Preparation of sterile multi-component SA-SCS/PMCG microcapsules for implantation studies

Multi-component microcapsules (Fig. 3 and Supplementary Fig. ^S14) were prepared by employing the continuous 2-step protocol developed for preparation of SA-SCS/PMCG microcapsules³² with two multiloop reactors³³ connected in a series. The SA-SCS solution (0.9 wt. % SA and 0.9 wt.% SCS dissolved in saline, pH 7.4) was air-stripped into droplets at the flow rate of 0.564 g/min using a custom designed coaxial nozzle. The droplets first fell into the funnel of the first reactor which was filled with a cation gelling solution of 1 mM BaCl₂ and 50 mM CaCl₂ dissolved in either 0.3 M D-mannitol (Fig. 3) or in saline (Supplementary Fig. ^S15), both at pH 7.4 (flow rate 23.3 g/min). The gelling time for each droplet in the first reactor was 20 s. Upon exiting the first reactor, the formed SA-SCS microbeads were carried by the gelling solution into the funnel of the second reactor that was continuously filled with a solution of 2.87 wt.% PMCG in saline (flow rate 22.8 g/min). The resulting PMCG concentration in the second reactor was 1.58 wt.%. The gelling time in the second reactor was 50 s. Microcapsules were collected in 200 mL of saline to stop the complexation reaction. This collecting solution was replaced by a fresh solution every 1 min. Pooled microcapsules after all collections were washed three times in saline and exposed to 50 mM citrate solution in saline at pH 7.4 for 10 min. Microcapsules were then washed three times in saline and stored at 4 °C. All the used solutions were 0.22 μm filter-sterilized using the syringe filter (SA-SCS solution) and the 0.22 μm bottle-top filters (gelling, washing and citrate solutions).

CRM intensity profiles (equatorial cross-section) of a multi-component microsphere (SA/SCS-PMCG) after preparation (a–c), and after explantation from the intraperitoneal space of C57bl/6 mice 2 weeks post-implantation (d–f). (a,d) Optical image (bar equals to 200 µm). Spatial distribution of individual polymeric components within the entire microcapsule (b,e) and in the outermost region only (c,f) obtained from Raman signal normalized to the maximum intensity.