**5.2 Membrane domain structure of liposomal OPP formulations**

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; Koklic et al., 2008). Typical spectra are presented in Fig. 5A.

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

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

N5 PEG 10:5:2:1 55.6 27.8 11.1 18 ± 7 179 ±23 23 ± 1 N5 10:5:2:0 58.8 29.4 11.8 20 ± 9 126 ± 30 18 ± 3 N7.5 10:7.5:2:0 51.3 38.5 10.2 11 ± 4 147 ± 32 18 ± 3 N10 10:10:2:0 45.5 45.5 9 5 ± 2 127 ± 29 28 ± 5 P10 10:10:2:0 45.5 45.5 9 0.5 ± 0.6 nd 19 ±2 N15 10:15:2:0 37.0 55.6 7.4 0 ± 1 nd n.d. Micelles 100:0:0:0 100 0 0 100 248 ± 11 17 ±9

**(%)** 

**Hemolysis increase# (%)** 

**Cytotoxicity IC50 MT-3#**

**OPP:CH:X:PEG OPP\* CH\* X\* Micelles\*\*** 

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

**5.2 Membrane domain structure of liposomal OPP formulations** 

Koklic et al., 2008). Typical spectra are presented in Fig. 5A.

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