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Stimuli-triggered activity of nanoreactors by biomimetic engineering polymer membranes Tomaz Einfalt, Roland Goers, Ionel Adrian Dinu, Adrian Najer, Mariana Spulber, Ozana Onaca-Fischer, and Cornelia G. Palivan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.5b03386 • Publication Date (Web): 02 Oct 2015 Downloaded from http://pubs.acs.org on October 7, 2015

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Nano Letters

Stimuli-triggered activity of nanoreactors by biomimetic engineering polymer membranes

Tomaž Einfalt1, Roland Goers1,2, Ionel Adrian Dinu1, Adrian Najer1, Mariana Spulber1, Ozana Onaca-Fischer1, Cornelia G. Palivan1*

1

Department of Chemistry, University of Basel, Klingelbergstrasse 80 CH-4056 Basel,

Switzerland 2

Department of Biosystems Science and Engineering, ETH Zürich, CH-4058 Basel,

Switzerland

1 ACS Paragon Plus Environment

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Abstract The development of advanced stimuli-responsive systems for medicine, catalysis or technology requires compartmentalized reaction spaces with triggered activity. Only very few stimuli-responsive systems preserve the compartment architecture, and none allows a triggered activity in situ. We present here a biomimetic strategy to molecular transmembrane transport by engineering synthetic membranes equipped with channel proteins so that they are stimuli-responsive. Nanoreactors with triggered activity were designed by simultaneously encapsulating an enzyme inside polymer compartments, and inserting protein “gates” in the membrane. The outer membrane protein F (OmpF) porin was chemically modified with a pH-responsive molecular cap to serve as “gate” producing pH-driven molecular flow through the membrane, and control the in situ enzymatic activity. This strategy provides complex reaction spaces necessary in “smart” medicine and for biomimetic engineering of artificial cells.

Keywords:

nanotechnology; polymersomes; confinement; membrane protein; polymer

chemistry; membrane permeability

In various scientific domains, such as medicine, technology or environmental sciences, there is a need to introduce new systems that can provide reaction spaces at the nano-scale with high space and time precision responses. For example, scientists have already anticipated that the design of molecular factories, such as artificial organelles, will provide a new frontier in medicine.1 These systems are required to support sensitive local diagnostic approaches and to minimize the doses of active compounds for patient-oriented solutions, to detoxify traces of pollutants traces when high purity water is required, or to offer specific products “on demand” in technological or catalytic applications.


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Inspired by biosynthetic processes in nature, researchers have become increasingly interested in designing reaction spaces based on supramolecular assemblies (dendrimers, polymer vesicles, PICsomes, or LBL capsules), with sizes in the nanometer range and containing active compounds so that they can function as nanoreactors.2 The advantage of nanoreactors is their ability to simultaneously protect catalysts from their environment, whilst preserving their activity inside the confined space of the supramolecular assembly. 2,3 Of particular interest in the generation of nanoreactors are polymer vesicles, named polymersomes, because of their greater stability than their lipid-based counterparts (liposomes),4 and the ability to modulate their properties (size, permeability, polarity, stimuli responsiveness) by changing the chemical nature of the copolymers or their functionalisation.5,6 In addition, their architecture offers three topologic regions: the inner cavity, the membrane, and the external interface with the environment, which support the simultaneous loading of both hydrophilic and hydrophobic molecules ranging from small weight molecules up to proteins.6 Polymersomes have been used either as single nanocompartments3,6,7 or as compartment-in-compartments8 by changing the chemical nature of the block copolymers or the encapsulated/co-encapsulated active molecules with the aim of developing model confined spaces for reactions (single enzyme3 or cascade reactions7,8). More recently, nanoreactors have been developed for translational applications, such as the production and release of desired compounds (antibiotics,9 other drugs10), detoxification of free radicals involved in oxidative stress,11 simultaneous degradation of peroxynitrites,12 storage of oxygen, and enzyme replacement therapy.13 A biomimetic approach was employed to design nanoreactors with tandem operation of enzyme-mediated reactions that acted as artificial organelles inside cells14 or as simple models of cell compartments.8 An essential molecular parameter for an in situ reaction inside the cavity of a nanoreactor is membrane permeability to allow transport of the substrates required for the reaction, and the


