**4.2 Transport of Perifosine (OPP) into the cell**

To get information about the transport of Perifosine (OPP) into breast cancer cells by EPR, several spin labeled OPPs were synthesized in our group (Mravljak et al., 2005), which have an EPR detectable nitroxide group at various positions along the alkyl chain. We have chosen spin labeled OPP (5P) (structural formula in Fig. 3) with the lowest critical micellar concentration (CMC) of all synthesized spin labeled OPPs (Mravljak et al., 2005). Its CMC is around 10 μM, while 14P (spin labeled OPP, containing the nitroxide group at the 14th C atom) exhibited the CMC of around 200 μM (Mravljak et al., 2005). Edelfosine and OPP disperse in water in the form of micelles, due to their inverted-cone shape (Busto et al., 2007), displaying a CMC at 2.5 - 3 μM (Rakotomanga et al., 2004). It appears that addition of the doxyl group to OPP distorts the inverted-cone shape of molecule to greater extent when it is placed further away from the polar head group, which results in increased CMC. Since the CMC of 5P is similar to CMC of other similar alkylphospholipids one can assume that

Interaction of Alkylphospholipid Formulations with Breast Cancer

cells (Podlipec et al., manuscript in preparation).

formulations with a lower CH content (Zeisig et al., 1998).

formulations increases with decreasing amount of cholesterol (Table 2).

**interaction with breast cancer cells** 

Cells in the Context of Anticancer Drug Development 369

described previously (Koklic et al., 2008). The rate of nitroxide reduction inside the cells does not differ significantly between the two cell lines and is faster at physiological temperature as at room temperature, while by increasing the temperature from room to physiological temperature the rate of transfer remains in the range of error for MT-3 cells (estrogen receptor negative), but increases significantly for estrogen receptor positive MCF7

**5. Interaction of liposomal Perifosine (OPP) with breast cancer cells** 

**5.1 Effect of supramolecular organization of liposomal OPP formulations on their** 

Alkylphospholipids are amphiphilic molecules and usually form micelles under physiological conditions. Unfortunately, administration of free (micellar) alkylphospholipids results in unwanted side effects, reflected in gastrointestinal toxicity and hemolytic activity, which limits the application of higher doses of alkylphospholipids. To achieve better therapeutic effects of alkylphospholipids *in vivo* with less side effects, different liposomal formulations of alkylphospholipids were prepared. This is possible only in the presence of lipids or other amphiphiles with a complimentary molecular shape. Usually cholesterol fulfills this role and enables the preparation of stable liposomal formulations from alkylphospholipids and lipids of different chain length and head groups. Among different alkylphospholipids, most investigations with liposomal formulations were performed with Perifosine (OPP). *In vivo* data show that the hemolytic effect of OPP is significantly diminished in liposomal formulations, but unfortunately in most cases, cytotoxic activity of OPP liposomes was also lower than of free OPP (Zeisig et al., 1998). In an early study (Zeisig et al., 2001) we investigated the influence of cholesterol in liposomes consisting of Perifosine (OPP), dicetylphosphate and cholesterol (CH) on liposome stability and in vitro cytotoxicity. It was found that the ratio between the alkylphospholipid and cholesterol affects the cytotoxicity of the liposomes (Table 2). An increase in the OPP/CH ratio correlated directly with an increase in cytotoxicity against breast cancer cells. In the same time it was shown that a portion of 10 – 30% of OPP was present as micelles in liposomal formulations with OPP/CH ratio between 10:10 and 10:5, while the remaining OPP was stabilised by CH and forms liposomes. This was concluded, using 1H-NMR spectroscopy, by the analysis of lipid composition after centrifugation of liposomal formulations, where micelles remain in supernatant in comparison to the initial sample. This micellar part of OPP molecules can easily be exchanged with the external environment and is able to become incorporated into other (bi)layers, as monolayer incorporation experiments demonstrated. It was assumed that this part of OPP is also mainly responsible for the cytotoxicity against tumor cells, which are not able to internalize the vesicles very well (Zeisig et al., 2001). A similar composition dependent effect was found *in vivo*, when the hemolytic effect of differently composed liposomes was followed. Again, liposomes with higher OPP/CH ratio, and thus containing a higher proportion of micellar OPP, were more hemolytically active than liposomal OPP

Recently we developed a new method achieving more accurate estimates of the relative proportion of micelles, in comparison to the previously used methods (Koklic et al., 2010). The method is based on the spectral decomposition of EPR spectra. We confirmed findings of previous studies, which showed that the amount of micelles in liposomal OPP

the disturbance caused by attaching doxyl group to OPP is small. Therefore we asumed that from all of synthesized spin labeled OPPs, 5P is the best candidate as a model molecule for studying behavior of Perifosine (OPP).

