424 CHIMIA 2016, 70, No. 6 NCCR MoleCulaR SySteMS eNgiNeeRiNgdoi:10.2533/chimia.2016.424 Chimia 70 (2016) 424–427 © Swiss Chemical Society

*Correspondence: Prof. W. Meier, Prof. C. G. PalivanE-mail: wolfgang.meier@unibas.ch,cornelia.palivan@unibas.chUniversity of Basel, Department of ChemistryKlingelbergstrasse 80, CH-4056 Basel

Artificial Organelles: Reactions insideProtein–Polymer SupramolecularAssemblies

Martina Garni, Tomaž Einfalt, Mihai Lomora, Anja Car, Wolfgang Meier*, and Cornelia G. Palivan*

Abstract:Reactions insideconfinedcompartmentsat thenanoscale representanessential step in thedevelopmentof complex multifunctional systems to serve as molecular factories. In this respect, the biomimetic approachof combining biomolecules (proteins, enzymes, mimics) with synthetic membranes is an elegant way to createfunctional nanoreactors, or even simple artificial organelles, that function inside cells after uptake. Functionality isprovided by the specificity of the biomolecule(s), whilst the synthetic compartment provides mechanical stabilityand robustness. The availability of a large variety of biomolecules and synthetic membranes allows the propertiesand functionality of these reaction spaces to be tailored and adjusted for building complex self-organized systemsas the basis for molecular factories.

Keywords: Amphiphilic copolymers · Artificial organelles · Enzymatic reactions · Nanoreactors

Introduction

Biological cells are complex structureswith well-organized molecular machiner-ies that work with high precision and effi-ciency.[1] In order to develop novel artificialmolecular systems with similar complex-ity and multifunctionality, it is necessaryto understand and mimic at the molecularlevel the various reactions involved in cellmetabolism. Reactions inside the confinedspaces of nanocompartments represent thecore for the development of molecular fac-tories for use in various domains, either inthe biological environment (tissue, cells,etc.) or in environmental or technologicalapplications.An essential step in the devel-opment of complex systems in terms of ar-chitecture and functionality is the bottom–up approach in which different modulesare combined in a more complex topology.In this respect, knowledge of self-assemblyacquired in biological systems is exploitedin artificial architectures, because it is con-venient for understanding conformational

changes and functionality at the nanoscale,and serves for translational applications inthe domains of medicine, catalysis and en-vironment.[2,3] In particular, amphiphilicblock copolymers are ideal candidates fordesigning complex systems, because theyself-assemble in a variety of supramo-lecular 3D assemblies (micelles, nanopar-ticles, tubes, polymersomes) and planarmembranes.[4,5] In addition to nanostruc-tures, amphiphilic copolymers can alsoself-assemble in giant unilamellar vesicles(GUVs) as simple compartment modelswith µm-range sizes (Fig. 1).[6] However,control of self-assembly into the desiredsuprastructures is a challenging task. Theirarchitecture and size strongly depend onthe molecular properties of the block copo-lymer itself, such as hydrophilic-to-hydro-phobic ratio, total molecular weight (Mw)and polydispersity index (PDI),[7] and notall kinds of supramolecular nanostructurescan be applied as platforms for developingconfined reaction spaces at the nanoscale,such as nanoreactors, artificial organelles,or simple cell models. Polymersomes andGUVs are of particular interest in the de-sign of compartmentalized reaction spac-es, because their architectures mimic natu-ral biocompartments (cells, organelles). Inthis respect, these vesicular structures areable to simultaneously host biomolecules(proteins, enzymes, nucleic acids) in theirmembranes, at their surfaces, and insidetheir inner cavities, and thus provide aprotective space for complex reactions.[3]Compartmentalization is achieved by en-capsulating active compounds during theself-assembly process of vesicle forma-tion, whilst membrane proteins are insert-ed during or after vesicle formation. The

