The introduction of poly(ethylene dioxythiophene) (PEDOT)/poly(styrene sulfonate) (PSS) polyelectrolyte into giant unilamellar phospholipid vesicles (GUVs) and cross-linking with Ca2+ ions to generate a hydrogel within the internal Compartment are reported. The aqueous colloidal suspension of PEDOT with excess PSS was microinjected into the internal compartment of liposomes as well as networks of GUVs and lipid nanotubes. The subsequent introduction of calcium ions as cross-linking agent in order to induce hydrogel formation was achieved by three different methods: vesicle fusion, electroporation, and direct microinjection. Gel formation was probed by coinjection of fluorescent nanoparticles and tracking of Brownian motion. Particle mobility was shown to be distinctly reduced in the gel-filled vesicles. Diffusion constants for the particles were calculated from the projected movement of the particles and compared to particles in reference gels and solutions.