Hollow capsules with multi-responsive valves for controlled enzymatic

Subscriber access provided by University of Sunderland

Article

Hollow capsules with multi-responsive valves for controlled enzymatic reactions Xiaoling Liu, Dietmar Appelhans, and Brigitte Voit J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b07980 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Hollow capsules with multi-responsive valves for controlled enzymatic reactions Xiaoling Liu†,‡, Dietmar Appelhans*,‡, Brigitte Voit*,‡,§ †

College of Polymer Science and Engineering, Sichuan University, 610065 Chengdu, P. R. China Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Straße 6, D-01069 Dresden, Germany § Organic Chemistry of Polymers, Technische Universität Dresden, D-01062 Dresden, Germany ‡

ABSTRACT: The current challenge for polymeric nanoreactors is to precisely control the membrane permeability between permeable, impermeable and semi-permeable at defined pH. Additionally, the synthetic methods are obstructed by tedious purification processes, especially when polymer multi-blocks are required in the membrane of capsule to achieve different responsiveness and functions. Here, we report a rapid one-pot synthesis of ABA-type triblock copolymer brushes on silica template via surface-initiated single electron transfer living radical polymerization (SI-SET-LRP). It is worth to note that there is no purification between the successive block formation steps, since each step is taken to fully translation within 20 min. After removing the template, hollow capsules with crosslinked membrane are obtained and have been used as multi-responsive nanoreactors for enzymatic reactions. Their membrane permeability is triggered primarily by temperature and secondarily by pH to allow controlled enzymatic reactions to be reversibly addressable between “permeable”, “semipermeable” and “impermeable” valve-like membrane status. These valvelike features highlight the significant potential of hollow capsules for example in the fields of synthetic biology and enzyme deficient disease therapeutic applications.

INTRODUCTION Recent advances in synthetic nanoreactors,1-4 mimicking enzyme regulatory circuitry outside of the cell, have demonstrated their potential utility in therapeutic applications ranging from self-regulating bioreactors to enzyme therapy.2,5-9 A primary focus is on nanoreactors used to provide enzymatic activity to support the synthesis of biologically active molecules missing in individuals suffering from enzyme deficient diseases.10 Polymeric hollow capsules are an ideal scaffold for use as nanoreactors due to their stable vesicular shape and their ability to house various enzymes. Over the years new thermo-,11-16 light-6 and pH-sensitive17-21 capsules and vesicles have been realized to use them as nanoreactors with selfregulating behavior. One of the limitations of current polymeric nanoreactors in this research field is the limited control over membrane permeability which is required for switching on and off enzymatic reactions at physiological pH.1,2,16 Membrane permeability should also be adaptable towards different physiological environments for the exchange of (bio)molecules. Thus, reactor systems would be more versatile if equipped with a multi-responsive valve to regulate enzymatic activity by controlling which substrates are presented to the enzyme without changing the shape or affecting the catalytic activity of the enclosed enzyme. This motivated us to search for hollow capsules with tunable membrane valve properties that would allow the membrane to be not only permeable or impermeable, but also semi-permeable, e.g. permeable only for substrates of specific sizes. Such stepwise switching of membrane permeability for polymeric capsules would open new possibilities in synthetic biology and systems biology to establish multi-

orthogonal-responsive membranes for spatially separated biological processes in multi-compartments. Among the current methodologies of constructing polymeric capsules, the template method using surface-initiated living radical polymerization (SI-LRP) from a particle surface is one of the most powerful approaches because of the robustness and versatility inherent to various LRP techniques.22-28 However, significant hurdles remain in the synthesis of multi-block polymer brushes from templates, as there are e.g. numerous purification steps,25,29 poor yields,25,30,31 low density polymer brushes25,28,30,31 and time-consuming polymerization.23,30,32 Recently we developed a novel surface-initiated single electron transfer living radical polymerization (SI-SET-LRP) approach for constructing copolymer brushes on a silica template in which full conversion was attained within 15 min of reaction time at ambient temperature.33 Here, we report the use of SI-SET-LRP to synthesize ABAtype triblock copolymer brushes with multi-responsive characteristics on a silica template in a facile, rapid and one-pot manner (Scheme 1). The A block consists of poly(Nisopropylacrylamide) (PNIPAM) chain, while the B block is a poly[2-(diethylamino)ethyl methacrylate-co-2-hydroxy-4(methacryloyloxy) benzophenone] (PDEAEMA-co-PBMA) chain. It is worth noting that there is no purification steps required during the synthesis of triblock copolymer brushes, since each block is accompanied by full monomer conversion within shortly defined period. After photo-crosslinking the polymer brushes and removal of the core template, a hollow capsule with a tunable valve-like membrane (permeable, impermeable and semi-permeable) is obtained. The resulting

1 ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

multi-responsive capsule functions as a novel enzymatic nanoreactor to reversibly control enzymatic reactions by adjusting temperature and pH. Temperature acts as a main valve control to reversibly switch “on” and “off” the enzymatic reaction, while pH changes function as a secondary sub-valve control to further regulate the enzymatic reaction in a “fully-on” and “half-on” manner. The development of these hollow capsules equipped with valve-like characteristics is not only beneficial for enzyme deficient therapeutic applications but also for other applications (e.g. synthetic biology and systems biology).

Scheme 1. Schematic representation of the formation of a pH and temperature dual-responsive hollow capsule with ABA-type triblock copolymer brushes by sequential in situ chain extensions via surface-initiated single electron transfer living radical polymerization (SI-SET-LRP). The A block as inner and outer shell consists of poly(Nisopropylacrylamide) (PNIPAM) chain, while the B block as intermediate membrane is a poly[2-(diethylamino)ethyl methacrylate-co-2-hydroxy-4-(methacryloyloxy) benzophenone] (PDEAEMA-co-PBMA) chain. EXPERIMENTAL SECTION Materials. All reagents and solvents were purchased from commercial suppliers and used as received unless otherwise noted. 3-(Ethoxydimethylsilyl) propylamine, 2bromoisobutyryl bromide, triethylamine, copper(I) bromide (CuBr), tris[2-(dimethylamino)ethyl]amine (Me6TREN), 2(diethylamino) ethyl methacrylate (DEAEMA), ethyl αbromoisobutyrate (EBiB), monosodium phosphate, disodium phosphate, hydrofluoric acid (HF), ammonium fluoride (NH4F), D-maltose monohydrate (Mal), rhodamine B isothiocyanate, sodium hydroxide (NaOH), hydrochloric acid (HCl), glucose oxidase from Aspergillus niger (GOx, lyophilized powder), myoglobin from equine skeletal muscle (Myo, essentially salt-free, lyophilized powder), guaiacol and D-(+)glucose were purchased from Sigma-Aldrich. 2-Hydroxy-4(methacryloyloxy) benzophenone (BMA, 99%) was purchased from Alfa Aesar. The monomer N-isopropylacrylamide (NIPAM, Sigma, 97%) was purified by recrystallization from a n-hexane/toluene mixture (90/10 vol.-%). SiO2 particles (5 wt.-%; Æ 500 nm) were obtained from Microparticles GmbH (Germany). Dialysis tubes, made from regenerated cellulose (MWCO 150 kDa), were purchased from Carl Roth. High purity and resistivity (> 18 M Ω cm) deionized water (MilliQ water) was obtained from an inline Milli-Q Reagent Water Purification System (Millipore Corporation) and was used in

