Polyion Complex-Templated Synthesis of Cross-Linked Single-Enzyme Nanoparticles

Abstract

Single-enzyme nanoparticles (SENs), which encapsulate individual enzymes in a thin polymer network, offer exciting possibilities for stabilizing enzymes and tuning their activity, but most approaches to date rely on covalent modification of the enzyme. We introduce here a new approach to their synthesis in which a pre-prepared polymer synthesized by reversible additionfragmentation chain-transfer polymerization is first tethered to the surface via electrostatic interactions. Isothermal titration calorimetry and asymmetric flow field-flow fraction (AF4) in combination with multiangle light scattering (MALS) reveal weak binding of 2− 5 chains/enzymes. The strength of binding can be tuned based on the charge density of the bound polymer. AF4-MALS and smallangle X-ray scattering confirm the formation of a thin cross-linked shell around the enzyme following chain extension of this polymer in the presence of a bis-functional monomer. The mild conditions of this method of SEN formation, which avoids any covalent modification of the enzyme, result in no loss in activity on our model enzyme (glucose oxidase) and 4-fold increase in thermal stability. It offers much greater control over the chemistry of the SEN, which we demonstrate by incorporation of trehalose between the enzyme and cross-linked shell.

On Complex Coacervate Core Micelles: Structure-Function Perspectives

“…Interestingly, both anionic and cationic proteins can be encapsulated herein, since the micelles comprise both neutral-anionic and neutral-cationic diblock copolymers. Aiming not to perturb the native enzyme and to retain its activity without chemical modifications, Chapman and co-workers instead crosslinked the polymeric shell around the enzymes [ 117 ]. First, glucose oxidase (GOx; net charge of −8) was complexed with a cationic-neutral block copolymer to form C3Ms.…”

“…This can be achieved in various ways, for example, by raising the salt concentration to values above the critical ionic strength, so that the C3Ms disassemble [ 97 , 123 ]. Alternatively, the thickness and density of the encapsulating matrix can be reduced, for example, by surface-tethering (short) polymers or growing thin, polymer shells around single enzymes to generate single enzyme nanoparticles (SENs), rather than trapping enzymes statistically into larger aggregates [ 117 ]. SENs were produced from lipase [ 124 ], GOx [ 125 ], horseradish peroxidase (HRP) [ 118 ], myoglobin [ 124 ], and ferritin [ 126 ].…”

100th Anniversary of Macromolecular Science Viewpoint: Attractive Soft Matter: Association Kinetics, Dynamics, and Pathway Complexity in Electrostatically Coassembled Micelles

“…Such poor salt stability makes them unsuitable for use under physiological conditions, corresponding to an ionic strength around 140 mM. The stability can be improved to overcome this impediment to biomedical applications, through the incorporation of auxiliary homopolymers, , protein supercharging, tethering of charged polypeptides, complementary nonelectrostatic interactions, and cross-linking of the polymeric shell. , The impact of these strategies on exchange dynamics and loading efficiency is yet to be investigated in-depth.…”

Enthalpy of the Complexation in Electrolyte Solutions of Polycations and Polyzwitterions of Different Structures and Topologies

“…The complexation of PB and P + in saline media was monitored primarily by isothermal titration calorimetry (ITC), ,,, a sensitive and robust method to measure the complexation enthalpy (Δ H ) as demonstrated by Schlenoff et al in the case of interpolyelectrolyte complexation. ,, In addition, we investigate the influence on Δ H of the polymer structure, topology, and solution salinity. Using PBs of different architectures, we evaluated the effect of polybetaine topology on PB/P + complexation with hydrophilic polycations, such as PMOTAC and hydrophobic polycations exemplified by CatPVI.…”

“…The complexation of PB and P + in saline media was monitored primarily by isothermal titration calorimetry (ITC), 24,25,32,33 a sensitive and robust method to measure the complexation enthalpy (ΔH) as demonstrated by Schlenoff et al in the case of interpolyelectrolyte complexation. 28,34,35 In addition, we investigate the influence on ΔH of the polymer structure, topology, and solution salinity.…”