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products generated by the reaction. Various approaches have been used to produce permeable membranes, including: (i) selection of a copolymer, which forms an intrinsic permeable membrane,15 (ii) chemical modification of membranes to create pores,16,17 (iii) incorporation of stimuli responsive polymer chains to induce membrane permeability in the presence of a stimulus,18 and (iv) reconstitution of channel porins.3,9,19 However, none of these approaches produced selective membrane permeability of nanoreactors that resulted only in a passive exchange of small mass molecules with a cut-off determined by the pore. Recently, in an analogous manner to biological cell membranes, where various proteins serve for ion transport, or stabilization of the internal environment of the cell, the first examples of polymersomes with selective membrane permeability were introduced by inserting membrane proteins, such as aquaporin Z,20 or biopores, such as gramicidin21 and ionomicin.22 The ability of the membrane of such polymersomes to selectively allow transmembrane transport of specific ions, or molecules opens the possibility of their use in constructing nanoreactors with triggered activity, but up to now no such examples have been reported. To maximize their effectiveness, especially when they are intended to serve as artificial organelles or simple mimics of cells, an additional step in nanoreactor design should be triggered activity of encapsulated enzymes to respond in a controlled manner or to produce and release molecules “on demand”. Significant efforts have been made to obtain stimulusresponsive polymersomes for use as drug carriers with enhanced efficacy by providing a homogenous spatial distribution, and increasing the localization of drugs in desired regions through triggered release.23 Polymersomes serving as drug delivery carriers were designed to respond to specific stimuli, such as pH,24 temperature,25 light,26 enzyme27 or a combination of thereof.28 The release from the carrier has been mainly obtained by dissociation or degradation of polymersomes into polymer chains or small molecules. However this strategy involving the dissociation of the polymer supramolecular assembly upon the presence of


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stimulus(i) is not compatible with the concept of a nanoreactor, in which the enzymatic reactions should take place in situ, inside their inner cavity. There are a few examples of stimuli responsive nanoreactors (to light,29,30 or pH changes18,31), but they are either permeable only to reactive oxygen species,29,30 or lack controlled permeability, and have limited stability.18,31 Therefore alternative strategies are required to design stimuli-triggered nanoreators in which polymersomes both preserve their integrity, and an in situ reaction is triggered by the presence of a stimulus. Here, we describe nanoreactors with triggered activity based on the encapsulation of enzymes inside polymersomes with a membrane rendered selectively permeable by inserting chemically engineered channel porins to act as “gates”. The protein “gates” reconstituted in the polymersome membrane are partially closed, and open at different pH values: thus pHcontrolled enzymatic activity inside the nanoreactors is mediated by the blockage/free diffusion of molecules through the porin channel (Scheme 1). Our biomimetic approach is inspired by protein functionality that is commonly found in nature, as for example the pHgated M2 protein found in the capsid of the Influenza B virus, which has very low conductance at physiological pH values, but increases 50 fold at lower pH.32 We selected the change in pH as a model stimulus for our triggered nanoreactors, because this is an essential signaling factor that accompanies pathological conditions.33 In medicine differences in pH are exploited by specific diagnostics or therapeutics agents.34,35 Nanoreactors were generated by encapsulating a model enzyme, horseradish peroxidase (HRP), inside the inner cavity of poly(2-methyloxazoline)-block-poly(dimethylsiloxane)block-poly(2-methyloxazoline)

(PMOXA6–PDMS44–PMOXA6)

polymersomes

with

a

membrane in which modified Outer membrane protein F, OmpF served as a protein “gate”. This type of amphiphilic polymer self-assembles in dilute solutions, and depending on the


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hydrophilic-to-hydrophobic ratio, generates highly mechanically stable and flexible vesicles36 with membranes permeable only to reactive oxygen species and oxygen.11 In order to obtain a protein gate, OmpF was chemically modified with a “molecular cap” to be partially blocked/blocked/open depending on the pH values of the environment of the nanoreactor. Differences in accessibility of the OmpF pore for molecules (substrates and products of the in situ enzymatic reaction) represent key factors for triggering the activity of nanoreactors. Use of the bottom-up approach of combining polymersome membranes with protein gates allows the optimization of nanoreactors with respect to: (i) membrane permeability, (ii) the mass of the molecules transported, and (iii) overall nanoreactor functionality. In contrast to polymersomes with membranes containing genetically modified OmpF, which served for pH-sensitive release of a payload,37 our nanoreactors represent an additional step in terms of functionality, because of the controlled modulation of the in situ enzymatic reaction. Developments in the design of nanoreactors with triggered activity, such as our system, will support the production of complex reaction spaces at the nanoscale in which the functionality is modulated in an analogous manner to that in cell membranes, and ultimately in the creation of artificial cells. In addition, the strategy we introduce here will provide solutions for personalized medicine by a simple change of combinations of active compounds (enzymes, proteins, mimics) encapsulated inside the nanoreactors.


