**2. Results**

### **2. Results**  *2.1. Rational Design*

*2.1. Rational Design*  Jeong et al. first reported the synthesis of triblock copolymers containing a central poly (lactic acid) (PLA) block bearing two sidechains of poly (ethylene glycol) (PEG) [24]. The synthetic method reported there comprised of two steps. First, poly(ethylene glycol) methyl ether Mn = 550 (mPEG 550) was used as a macroinitiator of lactide polymerization (via ring-opening polymerization in the presence of stannous octoate catalyst) to form a diblock copolymer. Two hydroxyl termini of two distinct PLA blocks were subsequently linked by reaction with diisocyanate, forming urethane linkages (Figure 2A). Whereas the original research focused on polymers with sol–gel properties, which required cooling to Jeong et al. first reported the synthesis of triblock copolymers containing a central poly (lactic acid) (PLA) block bearing two sidechains of poly (ethylene glycol) (PEG) [24]. The synthetic method reported there comprised of two steps. First, poly(ethylene glycol) methyl ether Mn = 550 (mPEG 550) was used as a macroinitiator of lactide polymerization (via ring-opening polymerization in the presence of stannous octoate catalyst) to form a diblock copolymer. Two hydroxyl termini of two distinct PLA blocks were subsequently linked by reaction with diisocyanate, forming urethane linkages (Figure 2A). Whereas the original research focused on polymers with sol–gel properties, which required cooling to move from sol form to gel morphology, subsequent reports focused on the development of sol–gel transitions, which occurred upon heating of the polymer solution. The enhancement of sol-to-gel properties from relying on cooling (gel at low temperatures and sol at high

temperatures) to being responsive to heating (sol at room temperature and gel at elevated temperature) was crucial, as the practical application of such gels relies on the incorporation of active agents in a room-temperature solution, which upon injection and exposure to physiological temperatures forms a semi-solid gel capable of extended release of the active agent. These sol–gel thermosensitive hydrogels were based on an amorphous PLA polymer block (as opposed to crystalline poly (L-lactic acid) polymer blocks employed in the original work). Gel properties, biodegradability, and drug release were all subsequently optimized, leading to over two decades of research and application of injectable sol–gel biodegradable polymer solutions [6,11,25]. In a similar fashion as the original work on PEG-PLA-PEG triblock copolymers, several synthetic steps were employed in order to incorporate a disulfide bond into the triblock copolymer structure. In the first synthetic step, 2-hydroxyethyl disulfide was used as the initiator of DL-lactide polymerization. In doing so, an amorphous DL-poly(lactic acid) (DL-PLA) was obtained bearing one embedded disulfide bond (PLA(SS)). In order to complete the triblock copolymer synthesis, mPEG with hydroxyl termini were linked to either terminus of the amorphous PLA diol by a 2:2:1 molar reaction between mPEG diisocyanate and PLA(SS), affording urethane bridges between PEG and PLA polymer blocks with an overall PEG-PLA-SS-PLA-PEG copolymer structure (Figure 2B).

solutions prepared thereof.

*2.2. Synthesis* 

*Gels* **2021**, *7*, x FOR PEER REVIEW 3 of 12

jectable sol–gel biodegradable polymer solutions [6,11,25].

move from sol form to gel morphology, subsequent reports focused on the development of sol–gel transitions, which occurred upon heating of the polymer solution. The enhancement of sol-to-gel properties from relying on cooling (gel at low temperatures and sol at high temperatures) to being responsive to heating (sol at room temperature and gel at elevated temperature) was crucial, as the practical application of such gels relies on the incorporation of active agents in a room-temperature solution, which upon injection and exposure to physiological temperatures forms a semi-solid gel capable of extended release of the active agent. These sol–gel thermosensitive hydrogels were based on an amorphous PLA polymer block (as opposed to crystalline poly (L-lactic acid) polymer blocks employed in the original work). Gel properties, biodegradability, and drug release were all subsequently optimized, leading to over two decades of research and application of in-

The PEG-PLA-PEG triblock copolymer described here is an analog of the original work performed by Jeong and co-workers. Its chemical structure bears the modular hydrophobic and hydrophilic units at the appropriate ratios for effecting sol–gel transitions near physiological temperatures, with the added benefit of one embedded disulfide bond. In doing so, the 'smart' hydrogel was imbued with secondary responsiveness to reduction agents in addition to its thermal responsivity. The hydrogel resultant thereof was therefore capable of injection as a liquid at ambient temperatures, gelling upon moderate heating, and subsequent collapse of the 3D gel structure upon exposure to strong reducing agents [26]. The following is a description of the synthesis and characterization of the novel triblock copolymer with a comprehensive study of the gel properties of aqueous

**Figure 2.** (**A**) Synthesis by Jeong et al. of a PEG-PLA-PEG triblock copolymer with urethane linkages [24]. (**B**) Synthesis of PEG-PLA-PEG with urethane linkages between polymer blocks and one embedded disulfide bond. **Figure 2.** (**A**) Synthesis by Jeong et al. of a PEG-PLA-PEG triblock copolymer with urethane linkages [24]. (**B**) Synthesis of PEG-PLA-PEG with urethane linkages between polymer blocks and one embedded disulfide bond.

The PEG-PLA-PEG triblock copolymer described here is an analog of the original work performed by Jeong and co-workers. Its chemical structure bears the modular hydrophobic and hydrophilic units at the appropriate ratios for effecting sol–gel transitions near physiological temperatures, with the added benefit of one embedded disulfide bond. In doing so, the 'smart' hydrogel was imbued with secondary responsiveness to reduction agents in addition to its thermal responsivity. The hydrogel resultant thereof was therefore capable of injection as a liquid at ambient temperatures, gelling upon moderate heating, and subsequent collapse of the 3D gel structure upon exposure to strong reducing agents [26]. The following is a description of the synthesis and characterization of the novel triblock copolymer with a comprehensive study of the gel properties of aqueous solutions prepared thereof.

### *2.2. Synthesis*

In a similar fashion as the original work on PEG-PLA-PEG triblock copolymers, several synthetic steps were employed in order to incorporate a disulfide bond into the triblock copolymer structure. In the first synthetic step, 2-hydroxyethyl disulfide was used as the initiator of DL-lactide polymerization. In doing so, an amorphous DL-poly(lactic acid) (DL-PLA) was obtained bearing one embedded disulfide bond (PLA(SS)). In order to complete the triblock copolymer synthesis, mPEG with hydroxyl termini were linked to either terminus of the amorphous PLA diol by a 2:2:1 molar reaction between mPEG diisocyanate and PLA(SS), affording urethane bridges between PEG and PLA polymer blocks with an overall PEG-PLA-SS-PLA-PEG copolymer structure (Figure 2B).

### *2.3. Characterization*

The formation of PLA(SS) and PEG-PLA-SS-PLA-PEG polymers was confirmed spectroscopically by 1H NMR. Polymer molecular weights (MW) were estimated by sizeexclusion chromatography (SEC). The presence of a disulfide bond was confirmed by elemental analysis (CHNS).
