Commonly, radicals and the reagents for radical polymerizations are considered cytotoxic. However, bacteria and yeast cells can withstand such challenging conditions. Even more, the microbes can catalyze radical polymerization starting from atom transfer radical polymerization initiators either by externally added metalloenzymes, by recombinantly expressed enzymes, or by endogenous enzymes of the cells. This opens up the possibility to engineer living cells on their surface and within their cytosol by biocatalytic radical polymerizations, thus opening up new possibilities for whole-cell biocatalysis, for communicating life-like biomaterials and synthetic biology systems, as well as for creating semi-synthetic engineered living materials.

The integration of polymer science and synthetic biology is revolutionizing biocatalysis by creating robust, recyclable, and adaptive catalytic systems. In this presentation, I will showcase our recent research on polymer-engineered living cells and enzymes, demonstrating how customized polymer strategies enhance biocatalytic performance and sustainability.

Our work begins with the development of protein–polymer conjugates, where catalytic polymers are chemically linked to proteins to form artificial polyenzymes [1]. These polyenzymes exhibit enhanced stability and reusability, facilitating complex reactions such as asymmetric aldol and click chemistry with improved efficiency and selectivity [1, 2].

Building on this foundation, we have engineered Escherichia coli (E. coli) cells with functional polymer coatings, transforming them into resilient and recyclable biocatalysts [3, 4]. By applying polydopamine and supramolecular polymers to E. coli cells, we strengthen the cells and enable them to perform reactions under harsh conditions [3, 4].

A pivotal aspect of our research is the integration of polymer photocatalysts with living cells (see Figure) [5]. Utilizing atom transfer radical polymerization (ATRP), we polymerize catalytically active species onto the surface of living E. coli cells. This polymer coating not only protects the cells but also combines polymeric catalysts with intracellular enzymes, enabling recyclable and active photoenzymatic cascade synthesis [5].

Throughout our studies, we have demonstrated the recyclability and adaptability of polymer-engineered biocatalysts across various processes [1-5]. These advancements highlight the transformative role of polymers in enhancing enzyme and cell-based catalysis, offering scalable and economically viable solutions for next-generation green chemistry and synthetic biology.

There are pro-fluorescent reagents that are used to label the thiol group of cysteines through a Michael addition reaction to a C-C double bond. These thiol-reactive probes show fluorescence switch-on during this reaction. These reagents include maleimide derivatives of BODIPY and coumarin as well as acrylic ester derivatives and acrylamide derivatives of coumarin. Even though maleimides, acrylates, and acrylamides are well-known to undergo radical polymerizations, only polycyclic aromatic hydrocarbon (PHA)-methacrylamides have been investigated as pro-fluorescent monomers in polymerizations. However, PHA-methacrylamides require high-energy excitation at 337 nm and substantial quantities of sodium dodecyl sulfate (SDS) to enhance solubility in the aqueous phase, which is harmful to living cells. Furthermore, conventional ATRP methods, rely on high concentrations of metal complex catalysts. Such limitations constrain the potential application of polymerizing pro-fluorescent monomers in biological contexts.
This work introduces enzymatic radical polymerizations combined with the pro-fluorescent monomer coumarin acrylamide (CAA; excitation wavelength: 406 nm) as a versatile technology platform (Figure 1a) for cellular imaging. Using bio-friendly monomers (such as DMA and NIPAM) as cosolvents and comonomers, instead of SDS, enhances the solubility of CAA in aqueous conditions. A strong blue-green fluorescence emission was obtained after polymerization. In live cells (Figure 1b-1e), the fluorescent copolymer exhibited robust imaging performance even after cell growth. Overall, we present the first demonstration of fluorescence switch-on during enzymatic radical polymerization and establish CAA fluorescent copolymer as a promising platform for long-term fluorescence retention in cellular imaging applications.

