Cyclic Polymers as Nanoscale Platforms for Enzyme Encapsulation and Transport

Cyclic polymers (CPs) make up a unique class of macromolecules characterized by their ring architecture without chain ends. Mimicking naturally occurring cyclic motifs, such as circular DNAs or viral plasmids, synthetic CPs, especially amphiphilic ones, can form supramolecular nanostructures capable of encapsulating small molecules, drugs, and nucleic acids. However, the interactions of CPs with proteins/enzymes have yet to be observed. Here, we demonstrate for the first time that poly(hydroxy butyrate)-based CPs can form nanoscale self-assemblies with a model enzyme, lysozyme, acting as enzyme/protein-stabilizing platforms. These assemblies are promoted when CPs interact with the enzyme under nonsolvent-induced phase separation conditions, displaying distinct hydrodynamic diameters (<i>D</i>_H), shapes, and surface charges that vary with the number-average molecular weight (<i>M</i>_n) of the polymer. The stability of lysozyme-CP nanoparticles depends strongly on the <i>M</i>_n of the CPs, with larger CPs forming nanoparticles at lower critical association concentrations. Encapsulation of lysozyme within the CPs does not alter the conformation or properties of the enzyme, as observed by circular dichroism spectroscopy. We also identify that CP nanoparticles can sustain the release/diffusion of the active protein, as evaluated by spectroscopy and the enzyme-linked immunosorbent assay. Lysozyme encapsulation in the CP was probed using site-directed spin labeling with electron paramagnetic resonance to pinpoint the presence of the enzyme spatially immobilized within the CP scaffold. Across all studied cell lines, nanoparticles composed of CPs exhibited minimal concentration-dependent cytotoxicity, regardless of the enzyme presence. While higher doses slightly reduced cell viability, it remained high at lower concentrations, indicating good biocompatibility. Meanwhile, CP nanoparticles encapsulating fluorescently labeled lysozymes promoted intracellular transport and cellular internalization of the enzyme. Collectively, this study is the first of its kind to report enzyme encapsulation within a CP scaffold, underscoring its potential in developing controlled-release enzyme/protein stabilization and transport platforms.