mild conditions for vesicle formation areessential for preserving the structural in-tegrity of biomolecule(s) and their activ-ity. Compared to phospholipids which arethe building block in cellular membranes,block copolymers have higher molecularweight and consequently form thicker andmore stable membranes. In situ reactionsin the cavities of polymersomes requirerapid exchange of substrates/productswith the environment, whilst keeping theactive compounds protected inside. Thus,membrane permeability plays a pivotalrole, and various methods have been re-ported to generate polymersomes withpermeable membranes: i) synthesis ofcopolymers that intrinsically form porousmembranes or membranes permeable toions, e.g. specific oxygen species, ii) poreformation with chemical treatments, andiii) permeabilization of membranes byUV-irradiation.[8–11] A completely differ-ent strategy for permeabilizing syntheticmembranes was developed in our group byapplying a biomimetic approach in whichmembrane proteins or biopores are insert-ed into the synthetic membrane, and servefor passive or active transport of moleculesthrough the membrane.[12] Reaction spacesbased on protein–polymer assemblies areat the early stage of research, but we havealready introduced models of syntheticfunctional systems that mimic artificialorganelles, and these will be further de-veloped and extended toward the design ofartificial molecular factories. The complexscenario of requirements needed in molec-ular factories will be achieved by interfac-ing multiple reactions at the nanoscale ina hierarchical self-organization to providethe necessary flow of molecules/signals.

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(2.5 nm in length) into the membranes ofpolymersomes and GUVs, we engineeredmembranes selectively permeable to pro-tons, Na+ and K+ ions (Fig. 3).[6] Indeedthe outflux of protons from GUVs upongA-insertion was proved by the change inthe fluorescence of the inner cavity of theGUVs (Fig. 3, right).[6]

In order to allow transport of mol-ecules, it is necessary to insert channelporins which allow diffusion of moleculeswith sizes smaller than the inner diameterof the pore. Outer membrane protein F(OmpF) was the first membrane protein wesuccessfully reconstituted with full func-tionality into PMOXA-PDMS-PMOXAtriblock copolymer membranes, and thisallowed diffusion of molecules up to 600Da.[17] In addition, we reconstituted FhuA,a channel protein with a large pore diam-eter of 39–46 Å elliptical cross-section,which ensured a rapid molecular flux intothe cavity of PMOXA-PDMS-PMOXApolymersomes.[18]

A step further was realized by geneticor chemical modification of OmpF withspecific molecules at key locations to in-fluence the pore permeability, substrate se-lectivity and to induce membrane respon-siveness. Such responsive synthetic mem-branes allow the design of nanoreactorswith triggered activity that depends on theexternal environment, whilst preservingthe vesicular architecture, and maintain-ing the encapsulated enzyme inside. (Fig.4).[19,20] Such nanoreactors with triggeredactivity represent ideal candidates whena specific step in the flow of a molecularfactory requires a reaction activated ‘ondemand’ by the presence of a stimulus.

Interestingly, functional insertion ofthe membrane protein aquaporin (AqpZ),a highly selective water channel, al-lowed us to develop synthetic membraneswith significantly higher water perme-ability than all commercial membranes.[21]Reconstitution of this membrane protein

synthetic membranes (Fig. 2). The expla-nation for this interesting behavior for themembrane proteins in synthetic thickmem-branes is the high flexibility of the PDMSdomain, which induces membrane fluiditysimilar to phospholipid bilayers.[15]

Reconstitution of membrane pro-teins/biopores to allow transport of ions/molecules is essential for reactions to beperformed in confined nanospaces, suchas nanoreactors and artificial organelles.In this respect, we were able to insert ina functional manner both biopores andmembrane proteins in synthetic flexiblemembranes based on PMOXA-PDMSand PMOXA-PDMS-PMOXA amphiphi-lic copolymers. For example, we insertedionomycin, a small ion transporter of 1.5nm diameter in membranes 6 times thickerthan its size. Interestingly, insertion ofionomycin rendered the polymersomepermeable only to Ca2+ ions, and resultedin synthetic membranes with selectivepermeability that might be necessary forspecific reactions in situ in polymersomecavities.[16] By successfully inserting thesmall pore forming peptide gramicidin

Bioinspired Permeabilization ofPolymer Membranes

Functional reconstitution of membraneproteins into synthetic membranes is com-plex, because they are normally denatur-ated in non-biological environments, anda key molecular factor is the membraneflexibility/fluidity for coping with theconformation and size of the membraneprotein.[14] However, we have shown thatpoly(2-methyloxazoline)-block-poly-(dimethylsiloxane) (PMOXA-PDMS) di-and tri-block copolymers generate poly-mersomes and GUVs by self-assemblyunder physiological buffer conditions withhigh flexibility and fluid membranes.[13]Although lateral diffusion properties ofvarious fluorescent-labeled membraneproteins (AqpZ, OmpF and KcsA) withinPMOXA-PDMS and PMOXA-PDMS-PMOXAGUVmembranes indicated a sig-nificant hydrophobic mismatch betweenthe membrane thickness (between 9 and13 nm) and the size of the proteins (3.5–5times), all these membrane proteins weresuccessfully inserted and functional in the

Fig. 1. Protein–polymer supramolecular assemblies and their use as reaction space at the nano-scale. Modified with permission from refs [3] and [13]. Copyright 2016 American Chemical Society.

