested that, above the CMC, alkylphospholipids can insert into both kinds of phases: as monomers into the condensed phase and as a group of monomers into the fluid phase. (Rakotomanga et al., 2005). The presence of albumin in the medium has the effect of increasing the CMC value by binding molecules of lipids and, hence, reducing the concentration of free monomers in the medium (Kim et al., 2007). Like albumin acts as an alkylphospholipid reservoir - it binds reversibly to the cell surface and may release the drug gradually - the role

N5 (29%)+MCF7

N15 (56%)+MCF7

cholesterol do not interact with cells. Fluorescence microscopy clearly shows that OPP liposomes with high amount of CH (N15) remain outside the cells, while low cholesterol – containing, N5 liposomes interact with cell membranes because the fluorescent probe distributes in the cell interior. In addition for liposomes with low amount of cholesterol (N5), for which C6-NBD-PC distributes inside the cells, maximum of fluorescence emission spectrum shifts for a few nanometers, indicating that either micelles or liposomes interacted with cells and delivered C6-NBD-PC into lipophilic compartments of MCF7 cells, where the environment of the fluorescent probe changed (Arsov et al., 2011).

Comparing liposome membrane characteristics, derived from EPR spectra (Fig. 5) and summarized in Table 2, with liposome cell interaction experiments (Fig. 6 and Fig. 7) we can see that the propensity of Perifosine (OPP) liposomal formulations for interaction with tumor cells and for delivery of OPP into cells coincides with the existence of disordered domains as well as with the existence of micelles. We have shown that by increasing the concentration of cholesterol above 50 mol% the domains with the lowest order parameter (between 0.06 and 0.03, Fig. 5) are transformed into a new type of domains with higher order parameter (S = 0.15). This suggests that disordered motion of lipid alkyl chains within the liquid-disordered domains, which coexist with liquid-ordered domains, is necessary for fast delivery of liposome encapsulated probe into the cells. On the other hand, the presence of micelles in liposome formulations with concentration of cholesterol below approximately 50 mol% suggests that micelles are necessary for efficient delivery of liposome encapsulated probe into cells.

#### **5.4 Transport of spin labeled Perifosine (OPP) from liposomes to cell membrane does not depend on the liposome cell interaction**

In order to get information about the rate of transfer of OPP from liposomes containing two different concentrations of cholesterol to cells, a portion of OPP molecules in liposomal OPP formulations was replaced with spin labeled OPP (5P), so that the final concentration of 5P in OPP liposomes was 17 mol%. Because of such high amount of P5 in the liposomal formulations, the EPR lines are highly broadened (Fig. 8A) due to the spin exchange interaction between paramagnetic probes.

EPR spectra in Fig. 8B are similar as obtained for MCF7 cells labeled with 5P, indicating that high amount of 5P from liposomal OPP formulation was transferred to cell membranes in a time shorter than 2 minutes. It was not possible to resolve any difference in the rate of transfer from N5 or N15 liposomes after 2 minutes of mixing liposomes with cells. Very fast transfer was also observed when giant liposomes (composition: POPC:POPE:POPS:CH molar ratios 40:20:10:40), which represent a model for cell membrane, were incubated with N5 or N15 liposomes (Mravljak et al., 2010), proving that spin labeled OPP molecules are transferred from one type of membrane to other membranes within several minutes, and the rate of transport does not depend significantly on the membrane composition. We can conclude from the above experiment that analkylphospholipid-like molecule can easily exchange between membranes and can accumulate in cells when they are in contact with liposomal OPP formulations. This is in agreement with lipid monolayer experiments, which showed that alkylphospholipids, below the critical micellar concentration (CMC), insert progressively into lipid monolayers as monomers from the aqueous medium, while above CMC, not only monomers but also groups of monomers (micelles) are transferred into the monolayers (Rakotomanga et al., 2004). It was also shown that, while the alkylphospholipid HePC is miscible with POPC, there is high affinity between HePC and sterols (ergosterol, and cholesterol) and that maximum

cholesterol do not interact with cells. Fluorescence microscopy clearly shows that OPP liposomes with high amount of CH (N15) remain outside the cells, while low cholesterol – containing, N5 liposomes interact with cell membranes because the fluorescent probe distributes in the cell interior. In addition for liposomes with low amount of cholesterol (N5), for which C6-NBD-PC distributes inside the cells, maximum of fluorescence emission spectrum shifts for a few nanometers, indicating that either micelles or liposomes interacted with cells and delivered C6-NBD-PC into lipophilic compartments of MCF7 cells, where the