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Scheme 1. Concept of a nanoreactor with triggered activity by a chemically engineered protein “gate” inserted in a polymersome membrane. A change in pH induces the release of the sensitive molecular cap (green dots) from the protein “gate” allowing the entrance of substrates (red dots), and the release of the products of the enzymatic reaction (yellow dots).

OmpF has already been used in its wild type form (trimer with a pore diameter that allows transport of molecules < 600 Da38) to render permeable the membrane of PMOXA-PDMSPMOXA polymersomes, and to develop nanoreactors for various applications.3,9,14 In order to design a pH responsive “gate” that triggers the in situ activity of nanoreactors, we selected to chemically modify the OmpF pore by attaching a molecular cap to reduce accessibility of the pore at pH = 7.4 (Figure 1). The cap is cleaved when the pH decreases to 5.5, and the “open gate” allows diffusion of molecules, and initiation or increase in activity of nanoreactors depending on the degree of pore blockage.


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Figure 1. Molecular representation of the pH responsive OmpF.

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A. OmpF with a pH

responsive cap (red molecular region) in the “closed state”, and B. OmpF after release of the pH responsive cap, in the “open state”. Image shown is a static representation.

There are various methods to modify the pores of channel proteins in order to reduce transport, such as replacement of native amino acids with ones selected to allow chemical modification (e.g. cysteins), modification of the amino acid sequence via trans-splicing, or introduction of novel amino acids at the genetic level.39 Here we have chemically modified the OmpF pore as the most straightforward approach. OmpF contains a constriction site with two amino acid half rings, which creates an electrostatic field that modulates solute fluxes and pore properties.38,40 Diffusion of molecules through the OmpF pore is determined by six amino acid residues located inside of the constriction zone of the pore.41 As accessibility of the K16 and K46 residues located inside the pore cavity favors interactions with small molecular weight molecules, we expect a binding of the pH sensitive cap to the these residues. Such an approach to the design of a pH responsive OmpF, has the advantage of being able to bind different porin “caps” that can block the pore to different degrees depending on their properties.


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First the residues were coupled with levulinic acid by a simple sulfo-NHS/EDC reaction (Scheme 2), and unreacted levulinic acid was removed by filtration and dialysis. This step was necessary to introduce reactive carbonyl groups, which favor the binding of a pH responsive cap cyanine5-hydrazide (Cy5-Hydrazide). Acid-liable chemical bonds, such as acetal, orthoester, hydrazone, imine and cis-aconyl bonds are well known to introduce pH responsiveness, and their degradation or hydrolysis in acidic media leads to the release of attached molecules.42 Cy5-hydrazide was selected as the second part of the pH responsive linker, because its molecular weight (569.6 g/mol) matches with the pore size at the constriction region (Figure 1). In addition, as the fluorescence intensity of Cy5 is not influenced by a change in pH, we can use the Cy5 fluorophore for an efficient assessment of the “gate” functionality by fluorescence correlation spectroscopy (FCS) (see below).

Scheme 2. Chemical modification of wild type OmpF with a pH sensitive molecular cap (by coupling with levulinic acid and Cy5-hydrazide).

After coupling Cy5-hydrazide to the carbonyl groups (CA) and the formation of a pH responsive hydrazone bond, any unreacted Cy5-hydrazide was removed by centrifugal filtration. To avoid the possible formation of multimers, and sedimentation in a non9 ACS Paragon Plus Environment

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amphiphilic environment, OmpF was stored in 3% octyl-glucopiranoside (OG). However, as the presence of OG interferes with the purification of Cy5-hydrazide, further purification through dialysis was necessary to lower the OG concentration below the critical micellar concentration. In this way we removed residual Cy5-hydrazide entrapped in OG micelles, and avoided any influence of OG on polymersome formation. In order to prove the introduction of CA, we compared carbonylated OmpF (OmpF-CA) with: wild type OmpF (OmpF-WT), wild type OmpF with added Cy5-hydrazide (OmpF-WT-Cy5) and carbonylated OmpF with added Cy5-hydrazide (OmpF-CA-Cy5). After purification procedure all these subsets of OmpF were tested with 2,4-dinitrophenylhydrazine (DNPH), a specific reagent to assess the presence of carbonyl groups. Successful introduction of carbonyl groups was found for OmpF-CA and a value of 56.5+/-16.8 nmol carbonyl groups per mg of protein was obtained by UV-Vis spectroscopy (Figure 2a, SI). This number of accessible carbonyl groups correlates with the theoretical number of carbonylated lysine residues of 61 nmol per mg of protein (SI). In contrast, OmpF-WT, OmpF-WT-Cy5, and OmpF-CA-Cy5 did not react with DNPH either because of the lack of CA (OmpF-WT and OmpF-WT-Cy5), or successful binding of Cy5-hydrazide to CA groups, which blocked further reactivity in the case of OmpF-CA-Cy5.


