Multifunctional and Programmable Modulated Interface Reactions on

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Surfaces, Interfaces, and Applications

Multifunctional and Programmable Modulated Interface Reactions on Proteinosomes Pei Zhou, Shuang Wu, Xiaoman Liu, Guangyu Wu, Mohammad Hegazy, and Xin Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11216 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 18, 2018

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ACS Applied Materials & Interfaces

Multifunctional and Programmable Modulated Interface Reactions on Proteinosomes Pei Zhou,† Shuang Wu,‡ Xiaoman Liu,*,† Mohammad Hegazy,† Guangyu Wu,† and Xin Huang*,† †

MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, The Key Laboratory of Microsystems and Microstructures Manufacturing, Ministry of Education, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China ‡ The First Affiliated Hospital of Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, China KEYWORDS: Protocells, multiresponsive, programmed release, enzymatic interface reactions, capture and release

ABSTRACT: A multiresponsive microcapsule has been synthesized by incorporating photoswitchable spiropyran units and the thermoresponsive monomer N-isopropylacrylamide into membrane lumens. By using functionalized light or thermoresponsive groups, this multifunctional microcapsule can modulate programmed release and interface reactions between lipase and fluorescein diacetate, alkaline phosphatase and fluorescein diphosphate, and others. Exposing this multifunctional microcapsule in a programmed controlled way allowed us to develop schematics to understand complicated interface interactions on protocells.

models are designed by controlling the movement and exchange of cargo between the outside and inside compartThe precise manipulation of biomembranes via external ments in response to external stimuli, leading to diverse stimuli is fundamentally important to cope with the comfunctions and applications related to light-switching effects, plex protocols of metabolic reactions for a broad series of acoustic signal capture, thermal energy transfer and magapplications, ranging from self-assembly to controlled carnetic signal response. Generally, these multifunctional progo transport. Notably, this operation is a precondition of all tocols can be achieved via the direct self-assembly of poliving systems and is becoming increasingly common in rous membranes, chemical modification of membranes, artificial cell prototypes. In addition, such manipulations insertion of biopores or reconstitution of channel pores. As contribute to the development of applications as diverse as a consequence, attractive and versatile smart microcapsule photosynthesis, metabolism and biological effects.1-2 Owmodels can be designed to selectively respond to external ing to their unique structure, well-defined artificial cell stimuli via programmable modulated interface reactions, models can act as a multifunctional platform and demonwhich could pave the way towards exploring complex biostrate promising applications in light, acoustic, temperature logical processes and phenomena. However, the precise and magnetic fields.3-8 To achieve multiple functions with positioning microcapsule with showing outside and inside these applications, an ingenious membrane design is curcommunications and interactions is still a challenge. rently attracting substantial attention in a diverse range of Compared with conventional acoustic, temperature and research communities and is capable of controlling the bemagnetic fields, light has a series of additional advantages havior of material exchange, cellular communication or in many regards. For example, light can be transmitted to a metabolism inside and outside the membrane.9-11 In this certain location in diverse wavelengths and then realize respect, an additional advantage of using multiple external photochemical reactions at specific sites. Furthermore, light stimuli is that such stimuli can be loaded and removed imenables the opening and closing of specific ligands, thus mediately; thus, the characteristics of biomembranes can be controlling extracellular and intracellular substance transidentified and understood with the goal of mimicking promission. Most importantly, light is a gentle means of enertocells by using programmable modulated properties or gy transfer and can transfer energy from ligands to metal structures. In recent years, strategies using amphiphilic cores, thereby revealing several distinctive properties of the building blocks that undergo spontaneous or directed asgenerated complexes.20-21 However, these models are based sembly in aqueous solution to construct capsules, e.g., lipoon the chemical modification of nanoparticles with a single somes,12-13 polymersomes,14-15 colloidosomes,16-17 and prolight-responsive molecular switch, such as azobenzenes22-24 tein-based microcapsules,18-19 have frequently been used to and spiropyrans,25-28 which can not only affect the perfordesign a variety of bioactive molecules, including nanoremance of switches due to electronic interactions with the actors, drug delivery systems and artificial cells. These metallic substrates but also significantly mimic the manACS Paragon Plus Environment

