# Characterization and Optimization of PLA Stereocomplexed Hydrogels for Local Gene Delivery Systems
## 3 Results and Discussion
### Gelation Mechanism and Sol-to-gel Phase Transition  
Thermo-responsive hydrogels were prepared using the stereocomplexation mechanism of enantiomeric polylactides. Stereocomplexed (physically crosslinked) hydrogels are attractive because they are free from potentially toxic cross-linkers typically required for chemically crosslinked hydrogels. Although the mechanical strength of physically crosslinked hydrogels are generally lower than chemically crosslinked hydrogels, the enantiomeric mixture of PLA-PEG-PLA micelles has shown significantly increased storage modulus of stereocomplexed hydrogels by controlling the polymer sizes and micelle packing parameters [28; 29]. The hydrogel formation is driven by the stereocomplexation of the PLLA and PDLA blocks of the micellar solution, which is the result of the chain exchange between neighboring micelles. The inter-micelle chain exchange predominates at higher temperatures and concentrations, leading to an irreversible hydrogel. The insight of hydrogel formation from 3 LMs was studied in this work. As depicted in Figure 1 above, the PDLA block is supplied by the blank D-micelles as one of the sources of stereocomplexation in the hydrogel formulations. Whereas, there are several sources the PLLA block can be obtained in the system. The intermediate hydrophobic layer of 3 LM is composed of two sets of PLLA, one derived from the DNA complexed PLLA-PEI-PLLA copolymer and the second from the corona forming PLLA-PEG-PLLA copolymer. The stereocomplexation ability of each PLLA segment was investigated by replacing the corona forming copolymer (outer polymer) with the racemic and amorphous PDLLA-PEG-PDLLA which has no ability to form stereocomplex crystals during the 3 LM-hydrogel preparation step. The presence/absence of the stereocomplexation diffraction peaks was determined through WAXS after lyophilized the mixtures of 3 LM and D-micelle solutions. The 3 LM containing PDLLA-PEG-PDLLA outer layer did not form a hydrogel after mixing with D-micelle solution at all temperatures and concentrations investigated. Justifying this result, we observed no stereocomplexation diffraction peak in the WAXS spectra for this mixture (Figure 3a) compared to the typical stereocomplex peaks observed in simple micelle mixture of PLLA-PEG-PLLA and PDLA-PEG-PDLA (Figure 3c). In contrast, the stereocomplex diffraction peaks at \(20=12^{\circ}\) and \(21^{\circ}\) were clearly observed in the WAXS spectra of the hydrogel forming 3 LM including semicrystalline PLLA-PEG-PLLA outer layer mixed with D-micelles (Figure 3b). Therefore, the PLLA blocks of the outer layer polymer are responsible for stereocomplexation event leading to hydrogel formation. For 3 LM with increasing _inner:outer_ ratios, 1:5, 1:10, 1:15 and 1:20, the stereocomplexation event is likely a combination of chain exchange between both 3 LM and excess of blank PLLA-PEG-PLLA micelles and blank PDLA-PEG-PDLA micelles, leading to hydrogel formation. Figure 2: DLS intensity analysis of: (**a**) 3 LM compositions prepared by different weight ratios of PLLA-PEI-PLLA (inner layer) and PLLA-PEG-PLLA (outer layer); (**b**) 3 LM sizes at _inner:outer_ ratio 1:1, but different PLLA block length of outer block copolymers. The phase transition (sol-to-gel) diagrams for the different formulations are shown in Figure 4. The phase diagram of 3 LM with the 1:1 _inner:outer_ ratio mixed with D-micelle (Figure 4a) shows a linear decrease in the sol-gel transition temperature with increasing polymer concentration. Hydrogels with clinically relevant sol-gel transition temperatures, 37 and 25 \({}^{\circ}\)C can be obtained at 20 and 25 wt %, respectively. The phase diagram of the different _inner:outer_ formulation in 3 LM is shown in Figure 4b. The concentration of all the solutions was kept constant at 20 wt % and thus only the effect of the increasing population of excess PLLA-PEG-PLLA micelles was investigated.