3.1 IR and 1H-NMR
PA-PSH was synthesized as reported (Fig. S1) 19,20. PSH as well as the crosslink of PA and PSH was confirmed by FT-IR, the C=O stretching vibrations of terminal thio-propionyl groups in PSH and PA-PSH were appeared at 1736 cm-1 and 1732 cm-1, respectively (Fig. S2). Furthermore, the structures of PA and PA-PSH were confirmed by 1H-NMR. As shown in Fig.2a,b, the increased signals at 1.2 ppm and 6.0-6.5 ppm comparing with the spectrum of Poloxamer 407 (P407) was the protons from acryl and propylene oxide units of PA.
For disulfide crosslinked P407 (PSS), three peaks appeared on the GPC curve at 21.19, 22.23 and 23.84 min; for PSH, the shoulder peak at 21.29 min disappeared; and for PA-PSH, the peak at 24.01 min broadened largely and a remarkable shoulder peak at 25.34 min appeared, which resulted from the larger molecular weight products based on the thiol-ene reaction (Fig. S3), suggesting the successful synthesis of PA-PSH 20.
3.2 Sol–gel transition
As shown in Fig. 2c,d, at 25 °C, pH 3.5, PA-PSH solution was a transparent solution (Fig. 2c), while at 37 °C, pH 7.4 (simulation of physiological conditions), the copolymer solution turned into a transparent gel (Fig. 2d).
3.3 Submucosal injection of PA-PSH in-situ gel in resected porcine stomachs
As shown in Fig. 3a, although adequate mucosal elevations occurred in each group, the PA-PSH in-situ gel had longer duration time than that of PBS or glycerol. Moreover, there was no significant collapse of SFCs elevated by the PA-PSH in-situ gel over 1 h. In contrast, the elevation created with PBS and glycerol dissapeared quickly in 15 min. Therefore, the ability of mucosal elevation and maintenance exhibited a sequence PA-PSH in-situ gel > glycerol > PBS.
3.4 Creation of SFCs in living minipigs
As shown in Fig. 3b, each agents elevated the SFCs immediately after injection. However, the SFCs in glycerol group collapsed fastly in 15 min and disappeared in 30 min. Contrastly, the SFCs of the PA-PSH in-situ gel maintained a clear shape, height and size in 30 min. Moreover, the elevation of the PA-PSH in-situ gel could be witnessed even after 9 days with no obvious ischemia or ulceration, while the SFCs of the controls was hard to find (data not shown).
3.5 In vivo ESD procedures
As shown in Fig. 4a,b, the ESD procedure could be accomplished in early stage with the guidance of the mucosal elevation created by the methylene blue labeled PA-PSH in-situ gel, without heavy bleeding or perforation. All of the procedures were successful and all the minipigs were sacrificed on 9th day, and the stomachs were dissected with ulcers displayed at early healing stage.
3.6 Biosafety of PA-PSH in-situ gel
The in vivo biodegradation of PA-PSH in-situ gel was examined in minipigs (Fig. 4c). The protrusion of SFCs created by PA-PSH in-situ gel maintained integrity even on 9th day, only with the reduction in height and size for the reason of the degradation of the matrix. The degradation was attributed the hydrolysis of ester bonds in polyoxypropylene segments, and all the final products were biocompatible 21. Additionally, there was neither obviously epithelial damage nor inflammatory infiltration was observed in the injection site (Fig. 4d). After 9 days of injection, the postoperative site was healed and re-epithelialized (Fig. 4e). All these results suggested that the PA-PSH in-situ gel was a biocompatible submucosal injection agent.