## 3 Results and Discussion
### Characterization of the Phase Transition  
The thermoresponsiveness of pEFS8-26 and pEFS8-46 in aqueous solutions at different concentrations ranging from 0.5 to 30 g/L was tested by turbidimetry using a UV-visible spectrophotometer. The transition due to aggregation phenomena was in general very sharp, being less defined for the lowest investigated concentration of 0.5 g/L (Figure 3a,b). The cloud point (\(T_{\text{cp}}\)) temperatures associated with the transition were found to decrease from 83 \({}^{\circ}\)C to 73 \({}^{\circ}\)C up to a plateau value of 71 \({}^{\circ}\)C by increasing the pEFS8-26 polymer concentration from 0.10 to 5 mg/mL up to 30 mg/mL (Figure 3c). A similar trend was also valid for pEFS8-46. \begin{table}
\begin{tabular}{c c c} \hline \hline  & \(D_{\text{h}}\) **at 25 \({}^{\circ}\)C (nm)** & \(D_{\text{h}}\) **at 80 \({}^{\circ}\)C (nm)** \\ \hline pEFS8-26 & \(6\pm 1\) & \(3000\pm 1000\) \\ (5 g/L) & \(7\pm 2\) & \(1500\pm 300\) \\ pEFS8-46 & \(7\pm 2\) & \(340\pm 60\) \\ (0.5 g/L) & \(7\pm 2\) & \(2000\pm 600\) \\ (10 g/L) & \(7\pm 2\) & \(2000\pm 600\) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Values of hydrodynamic diameters \(D_{\text{h}}\) (mean value \(\pm\) standard deviation) for pEFS8-x polymers. Figure 2: Intensity size distribution at 80 \({}^{\circ}\)C for (**a**) pEFS8-26 and (**b**) pEFS8-46 aqueous solution at 5 g/L and (**c**) for pEFS8-46 at various concentrations. Calorimetric measurements were performed on pEFS8-26 and pEFS8-46 aqueous solutions at different concentrations in order to obtain more information on the thermodynamic nature of the LCST. The thermal profiles of pEFS8-26 water solutions at concentrations of 0.5, 1, and 5 mg/mL are reported in Figure 4. The thermal profiles of pEFS8-46 are presented in Supplementary material Figure S1. The endothermic peaks observed provided information on the phase transition occurring during the thermal scan. The curves presented a complex profile that suggested the concurrence of different contributions (e.g., the breaking of solvent-polymer hydrogen bonds and the formation of polymer-polymer non-covalent interactions). The overall shape was highly asymmetrical, with a sharp rise of the curve from the onset temperature until the peak maximum and a slow decrease of the curve in the second part, forming a tail. A similar profile is also reported for other thermoresponsive polymers (e.g., PNIPAM [19] and poly(\(N\)-vinylcaprolactam) (PVCL)) [43] and the main contribution to the observed endothermic peak was attributed to the breaking of hydrogen bonds among the polymer chains and water, whereas the peak asymmetry was associated with the competition between the dehydration process and the polymer-polymer interchain aggregation in the heating scan [24]. In our case, the latter may have occurred by direct aggregation of self-folded unimers, or by the opening (or partial opening) of the self-folded unimers followed by their aggregation in a more extended conformation, in analogy with the thermal-induced aggregation of protein after the unfolding process [44; 45; 46; 47]. A recent small-angle X-ray scattering (SAXS) investigation on the self-assembly behavior in aqueous solution of amphiphilic (meth)acrylic random copolymers containing perfluoroalkyl and oligo(ethylene glycol) side chains highlighted that the aggregation process can be considered an assembling of self-folded unimers in an extended (partially opened) conformation, resulting in a rod-shaped structure [48]. Figure 3: Turbidimetry measurements at different aqueous solution concentrations for (**a**) pEFS8-26 and (**b**) pEFS8-46; (**c**) cloud point temperatures as a function of aqueous solution concentration for pEFS8-26. The onset (\(T_{\text{onset}}\)) and the maximum (\(T_{\text{max}}\)) temperatures in the process are reported in Table 3. Both temperatures decreased with increasing polymer concentrations, consistent with the existence of an LCST-type phase separation. The onset temperatures were associated with the beginning of the dehydration process.