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Figure 2. Characterisation of OmpF modification. a) carbonyl content determination of OmpF-CA, OmpF-WT, OmpF-CA+Cy5 and OmpF-WT+Cy5 protein pores. s determined by a DNPH protein carbonylation kit, and b.) Molecular brightness (CPM) of OG micelles with various OmpFs and/or free dye in 3% OG or PBS: (1) OmpF-CA+Cy5, 3% OG pH 7.2 (Dark blue), (2) OmpF-WT+Cy5, 3% OG pH 5.5 (Blue), (3) OmpF-WT+Cy5, 3% OG pH 7.2 (Dark red), (4) OmpF-WT+Cy5, 3% OG pH 5.5 (Red), (5) Cy5, 3% OG pH 7.2 (Dark grey), (6) OmpF-WT+Cy5, 3% OG pH 5.5 (Grey), and (7) Cy5 in 3% OG pH 5.5 (Black).

We also investigated the binding of Cy5-hydrazide to the carbonylated OmpF by native page (Figure S1, S2, SI). Lower electrophoretic migration of OmpF CA-Cy5 (35.9 ± 0.01 pixels) compared with OmpF-CA (37.7 ± 0.03 pixels) results from the trimer having a higher molecular weight when bound to Cy5-hydrazide. The modification did not affect the stability of the OmpF trimer, because no dissociation into mono or dimers was observed (Figure S2, SI). An important aspect of a functional pH-responsive protein gate is the release of its sensitive cap upon the pH change. To observe this we used fluorescence correlation spectroscopy (FCS), because it is able to assess the binding of fluorescent molecules to molecules with significantly higher molecular weight (protein, DNA, antibody), or to supramolecular assemblies (polymersome, nanoparticles),43,44 and their encapsulation/entrapment in such supramolecular assemblies.45 In addition, FCS has already been used to determine the release of small molecules through modified porins.37 FCS allows analysis of the binding of fluorescent molecules to molecules/assemblies with significantly higher molecular weight by comparing their diffusion property and molecular brightness (counts per molecule, CPM) in the free and bound states.45 In order to follow the release of Cy5 from OmpF-CA-Cy5 we compared the brightness of OmpF-CA-Cy5 with that


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of OmpF-WT+Cy5, and Cy5-hydrazide at different pH values, and in 3% OG to avoid aggregation and precipitation of OmpF. The presence of OG micelles complicated data interpretation because of interactions of dye molecules with the detergent micelles. Once solubilized in OG micelles, OmpF-CA-Cy5 had a diffusion time of τD = 350 ± 27 µs; both Cy5 in OG micelles with τD = 249 ± 34 µs, and OmpF-WT+Cy5 with τD = 266 ± 50 µs diffused similarly (Table S2, SI). Nevertheless, OmpF-CA+Cy5 micelles had a higher molecular brightness (CPM = 17 ± 1 kHz) compared to OmpF-WT+Cy5, (CPM = 4 ± 1 kHz) and therefore, on average, contained more dye molecules per micelle; this indicates additional interaction of the membrane protein with the dye, as expected for modified OmpFCA (Figure S4, Table S2, SI). A decrease of brightness from 17 ± 1 kHz to 12 ± 1 kHz was observed after keeping OmpF-CA-Cy5 at pH 5.5 for 1 h, which indicates cleavage of the pHresponsive hydrazone bond leading to the release of Cy5-hydrazide into the environment (Table S2, SI). Complete recovery of the free dye by diffusion was not possible due to the aforementioned interaction of the dye with the detergent micelles. No significant change in brightness was observed in OG micelles both for OmpF-WT-Cy5 and for Cy5-hydrazide at pH = 5.5 or pH = 7.2 (Figure 2B, Table S2, SI).

