Multiple-Enzyme Graphene Microparticle Presenting Adaptive

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Functional Nanostructured Materials (including low-D carbon)

A Multiple-Enzyme-Graphene Microparticle Presenting Adaptive Chemical Network Capabilities Xiangming Li, Zequn Ma, Yihe Zhang, Shaofeng Pan, Meng Fu, Chengjun He, and Qi An ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b13183 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 21, 2018

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ACS Applied Materials & Interfaces

A Multiple-Enzyme-Graphene Microparticle Presenting Adaptive Chemical Network Capabilities Xiangming Li, Zequn Ma, Yihe Zhang*, Shaofeng Pan, Meng Fu, Chengjun He, Qi An* Beijing Key Laboratory of Materials Utilization of Nonmetallic Minerals and Solid Wastes, National Laboratory of Mineral Materials, School of Materials Sciences and Technology, China University of Geosciences, Beijing, 100083, P. R. China. KEYWORDS. graphene, protein particles, layer-by-layer, NIR-responsive, reaction network ABSTRACT: Interrelated reaction networks steered by multiple types of enzymes are among the most intriguing enzyme-based cellular features. These reaction networks display

advanced

features,

such

as,

adaptation,

stimuli-responsiveness,

and

decision-making in accordance with environmental cues. However, artificial enzyme particles are still deficient of network-level capabilities, mostly because delicate enzymes are difficult to immobilize and assemble. In this study, we propose a general strategy to prepare enzyme-based particles that demonstrate network reaction capability. We assembled multiple types of proteins with a nanoscopic binder prepared from polyelectrolyte and graphene. After assembly, the enzymes all preserved their catalytic capabilities. By incorporating multiple types of enzymes, the particles additionally displayed network-reaction capabilities. We were able to use NIR irradiations to quasi-reversibly adjust the catalytic abilities of these enzyme-based particles. In addition,

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after a biomimetic mineralization process was used to wrap the protein complexes in a MOF shell, the particles were more robust and catalytically active even after being immersed in acidic (pH 4) or basic (pH 10) solutions for three days. This study provides an insight to study the network properties of functional enzyme-particles experimentally and enrich scientific understanding of multifunctional or stimuli-responsive behaviors at reaction network level. The building of artificial reaction networks possess high potential in realizing intelligent microparticles that can perform complicated tasks. INTRODUCTION Research concerning the assembly of proteins has gained strong interest due to the central role proteins play in cell functionalities.1-6 Interrelated reaction networks steered by multiple types of enzymes are among the most intriguing enzyme-based cellular features. In these reaction networks, the products of one or more reactions serve as the reactants of another one or more reaction, forming the nodes of the networks, and the chemical reactions, which transform one reagent to another, serve as the links in the network.7-8 Such reaction networks display advanced features such as adaptability and/or stimuli-responsiveness at levels not achievable by any single reaction. Although this phenomenon occurs in the field of bio-inspired artificial microparticles, it has been highly appealing to device micro-/nano-particles that resemble the intelligence displayed by cells. However, enzyme-based particles that presented the ability to steer a reaction network has not been available, as far as our knowledge.9-10 The big challenge of preparing enzyme-based materials mostly lies in the fragile nature of enzymes. Enzymes are delicate and easily lose their catalytic power if mishandled, such as being confronted by high temperatures, extreme pH values, or even high ionic strengths. Much effort has been exerted on the development of effective enzyme immobilization methods. Particular attention has been placed in preserving or even enhancing enzyme catalytic power by studying the immobilization chemistries, reaction


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parameters, and/or protein structure changes during immobilization.11-14 As a result, enzyme catalytic power was successfully harnessed in pharmaceutical, cosmetic, or fine chemical processes by using genetic engineering, covalent, or non-covalent immobilization strategies.15-17 These methods are usually applicable to a specific type of protein and, at the same time, are labor intensive and time consuming. In addition, most of these methods are effective in immobilizing enzymes onto a solid substrate and forming an enzyme layer on the surface of the substrate, but not very effective in the preparation of multicomponent protein based particles which can contain a vary of enzymes in single nanoparticle. Recently, research has demonstrated multi-component enzyme particles that presented cascade reaction capabilities.18 By applying multiple cycles of precipitation of CaCO3 or SiO2 or by integrating precipitation with polymer multi-layer assembly around particle surfaces, multi-compartment enzyme-based particles were obtained.11,19 Cascade reactions were realized in these particles. In these methods, however, these particles were usually limited: the loading capacity of proteins were low because of the massive inert materials and mass transportation was hampered by the inert component within these particles. Other studies also used liposomes or polysomes to prepare protein-containing particles that presented cascade reaction capabilities.20-21 In these studies the loading efficiency of proteins and the fragility of the particles were the main concerns. As opposed to the sequential nature of cascade reactions, network reactions require that multiple reactions are able to take place simultaneously within the same space domain. Their mutual influences on reaction fluxes form the basics of adaption, stimuli-responsiveness, and/or decision-making capabilities. The building of artificial reaction networks possess high potential in realizing intelligent microparticles that can perform complicated tasks. However, in contrast to the wide attention received by cascade reactions, the particles that present network reaction features remain under



researched. The enzyme-based reaction network includes several different reactions and has some conditions which will affect the activity of enzymes, such as extreme pH value or unsuitable temperature. In order to realize enzyme-based reaction networks, it is necessary to have multi-protein microparticles that are robust, highly loaded with enzymes, and allow for effective reagent diffusion within the particles. As a result of these requirements - in addition to the fragile nature of enzymes - the preparation of enzyme-based particles that display network reaction capabilities proved to be a very difficult task. Recently, remarkable progress has been reported in the preparation of enzyme-based materials, making it not only more facile but also more appealing than preparing particles from purely natural components.22-23 One important achievement was that noncovalent interactions between polyelectrolyte and proteins were successfully explored as an effective method for the preparation of catalytically active protein-based materials. This development allowed for the harnessing of the superior catalytic power of proteins not only in an aqueous environment but also in organic solutions. Another important result was to include nanocarbons with the photothermal effect in enzyme-based materials. These studies allowed for the activities of the enzyme-based materials to be remotely adjusted by IR irradiations, demonstrating appealing features in medical therapies.24-27 Inspired by such recent progress on enzyme-based materials, we propose in this study a general method to prepare enzyme-based microparticles that demonstrated the properties of reaction networks, were responsive in IR irradiation, and were more robust than natural enzymes. The materials were prepared using commercially available polyelectrolyte polyethylenimine (PEI) and poly (acrylic acid) (PAA), as well as a two-dimensional substrate graphene oxide, together with the various proteins. After a layer-by-layer wrapping of the PEI and PAA around the GO template, the two-dimensional composite was able to assemble with proteins and effectively retain its


