The use of giant vesicles as microreactors presents a novel approach to control biochemical reactions in confined spaces, offering advantages such as compartmentalization, tunable permeability, and potential for biomimetic applications. These constructs can serve as versatile platforms for catalysis, drug delivery, and synthetic biology by providing confined environments that mimic natural cellular compartments. We have successfully produced microvesicles (also referred to as giant vesicles) by means of the simple double emulsification method using five amphiphilic block copolymers comprising poly(ethylene oxide) (PEO) as hydrophilic segment and five disparate hydrophobic blocks: poly(caprolactone) (PCL), poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA), poly[2-(diisopropylamino)ethyl methacrylate] (PDPA), and poly[2-(heptamethyleneimino)ethyl methacrylate] (PHIA). The last two blocks are pH-responsive (PDPA, PHIA), while the first ones are not (PCL, PMMA, PLA). The resulting vesicles have average size ranging from 2.9 to 9.3 μm, with the pH-responsive vesicles exhibiting larger diameters, likely due to partial protonation of the hydrophobic blocks. The formation of the giant vesicles was confirmed via optical and fluorescence microscopy using Nile red as a hydrophobic marker. The ability of the vesicles to encapsulate larger molecules was demonstrated by loading Alexa-labeled bovine serum albumin (BSA-Alexa). Furthermore, the potential of these vesicles as microreactors was explored by encapsulating horseradish peroxidase enzyme (HRP) and evaluating the catalytic oxidation of o-dianisidine in the presence of hydrogen peroxide (H₂O₂), a reaction catalyzed by the HRP enzyme. The experimental data confirm that the pH-responsive vesicles are permeable to the reactants, as evidenced by colored product formation, whereas the permeability of the nonresponsive assemblies is negligible. The non-responsive vesicles exhibited particularly low permeability, even at the pH where the catalytic activity of the enzyme is optimum. These findings highlight the potential of pH-responsive vesicles for controlled molecular transport and catalytic applications, paving the way for their use in biocatalysis as microreactors.

Centro de Ciências Naturais e Humanas Universidade Federal do ABC Santo André Brazil

Centro de Ciências Naturais e Humanas Universidade Federal do ABC Santo André Brazil; Université Paris Saclay CNRS Laboratoire de Physique des Solides 91405 Orsay France

Equipe Chimie des Polymères Institut Parisien de Chimie Moléculaire Sorbonne Université Paris France

Institute of Macromolecular Chemistry Czech Academy of Sciences Prague Czech Republic

Institute of Macromolecular Chemistry Czech Academy of Sciences Prague Czech Republic; NanoBioMedical Centre Adam Mickiewicz University Wszechnicy Piastowskiej 3 61 614 Poznań Poland

Université Paris Saclay CNRS Laboratoire de Physique des Solides 91405 Orsay France

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