PAPER www.rsc.org/obc | Organic & Biomolecular Chemistry Enzymes containing porous polymersomes as nano reaction vessels for cascade reactions Suzanne M. Kuiper, Madhavan Nallani, Dennis M. Vriezema, Jeroen J. L. M. Cornelissen, Jan C. M. van Hest, Roeland J. M. Nolte and Alan E. Rowan* Received 3rd July 2008, Accepted 28th August 2008 First published as an Advance Article on the web 15th October 2008 DOI: 10.1039/b811196k Polystyrene 40 -b-poly(isocyanoalanine(2-thiophen-3-yl-ethyl)amide) 50 (PS-PIAT) polymersomes have the unique property of being sufficiently porous to allow diffusion of small (organic) substrates while retaining large biomolecules such as enzymes inside. Herein we report on the encapsulation and protection of glucose oxidase (GOx) and horse radish peroxidase (HRP) in PS-PIAT polymersomes and the successful employment of these functionalised nanoreactors in a cascade reaction. The demonstrated concept allows for further application in other enzymatic cascade reactions, bio-organic hybrid systems and biosensing devices. Introduction Cells consist of numerous different compartments that both physically and functionally separate vital biochemical processes. Communication and transport of molecules between these com- partments can occur via an active pathway, using membrane span- ning proteins, or a diffusional pathway. One approach to mimic this compartmentisation is by the use of lipid or amphiphilic block copolymer vesicles or within solgel matrices. 1–3 For biotechnology applications, polymer vesicles are superior to most of their lipid counterparts because of their higher mechanical and thermody- namical stability. 4 The drawback of this increased thermodynamic stability is that it is often more difficult for small substrate molecules, or even water, to diffuse in or out of the polymersome. A way to overcome this difficulty is by constructing stimulus- responsive membranes 5–9 that either decompose or reversibly open and close upon exposure, or by addition of channel proteins to the polymer membrane. 10–12 Another alternative, reported by Sukhorukov et al., makes use of a semi-permeable polyelectrolyte membrane. 13 This membrane allows small substrate molecules to enter and leave the capsules freely whilst retaining the larger biomolecules. Recently, we have reported on the synthesis and use of the diblock copolymer polystyrene 40 -b-poly(isocyanoalanine(2- thiophen-3-yl-ethyl)amide) 50 (PS-PIAT, Fig. 1) as a polymersome forming amphiphile. 14 This block copolymer consists of a rigid rod polyisocyanide headgroup and a flexible polystyrene tail, making it a rod-coil type of polymer. When injected into water, PS-PIAT readily forms nanometre sized polymersomes that have the unique property of being sufficiently porous to enable diffusion of low molecular weight molecules, whilst retaining large biomolecules such as enzymes inside. This porosity enables the enzyme to function as in solution, but now protected from a degrading environment, such as proteolytic Molecular Materials, Institute for Molecules and Materials, University of Nijmegen, Toernooiveld 1, Nijmegen, Gelderland, Netherlands 6500 GL Fig. 1 Chemical and schematic structure of PS-PIAT that forms vesicular aggregates when injected into water. enzymes and microbes, which are too large to penetrate the membrane pores. In addition, by the use of block copolymer lyophilisation followed by polymersome formation, it was demon- strated that different enzymes can be incorporated both in the central water pool and the membrane, respectively. This specific positioning enabled the efficient assembly of a cascade system in which horse radish peroxidase (HRP) and glucose oxidase (GOx) were chemically coupled within one polymersome. 15 This report further investigates the possibility of communica- tion between enzymes located in different polymersomes using the same GOx–HRP cascade reaction as mentioned above (Fig. 2). The first reaction in the cascade is the conversion of glucose by GOx into gluconolactone and H 2 O 2 . The formed H 2 O 2 then reacts further with HRP and added 2,2¢-azino-bis(3-ethylbenzthiazoline- 6-sulfonic acid) (ABTS) to form the ABTS radical cation. The cascade reaction can be easily monitored using UV spectroscopy due to a shift in the UV absorption spectrum from 340 nm to 405 nm when ABTS is oxidised to ABTS ∑ + . To the best of our knowledge, this is the first report on communi- cation between enzymes located in separate polymersomes without the addition of artificial transport mediators in the membrane or other external stimuli. Furthermore, it is a very easy and fast This journal is © The Royal Society of Chemistry 2008 Org. Biomol. Chem., 2008, 6, 4315–4318 | 4315