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enzyme activity. The model multi-component protein particle was prepared using superoxide dismutase (SOD), catalase (CAT), hemoglobin (Hb), and carbonic anhydrase (CA). These prepared protein particles were able to steer the superoxide radical into a collective output, including H2O2, O2, and oxyHb with adjustable fluxes, and, at the same time, adjust the biomineralization of CO2 into CaCO3. In addition, the chemical conversion capabilities of the reaction network could be tuned by using IR irradiation, taking advantage of the photothermal effect of rGO. The particles displayed negligible cytotoxicity, and enhanced cellular capabilities to survive oxidative stresses. After wrapping the multiple-component enzyme particles using a biomimetic mineralization process into ZIF-8 MOF, the robustness of the enzyme-based particle was remarkably enhanced and still active, even after a three-day rinsing in acidic solutions. RESULTS AND DISCUSSION The assemblies of the protein particles were inspired by the fact that promiscuous interactions effectively induced protein assembly in vivo. Polyelectrolytes were wrapped around a 2D substrate rGO28 (GO serves simultaneous two functions as (i) the assembly substrate to form the graphene-polymer composite which in turn served as building block for the enzyme assembly and (ii) as a photothermal agent to adjust enzyme activities. Reduced GO were used because of their effective photothermal effect. The polyelectrolytes were used to modify rGO interfaces which would not only bind with enzymes using supramolecular interactions but also creat a hydrophilic envirionment for enzyme to keep their activities.) to serve as a multivalent binder which then assembled with proteins to generate protein clusters. Polyelectrolytes ample of positive and negative charges were selected since they artificially resembled the protein surface. We expectated that they would provide a variety of functional groups capable of forming electrostatic interactions or hydrogen bonds to bind to proteins. In order to locate polyelectrolyte materials that could lead to functional protein assemblies, several combinations of



polyelectrolyte - including PEI/PAA, PEG/PAA, and PLL/PASP - were tested, and only the PEI/PAA pair assembled with enzymatic proteins to form nanoparticles that were catalytically active. In order to prepare the protein particles, PEI and PAA were first assembled alternately around the substrate rGO using the layer-by-layer method to form a multivalent building unit rGO/(PEI/PAA)3.5 (See Figure 1a and S1, detailed in SI). Afterwards, the rGO/(PEI/PAA)3.5 (denoted as rGP3.5) were mixed with a solution of protein in the existence of a surfactant Tween 80 (Figure 1b). A variety of protein was tested in this step in order to demonstrate the generality of the method to form protein particles. The proteins studied including carbonic anhydrase (CA), hemoglobin (Hb), catalase (CAT), superoxide dismutase (SOD), enhanced green fluorescent protein (EGFP), lipase (LIP), and bovine serum albumin (BSA). Within 20 minutes, the mixture of rGP3.5 and the corresponding protein became turbid, indicating the formation of nanoor micro-particles. The mixtures were kept for a further 100 min for the aging of the particles. (Preparation of each of the protein particles are detailed in Experimental Section.) TEM images in Figure 1g-j and 1o-q indicated that nanoparticles formed for all types of the proteins mentioned above: the proteins CAT, SOD, CA, Hb, LIP and EGFP all formed spherical particles with rGP3.5 with diameters ranging from 500-900 nm. BSA formed quasi-spherical clusters with bumps on their surfaces; the clusters’ average diameter was 800 nm. Because the spherical shape always has the minimum surface energy and the nano-materials tends to form in the direction of the lowest surface energy to achieve optimal steady state. Thus, the protein particles formed the spherical or clustered shape29-30. Subsequently, the functionalities of the assemblies were tested to verify that the composite rGP3.5@protein (protein: CA, SOD, CAT, Hb or EGFP) particles were effective in catalyzing corresponding reactions. First, UV spectra (as shown in Figure S2a-b) were used to verify that the single-type-protein composite particles presented absorbance from both rGO and the corresponding proteins, indicating the coexistence of


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the components in the particles. The absorbance bands around 270 nm were characteristic of rGO. The absorbance bands from 260 nm to 280 nm indicated the tyrosine and tryptophan residues of proteins. The Soret bands at 406 nm indicated the heme auxiliary of catalase and hemoglobin. ATR-FTIR spectra further supported the successful fabrication of the rGP3.5@protein particles, as shown in Figure S2c-d. For both the proteins and the single-type-protein composites, the position of C-O stretching vibrations (amide I) were located at 1655 cm−1, and N-H bending vibrations (amide II) were located at 1541 cm−1, suggesting that the enzymes preserved their functional structures in the composites. It is worth mentioning that the rGP3.5@SOD has no obvious vibration peaks at 1655 and 1541 cm-1, but there is an obvious stretching vibrations at 1082 cm-1, indicating that the other C=O vibrations (amide I) of amino acid. We further studied the catalytic functions of the protein composites. The composite rGP3.5@CAT was effective in catalyzing the decomposition of H2O2 (Figure 1c). Compared with the catalytic activity of pristine CAT, the efficacy of the rGP3.5@CAT composite was slightly lower, probably restricted by the decreased contact area between CAT and H2O2, limited diffusion of H2O2 within the composites and the surface concentration of the enzyme (Figure 1k). Interestingly, the catalytic efficacies were significantly influenced by the number of bilayers of PEI/PAA around the rGO template. The relative activity (calculation method detailed in EXPERIMENTAL SECTION) of the composite increased with the growth of the number of bilayers. The relative activity of rGO3.5@CAT was 1.74 times higher than rGP0.5@CAT, and was 1.10 and 1.12 times higher than rGP1.5@CAT and rGP2.5@CAT, respectively (Figure S3a). The optimal performance of rGP3.5 in obtaining the enzyme complex with high activity compared with rGP1.5 and rGP2.5 was attributed to two possible factors. One is that the thicker polyelectrolyte matrix should provide a more hydrophilic microenvironment beneficial to protein functionality, and the other is that they provide more ample functional groups which interact with protein with higher valency and pertube less of protein configrations. However, deeper understanding of this