INTRODUCTION

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agement of light control in aqueous solution. Inspired by this interaction, we designed a new multifunctional proteinosome with light-switchable and thermoresponsive ligands in aqueous solution. We hypothesized that, in photoresponsive and thermosensitive environments, these distinctive molecules, which are capable of capturing and releasing cargos, could guide the occurrence of interface reactions and thereby create attractive and versatile artificial cell models to understand extracellular and intracellular communications as well as the interactions of biomembranes.

Scheme 1. The concept of multifunctional self-assembly of proteinosomes. A multifunctional medium that contains temperature, ultraviolet and redox conditions can govern the assembly state of capsules functionalized with photoswitchable spiropyran and thermoresponsive NIPAAm ligands on the surface of the membrane. Modulating the temperature (LCST ≈ 32 °C), exposing the membrane to ultraviolet irradiation (λ < 410 nm) and introducing redoxactive compounds (GSH) could not only successfully trigger the programmed release of encapsulated substances but also modulate interface reactions between lipase and FDA, lipase and 4-MU-Bu, and ALP and FDP, which are held together by the reversible phototriggered isomerization of spiropyran ligands. An ideal candidate to realize this goal is the utilization of photoresponsive spiropyran and thermosensitive Nisopropylacrylamide (NIPAAm) monomers to construct a multiresponsive polymer that is capable of capturing and releasing hydrophobic substances. These polymers and a cationized BSA conjugate linked via a cross-linker (BS(PEG)5 ester) can successfully self-assemble into microcapsules (proteinosomes). This strategy not only yields multistimuli behavior with continuous modulation of membrane permeability but can also guide interface reactions between lipase and fluorescein diacetate (FDA) and between alkaline phosphatase (ALP) and fluorescein diphosphate (FDP) in the presence of light (Scheme 1). In this study, four stages were developed to programmably modulate membrane permeability by applying the following conditions: (i) body temperature; (ii) room temperature; (iii) ultraviolet light; and (iv) redox. Hence, such sequential modulation of membrane permeability was well demonstrated by studying the programmed release of encapsulated rhodamine-labeled ALP (ALP-RITC, 56 kDa) and DNA stained by SYBR green I (DNA & SYBR, 106 kDa). Then, a series of FITC-dextran (10 kDa, 20 kDa, 40 kDa, 70 kDa, 150 kDa, 500 kDa and 2000 kDa) were used to precisely

investigate the membrane permeability. The molecular weight cutoff of the constructed microcapsules could be continuously modulated from 18 kDa to 29 kDa, 41 kDa and 63 kDa by the four stages of continuous regulation. Moreover, the constructed microcapsules could allow different molecular weights of cargo to be offloaded batch by batch, especially in the presence of ultraviolet light, which enabled the release of hydrophilic (ALP-FITC) and hydrophobic (Nile red) substances. More importantly, the interface reactions between lipase and FDA or 4methylumbelliferyl butyrate (4-MU-Bu) and between ALP and FDP, as well as several enzymatic reactions. were investigated and the designed microcapsules could also facilitate the release and capture of hydrophobic products by opening and closing the photoresponsive spiropyran ligands.

Figure 1. (a) Optical microscopy image of BSA-SSPolymer proteinosomes in the water phase. (b) SEM image of the as-prepared proteinosomes showing hollow and structurally intact microcapsules. Scale bar is 50 μm for optical microscopy and 20 μm for scanning electron microscopy. (c, d) CLSM images showing the proteinosomes encapsulating the enzymes ALP-RITC (c) and DNA & SYBR (d). Scale bars are 20 μm. (e, f) CLSM images of the 3D structures of the corresponding encapsulated enzymes. The typical z-axis direction indicates the hemispheric height by gradient color, while the damp-dry 3D structures confirm that the hollow proteinosomes were robust and filled with a flow phase in the inner cavum.