In order to generate nanoreactors with pH triggered activity, we simultaneously encapsulated HRP and reconstituted OmpF-CA-Cy5 during the self-assembly of PMOXA6–PDMS44– PMOXA6 copolymers by thin film rehydration method. This preparation method has the advantage of avoiding organic solvents, which are difficult to be remove completely, and can also affect the biomacromolecules or be problematic in a later application of this system in vivo.46 In addition, the film rehydration method is known as a mild method of enzyme encapsulation, as already reported for nanoreactors based on encapsulation of HRP.2 Also, we inserted OmpF-WT and encapsulated HRP in supramolecular assemblies of PMOXA6–


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PDMS44–PMOXA6 copolymers in order to create a non-pH responsive nanoreactor, and to compare it with the activity of that intended to be a pH trigger. PMOXA6–PDMS44–PMOXA6 copolymers were selected here because they have an appropriate hydrophilic-to-hydrophobic ratio, and have been reported to self-assemble into polymersomes with sizes of 100-200 nm.21,22 We were interested in establishing whether the insertion of modified or non-modified OmpF, as well as the decrease in pH, affects polymersome morphology and stability. Light scattering experiments indicated the formation of spherical nano-objects with a RH of 77 nm for assemblies containing OmpF-WT and HRP, and 79 nm for those with-CA-Cy5 and HRP. At pH 7.2 the structural parameter ρ (ρ = RG/RH) has values of 0.90 – 0.96 for un-permeabilised HRP-loaded polymersomes, OmpFCA-Cy5 permeabilised HRP-loaded polymersomes, and OmpF-WT permeabilised HRPloaded polymersomes, whereas at pH 5.5 ρ values are 0.96 - 1.05. These values are all close to 1.0, which is typical for hollow spheres; therefore the polymersome self-assembly process was not affected by the presence of HRP and/or OmpF(s) (Table 2, Table S1, SI). TEM and cryo-TEM micrographs indicate the formation of spherical structures, with radii around 70 nm at pH = 7.2, both after self-assembly of PMOXA6–PDMS44–PMOXA6 copolymers, and upon insertion of OmpF and encapsulation of HRP in the polymer assemblies (Figure 3 and Figure S3b, SI). Therefore, insertion of OmpF-WT / OmpF-CACy5 and simultaneous encapsulation of HRP did not affect the self-assembly process of PMOXA6–PDMS44–PMOXA6 copolymers into polymersomes. A slight decrease in size compared to un-permeabilised polymersomes was observed, as already reported for such copolymers.21,22 The decrease of pH to 5.5 neither affected the architecture nor the size of OmpF-WT- and OmpF-CA-Cy5 equipped nanoreactors (Table S1, SI). After 14 days OmpFWT- and OmpF-CA-Cy5-equiped nanoreactors still preserved their structures, and did not aggregate.


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Figure 3. Cryo-TEM micrographs of: A. nanoreactors without OmpF, B. nanoreactors with reconstituted OmpF-WT, and C. nanoreactors with reconstituted OmpF-CA-Cy5. Scale bar = 200 nm.

Table 1. Molecular characteristics of PMOXA6–PDMS44–PMOXA6 assemblies without and with reconstituted OmpF at pH 7.2. Mw = 4500 g/mol, PDI = 1.8 fhydrophilic = 25 %.

DLS/SLS Nano-assemblies

Rg

Rh

[nm]

[nm]

95

106

0.90

77

80

0.96

79

84

0.95

ρ = Rg/Rh

HRP-loaded nano-assemblies HRP-loaded nano-assemblies with OmpF-CA-Cy5 HRP loaded nano-assemblies with OmpF-WT

In order to evaluate the insertion and number of OmpF gates/polymersome (OmpF-CACy5/vesicle) we used FCS. FCS autocorrelation curves show a significant difference in τd values between freely diffusing Cy5-hydrazide (τd = 67 µs), OmpF-CA-Cy5 in 3% OG micelles (τd = 266 µs) and polymersomes with reconstituted OmpF-CA-Cy5 (τd = 2914 µs) 14 ACS Paragon Plus Environment

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(Figure 4). The large increase in τd of OmpF-CA-Cy5-equipped polymersomes indicates that the modified protein gate was inserted in the polymer membrane. Furthermore, we calculated a mean value of 55 OmpF-CA-Cy5 pores per polymersome by comparing the molecular brightness (CPM) of OmpF-CA-Cy5 in OG micelles (CPM = 17+/-1 kHz) with the molecular brightness of OmpF-CA-Cy5 equipped polymersomes (CPM = 945 +/-33 kHz).

Figure 4. FCS autocorrelation curves 100 nM Cy5-hydrazide in PBS (Black), OmpF-CACy5 in 3% OG (Red) and OmpF-CA-Cy5 in the membrane of polymersomes (Blue). Experimental autocorrelation curves (dotted line), and their fit (full line). Curves normalized to 1 to facilitate comparison.

The hydrodynamic radious (RH) of the OmpF-CA-Cy5 nanoreactors at pH 7.2 was calculated by Stokes-Einstein equation as 47+/- 4 nm, in agreement with the values obtained from TEM, Cryo-TEM, and light scattering experiments.


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A crucial point in demonstrating that a nanoreactor has triggered activity is to evaluate its activity as function of pH. In this respect we used two different enzymatic assays, the TMB (3,3’,5,5’- tetramethylbenzidine) colorimetric assay and the Amplex Red® fluorimetric assay. We selected these two enzymatic substrates (Amplex Red and TMB), because they differ in their molecular properties, as for example the charge or geometrical arrangement, and therefore are able to serve as models to asses pore closure towards different types of molecule. It has been already reported that the molecular influx inside OmpF pores strongly depends on the properties of the molecules passing through them, which support an interaction, especially with the constriction zone.47 Therefore pore closing of a modified OmpF is based on two factors, which have to be considered together: the closing of the pore due to modification with a specific molecular cap (for example in our case CA-Cy5), and interaction of the molecules penetrating the pore with the constriction region. First, free HRP was tested to establish whether exposure to pH 5.5 affects its activity. No significant change in HRP activity was observed between incubation at pH 5.5 and pH 7.4 when TMB and Amplex Red were used as substrates (Fig. S7, S8 SI);47 thus any change in HRP activity inside nanoreactors is due to a triggered diffusion of substrates through the modified OmpF pore. When TMB (predicted polar surface area = 52 A2, MW = 240 g mol-1, Log P = 3.40, neutral at pH 7.4) was the substrate used to penetrate the modified OmpF gate, a smaller enzymatic turnover (around 38%) was obtained in nanoreactors equipped with OmpF-CA-Cy5 at pH = 7.4, compared with the OmpF-WT nanoreactors at the same pH value (Figure 5). This decrease in in situ enzymatic activity of HRP for nanoreactors equipped with OmpF-CA-Cy5 indicates a partial closing of the pore, which decreases the molecular influx of TMB molecules. As TMB has Mw = 240g mol-1, it is expected that this partial closing of the OmpF pore is serving as a barrier, and thus will block molecules with higher Mw.


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At pH = 5.5, the activity of nanoreactors equipped with OmpF-CA-Cy5 increased by 23%, indicating successful hydrolysis of the hydrazone bond, and the release of the Cy5-hydrazide molecular cap. The increase in the influx of TMB through the pore resulted in an increase in in situ nanoreactor activity. This allowed a comparable transport through the pore to that with carbonylated OmpF, and therefore a similar value for the in situ enzymatic reaction rate.

Figure 5. TMB conversion kinetics measured at pH = 7.4 of nanoreactors prepared with different OmpF subsets: (1) unpermeabelised nanoreactors incubated at pH = 5.5 (Orange), (2) Unpermeabelised nanoreactors incubated at pH 7.4 (Green), OmpF-CA-Cy5 nanoreactors incubated at pH 5.5 (Blue), (3) OmpF-CA-Cy5 nanoreactors incubated at pH 7.4 (Grey), (4) OmpF-CA nanoreactors incubated at pH 5.5 (Red), (5) OmpF-WT nanoreactors incubated at pH 5.5 (Black), (6) OmpF-WT nanoreactors incubated at pH 7.4 (Blue).

The enzymatic turnover of the Amplex Red substrate (predicted polar surface area 72 A2, MW = 257 g mol-1, Log P = 0.89, charge = -0.1 at pH 7.4) was drastically reduced (down to 14%), compared to OmpF-WT nanoreactors acting in similar conditions at pH = 7.4. The blockage of Amplex Red influx through the pore is considered to be due to a combination of effects: (i) closing of the pore by the molecular cap, and (ii) electrostatic interaction of Amplex Red with the molecular cap. Such interactions of charged molecules with the positively charged 17 ACS Paragon Plus Environment

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constriction region of OmpF have been already reported for ampicillin.48 After decreasing the pH to 5.5, the activity of nanoractors equipped with OmpF-CA-Cy5 increased to 74% in one hour. This indicates a successful pore opening, which allowed a significant increase in the influx of substrates through the pore (Figure 6). The slight increase in activity of the nanoreactors equipped with OmpF-CA with time is explained by the concentration gradient of the substrate at the beginning of the enzymatic reaction.

Figure 6. Amplex red conversion kinetics of nanoreactors equipped with different OmpFs: (1) unpermeabilised nanoreactors (orange), (2) OmpF-CA-Cy5 (blue), (3) OmpF-CA (grey), and (4) Ompf-WT (black) at pH 5.5, at time 0 (a) and after 1 hour (b).

Nanoreactors without inserted OmpF had a very low enzymatic turnover for both substrates at pH = 5.5, probably due to the presence of traces of HRP not removed by dialysis. As expected, no increase in activity was observed in nanoreactors without reconstituted OmpF at pH 5.5. This indicates that the permeability of polymersomes was not affected by the change in pH (Figure 5, Figure 6, Table S1, SI), in agreement with previous reports on the stability of PMOXA-PDMS-PMOXA polymersomes at different pH values.21 The difference in the decrease of the in situ activity of nanoreactors equipped with OmpFCA-Cy5 at pH = 7.4 as function of the molecular nature of the substrates results from a modulation of the influx through the pore due to the combined effect of pore closing and intermolecular interactions of the substrates in the “spatially reduced” constriction region. 18 ACS Paragon Plus Environment

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Whilst the influx of the neutral TMB is only reduced by the pore closing, that of the slightly negatively charged Amplex red is blocked by intermolecular interactions with the positively charged Cy5 flourophore. Our results are in agreement with previously reported data, in which mutations of key OmpF residues (K16 and K46) substantially altered the diffusion of molecules through the pore.47,48 Therefore, depending on the desired application, the “gate” can be partially or fully closed at pH = 7.4, and opened at pH = 5.5, resulting in a modulation of the enzymatic activity inside the cavity of nanoreactors. The decrease in the functional diameter of the OmpF pore defining the size exclusion limit for diffusion represents the driving force for a triggered in situ activity of these nanoreactors. Our stimuli-responsive switch is one-time stimulus responsive, especially designed to be released and open the pore upon a pH change. Reversible control of opening/closing the OmpF channel could be engineered by a gating moiety, near the constriction site of the protein, which should be covalently attached, stable in acidic pH, and present specific properties dependent on the pH of the environment. Ongoing experiments are dedicated to engineer this type of sensitive molecular cap, which could influence in a reversible manner the influx of substrates through the pore.

In cellular and subcellular environments, reaction catalysts (enzymes or small molecular weight compounds) are often protected in specific compartments where they act in situ when necessary, as for example in the presence of a stimulus.

We have now introduced a

biomimetic strategy for developing nanoreactors as protected reactions spaces at the nanoscale, and possessing triggered activity. Modulation of enzyme activity inside nanoreactors was successfully implemented by inserting chemically-modified channel proteins to act as pH-controlled “gates” in the membrane of enzyme-loaded polymersomes. Together with a preserved architecture of the polymersomes, the decrease/blocking of the


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flow of substrates through the membrane at neutral pH, and its unblocking at a lower pH resulted in very efficient stimulus-driven active nanoreactors. This system produces pHtriggered enzymatic activity, and therefore represents an additional step in the design of active systems capable of responding “on demand”. In addition, we present for the first time an example of chemical modification of OmpF that results in both tuning the pore cut-off size (from 600Da in the case of OmpF- WT to around 240Da for OMPF-CA-Cy5) and molecular selectivity. The design of nanoreactors with triggered activity opens new possibilities for advanced control of reactions at the molecular level, and is expected to have a significant impact in domains, such as medicine or controlled catalysis.

Methods OmpF expression, extraction The outer membrane protein F (OmpF) was expressed in BL21 (DE3) Omp8 Escherichia coli cells following a protocol described previously

34

with the following modifications: During

the purification OmpF was solubilized in a 3% Octyl-glucopiranoside solution (OG, Anatrace) to remove residual lipids. The detailed procedure is described in ESI. The extracted fraction was analyzed by 12% SDS-PAGE (CBB stained) to confirm the protein purity, and the protein concentration was determined using a BCA kit (Pierce Chemical Co, Rock- ford, USA). OmpF was stored at 4ºC in 3% OG at a concentration of 1.2mg/ml for several weeks.

OmpF modification OmpF was first coupled with levolunic acid. Levolunic acid was activated by sulfo/NHS and EDC (Sigma Aldrich) at pH 5.5 and then 75µL of 9.4µM activated levolunic acid was added to 600µL 1 mg mL-1 OmpF solution. OmpF was incubated with activated levolunic acid for 3h at pH 7.2. The OmpF-CA was immediately washed 10 times by 3% OG in PBS pH 7.2 in 20 ACS Paragon Plus Environment

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Amicon Ultra-0.5 mL centrifugal filters for protein purification and concentration, molecular cutoff: 30kDA (Millipore). The volume was adjusted to 475µL PBS pH 7.2 and 75µL of DMSO solution of Cyanine5-hydrazide (Cy5-hydrazide) (Lumiprobe) was added. The mixture was incubated in the dark for 24h at RT. The volume was adjusted to 1ml and the reaction mixture washed 25 times in Amicon Ultra-0.5 ml centrifugal filters with 3% OG to remove excess dye. The volume was adjusted to 500µL and the protein dialysed against 1L of 0.05 % OG. The protein concentration was determined by UV-Vis spectroscopy and adjusted to 0.5 mg mL-1 with 0.05% OG.

Carbonylation assay of OmpF A commercially available protein carbonyl content assay kit (Sigma Aldrich) was used to determine the carbonyl content of OmpF subsets. The method and calculations are described in detail in the SI.

Characterisation of OmpF A 20% SDS polyacrylamide gel was cast. OmpF-CA-Cy5 and OmpF-CA were mixed with BN-PAGE loading buffer and 15µL of the final OmpF solution was added to the gel. The gels were run at 300V for 6 hours. (Figure S1, SI)

Fluorescence correlation spectroscopy All FCS measurements were carried out using the ConfoCor2 instrument (Carl Zeiss, Germany) with a HeNe laser (633nm) using a 40x, 1.2 N.A. water immersion C-Apochromat objective lens. The measurements were carried out at room temperature using a sample volume of 20µl on a covered eight-well Lab-Tek chambered borosilicate cover glass (Nalage Nunc International). Measurements were recorded over 10sec and each measurement was


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repeated 30 times. The structural parameter and diffusion time of the free dye (100nM Cy5hydrazide) and measured probes were determined independently. The autocorrelation function was calculated using a software correlator and fitted with a one component fit (LSM 510 META-ConfoCor 2 System).

Preparation of pH triggered nanoreactors To produce pH triggered nanoreactors PMOXA-b-PDMS-b-PMOXA triblock copolymer films with different subsets of Outer membrane protein F (OmpF) were rehydrated with HRP. The detailed preparation technique and control experiments are described in the SI.

Characterisation of nanoreactors The size and morphology of the pH-triggered nanoreactors were characterized by a combination of light scattering (SLS, DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (Cryo-TEM) and fluorescence correlation spectroscopy (FCS). Quantification of reconstituted OmpF-CA-Cy5 was carried out with the FCS measurements. Detailed procedures are described in detail in the SI.

Enzymatic assay TMB The final nanoreactor solutions incubated at pH 7.4 or 5.5 were added to a preprepared TMB/H2O2 PBS solution at pH 7.4 and measured in 96-well plates. The absorbance of each well mixture was measured with a Spectromax M5e microplate reader (Molecular Devices) at 370 nm. The detailed procedure and calculation of the enzyme kinetics is described in the SI.


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Enzymatic assay Amplex red The emission fluorescence intensity was determined using a LS 55 Flourescence Spectrometer (Perkin Elmer). Samples containing the nanoreactors were excited at 535 nm and the emission intensity was monitored at 590 nm (15nm slits). Final nanoreactor solutions equipped with different OmpFs were adjusted to pH 5.5. Immediately and after 1 hour of incubation time, the samples were injected into a freshly prepared Amplex Red/H2O2 PBS solution and measured in PBS at pH 7.4. Fluorescence was expressed as arbitrary fluorescence units (AU) and was measured at the same instrument setting in all experiments. The detailed procedure and calculation of enzyme kinetics are described in the SI.

ASSOCIATED CONTENT Supporting Information Detailed experimental methods regarding the expression, purification and modification of OmpF, preparation and characterization of the nanoreactors, FCS data and enzyme kinetics data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. 23 ACS Paragon Plus Environment

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ACKNOWLEDGMENT We gratefully acknowledge the financial support provided by the Swiss Nanoscience Institute, the Swiss National Science Foundation, and the National Centre of Competence in Research Molecular Systems Engineering. TE thanks, S. Löcher, C. Edlinger (University of Basel) for fruitful discussions, and G. Persy (University of Basel) for TEM-measurements. Dr. M. Chami from the University of Basel, BSSE is acknowledged for the cryo-TEM experiments, and Dr. B.A. Goodman for editing the manuscript.

ABBREVIATIONS DLS,dynamic light scattering; DNPH, 2,4-Dinitrophenylhydrazine; FCS, Fluorescence correlation spectroscopy; HRP, Horseradish peroxidase; OG, Octyl-glucopiranoside; OmpF, Outer Membrane protein F; PMOXA-b-PDMS-b-PMOXA, poly(2-methyloxazoline)-blockpoly(dimethylsiloxane)-block-poly(2-methyloxazoline); SLS, Static light scattering; TMB, 3,3’,5,5’-Tetramethylbenzidine.

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