# Characterization and Optimization of PLA Stereocomplexed Hydrogels for Local Gene Delivery Systems
## 3 Results and Discussion
### Effect of Formulation on Micelle Size and Composition  
The effect of the _inner:outer_ polymer ratios on the 3 LM particle size and structure was determined by DLS measurements. The hydrodynamic radius is given for the 3 LM solutions from four _inner:outer_ ratios investigated, 1:1, 1:5, 1:10 and 1:20 (Figure 2a). The _\"inner\"_ value corresponds to the weight fraction of PLLA-PEI-PLLA, whereas the \"_outer_\" value corresponds to that of PLLA-PEG-PLLA which makes up the corona of 3 LM. As agreed with the previous reported 3 LM data, the 1:1 ratio formulation gave the particles with monodispersed sizes of 100-200 nm. However, with increasing _outer_ ratio values in the 3 LM formation process, a second peak at \(<\)50 nm was observed. The intensity of the second peak increased with increasing _outer_ ratio, while the intensity of the original peak decreased. The second peak at sizes \(<\)50 nm corresponds to the simple flower-micelles composed of simple PLLA-PEG-PLLA triblock copolymers (L-micelle); and the hydrodynamic sizes observed are comparable to the micelles from triblock copolymers with same molecular weight [35]. Therefore, with increasing _inner:outer_ ratios, the micellar solution is populated with two sets of micelle particles, 3 LM (containing DNA) and the flower micelles (DNA free). The DLS data indicates the size of 3 LM is not significantly affected by the increasing concentration of the PLLA-PEG-PLLA outer layer. Even though the excess of PLLA-PEG-PLLA outer polymers has a negligible effect on the 3 LM self-assembly properties, the presence of L-micelles may affect the sol-to-gel transition behavior and gel properties considerably and that will be discussed below. We have also examined the 3 LM size using the longer PLLA block of the outer polymer, PLLA-PEG-PLLA (1200-2000-1200) (Figure 2b). Interestingly, the thickness of hydrophobic intermediate layer created by both inner and outer PLLA blocks did not affect to the overall diameter of 3 LMs.  
\begin{table}
\begin{tabular}{l c c c c} \hline \hline \multirow{3}{*}{**Micelle ID**} & \multicolumn{2}{c}{**3 LM Composition I/O Polymer Weight Ratio (\%) 1**} & \multicolumn{2}{c}{**D-Micelle Polymer Weight Ratio (\%)**} \\ \cline{2-5}  & **Inner Polymer** & **Outer Polymer** & **short-PEG** & **long-PEG** \\ \cline{2-5}  & **PLLA-PEI-PLLA** & **PLLA-PEG-PLLA** & **PLLA-PEG-PLLA** & **PDLA-PEG-PDLA** \\  & **(1700-2000-1700)** & **(800-2000-800)** & **(1200-2000-1200)** & **(800-2000-800)** & **(800-3350-800)** \\ \hline
3 LM-1/1 & 50 & 50 & & & \\
3 LM-1/5 & 17 & 83 & & & \\
**3 LM-1/10 b** & **9** & **91** & & & \\
3 LM-1/20 & 5 & 95 & & & \\
3 LM-1/1-1200 & 50 & & 50 & & \\ \hline
**D-micelle–S c** & & & & **100** & **0** \\ D-micelle–70S & & & & 70 & 30 \\ D-micelle–50S & & & & 50 & 50 \\ D-micelle–30S & & & & 30 & 70 \\ D-micelle–0S & & & & 0 & 100 \\ \hline \hline \end{tabular}  
* Unless specified, the N/P ratio was kept as 12 by adjusting amount of DNA and PLLA-PEI-PLLA. b Unless specified, 3 LM used for most of hydrogel evaluations was 3 LM-1/10. c Unless specified, D-micelle used for most of hydrogel evaluations was D-micelle-S.  
\end{table}
Table 1: Composition ratios of _inner:outer_ polymers in 3 LM and mixed PEG block sizes in D-micelles.  
Figure 1: Schematic representation of 3 LM and hydrogel components.