RESULTS AND DISCUSSION Programmable Modulation of the Membrane Permeability of a Synthetic Proteinosome upon Multiple Stimuli Responses. To realize the above-discussed membrane modulation, we worked with a building block consisting of protein-polymer conjugates functionalized with photoresponsive spiropyran and thermosensitive NIPAAm mono-

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Figure 2. (a-d, e-h) Fluorescence microscopy images of constructed multifunctional proteinosomes showing the differences among four stages (body temperature, room temperature, ultraviolet light and redox) in the presence of ALP-RITC (≈ 56 KDa) and DNA & SYBR (≈ 106 KDa) by reverse osmosis (RO). (i-l) Corresponding intensity profiles for the single proteinosomes shown in a-d (red line) and e-h (green line). The intensity curves show the diffusion capability of diverse molecular weights after incubation for 30 min with proteinosomes. Scale bars are 100 µm. mers using reversible addition-fragmentation chain-transfer (RAFT) polymerization (Figure S1-S3). Interestingly, we found that the two functional monomers (NIPAAm and spiropyran) were difficult to polymerize together, presumably because of the strong steric hindrance when multiple benzene rings are involved in polymerization. However, the complex 60-repeat NIPAAm with 2 repeated spiropyran monomers is sufficient to regulate the compactness of the membrane; theoretically, the two types of functional monomers have equal performance in terms of the MWCO of the membrane. However, a protein was cationized by cystamine dihydrochloride according to a previously reported method29-31 and yielded a total of approximately 20 surface-accessible amines according to an amine titration measurement (Figure S4, table S1). Subsequently, by mixing the modified BSA-SS-NH2 with the obtained photoresponsive and thermosensitive polymer in pH 8.0 aqueous solution, the building block, a protein-polymer conjugate, was obtained. The obtained conjugates consisted of 1.22 polymer chains per BSA molecule on average (Figure S5, table S2), and owing to the strong steric hindrance, less of the conjugated polymer was formed than in the previous work. Zeta potential and dynamic light scattering measurements clearly demonstrated the successful construction of protein-polymer conjugates: BSA (-4.42 mV, 5.62 nm), BSA-SS-NH2 (25.8 mV, 7.53 nm) and BSA-SS-Polymer (12.8 mV, 10.5 nm) (Figure S6, S7). Significantly, aqueous BSA-SS-Polymer exposed to 40 °C (295.3 nm), ultraviolet light (8.72 nm) and GSH (blue, 6.50 nm) also confirmed the thermosensitive, ultraviolet and redox-responsive prop-

erties, respectively, of the obtained conjugates. Finally, mixing an aqueous solution of BSA-SS-Polymer with 2ethyl-1-hexanol at an aqueous/oil volume fraction of 0.08 produced a well-dispersed solution of microcapsules (proteinosomes), and in the presence of the cross-linker BS(PEG)5, the generated proteinosomes could be transferred into aqueous solution and showed diameters in the range of 10-30 μm (Figure 1a). A typical scanning electron microscopy (SEM) image of the synthesized proteinosomes is shown in Figure 1b, revealing that the hollow proteinosomes were collapsed but structurally intact microcapsules. We then investigated the stability of the proteinosomeencapsulated enzymes using ALP-RITC (Figure 1c) and DNA & SYBR (Figure 1d), described above by confocal fluorescence microscopy. The corresponding enzymeencapsulating 3D structures clearly showed the size of the hollow structures, discernible along the z-axis by gradient color (Figure 1e), while the damp-dry proteinosomes were structurally robust and filled with a flow phase in the inner cavum, which could enable free communication with the outer environment (Figure 1f). With the successful fabrication of proteinosomes in aqueous solution, considering the thermosensitive and photoresponsive characteristics of the polymer, the precise manipulation of multistimuli behavior was explored. As we know, NIPAAm, as a diverting monomer with a lower critical solution temperature (LCST) (ca. 32 °C), has attracted increasing interest from scientists. In our studied system of individual water-dispersed proteinosomes, thermosensitive stimulus modulation was monitored from high (40 °C) to



