# Characterization and Optimization of PLA Stereocomplexed Hydrogels for Local Gene Delivery Systems
## 3 Results and Discussion
### Hydrogel Degradation Study  
The progress of hydrogel degradation was monitored periodically by the weight loss of hydrogels in both neutral and acidic environment with addition of the enzyme. Proteinase K is known to selectively degrade PLLA with relatively low crystallinity. As shown in Figure 8, the hydrogel in the Tri-HCl buffer at pH 7.4 maintained a fairly consistent weight up to 2 weeks with 83% in average of the original weight. In contrast, the hydrogel in the acetate buffer at pH 4.5 showed a steady decline and 48% in average of the initial weight remained as a gel after 2 weeks. It should be noted that the hydrogels physically disintegrated after 2 weeks, and no reliable trend on the degradation profile was obtained. Separately, enzyme-free degradation samples exhibited relatively slower rate. \begin{table}
\begin{tabular}{l l l l} \hline
**3 LM (inner/outer)** & **D-micelle** & **Sol-to-Gel Transition** & **Time before** \\
**PLLA-PEI-PLLA** & **PDLA-PEG-PDLA** & **Temperature** & **Starting to Flow c** \\
**/PLLA-PEG-PLLA b  & (**c**) & **(min)** \\ \hline \end{tabular}
\end{table} 
Table 2: Comparison of so-to-gel transition and stability of 10 wt % stereocomplexed hydrogels using D-micelles with mixed PEG length (_imercouter_ of 3 LM kept 1/5). Figure 7: Schematic illustration of D-micelles with mixed PEG block length. To study the degradation process of 3 LM-hydrogel, the supernatants of the hydrogel test solutions after separation and lyophilization were analyzed by GPC and NMR. As the GPC curves of the accurately re-dissolved samples in Figure 9 show, the PEG (MW = 2000, polydispersity = 1.08) peak was detected after 24 h incubation in both neutral and acidic conditions with the presence of the enzyme. Therefore, it was speculated that some degree of PEG blocks was initially cleaved and released into the solution within one day regardless of the pH by enzymatic degradation at the junction with the PLLA blocks. However, the released components at neutral condition remained unchanged whereas the degradation in an acidic solution progressed in time (Figure 9b); the PEG peak appeared immediately, followed by the smaller molecular weight oligomers from PLLA block were released. Additionally, the higher molecular weight polymers were released into the buffer after 1 week. Those polymers, identified as PLLA-PEG-PLLA and PDLA-PEG-PDLA, are believed to be diffused into the solution as a result of the disintegration of the micelle-hydrogel matrix. Based on these results, the degradation process and mechanism of the 3 LM-hydrogels are proposed as follows: (1) the enzyme, proteinase K hydrolyzed the PLLA chain at the PLLA-PEG junction (the outer most PLLA of the micelle core) and released some PEG blocks into the solution, (2) further enzymatic degradation of PLLA proceeded slowly within 3 LM, and (3) physical disintegration of hydrogel resulted in the release of remaining block copolymers. Furthermore, acidic hydrolysis of the PLLA chain in pH 4.5 played a significant role to promote the degradation process. The supernatants of hydrogel degradation samples were also analyzed by \({}^{1}\)H NMR. The NMR spectra in Figure 10 show a PEG peak at 3.47 ppm and multiple peaks corresponding to CH\({}_{3}\) and CH for PLLA and its degradation products such as lactic acid and oligomers in the buffer solutions at different pH after 1 and 7 days. The intensity of all spectra was normalized using internal reference peak. It revealed that the concentration of degraded PLLA products was significantly higher in the acidic solution (red) than in the neutral solution (green) after 1 week. For the supernatants after 1 day (blue) and 7 days (red) in the acidic solution, the PEG peak was detected since day 1, and the PEG peak (see the enlarged spectra) and degraded PLLA species increased significantly after 1 week. These data correlate to the GPC results and support the proposed degradation mechanism.