Protein-Protected Porous Bimetallic AgPt Nanoparticles with pH

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Letter

Protein-Protected Porous Bimetallic AgPt Nanoparticles with pH-switchable Peroxidase/Catalase-Mimicking Activity Mustafa Gharib, Andreas Kornowski, Heshmat Noei, Wolfgang J Parak, and Indranath Chakraborty ACS Materials Lett., Just Accepted Manuscript • DOI: 10.1021/acsmaterialslett.9b00164 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Materials Letters

Protein-Protected Porous Bimetallic AgPt Nanoparticles with pH-switchable Peroxidase/Catalase-Mimicking Activity Mustafa Gharib,1,2 Andreas Kornowski,3 Heshmat Noei,4 Wolfgang J. Parak,1,5* Indranath Chakraborty1* 1Fachbereich

Physik, Center for Hybrid Nanostructures (CHyN), Universität Hamburg, Hamburg,

Germany 2Radiation Biology Department, Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt 3Fachbereich Chemie, Universität Hamburg, Hamburg, Germany 4DESY NanoLab, Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany 5CIC Biomagune, San Sebastian, Spain *corresponding authors: [email protected] [email protected]

Abstract Bovine serum albumin protected AgPt bimetallic nanoparticles have been synthesized through a galvanic replacement reaction using silver nanoprism as a template. Synthesis was carried out without the use of synthetic chemical surfactants, using proteins as surface coating. Structural and compositional characterizations were performed using several spectroscopic and microscopic techniques. The protein protected AgPt nanoparticles show effective pH-switchable enzyme mimicking (‘nanozyme’) activity similar to catalase and peroxidase. The content of Pt in the AgPt nanoparticles is playing an important role in determining the enzymatic catalysis rate of the nanoparticles. Due to their porous structure, also molecular payloads such as calcein can be incorporated into the AgPt nanoparticles, which can be released on demand by chemical triggers, such as hydrogen peroxide.

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During the last few decades, considerable interest has been devoted to bimetallic and multimetallic nanoparticles1 due to their different catalytic performance,2-3 electronic,4 magnetic,5 and optical6 properties relative to their corresponding monometallic counterparts.7 For instance, previous studies reported an enhanced surface enhanced Raman scattering (SERS) activity as well as superior catalytic performance of AgAu, AgPd, and AgPt bimetallic nanoparticles (NPs) compared to the original Ag NPs.8-12 Bimetallic NPs are mainly synthesized by either of two ways; i) through the galvanic exchange route, taking advantage of a well-defined morphology of a sacrificial template of one metal and the difference in reduction potential to deposit the other metal,13-15 or ii) via the co-reduction approach, during which two or more metal precursors are simultaneously co-reduced by strong reducing agents.16-18 However, these approaches usually involve the use of hazardous and noxious chemicals, in particular surfactants. For many biological applications nonbiocompatible surfactants have to be removed in tedious post-synthesis purification processes, whereby often complete removal/exchange cannot be granted.19-20 Therefore, synthesis approaches avoiding non-biocompatible surface coatings would be of interest. Proteins used as reducing agents and surface coatings to warrant colloidal stability of the NPs can be a good choice for such biocompatible NPs.21 Mimicking nature’s design by using nanomaterials is always a fascinating research interest. Recently, nanomaterials such as metal NPs, metal oxide NPs, carbon-based NPs, etc. with intrinsic enzyme-mimicking activity, the so-called “nanozymes”22-28 have been discovered. Nanozymes offer certain advantages over natural enzymes, such as the possibility for low-priced large-scale controlled synthesis, overcoming the time-consuming preparation and purification processes, as well as the high stability against denaturation and deactivation under stringent environmental changes. Since the early stages in the discovery of Fe3O4 NPs as peroxidase mimics29 and the catalase-like activity of nanoceria,30 enormous progress has been made in the field of nanozymes27. Encouraged by this, some efforts have been made to evaluate the catalytic performance of bimetallic and multimetallic NPs. For instance, He et al. reported the synthesis of AgM bimetallic NPs (M= Au, Pd and Pt) with inherent peroxidase-like activity, and they were able to tune the enzymatic activity of such NPs by simply varying their elemental composition (Ag:M).16 Shah et al. have also synthesized polyoxyethylene cholesteryl ether coated Fe-Pt NPs which show oxidaselike activity.31 Furthermore, Zhang et al. reported the synthesis of Au@PdPt alloy nanorods which showed tunable oxidase-like activity by proper alloying of Pd and Pt into the trimetallic nanocomposite.32 The performance of nanozymes offers great promise in biosensing as well as other biomedical applications.33-37 However, selectivity, stability, and biocompatibility are still remaining great challenges, particularly when it comes to biological applicability. Ligands play a vital role in determining the surface properties of NPs38 and in previous reports,39-40 we found that acute toxicity of NPs composed out of intrinsically non-toxic materials largely depends on the surface coating of the NPs. In this context, nanozymes with intrinsically non-toxic surface chemistry would be of utmost value.


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Herein we report a facile synthesis of biocompatible hollow AgPt NPs, using bovine serum albumin (BSA) as biotemplate and shape-directing agent, with a pH-switchable artificial peroxidase-/catalase-mimetic activity. BSA clearly forms an intrinsically non-toxic surface chemistry. Moreover, due to the porosity of the AgPt NPs they can also be utilized as cargo-carriers with triggered payload release behavior connected to their intrinsic peroxidase-mimicking activity. The synthesis of AgPt NPs involves a three-step process as demonstrated in Figure 1a (details of experimental methods are mentioned in the Supporting Information, SI). First, Ag seed-NPs (yellow colored) have been prepared in step 1, then BSA-coated prism-shaped Ag NPs (purple colored) were synthesized in step 2 (characterizations are mentioned in Figure S1), using our previously reported method.40 Then in step 3, AgPt NPs were synthesized by means of a galvanic exchange reaction. The preformed Ag NPs served as the sacrificial template for the controlled deposition of Pt, giving ultimately rise to deep navy blue colored AgPt NPs. Several control experiments were carried out to explore the role of BSA in dictating the shape of the NPs and to optimize the fabrication method (cf. Figures S8-S11). The transmission electron microscopy (TEM) images reveal a porous prism-shaped morphology of the AgPt NPs (Figure 1) with an average edge length (LE) of 44.3 ± 1.22 nm (averaged from 100 NPs from TEM images, cf. Figure S2). The high resolution TEM images of AgPt NPs show two crystals with different lattice orientations with d-spacing values between lattice fringes of 0.242 and 0.222 nm, which matches well with the interplanar spacing of the (111) planes of Ag (0.2359 nm, JCPDS no. 04-0783) and Pt (0.2265 nm, JCPDS no. 04-0802), respectively (Figure S3 a and b). The electron diffraction (ED) pattern of the AgPt NPs shows multiple rings with superimposed diffraction spots around the central bright spot (diffuse ring pattern), which indicates that different grains are lying in different orientations while the electron beam passes perpendicularly through the sample, which in turn reveals the polycrystalline nature of the AgPt NPs (Figure S3c). We hypothesize that the formation of AgPt NPs involves two parallel reduction reactions of the Pt precursor, followed by the deposition of Pt into the preformed sacrificial Ag NP template and the subsequent formation of porous AgPt NPs. The involved competing reduction reactions are governed by the remaining ascorbic acid (AA), which was used as reduction agent for the formation of Ag NPs, as well as the sacrificial Ag NPs (galvanic exchange route). As the concentration of AA is relatively low in such reaction conditions, both reduction pathway may proceed simultaneously. The major contribution, however, is thought to come from the galvanic exchange reaction during which the preformed Ag NP template is partially oxidized by the Pt precursor, followed by Pt atom deposition and the partitioning between the core (major part) and the surface (minor part). The oxidized silver ions (Ag+) are then dissolved in the solution and have two possible reaction pathways: they can be reduced again by the AA and subsequently redeposited on the surface or in the core, or they may be dissolved in solution and remain as Ag+ if the AA vanishes in the reaction mixture. We postulate that the rate of Ag+ reduction by AA is much lower than the rate of oxidative dissolution of the Ag template by the Pt precursor. The later mechanism counts for the porous structure of the resulting AgPt NPs. The X-ray photoelectron spectroscopy (XPS) survey data (Figure S4) of the AgPt NPs and Ag NPs show the expected elements including Ag and Pt. The deconvoluted XP spectrum of



AgPt NPs shows a broad Ag 3d peak compared to Ag NPs, which is expected due to the bimetallic NP formation. In both cases, the peak is centered around 368.2 eV, a state closer to Ag (0) (Figure S5a). Note that there is not much difference in the binding energy between Ag (I) and Ag (0) states. However, a small XP peak at 367.1 eV shows the presence of Ag (I). This can be explained with the presence of trace amounts of chlorine at around 200 eV in the survey spectra (cf. Figure S4). The oxidation state of Pt (0) can be confirmed from the spectrum which shows the metallic Pt 4f peak at 71 eV for the AgPt NPs (Figure S5b). Elemental mapping and corresponding spectra using EDS confirmed the coexistence and the homogeneous distribution of Ag (48.5%) and Pt (51.5%) in the AgPt NPs (Figure 1 d-f). The shape and composition of AgPt NPs can be controlled by changing the molar ratio of Ag:Pt precursors (cf. Figure S11). Considering the morphological and compositional aspects, we set the Ag:Pt molar ratio of 1:0.25 as the optimal conditions for the synthesis of AgPt NPs and were considered for further investigations. Considerable change in absorption spectra was seen during the transformation from Ag NPs to AgPt NPs (cf. Figure S9a). Once the Pt precursor was introduced to the Ag NP reaction mixture, a significant red-shift from λ ