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

### **5.1 Effect of supramolecular organization of liposomal OPP formulations on their interaction with breast cancer cells**

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

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

Interaction of Alkylphospholipid Formulations with Breast Cancer

**A**

Cells in the Context of Anticancer Drug Development 371

**order parameter (1)**

**[CH]=29 mol%**

**[CH]=45 mol%**

**[CH]=50 mol%**

**[CH]=56 mol%**

**1 mT**

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).

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

(republished with permission from (Koklic et al., 2008)).

**relative domain**

 **proportion (%)**

**rotational correlation**

 **time (ns)**

**polarity correction (1)**

**0.04 0.08 0.12 0.16**

**A**

**B**

**B**

**C**

**C**

**D**

**D**

**E**

**0.8 1.2 1.6 2.0**

**0.92 0.94 0.96 0.98** [CH] (mol%)

**30 40 50 60**

**[cholesterol] (mol%) 30 40 50 60**

According to the results presented in Table 2 we concluded that the amount of micelles in OPP liposome formulations is too small to be the main reason for better efficiency of liposomes with low amount of cholesterol in experimental breast cancer therapy (Koklic et al., 2010). Therefore we proposed that better efficiency of liposomes with lower amount of cholesterol depends also on the physical and chemical characteristics of liposome membranes and their interaction with cells.


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

X is a charged compound (DCP (dicetylphosphate) for N formulations and DDAB

(dimethylioctadecylammonium bromide) for P formulation)

PEG are stearically stabilized liposomes with PEG2000DSPE (1,2-distearoyl-sn-glycero-3-

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

\* mol% of total lipids

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