low (25 °C) by optical microscopy (Figure S8). The change in size showed an obvious critical point at 33 °C, indicating that during the decrease in temperature, the diameter of the proteinosomes increased gradually, especially after the lower critical solution temperature (ca. 32 °C), which contributed to the phase change of the thermosensitive polymer in the membrane. The photoresponsive character was further studied by ultraviolet and fluorescent spectrometry. It is well known that spiropyran is a light-responsive molecular switch, and the circular reaction of photoresponsive polymers from SP to McH+ to SP was adjusted by ultraviolet and visible light. To better understand the photoswitchable process, the changes in the absorption spectra of the polymer solutions were studied by ultraviolet–visible spectroscopy. As shown in Figure S9a-c, the process showed a gradual increase and decrease in the absorbance intensity at 500 nm with the SP-to-McH+ and McH+-to-SP reaction under ultraviolet and visible light, respectively. The color of the photoresponsive polymer solution also showed a change from colorless to pink. It should be mentioned that the McH+-to-SP process took more time to reach equilibrium in visible light. However, even after more than 20 minutes in visible light, the ring-closing reaction of MCH+ was still difficult to complete and enable the transformation to SP (Figure S9d, 9e). Once the first cycle was completed, the photoswitchable process could be repeated many times, and the ring-opening and the ring-closing reactions were triggered by ultraviolet and visible light, respectively, after five cycles. The absorption intensity at the wavelength of 500 nm could be modulated reversibly between ringopening and ring-closing processes via turning on and off the visible light (Figure S9f). The changes in fluorescent spectra were also demonstrated to assess the photoswitchable process. This excellent reversibility also appeared unexpectedly with the change in fluorescent spectra by controlling the visible light, given the pronounced emission wavelength at 580 nm, which is typically associated with switches in the photoresponsive compound spiropyran (Figure S10a, 10b). After 24 minutes in visible light, the fluorescence intensity was decreased to a minimum, indicating that the intensity of the SP-to-McH+-to-SP process approached that of the original spiropyran (Figure S10d, 10e). Similarly, the ring-opening and ring-closing reactions were also studied in five cycles, whereas the change in fluorescence intensity could be cycled with the use of ultraviolet light (λ ≈ 365 nm) and visible light to induce the isomerization reaction (Figure S10c). Switching was achieved successfully by changing light, but much less equilibration appeared in multiple cycles (Figure S10f). Consequently, ultraviolet and fluorescent spectrometry showed no significant changes in the polymer after fewer than 5 cycles, and by changing both the ultraviolet and visible light, the photoresponsive polymer in the membrane could be well modulated in permeability and hydrophobicity.

of the abovementioned stages by using the reverse osmosis (RO) method to measure the difference in fluorescence intensity inside and outside the proteinosomes. As anticipated, ALP-RITC showed no osmosis and weak osmosis at body and room temperature, respectively, but strong osmosis in ultraviolet or redox environments (Figure 2a-d). On the other hand, DNA & SYBR showed no osmosis in all these stages and could not pass through the membrane channels, owing to the high molecular weight of the complex; therefore, DNA & SYBR revealed a very weak fluorescence intensity in all proteinosomes (Figure 2e-h). This fluorescence intensity was observed systematically for diverse stages via comparing ALP-RITC with DNA & SYBR by ImageJ software. Upon plotting the diffusion distributions in the proteinosomes, the intensity showed different concave-convex curves near the horizontal (Figure 2i-l). From the change in these curves, the fluorescence intensity of osmosis could be observed directly. ALP-RITC could pass through the membrane in the transformations in diverse stages, while DNA & SYBR showed no osmosis effect despite the extreme opening of membrane channels, indicating that the membrane permeability of the constructed proteinosomes was limited for molecular weights ranging from 10 to 100 kDa.

First, to further quantitatively evaluate the membrane permeability of our constructed proteinosomes in response to multiple stimuli, ALP-RITC (56 kDa) and DNA & SYBR (106 kDa) were employed to assess the permeability

To further precisely assess these sequential modulation behaviors following diverse stimuli responses, different molecular weights of FITC-dextran ranging from 10 to 2000 kDa were selected to measure the membrane permea-

Figure 3. (a) The diffusion of different molecular weights of FITC-dextran from solution to the proteinosomes after 30 min incubation in four stages: treatment with 37 °C, 25 °C, 365 nm irradiation and GSH. The MWCO of the capsules against FITC-dextran could be continuously modulated from 18 kDa to 29 kDa, 36 kDa and 44 kDa under the four stages, respectively. (b) The catalytic reaction rates of glucose oxidase (GOx) were determined by monitoring the absorption intensity of the generated ABTS- derivative in the initial 30 s while undergoing transformation in the three stages. (c) Schematic illustration of the proteinosomeencapsulated GOx and free horseradish peroxidase (HRP)mediated catalytic reaction for trapping the ABTS- derivative that accompanies the generation of H2O2 driven from glucose introduced in an aqueous solution.


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ACS Applied Materials & Interfaces bility for each mentioned stage (Figure S11a-g). By reverse osmosis, the membrane permeability showed a relative molecular weight cutoff (MWCO) of 18 kDa, 29 kDa, 36 kDa and 44 kDa in the four stages corresponding to body temperature, room temperature, ultraviolet light and redox medium, respectively (Figure 3a). The above results indicated well that the membrane permeability could be modulated successfully under different stimuli. Differing from the study of the molecular weight cutoff of the membrane, exploiting an enzyme cascade reaction is another direct and efficient manner to evaluate these sequential modulation behaviors. In this system, an enzymatic reaction occurred in which glucose was oxidized to gluconic acid and hydrogen peroxide by encapsulated glucose oxidase (GOx) inside the proteinosome, then the hydrogen peroxide guided the peroxidation of 2,20-azino-bis-(3-ethylbenzothiazoline-6sulfonate) ([ABTS]2-) by free horseradish peroxidase (HRP) (Figure 3c). In detail, glucose was added to the encapsulated GOx proteinosomes suspension and thus initiated the GOx-mediated formation of H2O2 in a short time. Meanwhile, the generated H2O2 initiated the continuous reaction of [ABTS]2- to [ABTS]- by HRP, and as evidence, the distinct green coloration of [ABTS]- was determined in diverse stages by UV-vis spectroscopy (Figure 3b). Correspondingly, the two stages of ultraviolet light and redox medium after the addition of glucose showed a progressive increase in the initial formation rate of [ABTS]- (V0) associated specifically with the amplification of membrane channels (V0 values, 4.38 and 5.69 µM mg-1 s-1), while the room-temperature model showed a relatively slow reaction rate at 3.8 µM mg-1 s-1. Accordingly, we attributed the multistimuli behavior of those constructed proteinosomes to the increase in enzymatic activity associated with opening the membrane channels.

Figure 4. (a) Schematic representation of the regulated corelease of hydrophilic (ALP-FITC) and hydrophobic (Nile red) substances via UV light (λ ≈ 365 nm). (b) Release profiles of encapsulated hydrophilic ALP-FITC from the asprepared proteinosomes actuated by 10 min UV irradiation and GSH. (c) Corresponding release capability of adsorbed hydrophobic Nile red from the hydrophobic layer of the membrane driven by converting hydrophobic SP to hydrophilic McH+ using UV light irradiation (λ ≈ 365 nm). Multifunctional Modulated Interface Reactions on Synthetic Proteinosomes. The above studies revealed that