issue would need systematical further studies. In addition, the stabilities of catalytic efficacy also enhanced with an increase of the number of bilayers. After the composites were kept overnight, the catalytic activity of rGP0.5@CAT decreased by 30% when compared with freshly prepared samples (aged for 2 h), while the rGP3.5@CAT composites kept overnight decreased less than 10%, indicating that this composite could be preserved for a remarkably longer period (Figure S3b-c). Based on these results, we chose rGP3.5 for the preparation of protein composites. rGP3.5@SOD particles of around 400 nm in diameter could effectively convert O2.into H2O2 and O2 (Figure 1d). The mixture of xanthine, xanthine oxidase and NBT presented a strong fluorescence emission at around 580 nm after white light irradiation, demonstrating a high level of O2.- (Figure 1l). Once the rGP3.5@SOD were added into the mixture, the signal decreased dramatically, indicating the efficacy of rGP3.5@SOD in scavenging O2.-. The inhibition rate of O2.- could be adjusted by the doses of the rGP3.5@SOD. The inhibition fraction of the O2.- was 90.3% upon adding 600 μL dispersion of the rGP3.5@SOD (20 μg/mL), 86.4% and 76.9% upon 300 μL and 100 μL dispersions, respectively (Figure S4). rGP3.5@CA spheres with diameters around 500 nm were able to enhance the efficiency of carbon dioxide hydration (Figure 1e). The mass of the product CaCO3 was 91.2 mg with the existence of the rGP3.5@CA and 86.6 mg without the rGP3.5@CA (Figure 1m). The morphologies of the CaCO3 were also remarkably different; they were quasi-cubic with the existence of CA, and appeared as spherical particles with rGP3.5@CA (SEM images in Figure 1m). rGP3.5@Hb spheres of diameters around 900 nm were able to reversibly bind and release oxygen (Figure 1f). The deoxyHb, obtained by flowing CO2 into the mixture, presented a typical absorbance band at around 419 nm; while the absorbance of oxyHb, generated by introducing O2 into the mixture, blue-shifted to 406


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nm, as shown in Figure 1n. In order to demonstrate that the bound oxygen by the rGP3.5@Hb could be reversibly released, CO2 gas was again blown into the rGP3.5@Hb dispersion, and the absorbance of the composite shifted back to 419 nm, demonstrating that the rGP3.5@Hb transformed to the deoxidized form, and the bound oxygen was released from the composite. rGP3.5@EGFP presented enhanced fluorescence intensity compared with the EGFP solution (Figure S5a), probably due to the fluorescence enhancement effect of rGO.31-33 The extent of FL enhancement also increased with higher ratios of rGP3.5 (20, 60, 100 μg/mL, Figure S5b) in the preparative mixtures. These experiments demonstrated that the rGP3.5 was a universal building unit for the formation of a composite that would have a wide variety of proteins that could preserve their catalytic functions. To determine the stability of the rGP3.5@protein, the rGP3.5@protein dispersion was prepared and incubated in PBS buffer solution for 7 days. After 7 days, the rGP3.5@protein still keep the relative activity of catalyzing H2O2, inhibiting superoxide radicals, maintaining the spherical morphology of CaCO3 and loading O2 (shown as in Figure S6). Encouraged by the generality of the method to prepare a variety of protein composites, we wondered whether it would be possible to prepare a composite particle that contained simultaneously three or four types of proteins. The query was inspired by biological systems such as cells metabolisms and respiration processes where the coexistence of varying types of enzymes generate a reaction network. The reactants/products of the enzymatic reactions are the nodes and the enzymatic reactions are the links in such reaction networks. Such a connected reaction system would present emergent systematic properties of responsiveness and adaptations beyond the functional space of remote, singular reactions. However, because of the difficulties of producing functional protein assemblies, composite protein aggregates that present such network-scale functionalities



have been very rare. To meet this challenge, we began with the preparation of a ternary protein composite of rGP3.5@SOD@CAT@Hb by mixing the dispersions of rGP3.5 with the corresponding proteins. Such a composite particle was capable of transferring the input of proton and O2.- into H2O2, O2 and H2O, as shown in Figure 2a. The ability to adjust the levels of O2.and H2O2 is very important in living cells. This is because O2.- and H2O2 serve as important signal species or intermediate reactants for cells, calling on them to adjust their property states in response to external stimuli. At the same time, they can cause irreversible damages if their concentrations exceed the processing capacity of the cells. Hence, it is essential to maintain the balance of these species from the prespective of both fundamental study and therapeutic application. The composite particles of rGP3.5@SOD@CAT@Hb were spheres with diameters ranging from 500 to 900 nm, similar to the single-type-protein composites described above (Figure 2e). UV−vis absorption bands at 270 nm, 275 nm and 406 nm indicated coexistence of rGO and proteins in the rGP3.5@SOD@CAT@Hb complex (Figure S7a). ATR-FTIR spectra of the rGP3.5@SOD@CAT@Hb (as shown in Figure S7b) maintained the peaks of amide I and amide II, indicating the integrality of the enzymes’ chemical structure after encapsulating. The chemical formulae of the catalytic reactions and the possible reaction pathways of the reaction network appear in Figure 2b-d. The stoichiometric matrix of the reaction network was generated following the constraint-based reconstruction and analysis (COBRA) method. In the matrix, each metabolite occupied a row and the stoichiometric flux rate of a reaction was displayed in a column. The negative and positive values represent reactants and products, respectively. From the matrix, we clearly see that O2 participates in four reactions, being the most densely linked node in the network and capturing the inter-related nature of the reactions in the network.


