**7. Conclusions with respect to experimental breast cancer therapy with alkylphospholipids**

For efficient application of liposomes as nanocarriers in breast cancer therapy it is not only necessary to investigate in detail the physical properties of the nanocarrier, which has to transport the drug to the (target) cell, but also the properties of the target cell. In the case of application of alkylphospholipids, where plasma membrane is the specific target, one has to know the properties of the plasma membrane and differences among membranes of different breast cancer cell lines. We have shown that plasma membrane of MT-3 cells is more fluid (lower order parameter) as the membrane of MCF7 cells and was influenced more by OPP. Besides, it should be taken into account also that the properties of plasma membrane depend on external factors. For example, it has been shown that confluent MT-3 breast cancer cells have significantly higher membrane fluidity and higher relative proportion of disordered membrane domains as compared to cells harvested during exponential growth (Koklic et al., 2005). The fluidity of plasma membrane of MT-3 breast cancer cells also has an important role in metastasis development. The increase in membrane fluidity of MT-3 breast cancer cells was correlated with 2-fold increase in sialyl Lewis X and/or A ligand-mediated adhesion of these cells and a higher motility of ligands in the membrane of confluent cells, together with an accumulation of these ligands in distinct areas on a cell membrane (Zeisig et al., 2007).

In order to better understand the interaction of OPP micelles or liposomes with breast cancer cells, one has to take into account the following main characteristics of alkylphospholipids and of liposomal formulations:

2. OPP increases plasma membrane fluidity of both cell lines at concentrations higher than 50 µM. The influence of Perifosine (OPP) is less pronounced for MCF7 as for MT-3 cells. This indicates that OPP either doesn't incorporate into alkylphospholipid resistant, ER+ MCF7 cells as well as it incorporates into alkylphospholipid sensitive, ER- MT-3 cells, or it doesn't concentrate in plasma membrane of MCF7 cells at such high concentrations as

3. Transport of alkylphospholipids across plasma membrane and subsequent reduction in breast cancer cells (Fig. 4) showed that the transport and the reduction of spin labeled OPP is faster for MT-3 than for MCF7 cells at room temperature, whereas it is just the opposite at physiological temperature. The main difference between MCF7 and MT-3 cells is the transport of OPP across the plasma membrane, which increases significantly for MCF7 cells at physiologic temperature, but remains almost unchanged for MT-3 cells. Because of this we suspect 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 lipid transporter. This observation could explain lower influence of OPP on cell membrane fluidity of MCF7 cells and support the hypothesis that OPP doesn't concentrate in the

4. Liposomal OPP formulations with low CH concentration (N5) quickly release a portion of their content when mixed with breast cancer cells. At room temperature the release is comparable for MT-3 and MCF7 cells (Fig. 6). However, at physiological temperature the amount of released content increases for OPP sensitive MT-3 cells, but remain in the

For efficient application of liposomes as nanocarriers in breast cancer therapy it is not only necessary to investigate in detail the physical properties of the nanocarrier, which has to transport the drug to the (target) cell, but also the properties of the target cell. In the case of application of alkylphospholipids, where plasma membrane is the specific target, one has to know the properties of the plasma membrane and differences among membranes of different breast cancer cell lines. We have shown that plasma membrane of MT-3 cells is more fluid (lower order parameter) as the membrane of MCF7 cells and was influenced more by OPP. Besides, it should be taken into account also that the properties of plasma membrane depend on external factors. For example, it has been shown that confluent MT-3 breast cancer cells have significantly higher membrane fluidity and higher relative proportion of disordered membrane domains as compared to cells harvested during exponential growth (Koklic et al., 2005). The fluidity of plasma membrane of MT-3 breast cancer cells also has an important role in metastasis development. The increase in membrane fluidity of MT-3 breast cancer cells was correlated with 2-fold increase in sialyl Lewis X and/or A ligand-mediated adhesion of these cells and a higher motility of ligands in the membrane of confluent cells, together with an accumulation of these ligands in

In order to better understand the interaction of OPP micelles or liposomes with breast cancer cells, one has to take into account the following main characteristics of

**7. Conclusions with respect to experimental breast cancer therapy with** 

it does in MT-3 cells.

plasma membrane of MCF7 cells.

distinct areas on a cell membrane (Zeisig et al., 2007).

alkylphospholipids and of liposomal formulations:

same range for MCF7 cells.

**alkylphospholipids** 


Interaction of Alkylphospholipid Formulations with Breast Cancer

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Based on all of the above mentioned properties, there are two competing hypothesis of OPP liposome formulation – breast cancer cell interaction, which still need further experimental validation. Either OPP liposome formulations with low cholesterol concentration are able to deliver OPP into breast cancer cells by fusing with plasma membrane of breast cancer cells, due to liposome membrane properties, or alkylphospholipids whether in free or micellar form insert with high affinity into cholesterol containing target membranes (cells) as long as liposomal carriers contain low amounts of cholesterol. Once the remaining liposomal carriers have cholesterol concentration above around 50 mol% the remaining alkylphospholipids are stabilized in liposomes and do not interact with cells anymore. In this way liposomes serve as a reservoir capable of releasing alkylphospholipids and preventing side effect associated with high alkylphospholipid concentrations.
