# Assessment of DPPC Liposome Disruption by Embedded
## 4 Discussion  
The information regarding properties of TM in DPPC monolayers, or its destructive effects on cancer cell lines, were revealed previously [9, 48]. In this work, we present the results obtained in DPPC liposomes, which serve as a model for biological membranes. The ANS, DPH, and TMA-DPH fluorescence, together with a DSC method, confirmed their potential to detect structural changes in DPPC liposomes [21, 42]. The obtained results using ANS fluorescence indicate that with increasing TM concentration, the bilayer structure undergoes alterations in phase transition, ordering, and hydration in the interphase region. The obtained parameters suggest that the alterations may have arisen from the interactions at the interfacial region of the bilayer. Such modifications of the membrane structure as increase the volume of the head group were reported for alcohols [49, 50, 51], phenols [52], or sterols [38, 53]. Additionally, increasing hydrocarbon tilt and increasing molecular area lead to reordering of the acyl chain packing [46, 53, 54]. The kinetic results of ANS binding to DPPC revealed the mechanism for this process. The binding occurs fast by an adsorption of ANS dye present in bulk water onto accessible places on liposome surfaces. Because ANS binds by electrostatic forces, the binding to the surface occurs by interactions with head group atoms of phospholipid, phosphate, or choline moiety. A slow component characterized by longer times and stronger fluorescence indicates the motion of ANS into the liposome hydrophobic interior and an increasing number of binding sites due to added TM, which decreases membrane ordering. Thus, bilayer reorganization is giving opportunity that more ANS molecules bind to membrane, what is observed as the increase of FI. Increasing temperature leads to the formation of the ripple phase, where an ordered membrane transforms into a periodically undulated bilayer. Such reorganization opens more binding sites inside bilayer interior. Thus, more ANS molecules may bound to newly formed sites, what is observed as the increase of FI. The highest FI is observed at temperature of the phase transition from a rippled ordered state to a fluid phase, which is a less ordered state. Such a combination of ordered and fluid phases gives ANS molecules the highest possibility of membrane penetration and binding. Further loosening of the structure, which is connected with an increasing surface area of the lipid head group and changing packing order, also allows for the influx of water molecules, leading to the fluorescence quenching of ANS [25; 26; 41], which is observed as decreasing FI as temperature increases. The results regarding permeations of other dyes and drugs at the temperature of the main phase transition have also been reported [43; 44]. The presented results of ANS fluorescence indicate that TM embedded into DPPC modifies its properties mainly because of an increased influx of water molecules, which increases surface/volume per head group and introduces disordering in the acyl chain region. Such modifications in the membrane structure finally led to the further fluidization of the bilayer at lower temperatures. The molecular mechanism of these processes is connected with interactions between TM, which possesses a long anionic malonate moiety and the electrostatic potentials of the membrane surface arising from DPPC head group moieties where each possess lone pair electrons [55]. Interactions between negatively charged phosphate groups in the phospholipid head group and anionic malonate moiety from embedded TM modify membrane interface region, which leads to further processes. The anisotropy results of DPH and TMA-DPH indicate that TM inserted into DPPC increases its disorder, decreasing cooperativity and making the structure less rigid and fluid. To simultaneously follow the changes in two measured properties with the opportunity to observe the variation of interior membrane properties at each stage of transformation induced by the presence of TM, we apply the Cartesian plot.