Incompatibilities encountered in multienzyme syntheses often arise from inhibition or inactivation of individual enzymes by low-molecular-mass compounds. Polymersomes have the tremendous yet unproven potential to enhance the performance of cascade reactions by spatial separation of enzymes from the respective source of incompatibility. A main challenge is the requirement to reduce mass-transport limitations across the polymer membrane with sufficient selectivity to maintain the compartmentalization. We demonstrate that cross-inhibitions in cascade reactions can be avoided by reconstituting highly selective channel proteins into the membrane. Thus, the three-step synthesis of CMP-N-acetylneuraminic acid was improved 2.2-fold compared to the noncompartmentalized reaction.

Polymer vesicles, so-called polymersomes, gain more and more attention as potential carriers for medical and biotechnological applications. To put the production of these nanocompartments into action at an industrial scale, an efficient and scalable process has to be established. Moreover, being able to control the resulting particle size distribution (PSD) is vital. In this work, the amphiphilic triblock copolymer poly(2-methyloxazoline) 15 -poly(dimethylsiloxane) 68 -poly(2-methyloxazoline) 15 is formed into polymersomes in miniaturized stirred-tank reactors. Varying flow conditions have a huge impact on the resulting PSD. Dynamic light scattering measurements show that driving a S-shaped stirrer at 4000 rpm in unbaffled reactors leads to a monomodal PSD with a low polydispersity index (PDI<0.2). Vesicles with a mean diameter of 200 nm are achieved within less than 1 h in a single production step. The robustness of the established process is shown by producing uniform polymersomes at different temperatures and varying pH and buffer molarities.

Polymersomes have some fundamental advantages compared to their liposomal counterparts. Due to the increased stability of the polymeric membrane, polymersomes are intended to be reasonably applicable as carrier‐systems and universal reaction compartments for diverse medical and biotechnological applications. Regardless of the application area, suitable methods to produce large vesicle quantities in a controlled and cost‐effective manner have to be developed to put polymersome technology into action at the industrial scale. In this work, the amphiphilic triblock copolymer poly(2‐methyloxazoline)15‐poly(dimethylsiloxane)68‐poly(2‐methyloxazoline)15 was formed into uniform polymersomes. A recently established production process, based on the use of miniaturized stirred‐tank reactors at the milliliter‐scale (12 mL), was successfully scaled‐up to the liter‐scale (1.5 L) based on solid process engineering parameters. Dynamic light scattering measurements show that using standard propeller stirrers with a dimensionless diameter dD−1≥0.65 in an unbaffled stirred‐tank reactor led to a narrow particle size distribution when providing a Froude number of Fr= 6.52 at the same time. Polymersomes with a mean diameter of 180 nm and a low polydispersity index (PDI<0.2) were generated within about 1 h in one single production step. Thus, this work provides the fundamental basis for further scale‐up purposes, regarding polymersome production in stirred‐tank reactors at the industrial scale.

BackgroundHollow vesicles formed from block copolymers, so-called polymersomes, have been extensively studied in the last decade for their various applications in drug delivery, in diagnostics and as nanoreactors. The immobilization of proteins on the polymersomes’ surface can aid in cell targeting, lead to functional biosensors or add an additional reaction space for multistep syntheses. In almost all surface functionalization strategies to date, a chemical pre-conjugation of the polymer with a reactive group or ligand and the functionalization of the protein are required. To avoid chemical pre-conjugation, we investigated the simple and quick functionalization of preformed poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) polymersomes through the spontaneous insertion of four hydrophobic, non-antibacterial peptide anchors into the membrane to display enhanced green fluorescent protein (eGFP) on the polymersomes’ surface.ResultsThree of the four hydrophobic peptides, the transmembrane domains of a eukaryotic cytochrome b5, of the viral lysis protein L and of the yeast syntaxin VAM3 could be recombinantly expressed as soluble eGFP-fusion proteins and spontaneously inserted into the polymeric membrane. Characterization of the surface functionalization revealed that peptide insertion was linearly dependent on the protein concentration and possible at a broad temperature range of 4–42 °C. Up to 2320 ± 280 eGFP molecules were immobilized on a single polymersome, which is in agreement with the calculated maximum loading capacity. The peptide insertion was stable without disrupting membrane integrity as shown in calcein leakage experiments and the functionalized polymersomes remained stable for at least 6 weeks.ConclusionThe surface functionalization of polymersomes with hydrophilic proteins can be mediated by several peptide anchors in a spontaneous process at extremely mild insertion conditions and without the need of pre-conjugating polymers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0205-x) contains supplementary material, which is available to authorized users.

The increased membrane stability of polymersomes compared to their liposomal counterparts is one of their most important advantages. Due to this benefit, polymer vesicles are intended to be used not only as carrier systems for drug delivery purposes but also as nanoreactors for biotechnological applications. Within this work, the stability of polymersomes made of the triblock copolymer poly(2‐methyloxazoline)15‐poly(dimethylsiloxane)68‐poly(2‐methyloxazoline)15 (PMOXA15‐PDMS68‐PMOXA15) toward mechanical stress, typically prevailing in stirred‐tank reactors being the most often used reactor type in the biotechnological industry, was characterized. Dynamic light scattering and turbidity measurements showed that stirrer rotation causing a maximum local energy dissipation of up to 1.23 W/kg−1 did not result in any loss of vesicle quality or quantity. Nevertheless, most probably due to local membrane defects, 6.6% release of the previously encapsulated model dye calcein was recognized at 25°C within 48 h. Moreover, increased temperature, leading to decreased membrane viscosity and increased membrane fluidity, respectively, led to a higher molecule leakage. Besides, the stability of polymersomes in two‐phase systems was investigated. Although alkanes and ionic liquids were shown not to lead to complete vesicle damage, no efficient calcein retention was achieved in either case.