A microfluidic platform for the synthesis of polymer and polymer-protein-based protocells.

In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.

Asymmetry-Enhanced Motion of Urease-Powered Micromotors from Double Emulsion-Templated Microcapsules.

Autonomous motion of enzyme-powered motors has important implications for drug delivery, cell-cell communication, and protocell engineering. Although many of these systems are inspired by the motion of biological cells, most of them lack key structural features, like micrometer-sized boundaries and aqueous compartments, and rely on bubble propulsion to generation motion. In this study, we use droplet microfluidics to generate large populations of cell-sized microcapsules with poly(lactic-co-glycolic acid) shells and functionalize their surfaces with the enzyme urease to drive their motion. We adjust the number of surface functional groups for urease conjugation by preparing microcapsules with two different surfactants, poly(vinyl alcohol) (PVA) and poly(ethylene-alt-maleic anhydride) (PEMA). We also tune the surface roughness of the microcapsules by varying the concentration of silica nanoparticles in the droplet middle phase. We find that PEMA plays a crucial role in increasing the grafting density of urease on the surface of smooth microcapsules, leading to active motion in the presence of urea. In addition, rough microcapsules prepared with PEMA and loaded with comparable amounts of urease move up to three times faster than their smooth counterparts, which we believe is due to an asymmetric distribution of urease on the surface, giving rise to a preferred direction of motion. Taken together, these results provide new insights into the role that various stabilizing agents play in the induction of motion by enzymatic motors prepared from microfluidics, which is a potentially powerful tool for future preparation of motile protocells in biomedicine.

Low pKa of Lys promotes glycation at one complementarity-determining region of a bispecific antibody.

Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8-10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C-45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pKa on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.