Abstract:

Enzymes are proteins that play critical roles in regulating metabolic pathways and maintaining homeostasis by catalyzing in vivo biochemical reactions with superior efficiency. While natural enzymes usually suffer from low stability in the ambient environment and high cost in preparation and purification, artificial enzymes (AEs) are synthetic molecules/materials that aim to achieve similar catalytic activities as their natural counterparts but circumventing the abovementioned drawbacks. Inspired by nature, metalloporphyrins (MPs) and their derivatives have been extensively studied as potential candidates for AEs due to the fact that they are ubiquitously found in some enzymatic hemeproteins, such as Cytochrome P450s and nitric oxide synthase. In our work, we aim to explore the versatility of using water-soluble MPs as AEs to catalyze a wide range of biochemical reactions, immobilize them in polymer-based carriers as nano/microreactors, and examine their inter/intracellular activity with mammalian cells. First, a variety of water-soluble MPs coordinated with different metal ions and solubilizing groups were designed and synthesized. Their enzymemimicking activities, such as the dealkylation of resorufin ethyl ether to form resorufin, the hydroxylation of coumarin to obtain 7-hydroxycoumarin, and the oxidation of L-arginine to produce nitric oxide (NO), were screened in buffer solutions with hydrogen peroxide (H2O2) as the oxygen donor. Then the active MPs were anchored to different biocompatible polymer-based carriers, such as micelles prepared from an amphiphilic block copolymer poly(cholesteryl methacrylate)-blockpoly(2-carboxyethyl acrylate) and hydrogels prepared from a polysaccharide sodium alginate, via covalent conjugation or encapsulation. The polymers not only functioned as a carrier for the AEs but also provided a shielding effect for the MPs from the oxidative degradation of H2O2, leading to improved stability. Finally, the inter/intracellular interactions of the nano/microreactors with mammalian cells were studied, focusing on different cellular functions, such as cellular communication and intracellular NO production. Our work represents a paradigm of using small organic molecule-based AEs as active components in nano/microreactors to perform enzymatic reactions and study their interactions with mammalian cells, which could be potentially useful for treating deficient cells with lost functions for therapeutic purposes.