Page 2 of 11

all reactions, solution preparations, and polymer isolations. All other chemicals were obtained from Acros Organics, while anhydrous solvents were stored over molecular sieves. Methods. Gel permeation chromatography - The molecular weight distributions of the copolymers were measured at 40°C using a Polymer Laboratories PL-GPC50 Plus Integrated GPC system (Varian Inc., UK), equipped with a Polymer Laboratories pump, a PL ResiPore column (300 × 7.5 mm), a PL data stream refractive index detector, and a PL-AS-RT autosampler. The calibration was carried out using 12 polystyrene standards with Mn values ranging from 162 to 371,100 g mol-1 (Varian Inc., UK). The eluent was N,Ndimethylacetamide (DMAc), and the flow rate was 1.0 mL min-1. The data were processed using Cirrus GPC offline GPC/SEC software (version 2.0). NMR spectroscopy - 1H NMR spectra were recorded on Bruker Avance III 500 spectrometer operating at 500.13 MHz using CDCl3 as solvent at room temperature. Monomer conversions were determined via 1 H NMR spectroscopy by comparing the integrals of monomeric vinyl protons to polymer signals. Thermogravimetric analysis (TGA) - The samples were carried on a TGA Q 5000 instrument (TA Instruments). The samples were heated from room temperature to about 900°C at a heating rate of 10°C /min under a dry nitrogen atmosphere. Fourier transform infrared (IR) spectroscopy - The samples were carried out on a Bruker Equinox 55 Fourier transform infrared spectrophotometer, and the diffuse reflectance spectra were scanned over the range of 600-4000 cm-1 (resolution = 2 cm-1, 100 scans per measurement). The samples were mixed with potassium bromide powder (≈ 4 mg sample/500 mg KBr), while KBr powder is used as background. Photo-crosslinking of the polymer layers - The UV irradiation was performed using UVACUBE100 (honle UV Technologies, Germany) equipped with a low intensity (0.1 Wcm-2) iron lamp as UV source. Dynamic light scattering - The hydrodynamic size of capsules was determined by dynamic light scattering (DLS) using Zetasizer Nano-series instrument (Malvern Instruments, UK) equipped with a 633 nm He-Ne laser at fixed scattering angle of 173°. The data were analyzed using software version 6.12. Transmission electron microscopy - The diameters and morphologies of the capsules were observed using a transmission electron microscopy (TEM) Libra 120 equipped with a charge coupled device (CCD) camera at an accelerating voltage of 120 kV. 2 μL of the capsules or particles were dispersed in water with the concentration of 1 mg mL-1 and allowed to adsorb for 2 min. onto a 300 mesh, carbon film coated copper grid, and the specimen was dried at room temperature or the grid was blotted dry using filter paper. For the cryo-TEM measurement was conducted on the same instrument, except the sample was frozen in liquid ethane at -178°C. The blotting with the filter paper and plunging into liquid ethane was done in a Leica GP device (Leica Microsystems GmbH, Wetzlar, Germany). All images were recorded in a bright field at 172°C. UV/vis spectroscopy - The UV-Vis absorption spectra were recorded on a SPECORD 210 Plus (Analytic Jena, Germany). All investigations were performed in 1.5 mL semimicro cuvettes of PMMA (Brand, Germany). Fabrication of ABA-Triblock copolymer brushes on silica particles. Process for the synthesis of “first A block” PNIPAM (PN) from initiator-functionalized SP by SI-SETLRP - To a Schlenk tube fitted with a magnetic stir bar and a rubber septum, DMSO/H2O (1.5 mL, v/v 15 %) and tris[2(dimethylamino)ethyl]amine (Me6TREN, 0.0011 mmol) were


Page 3 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


charged and the mixture was bubbled with nitrogen for 30 min. In an additional flask, CuBr (0.0022 mmol) was dried for 30 min in vacuum and flushed with nitrogen before transferring the Me6TREN solution of the first flask to this flask. The mixture immediately became blue (CuII) and a purple/red precipitate (Cu0) was observed. Then the blue suspension with purple/red colored copper (0) powder was allowed for stirring at 480 rpm for 30 min. At the same time, DMSO/H2O (1.5 mL, v/v 15 %), N-isopropylacrylamide NIPAM monomer (0.65 mmol), and EBIB (32.0 μmol) were collected in another vial, fitted with a magnetic stir bar and a rubber septum. To this reaction mixture a DMSO/H2O (v/v 15 %) solution, containing the SI-SET-LRP initiator anchored silica nanoparticles 5 mL (20 mg/mL), was added. Then, the resulting mixture was bubbled with nitrogen for 30 min. After that, the degassed monomer/initiator solution was transferred through the septum to the Schlenk tube with Cu(0)/CuBr2/Me6-TREN catalyst under nitrogen protection. The Schlenk tube was sealed and the mixed solution was allowed to set at room temperature for the 20 min. Samples were taken periodically and conversions were measured using 1H NMR. An aliquot of the polymer grafted silica particles were dried and subjected to thermal gravimetric analysis (TGA) and IR. From the TGA, the graft density (σ) was estimated from the following equations: σ = (w/Mn)Av/(πR2), where w is the mass of polymer grafted on the SP, Mn is the number-average molecular weight of the grafted polymer, Av is Avogadro’s number and R is the diameter of the silica core. By calculating the σ values, the grafting density was determined to be about 0.66 polymer chain per nm2 (assuming the density of the silica nanoparticle 2.07 g/cm3). Process for the chain extension of PDEAEMA-coPBMA (PDB) based on PNIPAM grafted SP by SI-SET-LRP - After full conversion of the first block polymerization, a solution of DEAEMA monomer (0.325 mmol) and the crosslinker monomer BMA (0.0325 mmol) in 1.5 mL DMSO/H2O (v/v 15 %), previously degassed by nitrogen purging for 30 min, was directly transferred via cannula to the Schlenk tube under nitrogen protection. The reaction mixture was allowed to set at room temperature for 20 min. An aliquot of the copolymer grafted silica particles was dried and was subjected to thermal gravimetric analysis (TGA) and IR. Process for the additional chain extension of PNIPAM based on PNIPAMb-PDEAEMA-co-PBMA (PN-DB) grafted SP by SI-SETLRP - After full conversion of the second block polymerization, a solution of NIPAM monomer (0.65 mmol) in 1.5 mL DMSO/H2O (v/v 15 %), previously degassed by nitrogen purging for 30 min, was directly transferred via cannula to the Schlenk tube under nitrogen protection and polymerization for 20 min. The polymerization was stopped by in air, then the solution was centrifuged and the particles were collected. The cycle of centrifugation and redispersion in ethanol was repeated at least 5 times to ensure no free polymer remaining on the SP surface. An aliquot of the copolymer grafted silica particles was dried and was subjected to thermal gravimetric analysis (TGA) and IR. After dissolving the silica core, the polymer obtained was characterized by 1H NMR and GPC. From the TGA, the graft density (σ) was estimated from the following equations: σ = (w/Mn)Av/(πR2), where w is the mass of polymer grafted on the SP, Mn is the number-average molecular weight of the grafted polymer, Av is Avogadro’s number and R is the diameter of the silica core. By calculating the σ values, the grafting density was determined to be about 0.64

