# Synthesis of Covalently Cross-Linked Colloidosomes from Peroxidized Pickering Emulsions
## 2 Materials and Methods  
Styrene (St), butyl acrylate (BA), divinylbenzene (DVB) (Sigma-Aldrich, St. Louis, MO, USA), hexadecane (HD) (Alfa Aesar, Ward Hill, MA), 2,2'-azobis-isobutyronitrile (AIBN) (Aldrich, St. Louis, MO, USA) were used. Amphiphilic copolymers poly(2-_tert_-butylperoxy-2-methyl-5-hexen-3-ine-_co_-maleic anhydride) (PM-1-MA) or poly[N-(_tert_-butylperoxymethyl)acrylamide]-_co_-maleic anhydride (PM-2-MA) were synthesized as described elsewhere [28; 29; 30]. All monomers were purified by vacuum distillation. Other reagents and water (Milli-Q, 18 M\(\Omega\)) were used as received. Emulsion polymerization was carried out to synthesize peroxid oxidized latex particles. Briefly, 10 wt.% emulsions of styrene in an aqueous solution of PM-1-MAc (6-10 wt.% per styrene) at pH 10 or PM-2-MAc (1-7.5 wt.% per styrene) at pH 5.5-9.5 were prepared in a 50 mL single-neck round-bottom flask by magnetic stirring at 1200 rpm for 30 min. The emulsions were purged with argon under constant stirring for 20 min and subsequently placed in an oil bath heated to 85 \({}^{\circ}\)C. The polymerization was conducted under constant stirring to a conversion of 95%-98%. At the end of the reaction, the latexes were cooled and placed into a 5 mL dialysis bag (50 kDa molecular cut-off) to remove the non-grafted inisurf. The bag was submerged into a 2000 mL beaker filled with distilled water adjusted to pH 9.5 using NaOH. At intervals of 24 h, the water in the beaker was replaced to ensure a constant high concentration gradient to improve the dialysis process. After 14 days, the peroxididized latexes were removed from the dialysis bag and stored at 4 \({}^{\circ}\)C.  
The resulting polymer was then either (i) stored at 4 \({}^{\circ}\)C or (ii) precipitated by a freeze-thaw cycle and washed repeatedly with 0.1 N NaOH and hexane to remove non-reacted materials. The resulting polymer was collected, dried at room temperature, and stored at 4 \({}^{\circ}\)C to be used for further analysis. Volume mean particle size and zeta-potential measurements were performed in dilute aqueous dispersions of latex particles using Malvern Zetasizer Nano-ZS90 at 25 \({}^{\circ}\)C. The final numbers represent an average of a minimum of five individual measurements. A potentiometric titration (back titration using 0.1 N HCl as a titrant) of peroxididized latexes was used to calculate the amount of carboxyl groups (originated from PM-1-MAc or PM-2-MAc) on the surface of latex particles. A thermal analysis of peroxididized latexes was used to calculate the amount of peroxide groups on the surface of latex particles. For this purpose, latex samples (15 mg) were heated at an underlying heating rate of 10 \({}^{\circ}\)C/min to 400 \({}^{\circ}\)C in air using a TA Instruments Q500 (TA Instruments, New Castle, DE, USA). Gas-liquid chromatography was used to calculate the content of the peroxide monomer 2-_tert_-butylperoxy-2-methyl-5-hexen-3-ine (PM-1) units in both the copolymer PM-1-MA and the  
Figure 1: Schematic of the colloidosomes synthesis: (**A**) Aqueous and oil phases before homogenization; (**B**) Pickering emulsion; (**C**) Colloidosomes (polymeric shell covered with grafted peroxididized latex particles and solvent-filled hollow interior). peroxidized polystyrene latex particles. A quantitative analysis of the products of a complete decomposition of the PM-1 units was carried out using a Selmihrom chromatograph with columns (3 \(\times\) 3000 mm\({}^{2}\)) filled with an absorbent Inerton super (0.125-0.16 mm fraction) modified with 5 wt.% of Carbowax 40 M. The concentration of acetone and 2-methyl-2-propanol--the peroxide group decomposition products--was determined using chloroform as an internal standard. Pickering emulsions were formed by mixing 28 g of an aqueous phase (containing 0.3-1 wt.% of peroxid oxidized latex particles) with 2 g of an oil phase (consisting of HD, St and/or BA, DVB, and 0.06 g AIBN initiator).