Fig. 2. Left: Schematic representation of the hydrophobic mismatch between membrane thickness and membrane proteins (represented as greencylinder). Right: Relationship between membrane thickness, d, and diffusion coefficient, D, of polymer chains and AqpZ, OmpF, KcsA membraneproteins within self-assembled membranes from ABA34, ABA49, and ABA63 triblock copolymers. Modified with permission from ref. [15]. Copyright2015 American Chemical Society.

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responsive nanoreactors, in which a reac-tion starts under specific conditions, forexample a change in pH, or by irradiation.We developed a nanoreactor, which pro-duces ‘on demand’ reactive oxygen spe-cies (ROS) based on encapsulation of anefficient photosensitizer, Rose Bengal–bo-vine serum albumin conjugate (RB–BSA)in the cavity of PMOXA-PDMS-PMOXApolymersomes. Such nanoreactors wereable to produce ROS only when they wereirradiated with the appropriate wavelengthto induce singlet oxygen production bythe photosensitizer.[30] A step further wasachieved by simultaneously encapsulat-ing a set of enzymes that work in tandeminside polymersomes: the close spatialproximity and precise positioning of theenzymes leads to a highly efficient cascadereaction. When a nanoreactor is uptakenand preserves its activity inside cells it canbe considered as a simple artificial organ-elle that provides specific functionality tothe cell.[31]We introduced the first artificialperoxisome by co-encapsulating superox-ide dismutase and lactoperoxidase/catalaseinside the cavity of polymersomes. Uponuptake by THP-1/HeLa cells, this simpleperoxisome efficiently detoxified bothO

2– and H

2O

2.[32] Development of the ar-

tificial peroxisome using tandem enzymesfound in the natural peroxisomes opens a

form a mechanically more stable, but stillbiocompatible, polymer compartment.The role of the polymersome is to simul-taneously protect the encapsulated activecompounds from environmental condi-tions (e.g. proteolytic attack), and allowthem to act ‘in situ’. Polymer nanoreac-tors based on a single enzyme type havebeen developed with various proteins, suchas horseradish peroxidase,[11,20] nucleo-side hydrolase,[23] or acid phosphatase.[24]Interestingly, by encapsulating enzymeswith dual-functions, such as haemoglobin,it was possible to design multifunctionalnanoreactors to simultaneously transportoxygen and degrade peroxynitrites.[25]

Reactions inside nanocompartmentsshould be chosen to effectively meet thechallenges of the desired applications. Forexample, in the case of oxidative stressinvolving reactive oxygen species in highlevels that can damage cells, it is essentialto decrease their intracellular concentra-tion. We introduced a novel approach todetoxify superoxide radicals by designingnanoreactors with superoxide dismutase(SOD) or its mimics inside the cavity ofpolymersomes equipped with OmpF.[26–29]Another interesting approach for provid-ing appropriate conditions for the develop-ment of complex reaction systems, as inmolecular factories, is to design stimuli-

creates an opportunity for developing bio-hybrid desalination membranes that arenecessary for translational applications.

Nanocompartments as ReactionSpaces

The biomimetic approach for the for-mation of reaction spaces inside compart-ments (polymersomes andGUVs) involvesencapsulation of desired activemolecule(s)(biological or synthetic) together withmembrane permeabilization by reconstitu-tion of a membrane protein/biopore. Dueto the sensitive nature of encapsulated/in-serted biomolecules, it is essential to userelatively mild conditions, and to avoid or-ganic solvents and high temperatures. Oneof the first examples of NRs was based onencapsulating β-lactamase in liposomesto hydrolyze ‘in situ’ ampicillin to ampi-cillinoic acid, which was released fromthe nanoreactor through the OmpF pores.Interestingly, both OmpF and the encapsu-lated enzyme were not affected by intro-duction of methacrylate monomers into theliposome membrane to increase its stabil-ity by cross-linking polymerization.[17,22]Astep further was realized when completelysynthetic membranes based on PMOXA-PDMS-PMOXA copolymers were used to

Fig. 4. A) Cryo-TEM of OmpF equipped PMOXA-PDMS-PMOXA nanoreactors. Scale bar 100 nm. B) Schematic representation of pH responsiveOmpF. C) Substrate red conversion kinetics of nanoreactors equipped with different OmpFs: OmpF blocked with a pH responsive molecular cap(blue), OmpF with pH responsive molecular cap released (grey), Ompf-WT (black), and unpermeabilized nanoreactors (orange) at pH 5.5, at time 0(left) and after 1 hour (right). Modified with permission from ref. [20]. Copyright 2015 American Chemical Society.