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Experimental results confirmed the existence of the multiple reaction pathways. The particles presented the catalytic functions of the three enzymes simultaneously, SOD, CAT, and Hb (Figure 2f-h). With the existence of the composite ternary protein particles, the concentration of O2.- decreased, H2O2 was decomposed, and the absorbance of deoxyHb at 419 nm blue-shifted to 406 nm. This result corresponds to oxyHb, indicating the particles’ oxygen-binding capacity. Especially, the composite particle was able to transfer O2.- into bound oxygen even without the input of O2 into the reaction system. The source of the bound oxygen was the decomposition product of H2O2. These results demonstrated that the composite protein particles were able to transfer the chemical inputs (O2.- and proton) into chemical output across network nodes, which is a unique property of reaction networks - the ability to go beyond the level of a single reaction. Simulations indicated that the diffusion-based mass transport in a typical particle with a diameter of 1 μm was swift. It took only milliseconds for reagents generated at the surface of the particles to reach the particle center (Figure 2i). We further experimented with adjusting the process capabilities of the reaction network by manipulating the components in the composite particles. It is noteworthy that the reaction network of the rGP3.5@SOD@CAT@Hb could be broken if the key component was absent. When the SOD component was absent, O2.- was not able to be converted into H2O2 or O2. As shown in Figsure S8a-b, no oxyHb was obtained and the absorbance of the mixture remained at 419 nm without any shift because of the absence of O2 in the reaction system. Similarly, H2O2 could not be catalitically decomposed by the rGP3.5@SOD@Hb when the CAT component was absent (Figure S8d), and the system generated a high amount of H2O2 as its output (Figure S8c). However, oxyHb was still generated (see Figure S8d, the absorbance peak shifted from 421 nm to 406 nm) because in such circumstances O2 could be generated through the flux of O2.-



decompostion catalyzed by SOD. Despite the disfunction of the CAT flux, the generation of oxyHb demonstrated the robust and adaptable properties of the reaction network. In addition, the flux of a specific path (the decomposition efficiency of O2.-) could be adjusted with an abundant amount of the corresponding enzyme, that is, by varying the concentration of the preparative solution of the enzyme, see Figure S10a. By increasing the concentration of SOD in the preparative cocktail from 0.5 to 1 mg/mL, the absorbance of O2.- at around 580 nm decreased from 0.3 to 0.2 (a.u.) after a 10 min reaction, indicating an enhanced pathway flux (Figure S10b). However, the output of oxyHb did not alter remarkably, judging from the absorbance spectra shown in Figure S10c; both types of composite (SOD preparative solution 1 or 0.5 mg/ml) presented an absorbance shift to an identical extent and with identical peak intensity. These results indicated that the output of bound oxygen was not appreciably affected by the upstream flux fluctuation, probably due to the deficiency in the number of Hb as compared to that of SOD in the composite. While the surplus O2 generated by O2.- decomposition should be released as free O2, due to the low amount of the species, we were not able to detect the O2 output by gas chromatography. To demonstrate that the reaction network regulated by the rGPn@protein complex could be expanded, we added an additional enzymatic component into the composite - the CA. CA can promote the hydration rate of CO2 to produce bicarbonate and regulate the morphology of the generated CaCO3 via the regulation of the reaction kinetics. The produced proton should participate in the reaction path of catalytic decomposition of O2.regulated by SOD (Figure 2j). The possible pathways of the network and the stoichiometric matrix are shown in Figure 2k-m. The SEM EDX analysis (Figure S11) and TEM image (Figure 2n) demonstrated that the spherical enzyme nanoparticles with diameters of around 900 nm contained all the characteristic elements of the four enzymes (CA: Zn; SOD: Cu; CAT and Hb: Fe). The UV/Vis spectroscopy and ATR-FTIR spectra


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in Figure S12 further corroborated the existence of rGO and proteins. The quaternary protein particles presented all reaction fluxes of the ternary protein particles mentioned above. In addition, the morphologies of generated CaCO3 changed from cubes with a side length of around 3.5 μm to clusters formed by small particles with irregular shapes (Figure 2o-p). In order to show the advantage of the

rGP3.5@CA@SOD@CAT@Hb,

the relative activity of the enzymes mixture (native CAT, SOD, CA, Hb) has been tested, show as in Figure S13. Figure S13a verified that the enzymes mixture could catalyze H2O2, but the relative activity was much lower than the rGP3.5@CA@SOD@CAT@Hb. The

inhibition

fraction

of

the

O2.-

wasn’t

obvious

compared

to

the

rGP3.5@CA@SOD@CAT@Hb (Figure S13b). The SEM image in Figure S13c showed the morphologies of the CaCO3 were still quasi-cubic with the existence of enzymes mixture. Figure S13d indicated that the enzymes mixture possessed the O2 loading ability but wasn’t sensitive. These results indicating the rGP3.5@CA@SOD@CAT@Hb is more superior