Compartmentalization and location of enzymes play an important role in the design and fabrication of biomimetic organelles and cells for carrying out spatiotemporal, dynamic and feedback-controlled enzymatic reactions (e.g. imitation of pH homeostasis in protocells or extracellular matrix therapeutics [1,2]). Thus, membrane characteristics are deciding to being successful in organelle and protocell functions which is a main focus in our working group.
This contribution will present the design and fabrication of enzyme-loaded polymersomes for their use as biomimetic lysosomes and macrophages [3,4] as well as hydrolytic surface-active compartments [5]. In many diseases there is a lack of enzyme and organelle functions. To overcome the problems of enzyme displacement therapy, therapeutic approaches focus on the integration of enzymes in various polymeric materials (e.g. micelles, microgels, nanoparticles or polymersomes). With the concept of membrane- and lumen-integrated trypsin and other enzymes in polymersomes we are able to showcase the functions of artificial lysosomes to degrade model pathogens and amyloidogenic peptides, known from Alzheimer disease, in body-like fluids, but also their integration and desired function in proteinosomes as biomimetic macrophages.

References
[1] E. Geervliet et al.: J. Controlled Release 2021, 332, 594.
[2] F. Rajabasadi et al.: Adv. Mater. 2022, 34, 2204257.
[3] X. Xu et al.: Adv. Sci. 2023, 10, 2207214.
[4] X. Xu et al.: Small Methods 2023, 2300257.
[5] D. Wang et al.: Adv. Funct. Mater. 2023, 2306904.

Artificial cells, serving as biomimetic microstructures, emulate the functionalities of natural cells, becoming building blocks in molecular systems engineering and serving as vessels for synthetic biology. We unveil the creation of polymer-based artificial cells, synthesized enzymatically, with a capability for protein expression. The construction of artificial cells was accomplished utilizing biocatalytic atom transfer radical polymerization-induced self-assembly (bioPISA). To this end, myoglobin facilitates the synthesis of amphiphilic block copolymers, which self-organize into various structures including micelles, worm-like micelles, and giant unilamellar vesicles (GUVs). Throughout the polymerization process, the GUVs encapsulate diverse cargo, encompassing enzymes, nanoparticles, microparticles, plasmids, and even cell lysate. Consequently, the formulated artificial cells function as microreactors, facilitating enzymatic reactions and osteoblast-inspired biomineralization. Furthermore, upon being supplied with amino acids, they are able to express proteins, including a fluorescent protein and actin. Actin polymerizes within the vesicles, modifying the internal structure of the artificial cells by forming a cytoskeleton mimic. Therefore, GUVs produced via bioPISA can emulate bacteria, constituting a microscopic reaction compartment that holds genetic information, enabling protein expression upon induction. Artificial cells can also be further equipped with internal compartments , imitating the eukaryotic cell subdivisions, or be produced instead as membraneless coacervates.

Mass cytometry (MC) is a cutting-edge bioanalytical technique used to analyze biomarkers in individual cells. MC uses metal-tagged antibodies (Abs) as reagents to detect these biomarkers. Current MC reagents employ metal-chelating polymers (MCPs) that bind to isotopes of hard metal ions. MCPs bearing chelators for soft metal ions offer the promise of a large increase in multiplexing capabilities, but examples reported so far often have unacceptably high levels of non-specific binding (NSB). One strategy to reduce NSB is through attachment of antifouling materials to MCPs. Zwitterionic trimethylamine N-oxide (TMAO) is a new class of ultralow fouling biomaterials. There is growing interest in expanding its applications in biological systems as a material for reducing NSB.

In this work, we present a straightforward synthesis of TMAO small molecules with primary and secondary amine groups. We also synthesized a polymeric TMAO with an amine end group through post-polymerization modification. These TMAO molecules were then conjugated to lysine based dipicolylamine polymers via an amide coupling reaction. Additionally, Pt²⁺ was incorporated into the polymer to form a Pt mass tag for MC. The NSB levels of the Pt-modified polymer were then evaluated by incubating the polymer with peripheral blood mononuclear cells.

We found that Pt polymers modified with small molecule TMAO molecules have poor solubility in water and were not suitable for applications in biological media. Pt polymers modified with poly(TMAO) show good solubility and are remarkably effective at suppressing NSB. The corresponding polymer-Ab conjugate was effective in identifying cell populations in suspension mass cytometry.