polymer chain per nm2 (assuming the density of the silica nanoparticle 2.07 g/cm3). Fabrication of hollow capsules. Photo-crosslinking of the polymer brushes on silica particles - After the polymerization mentioned above, the copolymer grafted silica particles were isolated from the solvent by centrifugation and placed in a 10 mL of flask with ethanol. The solution of particles was sonicated for 30 min before UV light crosslinking started. Then the solution was placed in the UV chamber equipped with a low intensity (0.1 W cm-2) iron lamp (UVACUBE 100, honle UV Technologies, Germany) and irradiated for 20 min. Preparation of the hollow capsules - The particle suspensions were mixed well by vortexing and transferred to Eppendorf tubes. The particles were worked up by one centrifugation/redispersion cycles with MilliQ water (100 μL). The template silica cores were then removed to fabricate hollow capsules by the addition of a hydrogen fluoride (HF) buffered to pH 7.3 with ammonium fluoride (NH4F). (Caution! Note that hydrogen fluoride and ammonium fluoride are highly toxic. Extreme care should be taken when handling HF solution and only small quantities should be prepared.) The samples were tapped gently to dissolve the silica cores for about 30 min. The excess NH4F, HF, and SiF4 were removed from hollow capsules by three centrifugation/redispersion cycles with MilliQ water, followed by re-suspending in a suitable volume (typically 0.5 mL) of MilliQ water. Reversible swelling-shrinking behavior of the crosslinked hollow capsules. The photo-crosslinked hollow capsules were incubated with phosphate buffer possessing pH 6 and 8, respectively. At each pH state, hollow capsules were allowed to stand for 20 min. Then the temperature of the hollow capsules was switched between 25°C and 40°C and the particles diameters were determined by DLS. This process was repeated for several cycles as shown in the main text. No less than 20 measurements were taken on each temperature. Pure enzyme activity of glucose oxidase (GOx) and myoglobin (Myo) in pH 6 and 8 at different temperature. Stock solutions of GOx (0.1 mg/mL in phosphate buffer (0.1 M) at pH 6 or 8, respectively), Myo (1 mg/mL in phosphate buffer (0.1 M) at pH 6 or 8, respectively), guaiacol (0.1 M in phosphate buffer (0.1 M) at pH 7.4) and glucose (1 M in phosphate buffer (0.1 M) at pH 7.4) were prepared. The investigations were performed at different pH values by dissolving the enzymes in phosphate buffer at pH 6 and 8. To investigate the influence of temperature, the sample was treated and measured at different temperatures (25°C, 40°C). To a GOx sample (250 μL), Myo (50 μL), guaiacol (8 μL) and glucose (8 μL) were added. After preparation of bienzymatic reaction, then UV-vis monitoring at 470 nm for quinone formation was started and data points were recorded every second. The activity was determined after 200 seconds. For normalized activity of enzyme activity (refers to the production of quinone) presented in the relevant figures, the highest value was fixed as 100% to normalize the other determined enzyme activities within one experiment series or repeating experiment series. The enzymatic activity for free GOx in the cascade reaction with free Myo is given by Supplementary Figure S7. Preparation of enzyme-filled nanoreactors. Preparation of GOx-filled nanoreactors - After mixing 4 ml of hollow capsules (= nanoreactors) aqueous solution (pH 6, 1mg/ml) with 4 ml of GOx phosphate buffer solution (pH 6, 2.5 mg/4 ml), the mixture was shielded from light and stirred for 2 days



at room temperature. Then pH of the solution was adjusted to 8.0 with 1M NaOH, and dialysis against phosphate buffer solution (pH 8.0, 0.01M) at 40°C was carried out for 3 days (MWCO of the dialysis membrane 150 kDa) to remove the non-encapsulated GOx by analyzing the activity of enzyme in dialysate using UV-vis spectroscopy. Preparation of Myofilled nanoreactors - After mixing 4 ml of hollow capsules (= nanoreactors) aqueous solution (pH 6, 1mg/ml) with 4 ml of myoglobin phosphate buffer solution (pH 6, 1 mg/4 ml), the mixture was shielded from light and stirred for 2 days at room temperature. Then pH of the solution was adjusted to 8.0 with 1M NaOH, and dialysis against phosphate buffer solution (pH 8.0, 0.01M) at 40°C was carried out for 3 days (MWCO of the dialysis membrane 150 kDa) to remove the non-encapsulated myoglobin by analyzing the activity of enzyme in dialysate using UV-vis spectroscopy. Bienzymatic reactions of GOx-filled nanoreactors with extracellular free Myo (Case I). An aliquot of 250 μL GOxfilled nanoreactors was used for the following experiments. 3 aliquots were taken at pH 8, while 3 aliquots were taken after a pH switch to pH 6 and three aliquots after switch back to 8. For an activity experiment, the sample was treated with Myo (50 μL), guaiacol (8 μL) and glucose (8 μL). The sample was stirred for 5 minutes, and the UV-vis monitoring at 470 nm for quinone started subsequently. Data points were recorded every second. For normalized activity of enzyme activity (refers to the production of quinone) presented in the relevant figures, the highest value was fixed as 100% to normalize the other determined enzyme activities within one experiment series or repeating experiment series. Bienzymatic reactions of GOx-filled nanoreactors with Myo-filled nanoreactors (Case II). The prepared solutions of Myo-filled nanoreactors and GOx-filled nanoreactors were combined in a ratio of 1:1. An aliquot of 300 μL enzyme-filled nanoreactors was used for the following experiments. 3 aliquots were taken at pH 8, while 3 aliquots were taken after a pH switch to pH 6 and three aliquots after switch back to 8. For an activity experiment, the sample was treated with guaiacol (8 μL) and glucose (8 μL). The sample was stirred for 5 minutes, and the UV-vis monitoring at 470 nm for quinone started subsequently. Data points were recorded every second. For normalized activity of enzyme activity (refers to the production of quinone) presented in the relevant figures, the highest value was fixed as 100% to normalize the other determined enzyme activities within one experiment series or repeating experiment series. Determination of the loading efficiency of enzyme for hollow capsules. The calibration curve was plotted by the determination of the change in absorption of different mass concentrations of myoglobin (mass concentrations of 1, 2, 4, 6, 8, 10 mg/L in phosphate buffer 0.1 M pH 6). Stock solutions of guaiacol (0.1 M in phosphate buffer 0.1 M pH 7.4) and H2O2 (1 M in phosphate buffer 0.1 M pH 7.4) were prepared. The standard calibration is given in Figure S8. To a sample of Myo-filled nanoreactors (purified) (300 μL, pH 6), both substrates guaiacol (8 μL) and H2O2 (8 μL) were added. After addition, the sample was stirred for 5 minutes at 25°C and the UV-vis monitoring at 470 nm started subsequently. After 5 minutes at pH 6 and 25°C, change in absorbance was calculated according to the Myo calibration. In total, three measurements were conducted and the statistical average was used for the discussion. The loading efficiency was calculated from

Page 4 of 11

equation: Loading efficiency of Myo = [(Absorbance x 8)/0.02235 x 10-3] x 100%. A summary of the determination of the loading efficiency for nanoreactors is given in Table S1. RESULTS AND DISCUSSION Synthesis of ABA-type triblock copolymer brushes on a silica template by SI-SET-LRP. The well-defined sequencecontrolled ABA-type triblock copolymers brushes structure was achieved easily by a template approach using sequential in situ chain extensions by SI-SET-LRP33 (Scheme 1). The “first A block” polymerization onto silica particles (SP, 500 nm diameter) was initially performed by employing the insitu, fast and complete disproportionation of Cu(I)Br in the presence of Me6Tren to yield insoluble Cu(0) and Cu(II)Br2 in DMSO/H2O,34 followed by addition of both the SET-LRP initiator anchored SP (25 µmol SET-LRP initiator/g) and monomer N-isopropylacrylamide (NIPAM). Although 1H NMR study showed nearly complete conversion after 15 min (Figure S1), the polymerization was conducted in DMSO/H2O at ambient temperature for 20 min with the final molar ratio [NIPAM]0/[initiator]0/[CuBr]0/[Me6TREN]0 of 260: 1: 0.8: 0.4. This resulted in the homopolymer PNIPAM (PN) as the “first A block”. Kinetic results (Figure S1) showed a linear relationship of monomer consumption versus time and a concurrent linear increase in molecular weights of formed polymer with a narrow molecular weight distribution (Ð ≤ 1.06). This clearly indicates a high degree of control over the “first A block” polymerization. Typically, the polymerizations exhibited nearly full monomer conversion (> 97%) in 15 min. From the successfully obtained homopolymer PN grafted SP (SP-(PN)), a further chain extension was performed on the surface based on the PN grafted SP as chain transfer agent (CTA) and 2(diethylamino)ethyl methacrylate (DEAEMA) and 2-hydroxy4-(methacryloyloxy) benzophenone (BMA) as monomers by SI-SET-LRP using the same conditions (Scheme 1). The 2(diethylamino)ethyl methacrylate (DEAEMA) monomer was selected because of the pH sensitive behavior required of the future polymer brushes, while the monomer BMA was employed to enhance the stability of hollow capsules via photocrosslinking. This “second B block” copolymerization was performed by in-situ addition of monomers to the reaction solution at full conversion of NIPAM monomer for “first A block”, negating the need for purification between each block and further highlighting the high versatility of this one-pot reaction. Importantly, the chain extensions proceeded without a decrease in polymerization rate and near quantitative conversion (> 95%) was reached within 20 min. To realize the ABAtype triblock copolymers brushes structure, a second thermosensitive PNIPAM layer was further introduced by SI-SETLRP under the same conditions (Scheme 1). This additional chain extension was also carried out without any purification in-situ by adding again NIPAM monomer after completion of the “second B block” copolymer grafting. The final polymerization reached an overall conversion of > 90% in 20 min. FTIR and TGA confirmed well that each block polymerization takes place on the surface of silica particles (Figure 1a and 1b).


Page 5 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society (solid line) and derivative weight loss (dotted line) of SP as functions of temperature.

After dissolution of the silica core without crosslinking the polymer brushes, the isolated originally grafted block copolymers were subjected to characterization by 1H NMR (Figure S2, S3 and S4) and GPC. Analyzing the specific 1H NMR signals for NIPAM, DEAEMA and BMA, a molar ratio of PNIPAM: PDEAEMA: PBMA: PNIPAM in the triblock copolymer chain PNIPAM-b-PDEAEMA-co-PBMA-b-PNIPAM (PN-DB-N in Scheme 1) was determined to be 23: 10: 1: 20. From the GPC it was confirmed that the number-average molecular weight increased from 33300 g/mol with Ð of 1.06 for the grafted homopolymer PN to 55700 g/mol with Ð of 1.24 for the grafted diblock copolymer PNIPAM-bPDEAEMA-co-PBMA (PN-DB), and to 84000 g/mol with Ð of 1.40 for the grafted triblock copolymer PN-DB-N (Table 1).

Figure 1. Characterization of ABA-type triblock copolymer brushes. (a) IR spectrum of bare SP; PN = homopolymer grafted SP; PN-DB = diblock copolymer grafted SP; and PN-DB-N = triblock copolymer grafted SP. (b) TGA curves of bare SP; PN = homopolymer grafted SP; PN-DB = diblock copolymer grafted SP; and PN-DB-N = triblock copolymer grafted SP. Weight loss

Table 1. Characterization of the grafted polymers in this study. samplea)

reaction time (min)

Shell thicknessb) (nm)

Mn of grafted polymerc) (g mol-1)

Đc)

Monomer conversiond) (%)

SP-(PN)

20

21.5

33300

1.06

>97

SP-(PN-DB)

20

41.4

55700

1.24

>95

SP-(PN-DB-N)

20

60.0

84000

1.40

>90

a)

Silica nanoparticles of 500 nm were used as the template. b) The thickness of the core-shell structure was measured by TEM. c) Numberaverage molecular weight (Mn) and dispersity (Đ) of the grafted polymer were determined by GPC (polystyrene calibration) isolated after dissolution of the core before crosslinking. d) Monomer conversion was measured by 1H NMR.

Moreover, the formation of the polymer brushes structure on the silica core was observed by TEM (Figure 2). This clearly shows after chain extension that there was an obvious increase in the thickness of the shell (60.0 nm) for PN-DB-N (Figure 2c) compared with the diblock copolymer PN-DB grafted SP (41.4 nm) (Figure 2b) and the first chain extension for PNIPAM (21.5 nm) (Figure 2a). The increased thickness observed by TEM provides clear support that each block copolymer chain was successfully grafted onto the silica nanoparticles.

Figure 2. Morphology characterization of block copolymer grafted SP. (a,b,c) TEM images of PN grafted SP (a), PN-DB grafted SP (b) and PN-DB-N grafted SP (c), respectively.

Characterization of multi-responsive hollow capsules. Following our concept of multi-responsive hollow capsules (Scheme 1), a cross-linking step was applied before the removal of the template to ensure the integrity of swellable and shrinkable hollow capsules. In this study, the photocrosslinker (BMA), which had been demonstrated in our previous work to be effective for capsule crosslinking, was exploited.35 The exposure of the ABA-type triblock copolymer PN-DB-N brushes under the unfiltered light of a mercury lamp

for 20 min gave the desired crosslinked shell on the surface of SP. By subsequent etching out the silica particles in NH4F/HF buffer, hollow capsules, used later as polymeric nanoreactors, were produced. In order to confirm the shape of hollow capsules, transmission electron microscopy (TEM) and cryo-TEM measurements were performed to observe the morphology of the hollow capsules. The TEM (Figure 3a) were confirmed the existence of hollow capsules as collapsed structure. From the cryo-TEM images (Figure 3b) the regular spherical shape of the hollow capsules with uniform diameters of about 840 nm can be clearly observed. This is in accordance with the results obtained from dynamic light scattering (DLS), presented in Figure 3c. Also a shell thickness of about 64 nm for hollow capsules can be identified, which is consistent with the results obtained on the particles before the dissolution of silica core. After the successful formation of the ABA-type membrane, we next investigated the stability and membrane permeability of hollow capsules by performing their cyclic swelling and shrinkage at temperatures between 25 and 40°C at pH 6 and 8 (Figure 3c and 3d). In essence, the membrane of hollow capsules, consisting of triblock copolymer ABA (composition ABA 23-10-20), can undergo on/off switches for at least four cycles in response to both, pH and temperature, respectively. This implies that the tunable membrane of hollow capsules can be considered as a valve for controlling the traffic of small or larger molecules into or out of the capsule lumen depending on the pH and temperature stimulus (Figure 3e). Here, the membrane architecture of the hollow capsules consists of an ABA three layer structure with temperature-responsive inner


Journal of the American Chemical Society a

b

500 nm

500 nm

c

d

25 ℃

40 ℃

pH 6

pH 8

850

pH 6 pH 8

850

800 o

pH 6,25 6, 25°C C o pH 8,25 8, 25°C C

750

Diameter(nm)

and outer PNIPAM layers and a pH-responsive PDEAEMAco-PBMA central membrane layer. This specific stimuliresponsive membrane architecture allows the hollow capsules to adapt four swelling states resulting in nanoreactors with fine-tuned valve-like functions (Figure 3e). In detail, the hollow capsules were heated above the LCST of PNIPAM to experience a collapse of the inner and outer PNIPAM layer on both sides of the central membrane layer, so that the membrane of the hollow capsules acts as an “impermeable” valve. Using DLS measurements, as expected the size of the hollow capsules decreased sharply from 840 to 795 nm at pH 6 and from 745 to 695 nm at pH 8, respectively, after heating from 25 to 40°C and exhibited a return to previous diameters upon cooling. This behavior was reproducible for at least four cycles (Figure 3c). Beside this temperature tunable permeability of hollow capsules, the pH modulation of the membrane permeability was also investigated. The pH responsiveness is caused by the fact that the photo-crosslinked PDEAEMA chain as B block in the central membrane layer undergoes physicochemical changes going from a deprotonated, hydrophobic entangled state at a high pH value to a protonated, hydrated, repulsive state at a low pH value. As expected, the average diameter of hollow capsules increased from 745 nm at pH 8 to 843 nm at pH 6 with temperature held constant at 25°C, revealing 13% swelling as determined by DLS (Figure 3d). This is in accordance with the results obtained from cryo-TEM, presented in Figure S5. Also the membrane thickness of hollow capsules increased from 52 to 64 nm when changing pH from a basic state (pH 8) to an acidic value (pH 6). This impressively implies that the pHresponsive nature of the photo-crosslinked PDEAEMA-coPBMA central membrane layer causes the change of the membrane permeability of hollow capsules at different pH. Thus, this pH function paves the way for a secondary subvalve function to switch the capsule membrane between “permeable” and “semi-permeable” state (Figure 3e). Within this valve concept based on hollow capsules the inner and outer PNIPAM layer of the ABA membrane architecture (Figure 3e) should play the dominant role with its reversible switching between above and below LCST behavior for generating collapsed and swollen PNIPAM membrane layers, respectively. At 40°C the valve state should not be influenced by the pH-responsive PDEAEMA-co-PBMA central membrane layer, thus at 40°C the capsule membrane is impermeable at both pH 8 and pH 6. The pH-responsive PDEAEMA-coPBMA central membrane layer plays a secondary, but key role in our valve concept by fine-tuning the membrane to a semipermeable state at pH 8 and 25°C. The ability to control the density of the hollow capsule’s membrane via control of temperature and pH serves to provide a valve-like function in which the permeability of the capsule exhibits different states: permeable, semi-permeable and impermeable. Overall, this advancement in membrane architecture promises to be an important tool to stepwise control the permeability of such crosslinked hollow capsules for controlling the membrane traffic for small and large molecules as is of interest when encapsulating active moieties within the hollow capsules for creating nanoreactors.

Diameter(nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 11

o

6, 40°C pH 6,45 C o 8, 40°C pH 6,45 C

800

750

700 0

e

1

2 3 Number of cycles

4

5

0

1

2

3

4

5

Number of cycles

Hollow capsules with



semi-permeable valve

impermeable valve

permeable valve

T pH 8 + 25℃ Membrane – shrunken Inner/outer shell - swollen

T

pH 8 + 40℃ Membrane – shrunken Inner/outer shell - collapsed T pH 6 + 40℃ Membrane - swollen Inner/outer shell - collapsed

pH 6 + 25℃ T

Membrane - swollen inner/outer shell - swollen

Figure 3. Characterization of multi-responsive hollow capsules. (a,b) TEM (a) and cryo-TEM (b) image of PN-DB-N hollow capsules prepared using 500 nm diameter SiO2 particles (silica templates were dissolved with NH4/HF buffer). (c) Reversible swelling-shrinking of hollow capsules upon switching between 25°C and 40°C at different pH (6 or 8) (n=3). (d) Reversible swelling-shrinking of hollow capsules upon switching between pH 6 and 8 at 25°C (n=3). (e) Schematic representation of the valve concept for hollow capsules upon temperature and pH changes. The temperature changes trigger a reversible main valve function for hollow capsules; pH changes function as a secondary sub-valve control to further regulate the membrane of hollow capsules in “permeable” and “semi-permeable” state.

Nanoreactors with multi-responsive valve for enzymatic reactions. Combining the temperature responsiveness of the inner and outer PNIPAM membrane layer and the pH responsiveness of PDEAEMA-co-PBMA central membrane layer of our hollow capsules, we next set out to investigate whether the stimulus-dependent responsiveness is able to introduce an independent temperature and pH sensitive regulatory mechanism for controlling enzymatic reactions. One requirement for the success of our valve concept for controlling reactions of encapsulated enzymes is that the enzymes must be retained in the lumen of the swollen nanoreactors, at which enzymes were captured in capsules´ lumen during a post-loading process at pH 6 (below further details and Supporting Information). Only the trans-membrane diffusion of enzyme substrates and products can be allowed or prohibited. The trans-membrane trafficking smoothly depends on the applied valve functions (permeable, impermeable and semi-permeable).


Page 7 of 11

a

Valve in half-on

Valve in fully-off

Valve in fully-on

pH 6 / 40℃

pH 6 / 25℃

pH 8 / 25℃

pH 8 / 40℃

Case I H2O2

Enzyme 1 (Glucose oxidase)

90 60 30 0

Enzyme 2 (Myoglobin)

pH 8 pH 6 40oC 40oC

Product (quinone)

Substrate (guaiacol)

c

120

Normalized Activity (%)


b

H2O2

d 100

25oC 40oC

80 60 40 20 0 0

pH 8 pH 6 25oC 25oC

1

2 3 4 Number of cycles

5


Substrate (glucose)

100

pH 8 pH 6

80 60 40 20 0 0

1


5

Figure 4. Investigation of enzymatic activities for GOx-filled nanoreactors with extracellular free Myo (Case I). (a) Schematic representation of enzymatic reaction process at different physiological environment for Case I. (b) Enzymatic activities at different temperatures and in various pH medium. (c) Enzymatic activities recorded for repeated temperature changes at pH 8. (d) Enzymatic activities recorded for repeated pH changes at 25°C.

a

Valve in half-on

Valve in fully-off

Valve in fully-on

pH 6 / 40℃

pH 6 / 25℃

pH 8 / 25℃

pH 8 / 40℃

Case II H2O2

H2O2

90 60 30 0

Enzyme 2 (Myoglobin)

Substrate (guaiacol)

pH 8 40oC

pH 6 40oC

pH 8 pH 6 25oC 25oC

Product (quinone)

d

c

120


b

Enzyme 1 (Glucose oxidase)

100

25 oC 40 oC

80 60 40 20 0 0

1


5


Substrate (glucose)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

pH 8 pH 6

80 60 40 20 0 0

1


5

Figure 5. Investigation of enzymatic activities for GOx-filled nanoreactors with Myo-filled nanoreactors (Case II). (a) Schematic representation of enzymatic reaction process at different physiological environment for Case II. (b) Enzymatic activities at different temperatures and in various pH medium. (c) Enzymatic activities recorded for repeated temperature changes at pH 8. (d) Enzymatic activities recorded for repeated pH changes at 25°C.

In order to prove the technical relevance of our multiresponsive enzyme-loaded nanoreactors we decided to address the challenge of an enzymatic cascade reaction in different biomimetic environments. As the first approach, enzyme 1 is introduced into the cavity of the nanoreactors and an excess of free enzyme 2 is added later together with the substrate glu-

cose and guaiacol to the environment of nanoreactor (Figure 4a, Case I). Thus, the reaction product from enzyme 1 has to cross the nanoreactor membrane in “on” state to initialize the reaction of enzyme 2 outside of the nanoreactor. In a next approach (Figure 5a, Case II) a different type of enzyme is encapsulated separately into the nanoreactors. In this case, the



intermediate reaction product from enzyme 1 has to leave the first nanoreactor and then enter a second nanoreactor via membrane diffusion, both in “on” state to initialize the reaction of enzyme 2. As proof of the valve concept (Figure 3e) for controlling bienzymatic reactions (Figure S6), glucose oxidase (GOx) is used as enzyme 1, which turns D-glucose into Dglucono-δ-lactone and H2O2.36 Next, H2O2 acts as a cosubstrate for myoglobin (Myo) to oxidize guaiacol into a quinone,37,38 which is detected by UV-Vis spectroscopy.39 First, GOx (3.1 × 3.4 × 6.4 nm40) and Myo (6.0 × 5.2 × 7.7 nm41,42) were loaded into swollen nanoreactors in phosphate buffer pH 6 at 25°C, at which the enzyme can diffuse into the nanoreactors since the membrane is in the “fully-on” state. Afterwards, the non-encapsulated enzymes were removed by dialysis in phosphate buffer pH 8 at 40°C, at which the membrane was fully impermeable and the nanoreactors were completely switched into the “off” state, finally showing a loading efficiency of Myo slightly below 6 % (Table S1). Successful removal of non-encapsulated enzymes from enzymatic nanoreactor solution was demonstrated at pH 8 and 40°C (Figure 4b) as sufficient loading process to perform our original enzymatic study. Subsequently, these enzyme-filled nanoreactors solution were subjected to GOx and Myo enzymatic activity test at different temperatures (25 or 40°C) and in various pH medium (pH 6 or 8) (Figure 4 and Figure 5). For Case I (Figure 4a) GOx was encapsulated in nanoreactors and a corresponding concentration of free Myo was added later together with D-glucose and guaiacol. Under valve conditions with “off” state (impermeable) at 40°C and regardless of the pH value, the nanoreactors reject the membrane diffusion of enzymatic substrate from outside to inside. Consequently, no enzymatic activity of Myo could be monitored (Figure 4b). This indicates that enzymes are completely shielded in nanoreactors and the substrates cannot diffuse into the nanoreactors lumen where the enzyme is hosted. In contrast, when applying valve conditions with “on” state (permeable and semipermeable) at 25°C and regardless of the pH value, the substrate enter into the lumen of nanoreactors through the swollen membrane to reach the GOx, which catalyzes the reaction and causes an obvious activity of Myo (Figure 4b). It can be postulated that the main valve function to switch “on” and “off” the enzymatic reactions of nanoreactors is triggered by the temperature change. This confirms the postulated dominating role of temperature-responsive inner and outer PNIPAM membrane layers of our valve concept (Figure 3e). However, when at 25°C the environmental pH is decreased from pH 8 to 6, the state of nanoreactors switched from “half-on” (semipermeable) to “fully-on” (permeable). Consequently, the diffusion of substrate and reaction product H2O2 through the membrane of nanoreactors is faster, thus, an obviously higher enzymatic activity is available (Figure 4b). This implies that pH changes can function as a secondary sub-valve control to further increase/decrease the nanoreactors activity. So far, only pH responsive crosslinked polymersomes have been reported as nanoreactors for controlling enzyme reactions, where the enzyme activity was fully switched off at pH 8.37 In the present case, the dominating temperature-controlled valve function allows for a reduced enzyme activity at pH 8 at room temperature due to reduced permeability of the collapsed PDEAEMA-co-PBMA central membrane layer embedded between the swollen inner and outer PNIPAM layers.

Page 8 of 11

For Case II, GOx and Myo are encapsulated separately into nanoreactors and the two nanoreactor solutions are mixed afterwards. The only difference between Case I and Case II is that the free Myo is substituted by Myo-filled nanoreactors. This condition necessitates an additional membrane diffusion event of the product of enzyme 1 into the lumen of the nanoreactor containing enzyme 2 (Figure 5a). In Case II, analogous temperature- and pH-dependent enzymatic activity characteristics (Figure 5b) were observed as in Case I (Figure 4b). However, in contrast to Case I, especially in the “half-on” state for nanoreactors at pH 8 and 25°C (Figure 5b), the intermediate H2O2 from GOx has to cross the “half-on” membrane of nanoreactors twice to initialize the reaction of Myo. A lower activity for Myo (Figure 5b, 37%) was determined compared with the enzymatic conversion (Figure 4b, 54%) in Case I. It is important to note that the present concept, due to the main temperature-controlled valve function, allows for the control of enzyme activity over a broader pH range than previously reported.35,37,39 Furthermore, multiple swelling-shrinking cycles were tested to evaluate whether repeated changes in temperature or pH affect the functionality of the enclosed enzymes in nanoreactors. Thus, the enzymatic activity was monitored following several temperature switches between 25 and 40°C at pH 8 (Figure 4c and 5c). As expected, the enzymes encapsulated in the nanoreactors showed no activity at 40°C. In contrast to this, enzymatic activity was regained at 25°C due to the “on” state at this temperature. The enzymatic reactivity could be reinitiated for at least 5 cycles. This demonstrates that the temperature dependent “on-off” reaction-control cycle is repeatable and reproducible. In addition to the reproducible behavior observed for temperature cycling, we observed cycling of enzymatic activity due to pH changes at 25°C. Enzymatic activity was reproducible over four pH cycles (Figure 4d and 5d). Again, the enzymatic activity dropped down to its previous level once pH 8 was reinstated due to the “half-on” membrane for the nanoreactors. Hence, the nanoreactor membrane always goes back to its native state and exhibits a highly reproducible behavior following changes to both temperature and pH. CONCLUSION In summary, we demonstrated a fast one-pot approach for the fabrication of multi-responsive hollow capsules with an ABA-type triblock copolymers membrane by using SI-SETLRP polymerization technique. A notable feature of this synthesis of triblock copolymers on silica particles is that no purification is required between the successive block formation steps because each step was achieved with a full monomer conversion (> 96% conversion) and with high end group fidelity within 20 min at room temperature. After photocrosslinking and template removal, hollow capsules with very dense and thick polymer brush membranes (60 nm) based on high-molecular-weight triblock copolymers (84000 g/mol) were obtained. The membranes of the resulting hollow capsules can respond independently to temperature and pH due to the ABA three layer architecture with temperature-responsive outer and inner PNIPAM layers and a pH-responsive PDEAEMA-co-PBMA central membrane layer. This results in tunable permeability (permeable, semi-permeable and impermeable) for the hollow capsules. Using such hollow capsules as an enzymatic nanoreactor system, enzymatic reactions were


Page 9 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reversibly switchable “on” and “off” by adjusting temperature as a main valve control. Meanwhile, pH changes, as a secondary sub-valve control, further regulated the enzymatic reaction in a “half-on” to “fully-on” manner. This is a first example for nanoreactors with a tunable valve that can control its enzymatic reactions in a highly reproducible, efficient, specific and successive manner over a broader enzyme-friendly environment (between pH 6 and 8). This behavior has not been reported in previously described pH-switchable enzymatic nanoreactors18, 31, 37, 39 in which metabolites have to cross nanoreactors membrane without the help of transmembrane proteins4345 . The valve-like behavior of these nanoreactors also differs from steadily permeable enzymatic nanoreactors20, 46. Moreover, with this present valve-like behavior of hollow capsules the design and fabrication of novel multi-orthogonal responsive membranes (e.g. combining pH, temperature and light stimulus) is possible and is expected to have wide implications in the fields of synthetic biology and systems biology.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Synthetic details for initiator-modified silica particles and additional figures.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT X.L. is grateful for a scholarship under the Chinese government award for outstanding students abroad by the China Scholarship Council (CSC). We thank H. Komber for NMR measurements, M. Malanin for IR measurements, K. Arnhold for TGA and P. Formanek for SEM and cryo-TEM investigations.

REFERENCES (1) Vriezema, D. M.; Garcia, P. M. L.; Sancho Oltra, N.; Hatzakis, N. S.; Kuiper, S. M.; Nolte, R. J. M.; Rowan, A. E.; van Hest, J. C. M. Ange. Chem. Int. Ed. 2007, 46, 7378. (2) Liu, M.; Fu, J.; Hejesen, C.; Yang, Y.; Woodbury, N. W.; Gothelf, K.; Liu, Y.; Yan, H. Nat. Commun. 2013, 4, 2127. (3) Wilner, O. I.; Weizmann, Y.; Gill, R.; Lioubashevski, O.; Freeman, R.; Willner, I. Nat. Nanotech. 2009, 4, 249. (4) Fu, J.; Liu, M.; Liu, Y.; Woodbury, N. W.; Yan, H. J. Am. Chem. Soc. 2012, 134, 5516. (5) Schoonen, L.; van Hest, J. C. M. Adv. Mater. 2016, 28, 1109. (6) Yang, T.; Wang, Y.; Ke, H.; Wang, Q.; Lv, X.; Wu, H.; Tang, Y.; Yang, X.; Chen, C.; Zhao, Y.; Chen, H. Adv. Mater. 2016, 28, 5923. (7) Vriezema, D. M.; Comellas Aragonès, M.; Elemans, J. A. A. W.; Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M. Chem. Rev. 2005, 105, 1445. (8) Koblenz, T. S.; Wassenaar, J.; Reek, J. N. H. Chem. Soc. Rev. 2008, 37, 247. (9) Lin, Y.; Ren, J.; Qu, X. Acc. Chem. Res. 2014, 47, 1097. (10) Chang, T. M. S. Nat. Rev. Drug Discov. 2005, 4, 221. (11) Städler, B.; Chandrawati, R.; Price, A. D.; Chong, S.-F.; Breheney, K.; Postma, A.; Connal, L. A.; Zelikin, A. N.; Caruso, F. Angew. Chem. Int. Ed. 2009, 48, 4359.

(12) Chandrawati, R.; Hosta-Rigau, L.; Vanderstraaten, D.; Lokuliyana, S. A.; Städler, B.; Albericio, F.; Caruso, F. ACS Nano 2010, 4, 1351. (13) Hosta-Rigau, L.; Chung, S. F.; Postma, A.; Chandrawati, R.; Städler, B.; Caruso, F. Adv. Mater. 2011, 23, 4082. (14) Bolinger, P.-Y.; Stamou, D.; Vogel, H. Angew. Chem. Int. Ed. 2008, 47, 5544. (15) Huang, X.; Li, M.; Green, D. C.; Williams, D. S.; Patil, A. J.; Mann, S. Nat. Commun. 2013, 4, 2239. (16) Tu, Y.; Peng, F.; Sui, X.; Men, Y.; White, P. B.; van Hest, J. C. M.; Wilson, D. A. Nat. Chem. 2016, 9, 480. (17) Li, M.; Harbron, R. L.; Weaver, J. V. M.; Binks, B. P.; Mann, S. Nat. Chem. 2013, 5, 529. (18) Gaitzsch, J.; Appelhans, D.; Wang, L.; Battaglia, G.; Voit, B. Angew. Chem. Int. Ed. 2012, 51, 4448. (19) Kim, K. T.; Cornelissen, J. J. L. M.; Nolte, R. J. M.; van Hest, J. C. M. Adv.Mater. 2009, 21, 2787. (20) Comellas-Aragonès, M.; Engelkamp, H.; Claessen, V. I.; Sommerdijk, N. A. J. M.; Rowan, A. E.; Christianen, P. C. M.; Maan, J. C.; Verduin, B. J. M.; Cornelissen, J. J. L. M.; Nolte, R. J. M. Nat. Nanotech. 2007, 2, 635. (21) Huang, Y.; Lin, Y.; Ran, X.; Ren, J.; Qu, X. Nanoscale 2014, 6, 11328. (22) van Werne, T.; Patten, T. E. J. Am. Chem. Soc. 2001, 123(31): 7497. (23) Morinaga, T.; Ohkura, M.; Ohno, K.; Tsujii, Y.; Fukuda, T. Macromolecules 2007, 40, 1159. (24) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. Rev. 2004, 33, 14. (25) Huang, X.; Appelhans, D.; Formanek, P.; Simon, F.; Voit, B. Macromolecules 2011, 44, 8351. (26) Husseman, M.; Malmström, E. E.; McNamara, M.; Mate, M.; Mecerreyes, D.; Benoit, D. G.; Hedrick, J. L.; Mansky, P.; Huang, E.; Russell, T. P.; Hawker, C. J. Macromolecules 1999, 32, 1424. (27) Li, G. L.; Xu, L. Q.; Tang, X.; Neoh, K. G.; Kang, E. T. Macromolecules 2010, 43, 5797. (28) Li, G.; Lei, C.; Wang, C. H.; Neoh, K. G.; Kang, E. T.; Yang, X. Macromolecules 2008, 41, 9487. (29) Huo, F.; Li, S.; Li, Q.; Qu, Y.; Zhang, W. Macromolecules 2014, 47, 2340. (30) von Werne, T.; Patten, T. E. J. Am. Chem. Soc. 2001, 123, 7497. (31) Huang, X.; Appelhans, D.; Formanek, P.; Simon, F.; Voit, B. ACS Nano 2012, 6, 9718. (32) Li, D.; Sheng, X.; Zhao, B. J. Am. Chem. Soc. 2005, 127, 6248. (33) Liu, X.; Appelhans, D.; Zhang, T.; Voit, B. Macromolecules 2018, 51, 1011. (34) Zhang, Q.; Wilson, P.; Li, Z.; McHale, R.; Godfrey, J.; Anastasaki, A.; Waldron, C.; Haddleton, D. M. J. Am. Chem. Soc. 2013, 135, 7355. (35) Liu, X.; Appelhans, D.; Wei, Q.; Voit, B. Adv. Sci. 2017, 4, 1600308. (36) Kumar, C. V.; Chaudhari, A. J. Am. Chem. Soc. 2000, 122, 830. (37) Gräfe, D.; Gaitzsch, J.; Appelhans, D.; Voit, B. Nanoscale 2014, 6, 10752. (38) Matsuo, T.; Murata, D.; Hisaeda, Y.; Hori, H.; Hayashi, T. J. Am. Chem. Soc. 2007, 129, 12906. (39) Liu, X.; Formanek, P.; Voit, B.; Appelhans, D. Angew. Chem. Int. Ed.2017, 56, 16233. (40) Zelikin, A. N.; Price, A. D.; Städler, B. Small 2010, 6, 2201. (41) Wang, G.; Thai, N. M.; Yau, S.-T. Electrochem. Commun.2006, 8, 987. (42) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol.1993, 229, 153. (43) Palivan, C. G.; Fischer-Onaca, O.; Delcea, M.; Itel, F.; Meier, W. Chem. Soc. Rev. 2012, 41, 2800-2823. (44) Itel, F.; Najer, A.; Palivan, C.G.; Meier, W. Nano Lett. 2015, 15, 3871–3878.



Page 10 of 11

(45) Edlinger, C.; Einfalt, T.; Spulber, M.; Car, A.; Meier, W.; Palivan, C. G. Nano Lett. 2017, 17, 5790–5798. (46) Spulber, M.; Najer, A.; Winkelbach, K.; Glaied, O.; Waser, M.; Pieles, U.; Meier, W.; Bruns, N. J. Am. Chem. Soc. 2013, 135, 9204-9212.


Page 11 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fully-off valve pH 6 / 40℃ pH 8 / 40℃ Half-on valve

Fully-on valve

pH 8 / 25℃

pH 6 / 25℃

H2O2

pH 6

H2O2

pH 8

ACS Paragon Plus Environment

11

Hollow capsules with multi-responsive valves for controlled enzymatic

Recommend Documents