*3.2. Lipid Packing in the Presence of Sugars*

We probe the effect of glucose and sucrose at concentrations in the aqueous phase up to 400 mmol/L on the lipid packing in the bilayer at various temperatures ranging from 20 to 60 ◦C. Normalized fluorescence emission spectra of Laurdan/SOPC and Laurdan/POPC vesicles at different temperatures in bidistilled water as well as in 400 mmol/L glucose and sucrose aqueous solutions are depicted in Figure 5. The emission spectra at 20 ◦C of both control and sugar-containing POPC and SOPC vesicles exhibit two peaks of nearly equal intensities centered at 430 nm (blue-shifted) and 490 nm (red-shifted), respectively. The presence of two peaks indicates that Laurdan senses two environments, one ordered and another one, more disordered, which could be associated with the presence of saturated and unsaturated fatty acids in PC molecules.

**Figure 5.** Laurdan intensity profiles from (**a**) POPC and (**b**) SOPC LUVs in water (control) and in 400 mmol/L glucose or sucrose scanned at 20, 35, and 60 ◦C.

 20 60 ℃ At 20 ◦C the spectral profiles of POPC vesicles in sugar solutions resemble the profile of the control sample similarly characterized by two peaks centered at 430 and at 490 nm. At the same temperature, the disordered red-shifted peak is characterized with slightly lower intensity compared to the blue-shifted one in sucrose solutions both for POPC and SOPC vesicles (20 ◦C, red curves in Figure 6a,b). The temperature increase leads to the

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60 ℃

20℃ − 60 ℃

progressive disappearance of the more ordered blue-shifted peak reflecting that at 60 ◦C the mobility of the two fatty acids is undistinguishable for the fluorescent probe. 

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 **Figure 6.** Fluorescence spectroscopy data for POPC membranes: Laurdan *GP* as a function of temperature in: (**a**) glucose and (**b**) sucrose solutions; DPH anisotropy vs. temperature in (**c**) glucose and (**d**) sucrose solutions.

ு − ு Laurdan *GP* values for POPC LUVs in water (control) and in sugar-containing aqueous solution as a function of the temperature are presented in Figure 6a,b. For the sake of clarity only the data for the highest sugar concentration studied are displayed in the figures. The fluorescence emission spectrum of Laurdan-labeled LUVs is measured at 20 to 60 °C. The generalized polarization *GP* is calculated according to Equation (1) in the control sample as well as for the carbohydrate concentrations studied. As it is indicated above, *GP* scale values vary between −1 and 1. In our experiments, the control POPC LUVs exhibit *GP* values from 0.05 (20°C) to −0.35 (60 °C) corresponding to intrinsically loosely packed lipids in liquid-disordered phase (*L<sup>d</sup>* ). A similar trend is observed for Laurdan *GP* values for POPC LUV suspensions in the presence of glucose and sucrose. In sugar-containing aqueous environment, we report higher *GP* values compared to the control samples in the whole temperature range scanned.

We quantify the effect of glucose and sucrose on the molecular organization in membrane hydrophobic core using DPH. The fluorescence anisotropy of this molecular probe is related to the fatty acids mobility [31]. The steady-state DPH anisotropy, *rDPH*, within the bilayer is determined according to Equation (2). As discussed above, the scale values of the parameter vary between −0.2 and 0.4. Here, DPH fluorescence anisotropy changes from 0.14 to 0.06 for POPC and SOPC vesicles in water (Figures 6 and 7), thus indicating membranes in liquid-disordered phase in the temperature range studied from 20 to 60 ◦C.

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This representation allows for discerning the different trends in the thermotropic behavior of POPC and SOPC bilayers in glucose and sucrose solutions.

 **Figure 7.** Fluorescence spectroscopy data for SOPC bilayers: Laurdan *GP* as a function of temperature in: (**a**) glucose and (**b**) sucrose solutions; DPH anisotropy vs. temperature in (**c**) glucose and (**d**) sucrose solutions.

∆ ∆ Δ Δு 20℃ Δு Δ Upon increasing temperature, DPH fluorescence anisotropy is reduced both in POPC and SOPC LUVs, which corresponds to an increase in membrane fluidity. For the two PC studied, we observe different behavior of *rDPH* upon addition of glucose and sucrose in the aqueous phase. While in glucose solutions, no correlation between DPH fluorescence anisotropy of POPC vesicles and the monosaccharide concentration is found, the presence of sucrose in the aqueous surroundings leads to a decrease in DPH rotational diffusion. As far as DPH anisotropy is inversely proportional to membrane fluidity, the above results correspond to the formation of more ordered liquid hydrocarbon region of POPC bilayers in the presence of sucrose compared to the control sample. In sucrose solutions with increasing the temperature the value of DPH fluorescence anisotropy for both lipid compositions decreases differently in comparison to *rDPH* reduction of the control sample in water upon heating (Figures 6d and 7d). At 20 ◦C DPH anisotropy in POPC membranes is lower in glucose-containing solution than in water, which corresponds to higher membrane fluidity. Inverse effect of sucrose on *rDPH* is observed at higher temperatures corresponding to decreased rotational diffusion of the fluorophore (Figure 6c,d). For SOPC, the inverse picture is observed with higher fluorescence anisotropy at 20 up to 55 ◦C. Upon further heating, we measure increased fluidity of SOPC bilayers both in glucose and sucrose solutions compared to the control sample (Figure 7c,d).

Δ/Δு~2.3

A noteworthy finding is the qualitatively different behavior of DPH anisotropy in POPC and SOPC bilayers as shown in Figures 8 and 9. In Figure 8, fluorescence spectroscopy data for single-component POPC and SOPC vesicles in water and in 400 mmol/L sugar solutions (glucose and sucrose) are shown as the difference ∆ Laurdan GP between GP at 60 and 20 ◦C, and the difference ∆ DPH anisotropy between DPH fluorescence anisotropy at 60 and 20 ◦C, respectively. The change of lipid ordering and membrane fluidity of PC vesicles in water and sugar-containing solutions are displayed as the temperature increases. Sucrose solutions membranes (especially SOPC ones) are more thermostable compared to controls because of the smaller change between GP at 60 and 20 ◦C. Upon increasing the temperature, the effect of sucrose on the hydrophobic core fluidity is more pronounced for SOPC vesicles, while for POPC membranes, the fluidity alteration is weaker. ~1.7 Δ/Δு~ − 43 ~−5 ~1.7 Δ/Δு~ − 43 ~−5

∆ ∆ **Figure 8.** Lipid packing and membrane fluidity in single-component POPC and SOPC vesicles in water (controls) and in 400 mmol/L sugar solutions (glucose and sucrose): (**a**) ∆ Laurdan GP is defined as the difference between GP at 60 and 20 ◦C; (**b**) ∆ DPH anisotropy is defined as the difference between anisotropy at 60 and 20 ◦C. DPH anisotropy and Laurdan GP values represent the mean of three independent experiments. Error bars represent standard errors. ∆ ∆

Δ Δு 20 ℃ Δ Δு 20 ℃ **Figure 9.** ∆*GP* and ∆*rDPH* calculated as the difference between DPH anisotropy or Laurdan GP of (**a**) POPC and (**b**) SOPC vesicles in 400 mmol/L glucose or sucrose and in water (control) at 20 °C. DPH anisotropy and Laurdan GP values represent the mean of three independent experiments. The error bars correspond to the standard deviations.

Figure 9 represents ∆*GP* and ∆*rDPH*, calculated as the difference between Laurdan generalized polarization and DPH anisotropy, respectively, measured for POPC vesicles with and without sugars in the bulk phase at 20°C. The same quantities are calculated also for SOPC LUVs (Figure 9b). The plot allows for discerning the different strength of the effect for the two types of PC studied in the presence of the mono- and disaccharide. The comparison between the reported values of ∆*rDPH* and ∆*GP* suggests that the presence of glucose or sucrose in the aqueous surroundings affects the lipid packing in the bilayer more strongly at the glycerol level for both lipid compositions displayed in Figure 9. For SOPC bilayers in 400 mmol/L sucrose solutions, we obtain <sup>∆</sup>*GP*/∆*rDPH* ∼2.3 compared to ∼1.7 at the same concentration of glucose. The effect is more pronounced for POPC samples yielding <sup>∆</sup>*GP*/∆*rDPH* ∼−43 in sucrose-containing environment and ∼−5 in glucose solutions, respectively. Here, the negative values reflect the slight reduction of DPH anisotropy in POPC membranes upon addition of 400 mmol/L glucose or sucrose in the aqueous surroundings (Figures 8 and 9).