the continuous multistimuli manipulation of membrane permeability could be well modulated and led to programmably varied enzymatic reaction rates under the four stages. Subsequently, we investigated whether we could take advantage of ultraviolet light acting on phototriggered polymer chains to switch between hydrophilicity and hydrophobicity. This reversible switch effect could neatly induce a hydrophobicity-to-hydrophilicity transition in the proteinosome membrane under alternative ultraviolet light irradiation, which can be further used to trigger the corelease of hydrophobic and hydrophilic substances. To do so, hydrophilic FITC-labeled alkaline phosphatase (ALP-FITC) and hydrophobic Nile red were loaded inside the core and membrane lumens, respectively, of proteinosomes (Figure 4a). Since ultraviolet light can induce SP-to-McH+ isomerization, the controlled release of the two payloads would be driven by both increasing the pore size and changing the hydrophobicity in the membrane. As shown in Figure 4b, only a small portion of the hydrophilic ALP-FITC could be released under ultraviolet light irradiation. However, such release behavior could be changed significantly under the redox environment, with over 90% of the encapsulated ALP-FITC being released, due to the loss of the polymer from the membrane (Figure 4b). In contrast, approximately 80% of the hydrophobic Nile red could be released under ultraviolet light irradiation (Figure 4c). Compared to the release of 20% of the hydrophilic ALP-FITC from proteinosomes during the same time period, this result suggested that rather than being encapsulated inside proteinosomes, the hydrophobic Nile red was located in the hydrophobic layer of the membrane, and thus hydrophilic and hydrophobic cargo release processes could be actuated by different manners. Moreover, this corelease of hydrophobic and hydrophilic substances was further observed by confocal laser scanning microscopy (CLSM): hydrophilic FITCdextran (40 KDa) was mainly located in the core domain (Figure S12), and hydrophobic Nile red was strictly located on the membrane domain. The study of the release kinetics of the encapsulated hydrophilic FITC-dextran under ultraviolet light irradiation and in redox medium is summarized in Figure S12h, and there was an obvious leap in the release percentage of the encapsulated FITC-dextran from 30% to 80% when changing the stimulation from ultraviolet light to the redox medium. In contrast, the loaded hydrophobic Nile red showed a steady release profile even upon shifting into the redox environment (Figure S13a-h). These results clearly confirmed photoswitchable cargo release as well as switchable membrane permeability and hydrophobicity. Encouraged by the photoswitchable release of hydrophilic and hydrophobic substances, we attempted to realize the modulated interface reaction of the membrane with enhanced catalytic efficiency. A promising example is the lipase-mediated enzyme catalytic reaction. The encapsulated lipase could hydrolyze the substrate fluorescein diacetate (FDA) into hydrophobic fluorescein, which adsorbed onto the membrane; thus, the product was absolutely covered on the surface of this membrane in a short time (Figure S14). In contrast, when using another substrate, 4-meth-



membrane was attributed to the enrichment of hydrophobic green fluorescence products (Figure S16b-d). In contrast, the release of generated fluorescein was triggered by continuous ultraviolet irradiation accompanied with the photoisomerization of spiropyran (Figure S16e-f). After irradiation for 15 min by ultraviolet light, the SP-to-McH+ isomerization within membranes led to an obvious cessation of fluorescence due to the diffusion of fluorescein across the membrane (Figure S16g). Accordingly, the photosensitive membrane exhibited excellent advantages in the programmed modulation of membrane permeability and reversible transition of ultraviolet-mediated interface reactions.

