# Assessment of DPPC Liposome Disruption by Embedded
## 3 Results
### The ANS Emission in DPPC Liposomes
#### 3.1.2. Hydrophobicity and Water Permeability Evaluation in DPPC Using ANS Fluorescence  
The temperature dependencies of FI of ANS during heating and cooling of DPPC liposomes at different amounts of TM are presented in Figure 3a,b. As a reference to the results obtained for TM, we performed the same experiments for its parent compound the \(\mathrm{Toc}\); the results for the heating cycles are presented in Figure 3c. In pure DPPC during heating, we observe two maxima at 36 \({}^{\circ}\)C and at 42 \({}^{\circ}\)C, at temperatures that are characteristic for DPPC liposomes and assigned as the pretransition \(\mathrm{T_{p}}\) and the main phase transition \(\mathrm{T_{m}}\) temperatures, respectively. The biggest value of permeation of ANS occurs at temperatures close to the phase transitions and is similar to previously published results [43; 44]. After reaching the maximum at \(\mathrm{T_{m}}\), a monotonically decreasing FI up to 60 \({}^{\circ}\)C was obtained. In the gel phase, the presence of an increasing concentration of TM increases the FI value and results in a gradual disappearance of the pretransition temperature. Additionally, increasing the presence of TM widened the trace, flattened the curves, and simultaneously shifted the positions of the FI main maximum. At the highest concentration of TM, the curve was flattened with a shape incomparable to lower concentrations. The similar observations on the traces of \(\mathrm{Toc}\) were noted. Increasing temperatures above the main phase transition decrease the intensity with similar slopes for TM and \(\mathrm{Toc}\) samples, which may indicate that one mechanism is involved in the observed process. We suggest that the observed quenching is due to the increasing number of water molecules in the inner leaflet of the liposome modified by embedded TM. With an increasing number of TM or \(\mathrm{Toc}\) molecules, for both tocopherols, a similar decrease of the temperature at which the FI curve achieves a maximum was observed. Since \(\mathrm{T_{m}}\) is related to the structure of acyl chains, the presence of this same chromatol ring and phytol chain induced similar results. Additionally, it confirms that malonate moiety attached at 6 position of the chromatol ring does not influence this parameter. In the gel phase at 23 \({}^{\circ}\)C, FI increases by nearly 3-fold compared to pure DPPC, whereas at main phase transition at 42 \({}^{\circ}\)C, a FI increase is only 25% of the highest intensity observed for 2 mol% compared to DPPC. At increasing TM concentrations, FI lowers significantly, indicating its disruptive role. Figure 2: Time-course of ANS (16 \(\mathrm{\upmu M}\)) inclusion into DPPC (0.08 mg/mL) membrane at 25 \({}^{\circ}\)C for (a) malonate (TM); (b) ocopherol (\(\mathrm{Toc}\)). TM and \(\mathrm{Toc}\) concentrations are given in figures. The plots of the FI during the cooling of the samples from 60 \({}^{\circ}\)C to 20 \({}^{\circ}\)C for TM are given in Figure 3b. The results reveal that the traces observed during cooling differ, including the shape and the positions of the maxima are shifted to lower temperatures. We assume that the presence of TM induces structural changes depend on the homogeneity of the liposome and forms the defects in the regular DPPC structure, which decreased the cooperativity of the transition, thus lowering its phase transition temperature. We also notice that, after cooling, the FIs in the gel phase were 2- or 3-fold higher compared to those observed at the beginning of the heating (Figure 3a,b,d). These results may indicate that some of the ANS molecules were bound inside the modified liposome layer. The formation of the metastable rippled gel phase during cooling has been reported [33; 45]. In our studies with ANS fluorescence at increasing concentrations of TM and Toc, in DPPC during the course of heating and cooling, we observe that \(\lambda_{\text{max}}\) changes; these dependencies are given in Figure 4a-c.