Fig. 3. Left: Design of polymersomes engineered for ion selective permeability based on gramicidin (gA) biopore insertion. Right: Change in fluores-cence intensity inside GUVs between a non-permeabilized membrane (A) and a gramicidin permeabilized membrane (B). Modified with permissionfrom ref. [6]. Copyright 2015 Elsevier.

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[7] D. E. Discher, A. Eisenberg, Science 2002, 297,967.

[8] S. M. Kuiper, M. Nallani, D. M. Vriezema, J.J. Cornelissen, J. C. van Hest, R. J. Nolte, A. E.Rowan, Org. Biomol. Chem. 2008, 6, 4315.

[9] D. R. Arifin, A. F. Palmer, Biomacromol. 2005,6, 2172.

[10] D. Grafe, J. Gaitzsch, D. Appelhans, B. Voit,Nanoscale 2014, 6, 10752.

[11] M. Spulber,A. Najer, K.Winkelbach, O. Glaied,M.Waser, U. Pieles, W. Meier, N. Bruns, J. Am.Chem. Soc. 2013, 135, 9204.

[12] C. Nardin, S. Thoeni, J. Widmer, M.Winterhalter, W. Meier, Chem. Commun. 2000,1433.

[13] F. Itel, M. Chami, A. Najer, S. Lörcher, D. Wu,I. A. Dinu, W. Meier, Macromol. 2014, 47,7588.

[14] A. M. Seddon, P. Curnow, P. J. Booth, Biochim.Biophys. Acta 2004, 1666, 105.

[15] F. Itel, A. Najer, C. G. Palivan, W. Meier, NanoLett. 2015, 15, 3871.

[16] M. Lomora, F. Itel, I. A. Dinu, C. G. Palivan,PhysChemChemPhys 2015, 17, 15538.

[17] C. Nardin, J. Widmer, M. Winterhalter, W.Meier, Eur. Phys. J. E 2001, 4, 403.

[18] M. Nallani, S. Benito, O. Onaca, A. Graff, M.Lindemann, M. Winterhalter, W. Meier, U.Schwaneberg, J. Biotechnol. 2006, 123, 50.

[19] S. Ihle, O. Onaca, P. Rigler, B. Hauer,F. Rodríguez-Ropero, M. Fioroni, U.Schwaneberg, Soft Matter 2011, 7, 532.

[20] T. Einfalt, R. Goers, I. A. Dinu, A. Najer, M.Spulber, O. Onaca-Fischer, C. G. Palivan, NanoLett. 2015, 15, 7596.

[21] M. Kumar, M. Grzelakowski, J. Zilles, M.Clark, W. Meier, Proc. Natl. Acad. Sci. USA2007, 104, 20719.

[22] A. Graff, M. Winterhalter, W. Meier, Langmuir2001, 17, 919.

[23] A. Ranquin, W. Versées, W. Meier, J. Steyaert,P. Van Gelder, Nano Lett. 2005, 5, 2220.

[24] P. Broz, S. Driamov, J. Ziegler, N. Ben-Haim,S. Marsch, W. Meier, P. Hunziker, Nano Lett.2006, 6, 2349.

[25] D. Dobrunz, A. C. Toma, P. Tanner, T. Pfohl, C.G. Palivan, Langmuir 2012, 28, 15889.

[26] F. Axthelm, O. Casse, W. H. Koppenol, T.Nauser, W. Meier, C. G. Palivan, J. Phys. Chem.B 2008, 112, 8211.

[27] V. Balasubramanian, O. Onaca, F. Axthelm,D. Hughes, M. Grzelakowski, W. Meier, C. G.Palivan, J. Optoelectron. Adv. Mater. 2009, 1,1083.

[28] V. Balasubramanian, M. Ezhevskaya, H.Moons, M. Neuburger, C. Cristescu, S. VanDoorslaer, C. Palivan, PhysChemChemPhys2009, 11, 6778.