CONCLUSIONS

Figure 5. (a) Schematic illustration of the reversible trapping and release of an FDP derivative (fluorescein, insoluble, green) with encapsulated alkaline phosphatase (ALP), dominated by converting hydrophobic SP to hydrophilic McH+ through UV light (λ ≈ 365 nm) irradiation. (b-f) CLSM images of photoswitchably generated fluorescein upon continuous UV light (λ ≈ 365 nm) irradiation with increasing time intervals (60 s, 300 s, 500 s, 800 s, 1000 s, respectively). Scale bar is 20 µm. ylumbelliferyl butyrate (4-MU-Bu), which can be hydrolyzed into the relatively hydrophilic product 4-MU (blue fluorescence), it was observed that upon increasing the time intervals for the obtained proteinosomes (Figure S15), the blue fluorescent products were dispersed throughout the whole solution. The above results clearly indicated that membrane-mediated interface reactions could be well varied by the transition of the hydrophilic and hydrophobic layers. To further elucidate this issue, membrane-mediated dynamic measurements were constructed to assess the evolution of the generated product during ultraviolet irradiation. In this study, the enzyme ALP and the substrate fluorescein diphosphate (FDP) were employed. When regulated by ALP, the formation of insoluble fluorescein within the hydrophobic layer of the membrane would lead to the timedependent release of the product upon ultraviolet irradiation (Figure 5a). After 60 to 1000 s, the proteinosomes showed increasing release efficiency under continuous ultraviolet irradiation (Figure 5b-f). The corresponding formation of fluorescent product showed an initial abrupt increase at 300 s and a maximum extent at 800 s, as determined by ultraviolet irradiation. Such in situ observations are highly advantageous for understanding dynamic ultraviolet-mediated interface reactions. To provide further insights concerning the effect of interface reactions, the releasing process of the product was further confirmed by confocal fluorescence microscopy (Figure S16). After the encapsulated ALP proteinosomes were incubated for 0-4 h in the presence of FDP, the confocal fluorescence images indicated that the increasing fluorescence intensity on the

In summary, we have demonstrated an effective way to modulate the programmed release of proteinosomes, which showed a clear stimuli responsive behavior by allowing different-molecular-weight cargos to release under body temperature, room temperature, ultraviolet, and redox treatment. Moreover, reversible phototriggered SP-toMcH+-to-SP transitions in the membrane could allow the release of hydrophilic (FITC-dextran) and hydrophobic (Nile red) substances and guide interface reactions between lipase and FDA, lipase and MU-Bu, and ALP and FDP, as well as several enzymatic reactions. Most interestingly, the photosensitive membrane exhibited advantages in the capture and release of hydrophobic products by opening and closing photoresponsive spiropyran ligands. In general, this study clearly exhibited both the programmed release of cargos and dynamically modulated ultraviolet-mediated interface reactions in proteinosomes. In addition, the in situ observation of dynamic interface reactions was highly advantageous for understanding membrane-associated stimuli-responsive characteristics for future application in nanoreactors as well as artificial cells.

ASSOCIATED CONTENT Supporting Information. 1H NMR spectra were recorded on a Bruker Advance-400 MHz spectrometer with CDCl3 as a solvent at room temperature. Chemical shifts (δ) are expressed in ppm. UV/vis absorbance and optical transmittance experiments were conducted on a PerkinElmer spectrophotometer (Lambda 750S, USA). Fluorescence experiments were conducted on PerkinElmer spectrofluorometer (LS55, USA). A handheld UV lamp (365 nm, 1.5 mW/cm2) was used for UV light irradiation. The average particle size and size distribution of the capsules (0.1 mg/mL, pH 6.8, 5.0 mM PBS buffer) were characterized by dynamic light scattering (DLS) with an ALV-5000/E DLS instrument (Malvern Instruments, UK) at a fixed scattering angle of 90°, after being filtered by 0.45 μm Millipore filters. Zeta potential studies of sample solutions (0.2 mg/mL, pH 6.8, 5.0 mM PBS buffer) were carried out at 25 °C using a ZETASIZER Nano series instrument (Malvern Instruments, UK). Transmission electron microscope (TEM) measurements were conducted on a JEM-1400 electron microscope. TEM was performed using a LaB6 filament at 120 kV in bright-field mode. The samples for TEM observations were prepared by dropping 10 μL of an aqueous dispersion of