Comparing liposome membrane characteristics, derived from EPR spectra (Fig. 5) and summarized in Table 2, with liposome cell interaction experiments (Fig. 6 and Fig. 7) we can see that the propensity of Perifosine (OPP) liposomal formulations for interaction with tumor cells and for delivery of OPP into cells coincides with the existence of disordered domains as well as with the existence of micelles. We have shown that by increasing the concentration of cholesterol above 50 mol% the domains with the lowest order parameter (between 0.06 and 0.03, Fig. 5) are transformed into a new type of domains with higher order parameter (S = 0.15). This suggests that disordered motion of lipid alkyl chains within the liquid-disordered domains, which coexist with liquid-ordered domains, is necessary for fast delivery of liposome encapsulated probe into the cells. On the other hand, the presence of micelles in liposome formulations with concentration of cholesterol below approximately 50 mol% suggests that micelles are necessary for efficient delivery of liposome encapsulated

**5.4 Transport of spin labeled Perifosine (OPP) from liposomes to cell membrane does** 

In order to get information about the rate of transfer of OPP from liposomes containing two different concentrations of cholesterol to cells, a portion of OPP molecules in liposomal OPP formulations was replaced with spin labeled OPP (5P), so that the final concentration of 5P in OPP liposomes was 17 mol%. Because of such high amount of P5 in the liposomal formulations, the EPR lines are highly broadened (Fig. 8A) due to the spin exchange

EPR spectra in Fig. 8B are similar as obtained for MCF7 cells labeled with 5P, indicating that high amount of 5P from liposomal OPP formulation was transferred to cell membranes in a time shorter than 2 minutes. It was not possible to resolve any difference in the rate of transfer from N5 or N15 liposomes after 2 minutes of mixing liposomes with cells. Very fast transfer was also observed when giant liposomes (composition: POPC:POPE:POPS:CH molar ratios 40:20:10:40), which represent a model for cell membrane, were incubated with N5 or N15 liposomes (Mravljak et al., 2010), proving that spin labeled OPP molecules are transferred from one type of membrane to other membranes within several minutes, and the rate of transport does not depend significantly on the membrane composition. We can conclude from the above experiment that analkylphospholipid-like molecule can easily exchange between membranes and can accumulate in cells when they are in contact with liposomal OPP formulations. This is in agreement with lipid monolayer experiments, which showed that alkylphospholipids, below the critical micellar concentration (CMC), insert progressively into lipid monolayers as monomers from the aqueous medium, while above CMC, not only monomers but also groups of monomers (micelles) are transferred into the monolayers (Rakotomanga et al., 2004). It was also shown that, while the alkylphospholipid HePC is miscible with POPC, there is high affinity between HePC and sterols (ergosterol, and cholesterol) and that maximum

environment of the fluorescent probe changed (Arsov et al., 2011).

probe into cells.

**not depend on the liposome cell interaction** 

interaction between paramagnetic probes.

condensation is reached at a ratio of HePC/sterol around 50:50 (mol/mol) (Rakotomanga et al., 2004). This kind of behavior is generally known as the condensing effect of cholesterol towards phospholipids (Chapman et al., 1969; Ghosh & Tinoco, 1972). Micelles constituted a reservoir of monomers both for monomer insertion between condensed phospholipids and for insertion of groups of monomers between fluid phospholipids. Since biological membranes are composed of dynamically condensed domains surrounded by fluid domains, it has been suggested that, above the CMC, alkylphospholipids can insert into both kinds of phases: as monomers into the condensed phase and as a group of monomers into the fluid phase. (Rakotomanga et al., 2005). The presence of albumin in the medium has the effect of increasing the CMC value by binding molecules of lipids and, hence, reducing the concentration of free monomers in the medium (Kim et al., 2007). Like albumin acts as an alkylphospholipid reservoir - it binds reversibly to the cell surface and may release the drug gradually - the role of liposomes seems to be similar.

Fig. 8. EPR spectra of lipophilic spin-probe – spin labeled OPP (5P)

A) in the membrane of liposomal OPP formulation with different concentrations of cholesterol (the amount of cholesterol is indicated in mol%) in PBS buffer at room temperature. The arrows point to peaks corresponding to free 5P, which is neither incorporated in liposomes, neither in micelles. A broad spectrum corresponds to supramolecular structures of OPP (liposomes and micelles);

B) in the membrane of MCF7 cells. Spectra were recorded 2 minutes after mixing of spin labeled OPP liposomes (1.5 µL) with pellet of MCF7 cells (1.5 µL with 5 -10 x 106 cells).

At first glance these results are in stark contrast with the experiments with fluorescently labeled liposomal OPP formulations (Fig. 7) and with OPP liposomes with the entrapped hydrophilic probe (Fig. 6), since those experiments suggest that OPP liposomes with high amount of cholesterol almost do not interact with breast cancer cells. Fast transfer of OPP from liposomes to other membranes would lead to destabilization of liposomes, which was not the case for N15 liposomes with high amount of CH. It seems that OPP differs from spin labeled OPP (5P), which is not surprising with respect to the doxyl group attached to the alkyl chain, which probably prevents the condensing effect of cholesterol.
