## 3 Results and Discussion
### Drug Loading and Micelle Characterization  
Since the arborescent PBG-\(g\)-PEO copolymers have a hydrophobic PBG core and a hydrophilic PEG shell, these amphiphilic molecules could be useful as nanocarriers to physically encapsulate hydrophobic guest molecules through hydrophobic interactions (Scheme 3). Figure 3: Hydrodynamic diameter distributions for randomly grafted arborescent copolymers determined by DLS in DMF (**left**) and in PBS (**right**): (A) G1 PBG\({}_{15}\)-\(g\)-PEO, (B) G1 PBG\({}_{29}\)-\(g\)-PEO, and (C) G1 PBG\({}_{65}\)-\(g\)-PEO. Figure 4: TEM (**left**) and AFM phase scan (**right**) for randomly grafted arborescent copolymers: (**A**) G2 PBG\({}_{15}\)-\(g\)-PEO, (**B**) G2 PBG\({}_{29}\)-\(g\)-PEO, and (**C**) G2 PBG\({}_{65}\)-\(g\)-PEO. The ability to load the hydrophobic core of these unimolecular micelles with guest molecules was investigated to determine whether the differences in their core structure affected their ability to encapsulate and control the rate of release of the hydrophobic drug. Doxorubicin (DOX), an anticancer drug, was selected as a model drug to assess loading and release in vitro. DOX-HCl was treated with triethylamine before encapsulation to obtain DOX in its free base (more hydrophobic) form. DOX was encapsulated within the hydrophobic core of G1 PBG-\(g\)-PEO and G2 PBG-\(g\)-PEO by co-dissolution followed by dialysis. It was indeed determined that hydrophobic DOX could be solubilized in the amphiphilic arborescent copolymers, as shown in Figure 5. For example, the successful encapsulation of DOX within the hydrophobic core of G2 PBG-\(g\)-PEO is indicated by the enhanced absorption around 490 nm of the DOX-loaded micellar solutions (G2 PBG-\(g\)-PEO/DOX) as compared to the empty micelles (G2 PBG-\(g\)-PEO). The encapsulation of DOX specifically within the hydrophobic core of the micelles is also suggested by the blue shift in the maximum absorption for G2 PBG-\(g\)-PEO/DOX as compared to free DOX in PBS (\(\lambda_{\max}=483\) nm), which is attributed to the hydrophobic microenvironment created by the PBG core of G2 PBG-\(g\)-PEO [36]. Figure 5: UV Absorption of free DOX, G1 (**left**) and free DOX, G2 (**right**) arborescent copolymers in PBS. The existence of hydrophobic interactions was further confirmed by \({}^{1}\)H NMR analysis of the G2 PBG\({}_{15}\)-\(g\)-PEO/DOX unimolecular micelles in D\({}_{2}\)O (Figure 6). The complete disappearance of both the PBG and DOX signals for G2 PBG\({}_{15}\)-\(g\)-PEO/DOX in D\({}_{2}\)O (Figure 6c) in comparison to the characteristic signals observed of free DOX in D\({}_{2}\)O at the same overall DOX concentration (Figure 6a) confirms the restricted mobility of both the PBG and DOX components within the core of G2 PBG\({}_{15}\)-\(g\)-PEO. This again supports the solubilization and encapsulation of DOX molecules within the core of the micelles in aqueous solutions. The drug loading content (DLC) determined for the micelles ranged from 7.1-10.8 wt%, and the drug loading efficiency (DLE) from 42-65% for the different G1 PBG and G2 PBG copolymers (Table 3). Both the drug loading content (DLC) and the drug loading efficiency (DLE) increased with the generation number of copolymers. However, for the same generation, the minor variations observed do not seem to correlate with parameters such as differences in branching density among the samples. Sample G2 PBG\({}_{15}\)-\(g\)-PEO/DOX nevertheless has slightly higher values of DLC (10.8%) and DLE (65%) as compared with  
Figure 6: \({}^{1}\)H NMR spectra for (**a**) DOX in D\({}_{2}\)O, (**b**) G2 PBG\({}_{29}\)-\(g\)-PEO in D\({}_{2}\)O and (**c**) G2 PBG\({}_{29}\)-\(g\)-PEO/DOX in D\({}_{2}\)O. The insets show the appearance of the (1) DOX, (2) G2 PBG\({}_{29}\)-\(g\)-PEO, and (3) G2 PBG\({}_{29}\)-\(g\)-PEO/DOX samples in D\({}_{2}\)O.  
the two other arborescent copolymers. Common sense dictates that larger particles should be capable of loading more drugs.