# Light-Responsive Polymeric Micellar Nanoparticles with Enhanced Formulation Stability
## 3 Results
### Measurements of CMC, LE and LC, and Stability  
CMC is a key parameter to describe the self-assembly behavior of micelles. The CMC of \(\mathbf{NP_{DNQ}}\) was calculated by the pyrene fluorescence method [53]. Pyrene has a fluorescence profile that is dependent on its microenvironment. In hydrophobic environments, the fluorescent intensity ratio of the first (373 nm) to third (383 nm) emission peaks (Figure 3a), the \(I_{373}/I_{383}\) ratio known as polarity parameter, is low (close to 1); whereas in hydrophilic environments, the \(I_{373}/I_{383}\) ratio increases (close to 2). Using this method, we were able to determine whether pyrene was in the core of the micelle (hydrophobic environments) or in aqueous solution (hydrophilic environments). The polymer concentration at which the location of pyrene changed from solution to micelle is the CMC. Figure 3a shows the fluorescence emission spectra of pyrene encapsulated in \(\mathbf{NP_{DNQ}}\) and the change of the \(I_{373}/I_{383}\) ratio at the various concentrations of PEG-PLL-DNQ at room temperature. The curve was almost flat at low polymer concentrations but rapidly decreased at higher polymer concentrations and eventually reached a plateau. The CMC value of \(\mathbf{NP_{DNQ}}\) was determined by the intersection of two straight lines and the CMC was about 7.1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (Figure 3b), which is a relevantly low CMC compared to other reported amphiphilic diblock copolymers (>20 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) and four times lower than the control micelle system, \(\mathbf{NP_{Oct}}\) (25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, Figure 3c) due to \(\pi\)-\(\pi\) stacking interactions between neighboring DNQs on the polymer backbone leading to a more condensed core of \(\mathbf{NP_{DNQ}}\). The reduced CMC could contribute to improved thermodynamic stability of the micelle, reduced toxicity toward cells, and increased circulating time in blood.  
In addition to CMC, we evaluated the drug loading efficiency (LE) and capacity (LC) of \(\mathbf{DTX}\)-\(\mathbf{NP_{DNQ}}\) and \(\mathbf{DTX}\)-\(\mathbf{NP_{Oct}}\) using HPLC (Table S1). The results show LCs of 13.5% and 7.9% for \(\mathbf{DTX}\)-\(\mathbf{NP_{DNQ}}\) and \(\mathbf{DTX}\)-\(\mathbf{NP_{Oct}}\), respectively. These were associated with LEs of 90.3% and 79.1%, respectively. \(\mathbf{NP_{DNQ}}\) encapsulated approximately two times more DTX than \(\mathbf{NP_{Oct}}\), which can be presumably ascribed that \(\mathbf{NP_{DNQ}}\) has a better compatibility of DTX due to \(\pi\)-\(\pi\) stacking interactions between DNQs and aromatic DTX.  
Subsequently, we measured the stability of the micelles in blood plasma at 37 \({}^{\circ}\)C because the stability is greatly influenced by serum proteins. The hydrophobic Nile red dye (NR)-loaded micelles, \(\mathbf{NR}\)-\(\mathbf{NP_{DNQ}}\) and \(\mathbf{NR}\)-\(\mathbf{NP_{Oct}}\), were incubated in 10% bovine serum albumin (BSA) solution and the intensity of fluorescence emission was monitored. NR is a fluorescent indicator for the micropolarity; it shows strong fluorescence emission intensity in hydrophobic environments while it is quenched in a hydrophilic solution [54]. As shown in Figure 3d, \(\mathbf{NR}\)-\(\mathbf{NP_{DNQ}}\) was stable in BSA solution retaining their intensity up to 2 days at 37 \({}^{\circ}\)C (>85%) while \(\mathbf{NR}\)-\(\mathbf{NP_{Oct}}\) showed a dramatic decrease in intensity, indicating release  
Figure 3: (**a**) Normalized emission spectra of pyrene in solutions of PEG-PLL-DNQ in PBS; emission intensity of pyrene at 373 and 383 nm versus the concentrations of (**b**) PEG-PLL-DNQ and (**c**) PEG-PLL-Oct; (**d**) stability of \(\mathbf{NR}\)-\(\mathbf{NP_{DNQ}}\) (d-blue) and \(\mathbf{NR}\)-\(\mathbf{NP_{Oct}}\) (d-red) in BSA solution. Data are means \(\pm\) SD, \(N=4\), asterisks indicate \(p<0.05\).  
of NR into hydrophilic environments (Figure S4). These results indicate that the presence of **DNQ** on the polymer backbone increased the stability of the micelles.