When 5P is transported into the cell, the EPR spectra intensity decreases due to the reduction of the nitroxide group to the corresponding EPR non-visible hydroxylamine by oxy-redoxy systems inside the cells (Chen et al., 1988; Swartz et al., 1986; Ueda et al., 2003) and can be detected by measuring the amplitude of the middle line of EPR spectra with time. From the kinetics of EPR spectra intensity decrease, information about the transport and/or interaction of spin probe with cells can be obtained. Reduction kinetics of 5P was found to be much slower as for spin probes usually used in EPR investigations of cell membranes MeFASL(10,3) and HFASL(10,3) (Chen et al., 1988; Yonar et al., 2010) indicating that its transport into the cell cytoplasm and organelles is slower as for the other probes. This is not surprising due to the charge at the head group of 5P (Fig. 3), which prevents passive transport of OPP across the membrane. For human KB carcinoma cells it has been demonstrated that OPP is internalized by an ATP-dependent translocase activity across the plasma membrane (Munoz-Martinez et al., 2008).

In order to investigate whether there is a difference in the uptake of OPP by alkylphospholipid (APL) resistant MCF7 cells (estrogen receptor positive, ER+) and APL sensitive MT-3 cells (estrogen receptor negative, ER-), both cell lines were incubated with 5P and EPR spectra intensity decrease was measured with time after incubation (Fig. 4).

Fig. 4. EPR spectra intensity decrease with time after incubation of MCF7 cells (●), and MT-3 (○) cells with 5P at A) room temperature and B) 37 °C MCF7 (estrogen receptor positive) and MT-3 (estrogen receptor negative) breast cancer cells (5-7 x 106 MCF7 cells and 12-20 x 106 MT-3 cells) were incubated with 5P (2-3 µM concentration, depending on the estimated number of total cell membrane bilayer lipids), which was adsorbed to the wall of a glass tube in order to achieve gradual accumulation of OPP in cells during 10 min incubation at room temperature and EPR spectra intensity decrease was measured with time after incubation.

From the kinetics of EPR spectra intensity decrease (Fig. 4) the rate of transfer of spin labeled OPP (5P) across the cell membrane was calculated using a similar model as

the disturbance caused by attaching doxyl group to OPP is small. Therefore we asumed that from all of synthesized spin labeled OPPs, 5P is the best candidate as a model molecule for

When 5P is transported into the cell, the EPR spectra intensity decreases due to the reduction of the nitroxide group to the corresponding EPR non-visible hydroxylamine by oxy-redoxy systems inside the cells (Chen et al., 1988; Swartz et al., 1986; Ueda et al., 2003) and can be detected by measuring the amplitude of the middle line of EPR spectra with time. From the kinetics of EPR spectra intensity decrease, information about the transport and/or interaction of spin probe with cells can be obtained. Reduction kinetics of 5P was found to be much slower as for spin probes usually used in EPR investigations of cell membranes MeFASL(10,3) and HFASL(10,3) (Chen et al., 1988; Yonar et al., 2010) indicating that its transport into the cell cytoplasm and organelles is slower as for the other probes. This is not surprising due to the charge at the head group of 5P (Fig. 3), which prevents passive transport of OPP across the membrane. For human KB carcinoma cells it has been demonstrated that OPP is internalized by an ATP-dependent translocase activity across the

In order to investigate whether there is a difference in the uptake of OPP by alkylphospholipid (APL) resistant MCF7 cells (estrogen receptor positive, ER+) and APL sensitive MT-3 cells (estrogen receptor negative, ER-), both cell lines were incubated with 5P

Fig. 4. EPR spectra intensity decrease with time after incubation of MCF7 cells (●), and MT-3 (○) cells with 5P at A) room temperature and B) 37 °C MCF7 (estrogen receptor positive) and MT-3 (estrogen receptor negative) breast cancer cells (5-7 x 106 MCF7 cells and 12-20 x 106 MT-3 cells) were incubated with 5P (2-3 µM concentration, depending on the estimated number of total cell membrane bilayer lipids), which was adsorbed to the wall of a glass tube in order to achieve gradual accumulation of OPP in cells during 10 min incubation at room temperature and EPR spectra intensity decrease was measured with time after

From the kinetics of EPR spectra intensity decrease (Fig. 4) the rate of transfer of spin labeled OPP (5P) across the cell membrane was calculated using a similar model as

**● MCF7 ○ MT-3**

and EPR spectra intensity decrease was measured with time after incubation (Fig. 4).

studying behavior of Perifosine (OPP).

plasma membrane (Munoz-Martinez et al., 2008).

**A B**

incubation.

described previously (Koklic et al., 2008). The rate of nitroxide reduction inside the cells does not differ significantly between the two cell lines and is faster at physiological temperature as at room temperature, while by increasing the temperature from room to physiological temperature the rate of transfer remains in the range of error for MT-3 cells (estrogen receptor negative), but increases significantly for estrogen receptor positive MCF7 cells (Podlipec et al., manuscript in preparation).