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ACS Applied Materials & Interfaces self-assembled aggregates (0.1 mg/mL) onto a 300-mesh, carbon film-coated copper grid, and the specimens were then dried under vacuum for one day. Field-emission scanning electron microscope (SEM) observations were conducted on a HITACHI UHR FE-SEM SU8000 fieldemission scanning electron microscope. The samples for SEM observations were prepared according to a procedure similar to that used for TEM. Optical and fluorescence microscopy was performed on a Leica DMI8 manual inverted fluorescence microscope at 10x, 20x, 40x and 100x magnification. Confocal laser scanning microscopy (CLSM) images were acquired using a Leica TCS SP5 microscope (Leica TCS confocal microscope with a Nikon Eclipse TE2000-S objective (60x oil)). NIPAAm (Energy Chemical, 98%) was recrystallized twice in hexane and toluene before use. AIBN, (Energy Chemical, 98%) was recrystallized from methanol. ACVA (Energy Chemical, 98%), 2mercaptothiazoline (Sigma, 98%), cystamine dihydrochloride (Energy Chemical, 99%), DCC (Energy Chemical, 98%), DMAP (Energy Chemical, 99%), carbon disulfide (Sigma-Aldrich, 99%), 1,6-diaminohexane (Sigma-Aldrich, 98%), 1,3,3-trimethyl-2-methyleneindoline (Energy Chemical, 99%), 2,5-dihydroxybenzaldehyde (Energy Chemical, 99%), acrylyl chloride (Energy Chemical, 99%), TNBSA solution (5% (w/v) in H2O, Sigma), EH (Sigma, 98%), EDAC (Sigma-Aldrich, 98%), BS(PEG)5 (Mw 532, Sigma, 98%), and BSA (isoelectric point = 4.6) (Sigma, 98%, Mw ~66 kDa) were used as received without further purification. ALP (Sigma, 10 DEA units/mg), lipase (Aladdin, from porcine pancreas), GOx (Sigma, from Aspergillus niger), HRP (Sigma), DNA (salmon testes, Sigma), SYBR green I (Sigma, 10000x in DMSO), FITC (Sigma, 90%), RBITC (Sigma), GSH (Sigma, 98%), FDA (Aladdin, 97%), FDP (AAT Bioquest), 4-MU-Bu (Energy Chemical, 97%), Nile red (Energy Chemical, 95%), and FITC-Dextran (MW 10 kDa, 20 kDa, 40 kDa, 70 kDa, 150 kDa, 500 kDa, and 2000 kDa, Sigma, 98%) were also used. Milli-Q water was used to prepare all the solutions in this study. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]. *Email: [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. / Funding Sources The National Natural Science Foundation of China and the China Postdoctoral Science Foundation. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

We thank NSFC (21504020, 21871069), the Open Project of Key Laboratory of Microsystems and Microstructures Manufacturing (2016KM003), the China Postdoctoral Science Foundation (2015M571401; X.L.) and the Thousand Young Talent Program.

ABBREVIATIONS NIPAAm, N-isopropylacrylamide; AIBN, 2,2’-azobis-(isobutyronitrile); ACVA, 4,4’-azobis(4-cyanovaleric acid); DCC, N’-dicyclohexylcarbodiimide; DMAP, 4(dimethylamino)pyridine; TNBSA, 2,4,6-trinitrobenzene sulfonic acid; EH, 2-ethyl-1-hexanol; EDAC, N-ethyl-N’(3-dimethylaminopropyl) carbodiimide hydrochloride; BS(PEG)5, PEG-bis(N-succinimidyl succinate); BSA, bovine serum albumin; ALP, alkaline phosphatase orthophosphoric-monoester phosphohydrolase; GO, glucose oxidase; HRP, horseradish peroxidase; DNA, deoxyribonucleic acid sodium salt; FITC, fluorescein isothiocyanate isomer I; RBITC, rhodamine B isothiocyanate; GSH, reduced Lglutathione; FDA, fluorescein diacetate; FDP, fluorescein diphosphate tetraammonium salt; 4-MU-Bu, 4methylumbelliferyl butyrate; FITC-dextran, fluorescein isothiocyanate-labeled dextran (Mw 10 kDa, 20 kDa, 40 kDa, 70 kDa, 150 kDa, 500 kDa, and 2000 kDa).

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An effective way to modulate the programmed release of synthetic proteinosomes was demonstrated. Moreover, reversible phototriggered membrane transitions could monitor the release of hydrophilic or hydrophobic substances and guide enzymatic interface reactions, as well as exhibit advantages in the capture and release of hydrophobic products.

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Multifunctional and Programmable Modulated Interface Reactions on

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