than

the

enzymes

mixture,

probably

because

the

rGP3.5@CA@SOD@CAT@Hb could attract more target reactant with the abundant surface charge than the enzyme mixture that dispersed in the entire container volume. Furthermore, taking advantage of the photothermal effect of rGO34-39, we adjusted the catalytic activities of the enzymatic complex using a near-infrared (NIR) laser. We found that the catalytic capabilities of the enzyme complex could be adjusted by NIR irradiation, with extents dependent on the power of the laser and the period of the irradiation. Firstly, the composite of a single-type-protein was irradiated using the NIR laser. With a laser power of 1 W/cm2, the relative activity of the rGP3.5@CAT decreased to 66% of the original value after irradiation for 20 min and by 33.4% after 60 min. After removing the irradiation, the activities recovered to 89.7% and 76.9%, respectively, of the original value within 20 min. When the laser power was increased to 3 W/cm2, the catalytic activity decreased to 53.4% after 10 min and 10.0% after 30 min irradiation, followed by a maximum recovery of 87.1% and 34.7% (relative to the original value)



after removal of the irradiation (Figure 3a-d). Relative activity can be controlled by the irradiation time. The longer the irradiation, the greater the degree of inhibition (Figure S14). The fluorescence intensity of the rGP3.5@EGFP also decreased with time under 3 W/cm2 NIR laser irradiation. As shown in Figure S12a, the fluorescence intensity decreased by 25.1%, 40%, and 54% after 10, 30, and 60 min irradiation, respectively. The temperature rise of the composite dispersion (20 μg/mL) during the irradiation was 23.6 ºC when the irradiated time was 60 min. The activity adjustment was only slightly dependent on the concentration of the composite dispersion. When the concentration of the rGP3.5@EGFP was 100 μg/mL (Figure S15b), the fluorescence intensity decreased by 34.8%, 40.3%, and 64.4% after irradiation of 10, 30, and 60 min, respectively, with a temperature rise of 29.2 ºC. Then fluorescent intensities were also able to partially recover after removal of the irradiation (Figure S15c and S15d). The activities of the ternary-type-protein composite rGP3.5@SOD@CAT@Hb could also be adjusted using the NIR laser, as shown in Figure 3e-f. The catalytic efficiency of O2.- scavenging increased under irradiation as verified by lowered characteristic absorbance of O2.- at around 580 nm. The oxygen-loading efficiency also increased under irradiation, indicated by a more pronounced absorbance shift: the characteristic absorption peak shifted to 407 nm after irradiation and to 413 nm without irradiation. The phenomena observed were consistent with previous reports that enzyme activities could be either enhanced or decreased with NIR irradiation, and, upon removal of the irradiation, would partially recover. Comparing to the recently works, our study presents a facile and accelerated method to prepare the encapsulated multiple


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enzymes particles which possess the adjustable ability to participate in a quadruple cascade reaction under NIR laser. We further assessed the biocompatibility and a therapeutic effect of the composite rGP3.5@CA@SOD@CAT@Hb nanoparticles with regard to easing the oxidative stress of HLF cells (Figure 4a). As shown in Figure 4b, after incubated with the rGP3.5@CA@SOD@CAT@Hb spheres for 24 h, cell viability was higher than 95% when assessed by MTT assay, indicating low cytotoxicity of the nanoparticles. An illustrative therapeutic effect of the composite protein particles was demonstrated by their ability to increase cell viability under H2O2-induced oxidative stress. After incubation for 48 h, the cell viability decreased to 76.9%, 56.0% and 25.7% with the increase of the concentrations of H2O2 at 0.5, 1, and 20 mM, respectively, in the media. In contrast, when incubated with the existence of the rGP3.5@CA@SOD@CAT@Hb complex (20 μg/mL) in the media, the cell viabilities significantly rose to 94.2%, 90.8% and 83.6%. Lastly, we managed to increase the stability of the delicate protein complex using a biomimetic mineralization method40-41. Inspired by biomineralization process, for example, how hard shells protect the soft functional structures of mollusks, we used ZIF-8 to coat the MOF structure and protect the protein complex (Figure 5a). As shown in Figure 5b-d, the spherical or semi-ellipsoid morphologies of the complex were preserved after MOF coating, and the core-shell structure was observable using TEM and SEM. XRD patterns indicated that the MOF was well crystallized (Figure 5e). In addition, the FTIR indicated that the amino I and II structures (1652 and 1542 cm-1) were well-preserved for the proteins, and coexisted with the signals from the MOF structure (1400-1500 cm-1) corresponded to the imidazole ring stretching, and 1580 cm-1 corresponded to C=N stretching of imidazole; Figure 5f-g). The catalytic ability to eliminate O2.- and to scavenge H2O2 was preserved for the protein complex (Figure



S18a-c). However, after MOF wrapping, the ability of the protein complex to generate oxyHb was compromised. A possible reason might be the competitive binding of the oxygen of ZIF-8.42-44 The ZIF-8 coating remarkably enhanced the stabilities of the protein complex in extreme pH environments. After storage in solutions of pH 4 or pH 10 for 3 days, the rGP3.5@CA@SOD@CAT@Hb complexes without the ZIF-8 coating were completely destroyed into pieces with irregular shapes and lost their catalytic capabilities (Figure 5h-i). In contrast, the complexes protected by ZIF-8 not only preserved their morphology but also kept their catalytic abilities to eliminate O2.- and H2O2 (Figure 5j-k and Figs. S18d-i). The manuscript aims to establish a concept to view the multifunctional particles from a network perspective. One of the possible application of the multifunctional enzyme particles is to be used in medical therapy, and serve as a reaction sub-network to simultaneously regulate the flux in more than one metabolic pathways in the patient, and achieve the level of adjustability and interconnection that could not be reached by conventional particles. The other important application is for the application method to be used to include more enzyme and used as model materials to study experimentally the network properties of functional particles and enrich scientific understanding of multifunctional or stimuli-responsive behaviors at the network level. CONCLUSIONS In summary, we developed a general method to prepare catalytically active protein complexes. The binder, (PEI/PAA)3.5-protected rGO was able to assemble with a wide variety of proteins, including all our tested proteins - CAT, SOD, Hb, CA, EGFP, BSA, and LIP - to generate composite particles with diameters of several hundred nanometers. Furthermore, we used four types of proteins, CAT, SOD, Hb, and CA, to prepare a composite protein particle with rGP3.5 and establish a reaction network. The network


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reactions were able to channel the input of O2.- through its two pathways into various output, including H2O, O2, H2O2, and oxyHb, and simultaneously adjust the mineralization process of CO2 into CaCO3 particles. The fluxes of the pathways were adjusted by varying the amount of the included enzymes. Even if one of the two pathways to generate oxyHb (via CAT) was sabotaged, oxyHb was still generated via another pathway of the SOD catalytic reaction. In addition, the activities of the particles could be adjusted using NIR irradiation, taking advantage of the photothermal effect of rGO. A hard shell of MOF was coated onto the surface of the composite protein particles to enhance the stability of the particles, mimicking the biomineralization processes. This report demonstrates a general method to prepare a reactive catalytic network mimicking the systematically linked reactions in biological species, and provides a concrete material preparative platform to elevate the study of responsive or intellectual materials to the systematic reaction level. We expect that this report provides an insight to study the functions of artificial microparticles at reaction network level. The building of artificial reaction networks possess high potential in realizing intelligent microparticles that can perform complicated tasks. EXPERIMENTAL SECTION Materials. Graphene oxide was purchased from Nanjing XFNANO Materials Tech Co., Ltd. Polyethylenimine (PEI, MW 700,000), Poly (acrylic acid) (PAA, MW 100,000) was purchased from Alfa Aesar. Catalase (CAT, MW 250,000), Xanthine and H2O2 (30 wt%) were

purchased

from

Sigma

Aldrich.

Nitro-blue

tetrazolium

(NBT)

and

Ethylenediaminetetraacetic acid (EDTA) were purchased from Amresco. Carbonic anhydrase (CA) and Hemoglobin (Hb, MW 645,000) were purchased from Yuanye Bio-Technology Co., Ltd., Shanghai. Tween 80, CaCl2 • 2H2O, K2HPO4 and KH2PO4 were purchased from Xilong Chemical Co., Ltd., Guangzhou. Superoxide dismutase (SOD), Xanthine oxidase, Tris (hydroxymethyl) aminomethane (Tris) and Na2S2O4 were



Page 18 of 39

purchased from Xiya Chemical Industry Co., Ltd., Shandong. Zinc acetate dihydrate and 2-methylimidazole were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). Ultrapure water (higher than 18.2 MΩ) was used in all experiments. Fabrication of rGPn@X (X = CA, SOD, CAT or Hb). The rGO/(PEI/PAA)n films (rGPn) were prepared as the precursor according to our previously established methods28. Preparation of rGPn@X was based on the rGPn films. Firstly, 5 mL of the rGPn dispersion (20 μg/mL) was prepared and added into a beaker. Then 5 mL of the enzyme or protein solution (CA, SOD, CAT, Hb) with the concentration of 1 mg/mL and 5 mL of Tween 80 solution (1 wt%) were added into the beaker respectively. The mixture was stirred for 10 min and incubated at 4 ℃ for 2 h. After reaction, the mixture was purified by centrifugation and washing with ultrapure water (repeated three times). Fabrication of rGP3.5@SOD@CAT@Hb. Briefly, native SOD (5 mL, 1 mg/mL), CAT (5 mL, 1 mg/mL), Hb (5 mL, 1 mg/mL) and Tween 80 (5 mL, 3 wt%) were quickly added into the rGP3.5 dispersion (5 mL, 20 μg/mL) step by step. The mixture was incubated at 4 ℃ for 2 h after stirring for a while. After reaction, the mixture was purified by centrifugation and washing with ultrapure water (repeated three times). The rGP3.5@CA@SOD@CAT@Hb hybrid nanoparticles were obtained in a similar manner of the rGP3.5@SOD@CAT@Hb. Synthesis of the ZIF-8 Coated rGP3.5@CA@SOD@CAT@Hb. 5 mL of the rGP3.5@CA@SOD@CAT@Hb dispersions (20 μg/mL) was added into 10 mL aqueous solution of 2-methylimidazole (3.46 M). And then 1 mL aqueous solution of zinc acetate dihydrate (0.5 M) was rapid poured into the above mixture. The resulting solution was stirred at room temperature for 5 min. The product was washed with DI water for three times by centrifuging (4000 rpm, 10 min) and restored the original 5 ml volume. To repeat

the

above

steps

for

5

cycles

to

rGP3.5@CA@SOD@CAT@Hb.


obtain

the

ZIF-8

coated

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CO2 Absorption Studies. The influence of rGP3.5@CA in CO2 absorption was studied by the form of calcium carbonate (CaCO3). 30 mL of two samples (one was ultrapure water, the other contained 2 mL of rGP3.5@CA dispersion (20 μg/mL)) were loaded with CO2 by bubbling the samples with the CO2 gas on the same flow rate for 1 h. To start the reaction, the two samples were added into a Tris buffer solution (1.39 M, 7.5mL) contained 0.45g CaCl2 • 2H2O respectively. The reaction mixture was maintained at approximately 0 °C for 2 h. Then, the mixture was filtered and dried to measure the weight of precipitated CaCO3. SOD Activity Assay. The classical Fridovich method45 was used to determine the enzymatic activity of rGP3.5@SOD. Xanthine and xanthine oxidase were used to produce superoxide radicals. Nitro blue tetrazolium (NBT) was used as a scavenger indicator. For a typical test reaction, the solution containing 0.3 mL of phosphate buffer (50 mM, pH 7.4), 0.3 mL of EDTA (0.1 mM), 0.3 mL of xanthine (5 mM), 0.3 mL of xanthine oxidase (1 mg/mL) and 2.5 mL ultrapure water was prepared for 10 min. Then, the rGP3.5@SOD composites (20 μg/mL) of various volume and 0.3 mL of NBT (3 mM) were added into the above solution. The mixture was illuminated to reduce NBT by a lamp with a stable light intensity for 10 min at room temperature. The color of the mixture was changed from yellow to blue and the mixture was measured by a UV-vis absorption spectrometer. CAT Activity Assay. The catalytic activity of the rGP3.5@CAT was determined by catalyzing hydrogen peroxide. The absorption intensity at 240 nm would decline while H2O2 reduce. Briefly, 2 mL of H2O2 (0.1 wt%) was added into a small quartz cell. 1 mL of the rGP3.5@CAT composites (20 μg/mL) was then added into the quartz cell to start the reaction. The original concentration of H2O2 is keep the same all the time. The absorption was measured at 240 nm per 5 min. The relative activity (RA) of the rGP3.5@CAT was counted by using Eq (1):



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(1) where Abs0 representes the initial absorption strength at 240 nm, Abst is the absorption strength at 240 nm within time t. Oxygen-Carrying Capacity. The prepared rGP3.5@Hb composites were dispersed in 5 mL of PBS (pH 7.4). To obtain deoxy-Hb based the rGP3.5@Hb, CO2 gas was allowed to flow over the above dispersion. After about 1 h, 1 mL of sodium dithionite (Na2S2O4, 10 wt%) solution was added. The characteristic absorption peak of deoxyHb was measured by a U-3900H UV-vis absorption spectrometer with PBS as a reference solution. To verify whether the rGP3.5@Hb could reversibly bind and release O2, O2 gas and CO2 gas were then bubbled into the above mixture for 1 h, respectively. The light absorption was recorded by UV-vis absorption spectrometer. Catalytic

Activity

Assay

of

rGP3.5@SOD@CAT@Hb.

To

verify

the

rGP3.5@SOD@CAT@Hb hybrid nanoparticles possessed the activity of SOD, CAT, and Hb simultaneously, these nanoparticles were used to inhibit superoxide radical, H2O2 and carry O2. Briefly, CO2 gas flowed over 5 mL of the rGP3.5@SOD@CAT@Hb dispersion for 1 h and then 1 mL Na2S2O4 solution (10 wt%) was added, followed by measuring the UV-vis absorbance. Then, the test was divided into the following two aspects: i) 0.1 mL of the enzyme mixture was added into 3 mL of H2O2 (0.1 wt%) solution. The absorption was measured at 240 nm per 5 min. ii) The system contained 0.1 mL of phosphate buffer (50 mM, pH 7.4), 0.1 mL of EDTA (0.1 mM), 0.1 mL of xanthine (5 mM), 0.1 mL of xanthine oxidase (1 mg/mL) and 2.5 mL ultrapure water was used to produce superoxide radicals. Then, 0.1 mL of the enzyme mixture was added into the system. The mixture was kept in a vacuum for 10 min. The light absorption of the mixture was scanned from 200 to 800 nm by a UV-vis


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spectrometer. Finally, 0.1 mL of NBT (3 mM) was added into the mixture. After illuminating for 10 min, the absorbance was measured. The catalytic activity of the ZIF-8 coated rGP3.5@CA@SOD@CAT@Hb were measured in a similar manner. NIR Light Adjust Enzyme Complex Activity Assay. i) rGP3.5@CAT The rGP3.5@CAT dispersions was irradiated with different time periods by NIR laser with different power density (940 nm, 1.0 W/cm2 or 3.0 W/cm2), respectively. After irradiating, part of the irradiated dispersions stood at room temperature for 2 h to recover the initial temperature. The catalytic activity of the irradiated dispersions and recovered dispersions was determined by the same method mentioned above. The “100%” of RA plateau at X% level in Figure 3d-e referred to the activity of the rGP3@CAT without a NIR laser. We used this value as a control and the relative activity of the rGP3@CAT (different concentrations) after NIR laser irradiation was calculated relative to this standard. So the 100% RA plateau at X% level is a relative value and is use to facilitate the comparison between the enzyme activity before and after laser irradiation. ii) rGP3.5@SOD@CAT@Hb To prepare the deoxy-rGP3.5@SOD@CAT@Hb with CO2 gas. 0.1 mL of the mixture was added into the system containing xanthine and xanthine oxidase and irradiated by NIR laser (1.0 W/cm2) for 10 min. Then the light absorption was measured. Finally, the above mixture was illuminated for 10 min after adding 0.3 mL of NBT (3 mM). Biocompatibility Test. The HLF cells with a density of 4 × 104 cells/well were incubated with 20 μL of the rGP3.5@CA@SOD@CAT@Hb nanoparticles dispersion (20 μg/mL) for 24 h and washed three times with PBS. Alexafluor 488 was utilized to label with the



Page 22 of 39

rGP3.5@CA@SOD@CAT@Hb nanoparticles for 10 min, and then washing. Finally, the cells were observed by CLSM. In Vitro Evaluation of Intracellular ROS-Sensitivity. HLF Cells were seeded into 96-well plates with a density of 2.5 × 104 for 24 h. It could be divided into two groups. One was incubated with 10 μL of the rGP3.5@CA@SOD@CAT@Hb nanoparticles dispersion (20 μg/mL) or 20 μL of H2O2 with different concentration (0.5 mM, 1 mM, 20 mM).

