The bioPISA procedure was developed with biocompatibility and ease of use in mind. As such, we selected biocompatible chemicals according to the principles delineated by Matyjaszewski and coworkers, 51 and the reaction steps were simplified as much as possible. Mb was selected as biocatalyst because it is a small, robust and unimeric protein that functions as ATRPase at a biologically relevant pH of 7.4 (Figure ), unlike Hb which requires acidic pH to become an ATRPase. Moreover, Mb also displays peroxidase activity, 52 which is of relevance for the preparation of enzymatically active self-assemblies, as discussed below. In a typical bioPISA, the hydrophilic macromolecular ATRP initiator poly(ethylene glycol) methyl ether 2-bromoisobutyrate (mPEG-BiB) was chain-extended with 2-hydroxypropyl methacrylate (HPMA), which is the most commonly used monomer for PISA. The reaction was carried out in a phosphate buffer solution at pH 7.4 with 100 mM of NaBr (PBS-Br) containing 5 v/v% DMSO, myoglobin (Mb) and the reducing agent sodium ascorbate (NaAsc) at 37 °C (Figure ). The monomer concentration was varied between 10 wt% and 25 wt%, and various degree of polymerizations (DP) were targeted by adjusting the initiator:monomer ratio between 150 and 400 (Figure ). The reactions were run for 4 h before they were stopped by opening the reaction mixtures to air. The formation of nanostructures was easily recognised with the naked eye as after 1 h the reaction mixtures were already opaque (Figure ). H NMR spectroscopy showed that after less than 2 h the monomer consumption usually plateaued, indicating the end of the reaction (Figure ). Moreover, kinetic data displayed the characteristic shape for a PISA process with a hastening of the monomer consumption at the onset of aggregation (Figure ). The Mb-mediated PISA proceeded faster than Mb-catalysed ATRP in solution, as PISA conditions compared to conventional solution polymerisation often lead to high monomer consumption in shorter reaction time. When a monomer concentrations of >10 wt% was used in the reactions, the final monomer consumption, except for high DP and lower wt% conditions, exceeded 90% which indicated near complete conversion (Table ). In contrast, when a lower concentration of monomer was employed (5 wt%), a lower monomer consumption occured (< 60%). At the same time, the reaction for high DPs (i.e. lower initiator concentration) also yielded low monomer consumption which established that a certain threshold of monomer and initiator concentration was necessary for the reaction to reach high yields. bioPISA from a small molecule initiator (2-hydroxyethyl 2bromoisobutyrate) led to the formation of a macroscopic precipitation. This is certainly a consequence of the absence of mPEG as hydrophilic block in the forming polymers, which acts as stabilising hydrophilic corona for the forming self-assemblies. One of the key features of PISA is that it allows to tailor the morphology of the resulting structures by varying parameters such as the degree of polymerisation (DP) of either block or the monomer concentration in the reaction mixture. The case of bioPISA is no different, as the aimed DP of polyHPMA and the concentration of monomer had a clear effect on the morphology, as analysed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). These characterisations allowed to establish a phase diagram representing the morphology of the self-assembled structures as a function of the aimed DP and the initial monomer concentration (Figure and Figure S5-Figure ). Spherical objects with an average diameter of 227 nm, as determined from TEM, were obtained at DP 150 and 10 wt%. This size was confirmed by DLS analysis with a hydrodynamic diameter (Dh) of 238 ± 22 nm (Figure ). Worm-like micelles were obtained at DP 150 and 200 with ≥15 wt% and 15-20 wt% of monomer, respectively. The worm-like micelles had an average diameter of 73 ±15 nm as measured by TEM. Interestingly and in accordance with what has been reported before for worm-like micelles synthesised by PISA, the reaction mixtures containing worm morphologies thickened and became selfstanding gels (Figure -Figure ). Increasing the DP between 200 and 300, we could observe an intermediate region of mixed structures, between spheres, worms, and proper vesicles (polymersomes), as longer polymers could self-assemble into diverse shapes; however, at even higher DP and lower wt% (above 300 and below 20, respectively), the polymerization yield was <60%, with either mixed or spherical morphologies. Vesicles (polymersomes) were obtained with DPs of 250, to 400 at monomer concentrations of ≥20 wt%. The diameter of the polymersomes ranged from a few hundred nanometres up to several micrometres, as imaged by TEM and confocal laser scanning microscopy (CLSM) (Figure ). This broad range of sizes meant that it was not possible to fully analyse the suspensions by light-scattering. The membrane of the polymersomes is clearly visible in the TEM images with a thickness of 50 ± 11 nm. The micrometre-sized polymersomes are giant unilamellar vesicles (GUVs) and therefore ideally suited as shell of an artificial cell.