# Assessment of DPPC Liposome Disruption by Embedded
## 4 Discussion
In this presentation, we may also follow the dynamics of given bilayer structures induced by increasing presences of TM. Figure 8 presents the Cartesian diagram for DPPC liposomes modified with 2, 5, 10, and 20 mol% of TM, arranged from the data of membrane fluidity (DPH, TMA-DPH) and hydration (ANS intensity) at four characteristic temperatures: for gel, T\({}_{\text{p}}\), T\({}_{\text{m}}\), and crystalline phases. The vertical line at 170 is a threshold line between low and high hydration, whereas a horizontal line at 0.2 is a threshold for ordered and transition states. The DPH data, Figure 8a, indicate that in the gel phase increasing TM concentration decreases membrane order in its hydrophobic region. It is concurrently observed increasing hydration as a contradiction to the fluorescence mechanism of ANS emission. Thus, we have  
Figure 8: The Cartesian diagrams of TM at 25, 36, 41, and 50 \({}^{\circ}\)C; (**a**) DPH/ANS; (**b**) TMA–DPH/ANS. The x axis indicates hydration/polarity and y axis represents fluidity. Arrows are located at pure DPPC and indicate direction of changes with increasing TM concentration. to introduce another mechanism in accordance to the ANS fluorescence mechanism and call it polarity. The increasing intensity is related to electrostatic interactions between ANS and ionic groups of the phospholipid head. A disturbing presence of TM in the membrane leads to more binding places on the membrane surface. The ripple phase observed as pretransition undergoes membrane progressing disordering as the TM presence raises, and the recorded ANS intensity is ascribed to the aforementioned mechanism. At T\({}_{\text{m}}\), a pure DPPC is still in an ordered state. However, from 2 mol% to 5 mol% of TM, an increasing disorder in the dehydrated interior is observed. Increasing TM concentration induces hydration in this region, allowing for the water influx. In the liquid crystalline phase, the fluid state shows an increasing hydration, indicating a further influx of water molecules into the liposome interior. The results indicate that the presence of TM, even at 2 mol%, alters fluidity and/or hydration/polarity of the hydrophobic region of the membrane in the pattern. To observe and analyze behavior in the hydrophilic membrane region, we apply another fluorescent probe that is placed closer to membrane surface. TMA-DPH probes the interface region in the vicinity of the glycerol backbone and upper acyl chain region because of its cationic group, which anchors to water/a bilayer interface [46]. Figure 8b presents the Cartesian diagram in this same form as given for DPH, Figure 8a, to visually compare the results. We may notice in Figure 8b that in ordered states, the increasing concentration of TM decreases ordering in the interface region to the same extent as observed for DPH, and with this same molecular mechanism. At T\({}_{\text{m}}\), and in a liquid crystalline phase, the increasing TM concentration practically has no effect of interface fluidity, which remains still in an ordered state. However, the influx of water is observed leading to increased hydration. The parameters obtained from the DSC traces (T\({}_{\text{m}}\), \(\Delta\)T\({}_{1/2}\) and \(\Delta\)H\({}_{\text{m}}\)) are very sensitive indicators of the calorimetric properties characterizing the system. Our results show that in the presence of TM, the changes in the broadening of \(\Delta\)T\({}_{1/2}\), decreasing T\({}_{\text{m}}\), and diminishing \(\Delta\)H\({}_{\text{m}}\) and cooperativity were noted. Additionally, with an increasing TM, the new peaks appeared on thermograms. These findings indicate that the observed changes can be ascribed to lowering rigidity of phospholipid molecules in the liposomes and show that the presence of the TM molecules in DPPC liposomes lead to its fluidization.