# Assessment of DPPC Liposome Disruption by Embedded
## 3 Results
### Fluidity Evaluation--DPH and TMA-DPH Fluorescence Anisotropy  
Figure 5a displays the effects of temperature changes with an increasing concentration of TM in the DPPC liposomes on the fluorescence anisotropy of DPH. The plotted results show a sigmoidal shape with a decreasing slope with increasing TM concentration. With rising temperature, the anisotropy decreases, reaching a value of 0.06. When temperature are above 45 \({}^{\circ}\)C, it remains constant. Increasing TM concentrations flattened the plots' shapes and simultaneously shifted the onset of the main phase transition, as well as T\({}_{\text{m}}\) from 41.5 \({}^{\circ}\)C to lower temperatures. The changes in the shape and slope of the traces observed with increasing temperature and concentrations are indicators of membrane  
Figure 4: Fluorescence maxima (\(\uplambda_{\text{max}}\)) of ANS (16 \(\uplambda\)M) in DPPC (0.08 mg/mL) versus temperature with increasing concentrations of: (**a**) TM during heating; (**b**) TM during cooling; (**c**) Toc during heating.  
fluidization. This shows that doping with TM lowers T\({}_{\text{m}}\), and induces different membrane organization at higher concentrations.  
In the case of TMA-DPH, the temperature profiles of Figure 5b are similar to those observed for DPH. However, the range of observed anisotropy changes is much smaller than for DPH. In the gel phase, the amplitude of anisotropy decrease is similar for both dyes. This suggests that the presence of TM also perturbs the structure of the interphase region of the DPPC membrane concurrently with changes induced in the acyl chain region as measured by DPH. With temperature above T\({}_{\text{m}}\), the anisotropy decreases to a value of 0.22, indicating a loosening in the hydrophilic region of the membrane. However, it still remains structurally ordered.  
The DPH anisotropy profile reflects the modifications in the ordered molecular structure of the hydrophobic part of the bilayer induced by embedded TM in the DPPC. TMA-DPH, which is anchored close to the membrane surface, probes the glycerol backbone and upper part of acyl chains, thus reflecting membrane surface fluidity and membrane density [46]. When the molecular motion of DPH is constrained, it leads to high anisotropy values, which occur in the gel phase. Increasing the rotational motion of acyl chains with rising temperature gives more motion freedom to DPH, which results in lower anisotropy. In the gel phase, its anisotropy responds to increasing membrane disordering due to embedded TM molecules, observed as anisotropy lowering. The fact that, in a liquid crystalline state, the TMA-DPH shows a high value of anisotropy may indicate that, despite an observed loosening in the glycerol region of the membrane, the electrostatic interactions between head molecules and malonate moiety still preserve an ordered structure.  
Figure 6 summarizes the anisotropy results of DPH and TMA-DPH recorded at 25 \({}^{\circ}\)C and 48 \({}^{\circ}\)C with increasing concentrations of TM and Toc. In the gel phase, the increasing TM concentrations lead to progressive anisotropy lowering, which is opposite to the scenario when Toc is embedded into DPPC and anisotropy increases. More than 5 mol% slowly decreases anisotropy. An increasing presence of tocopherols in the fluid phase increases the DPH anisotropy, indicating membrane rigidification. The TMA-DPH anisotropy value at 0.21 remains constant, whereas the presence of Toc at this same condition increases anisotropy, indicating decreasing membrane fluidity. Increasing anisotropy in the liquid crystalline phase of DPPC for Toc was reported [23; 47].  
Figure 5: Temperature profiles of anisotropy values at increasing concentrations of TM in DPPC liposome (0.08 mg/mL): (**a**) DPH; (**b**) TMA–DPH. Concentrations of TM are given in the legend.