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

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

1. Plasma membrane fluidity is slightly larger for estrogen receptor negative (ER-) MT-3 as for estrogen receptor positive (ER+) MCF7.

Interaction of Alkylphospholipid Formulations with Breast Cancer

have cholesterol concentration below 50 mol%.

environment less accessible to water.

mixture to the aqueous environment (Busto et al., 2008).

lipid transporter.

(Zeisig et al., 1998).

Cells in the Context of Anticancer Drug Development 377

1. In mouse S49 lymphoma cells, alkylphospholipids accumulate in detergent-resistant, sphingolipid- and cholesterol-enriched lipid raft domains and are rapidly internalized

by clathrin-independent, raft-mediated endocytosis (van der Luit et al., 2007). 2. Alkylphospholipid uptake in KB carcinoma cells appears to be raft-independent and mediated by a yet unidentified ATP-dependent lipid transporter (Vink et al., 2007). 3. Lipid monolayer experiments 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). 4. While alkylphospholipid HePC is miscible with POPC, there is high affinity between HePC and sterols (ergosterol, and cholesterol) and that maximum 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).

5. Alkylphospholipid OPP increases membrane fluidity of both MCF7 and MT-3 cell line at concentrations higher than 50 µM, indicating that OPP incorporates into the membrane of breast cancer cells and is slowly transferred into the cell interior as it was detected by reduction kinetics of spin labeled OPP (Fig. 4). OPP transfer increases at physiologic temperature for OPP resistant MCF7 cells, but remains almost the same for OPP sensitive MT-3 cells. We believe that OPP uptake by OPP resistant MCF7 cells might be mediated, similarly as in the case of KB carcinoma cells (Vink et al., 2007), by a

6. Liposomal OPP formulations efficient in experimental breast cancer therapy should

7. *In vivo* data show that hemolytic effects of liposomal OPP formulations is diminished as compared to free OPP, but cytotoxic activity of liposomal formulations is also lower

8. Liposomal formulations with lower cholesterol/OPP ratio containing higher proportion of micellar OPP, are hemolytically more active than liposomal formulations with lower cholesterol concentration (Zeisig et al., 1998). At approximately 55 mol% cholesterol liposomal formulations do not contain any OPP micelles (Koklic et al., 2010). While there is almost no release of content from liposomal OPP formulations with 56 mol% cholesterol for both cell lines, liposomal formulations with low cholesterol concentration quickly release a portion of their content when mixed with breast cancer cells (Fig. 6). Similarly lipid phase of liposomal OPP formulations with low cholesterol concentration quickly enters and crosses plasma membrane of OPP resistant MCF7 cells, but remains outside of cells in the case of liposomal formulations with 56 mol%

cholesterol, as measured by C6-NBD-PC labeled liposomal formulations.

9. Fluidity characteristics of liposomal OPP formulations depend mainly on the amount of cholesterol, and only to a minor part on charge and sterical stabilization (Koklic et al., 2002). A sudden increase in order parameter of most disordered domain type occurs at cholesterol concentration around 50 mol%, while at the same time the polarity correction factor decreases, indicating that the spin probes are located in an

10. Surface activity of an alkylphospholipid Edelfosine is decreased by other lipids, especially sterols, which indicates that Edelfosine is slowly released from the lipid