The

other

group

was

firstly

incubated

with

10

μL

of

the

rGP3.5@CA@SOD@CAT@Hb nanoparticles dispersion (20 μg/mL). And then 20 μL of H2O2 with different concentration (0.5 mM, 1 mM, 20 mM) was added, respectively. The two groups were then cultivated for another 48 h. At the end of incubation, the tetrazolium salt (MTT) in PBS was added to each well (final concentration: 0.5 mg/mL) and kept for 4 h at 37 °C. Then 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan product for 20 min, followed by measuring the absorbance at 490 nm with a with a microplate reader (Multiskan Mk3; Thermo Labsystems, Vantaa, Finland). Diffusion Model of rGP3.5@SOD@CAT@Hb. The diffusion model of the rGP3.5@SOD@CAT@Hb simulated with Comsol was described as below: A microparticle with a diameter of 1 μm and a cube with the side length of 5 μm were set as the rGP3.5@SOD@CAT@Hb composites and the aqueous solution environment surrounding the composites, respectively. The microparticle was in the centre of the cube. The surface concentration of the microparticle was 1 μmol/L while the concentration of the other area of in the microparticle and cube was 0. The diffusion coefficient in aqueous solution and inside of the microparticle were 1*10^(-10) m^2/s and 1*10^(-11) m^2/s, respectively. Characterization. The samples were characterized by transmission electron microscopy


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(TEM, H-8100, Hitachi) and scanning electron microscopy (SEM, Zeiss Supra55). UV/vis absorption spectra were measured by Hitachi U-3900H spectrophotometer (double beam spectrophotometer). The zeta potential of the self-assembly films was measured on Malvern Instruments Zetasizer Nano-ZS90. Fourier transform infrared spectra (FTIR) were recorded in a PerkinElmer Spectrum 100. ASSOCIATED CONTENT Supporting Information. Fourteen supplementary figures are present as supporting information. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] Author contributions X. L, Y. Z. and Q. A. conceived, designed the experiments, and co-wrote the paper. X. L performed most of the experiments. Z. M, M. F., C. H assisted in some experiments and analysed the data. S. P. performed simulation for diffusion. All authors contributed to the discussion and analysis of the results. ACKNOWLEDGEMENT This work was supported by the NSFC (21673209, 51772279) and the Fundamental Research Funds for the Central Universities (2652015295). The authors gratefully acknowledge the financial support from China Scholarship Council.



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microwave-assisted solvothermal synthesis. J. Am. Chem. Soc. 2009, 131, 16000-16001. 45. Pavlovic, M.; Rouster, P.; Szilágyi, I. Synthesis and formulation of functional bionanomaterials with superoxide dismutase activity. Nanoscale 2016, 9, 369-379.



Figure 1. Schematic illustration to show: a) layer-by-layer modification of the rGO template by PEI and PAA. b) the fabrication process of rGP3.5@X nanoparticle based on the layer-by-layer modified rGO film and the adjustable multifunctional mechanism via NIR irradiation. Enzymatic reaction diagram of c) rGP3.5@CAT, d) rGP3.5@SOD, e) rGP3.5@CA, f) rGP3.5@Hb. TEM image of g) rGP3.5@CAT, h) rGP3.5@SOD, i) rGP3.5@CA, j) rGP3.5@Hb, o) rGP3.5@EGFP, p) rGP3.5@LIP, q) rGP3.5@BSA. k) The relative activity of native CAT and rGP3.5@CAT to catalyze H2O2. l) Scavenging efficiency of O2.- with rGP3.5@SOD in different conditions: (1) in the absence of rGP3.5@SOD and (2-3) in the presence of 100 μL and 600 μL of rGP3.5@SOD after illumination. m) The mass and SEM image of the product CaCO3 of the control group


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and the rGP3.5@CA. n) UV-vis spectra of rGP3.5@Hb after successively feeding: (1) CO2, (2) O2, and (3) again CO2 on the results in (2) for 1 h each.



Figure 2. a) The network reaction concept of the input and output in the


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rGP3.5@SOD@CAT@Hb system. b) The relevant reaction equations of the rGP3.5@SOD@CAT@Hb system. c) The reactions path of the rGP3.5@SOD@CAT@Hb in graphical form. d) The stoichiometric matrix corresponding to (c). e) TEM image of the rGP3.5@SOD@CAT@Hb. f) Scavenging efficiency of O2.- (1) without or (2) with rGP3.5@SOD@CAT@Hb. g) The relative activity of H2O2 with rGP3.5@SOD@CAT@Hb. h) UV-vis spectra of rGP3.5@SOD@CAT@Hb after successively feeding: (1) CO2, (2) O2 for 1 h each. i) Diffusion simulation model of the rGP3.5@SOD@CAT@Hb in an aqueous solution. j) The network reaction concept of input and output in the rGP3.5@CA@SOD@CAT@Hb system. k) The relevant reaction equations of the rGP3.5@CA@SOD@CAT@Hb system. l) The reactions path of the rGP3.5@CA@SOD@CAT@Hb in graphical form. m) The stoichiometric matrix corresponding to (l). n) TEM image of the rGP3.5@CA@SOD@CAT@Hb. EM image of the product CaCO3 (o) with and (p) without the CA component.



Figure 3. a-d) The relative activity of H2O2 with rGP3.5@CAT under different irradiation times with and without an infrared laser. The laser power in b) and d) is 1 W/cm2, in c) and e) 3 W/cm2. e) UV-vis spectra of rGP3.5@SOD@CAT@Hb after treating by O2.- with and without a NIR laser. f) Scavenging efficiency of O2.- with rGP3.5@SOD@CAT@Hb with and without a NIR laser (1 W/cm2).


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Figure 4. a) Schematic illustration of the coexistence of normal cells and the protein complex. b) The modulation of survival of HLF cells with and without rGP3.5@CA@SOD@CAT@Hb under an oxidant stress microenvironment with H2O2 for 48 h.



Figure 5. a) Schematic synthesis of ZIF-8-coated rGP3.5@CA@SOD@CAT@Hb. b) TEM image of ZIF-8-coated rGP3.5@CA@SOD@CAT@Hb. c) The enlarged image of (b). d) SEM image of ZIF-8-coated rGP3.5@CA@SOD@CAT@Hb. e-f) XRD patterns and ATR-FTIR spectra of pure ZIF-8 and ZIF-8-coated rGP3.5@CA@SOD@CAT@Hb. g) The enlarged characteristic peaks of (f). h-k) TEM images of the rGP3.5@CA@SOD@CAT@Hb and ZIF-8-coated rGP3.5@CA@SOD@CAT@Hb after storage in extreme pH solutions for 3 days.


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Multiple-Enzyme Graphene Microparticle Presenting Adaptive

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