Artificial cells are biomimetic microstructures that mimic functions of natural cells, can be applied as building blocks for molecular systems engineering, and host synthetic biology pathways. Here we report enzymatically synthesized polymer-based artificial cells with the ability to express proteins. Artificial cells were synthesized using biocatalytic atom transfer radical polymerization-induced self-assembly, in which myoglobin synthesizes amphiphilic block co-polymers that self-assemble into structures such as micelles, worm-like micelles, polymersomes and giant unilamellar vesicles (GUVs). The GUVs encapsulate cargo during the polymerization, including enzymes, nanoparticles, microparticles, plasmids and cell lysate. The resulting artificial cells act as microreactors for enzymatic reactions and for osteoblast-inspired biomineralization. Moreover, they can express proteins such as a fluorescent protein and actin when fed with amino acids. Actin polymerizes in the vesicles and alters the artificial cells’ internal structure by creating internal compartments. Thus, biocatalytic atom transfer radical polymerization-induced self-assembly-derived GUVs can mimic bacteria as they are composed of a microscopic reaction compartment that contains genetic information for protein expression upon induction.

Through the use of surface-displayed horseradish peroxidase, this work explores the enzymatic catalysis of both bioRAFT and bioATRP to prompt polymer synthesis on the surface of S. cerevisiae cells, with bioATRP outperforming bioRAFT polymerization.

The masking of therapeutic antibodies by the installation of a blocking module that can be removed under certain physiological conditions, is becoming increasingly important to improve their safety and toxicity profile. To gain access to such masking units, we used chicken immunization in combination with yeast surface display and a competition‐based FACS screening campaign to obtain anti‐idiotypic single‐chain Fv (scFv) fragments. This approach promotes the identification of functional masking units, since specificity and high affinity do not necessarily guarantee a paratope blocking effect. This strategy was used to isolate a scFv masking unit for the therapeutic antibody 6G11 (BI‐1206), which is currently in clinical trials for the treatment of B‐cell lymphoma to block the inhibitory Fcγ receptor IIB (CD32b). N‐terminal fusion of the anti‐idiotypic scFv to the 6G11 light chain successfully abolished binding to FcγRIIB in vitro. For conditional activation, a cleavable linker for the tumor‐associated protease MMP‐9 was implemented. To improve demasking efficiency, the affinity of the scFv mask was attenuated through rational design. The substitution of one key amino acid in the masking scFv reduced the affinity toward the 6G11 paratope by factor 10 but still mediated 9800‐fold blocking of receptor binding. Proteolytic demasking allowed full recovery of therapeutic antibody function in vitro, supporting the concept of conditional antibody activation using this anti‐idiotypic binding module.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a V_L domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

Artificial cells are biomimetic microstructures that mimic functions of natural cells and find application, e.g., as microreactors, as building blocks for molecular systems engineering, and to host synthetic biology pathways. Here, we report enzymatically synthesised polymer-based artificial cells with the ability to express proteins. They are created by biocatalytic atom transfer radical polymerization-induced self-assembly (bioPISA). The metalloprotein myoglobin synthesises amphiphilic block copolymers that self-assemble into structures ranging from micelles over worm-like micelles to polymersomes and giant unilamellar vesicles (GUVs). The GUVs encapsulate cargo during the polymerisation, including enzymes, nanoparticles, microparticles, plasmids and cell lysate. The resulting artificial cells act as microreactors for enzymatic reactions and for osteoblast-inspired biomineralization, and could express proteins when fed with amino acids, as shown by the expression of the fluorescent protein mClover and of actin. Actin polymerises in the vesicles and alters the artificial cell’s internal structure by creating internal compartments. Thus, bioPISA-derived GUVs mimic bacteria as they are composed of a microscopic reaction compartment that contains genetic information which is able to express proteins upon induction. bioPISA not only is a powerful tool in the pursuit of artificial cells but also for the easy and highly efficient encapsulation of biological molecules under mild conditions and in biologically relevant media. Therefore, it could have significant implications for the development of biomaterials and drug-delivery systems, as well as for cell encapsulation, and the in-situ formation of nano-objects.

Innovatively utilizing surface-displayed horseradish peroxidase, this paper explores the enzymatic catalysis of both bioRAFT polymerization and bioATRP to prompt polymer synthesis on the surface of Saccharomyces cerevisiae cells, with bioATRP outperforming bioRAFT polymerization. The resulting surface modification of living yeast cells with synthetic polymers allows for a significant alternation of yeast phenotype, including growth profile, aggregation characteristics, and conjugation of non-native enzymes to the clickable polymers on the cell surface, opening new avenues in bioorthogonal cell-surface engineering.