## 2 Results and Discussion
### Self-Assembly Behavior in Aqueous Solution  
#### 2.2.1 1H NMR spectra in D2O  
The prepared **UBD-1-3** underwent self-assembly in aqueous solution to form aggregates. To determine the critical aggregation concentrations (CACs), the concentration dependence of the 1H NMR spectra in D2O was investigated, and the spectral changes for **UBD-1** having a hexyl chain are shown in Figure 1a,b. The signals corresponding to the alkyl, ethylene glycol, and phenyl moieties were observed in the ranges of 1.1-1.6, 3.0-3.7, and 6.9-7.5 ppm, respectively. For concentrations between 0.25 and 20 mM, no chemical shift changes were observed, indicating that **UBD-1** was in the monomeric form in this concentration range. Above 20 mM, the proton peaks of the alkyl and ethylene glycol chains as well as some phenyl signals shifted to higher shielding and broadened, suggesting a concentration dependence. In contrast, the aromatic protons in the ortho position (H\({}_{o}\) in Scheme 1) were deshielded. These shielding [13,19,22] and deshielding shifts [13,19,23] typically derive from the formation of aggregates and the interaction via hydrogen bond between urea moiety and H\({}_{o}\) atoms in the phenyl ring, respectively. The chemical shift of the aromatic protons in the meta position (H\({}_{m}\) in Scheme 1) was plotted as a function of the concentration, and a breaking point was observed at 16 mM (Figure 1c), which corresponds to the CAC of **UBD-1**. Notably, compared to **UBD-1**, **UBD-2** and **-3** with heptyl and octyl chains, respectively (Figure S1), showed a similar concentration dependence but different CAC values (9.0 and 2.0 mM, respectively), indicating that the CAC depended on the hydrophobicity of the amphiphilic chain. As expected, a longer chain was associated with a smaller CAC value. For all **UBD**, the \({}^{1}\)H NMR spectral changes with concentration are shown in Figure 1 and Figure S1, and the chemical shift changes of the meta protons are shown in Figure 1c. Moreover, the CAC and LCST values are summarized in Table 1.  
2.2 Size Distribution and Morphology of **UBD-1-3** by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM)  
The size distribution and morphology of the resulting **UBD-1-3** aggregates in aqueous solution were determined by DLS and TEM. The DLS spectrum of a 20 mM **UBD-1** solution at 20 \({}^{\circ}\)C showed a broad peak corresponding to a hydrodynamic diameter (\(D_{\rm H}\)) of approximately 30 nm (Figure 2a). At 90 \({}^{\circ}\)C, the peak shifted to about 200 nm, indicating that the aggregate size (\(D_{\rm H}\) value) was temperature dependent, increasing with increasing temperature. The temperature dependence of the \(D_{\rm H}\) values for **UBD-1** is illustrated in Figure S2, which also shows the transmittance changes (vide infra). A similar temperature dependence of the size distribution was observed for **UBD-2** and **-3** aggregates (10 mM). At 20 \({}^{\circ}\)C, **UBD-2** and **-3** had a size of approximately 20 nm and 70 nm, respectively, and at 30 \({}^{\circ}\)C, the \(D_{\rm H}\) values shifted to larger sizes, namely, \(\sim\)1500 and \(\sim\)4000 nm, respectively (Figure 2b). Figure 1: \({}^{1}\)H NMR spectral changes with concentration of **UBD-1**. (**a**) \({}^{1}\)H NMR spectra in the chemical shift region of 0.0–7.8 ppm; and (**b**) enlarged aromatic region; (**c**) Plots of the H\({}_{m}\) chemical shift as a function of the concentration for **UBD-1–3**. H\({}_{m}\) indicates the aromatic protons in the meta position (Scheme 1). The temperature-dependent change in size distribution for **UBD-1** is shown in Figure 2a, and for all **UBD**, the temperature dependence of \(D_{\rm H}\) is shown in Figure 2c. The TEM images of **UBD** were obtained using uranyl acetate as a negative staining agent and the dried samples prepared according to previous reports [12; 13; 19]. As can be seen from the images shown in Figure 3, for all **UBD**, spherical nanoparticles with sizes in the 10-80 nm range were observed.