[29] V. Balasubramanian, O. Onaca, M. Ezhevskaya,S. Van Doorslaer, B. Sivasankaran, C. G.Palivan, Soft Matter 2011, 7, 5595.

[30] P. Baumann, V. Balasubramanian, O. Onaca-Fischer, A. Sienkiewicz, C. G. Palivan,Nanoscale 2013, 5, 217.

[31] P. Tanner, V. Balasubramanian, C. G. Palivan,Nano Lett. 2013, 13, 2875.

[32] P. Tanner, O. Onaca, V. Balasubramanian, W.Meier, C. G. Palivan, Chem. Eur. J. 2011, 17,4552.

erarchical systems based on self-organizedarchitectures: more complex reactions willbe possible in such complex reaction to-pologies. The first steps towards achiev-ing such molecular complexity needed fordeveloping nanoreactors and simple artifi-cial organelles have been presented here.However, for the design of molecular fac-tories there are still major challenges thathave to be solved, such as controlling theflow of molecules between different reac-tion spaces, the interconnectivity of sys-tems with complementary roles, and theoverall architecture – multifunctionalityof the final system. Last but not least, theexpected high-added value of translationalapplications have to be considered and ap-propriately introduced into the complexityof the molecular factory design. Futurechallenges for the design of model artifi-cial cells and complex molecular factoriesinclude the design of systems capable ofdivision and reproduction, systems withmetabolic activity switched on and off ‘ondemand’, and systems that can synthesizeor release naturally occurring substances.

AcknowledgmentsThe authors acknowledge the financial sup-

port from SNSF, NCCR Molecular SystemsEngineering and the University of Basel. Dr.B.A. Goodman is thanked for editing the manu-script.

Received: March 19, 2016

[1] S. M. Rafelski,W. F. Marshall, Nature Rev. Mol.Cell Biol. 2008, 9, 593.

[2] G. Gunkel-Grabole, S. Sigg, M. Lomora, S.Lörcher, C. G. Palivan, W. P. Meier, Biomater.Sci. 2015, 3, 25.

[3] C. G. Palivan, R. Goers, A. Najer, X. Zhang, A.Car, W. Meier, Chem: Soc. Rev. 2016, 45, 377.

[4] K. Letchford, H. Burt, Eur. J. Pharma.Biopharma. 2007, 65, 259.

[5] J. Kowal, X. Zhang, I. A. Dinu, C. G. Palivan,W. Meier, ACS Macro Lett. 2014, 3, 59.

[6] M. Lomora, M. Garni, F. Itel, P. Tanner, M.Spulber, C. G. Palivan, Biomater. 2015, 53, 406.

new direction for complex multifunctionalsystems that can be regarded as ‘cellularimplants’ (Fig. 5).[31] Despite the fact thatnanoreactors and artificial organelles arein their early stage of research, they provethat nanoscience-based solutions are ableto support the development of molecularfactories. Once nanocompartments are en-gineered to perform complex functions,such as cascade reactions, energy conver-sion, signal transduction, minimal me-tabolism and synthesis of molecular com-pounds, they can be assembled into moreelaborate systems that perform as basicmolecular factories.

From Nanoreactors and ArtificialOrganelles toward more ComplexReaction Spaces

Synthetic compartments that combinepolymer membranes with tailored perme-ability and catalytically active moleculesrepresent essential building blocks for mo-lecular factories. Molecular factories willinvolve interfacing different types of reac-tion spaces, one of them being presentedhere (polymersomes/GUVs) in a con-trolled manner to obtain the final productresponse. Additionally, the existence of aplethora of enzymes and enzyme mimicsfor encapsulation allows several differenttypes of catalytic reaction (e.g. detoxifica-tion of reactive oxygen species, pro-drugactivation, etc.), depending on the desiredtranslational application. Further, poly-mersomes can be functionalized to exposeat their external surfaces specific molecu-lar moieties, which serve for targeting ap-proaches, or surface immobilization: theseexposed molecular entities will serve aspoints for specific interactions with biolog-ical systems (inter- or intra-cellular). In ad-dition, such external molecular functional-ity will serve for zipping vesicles, which isanessential step for developingcomplexhi-

Fig. 5. Schematic drawing of a simplistic artificial peroxisome (AP) (left). ROS detoxification ki-netics in cells under oxidative stress (right) without APs (a), and after incubation with APs (b).Modified with permission from ref. [31]. Copyright 2013 American Chemical Society.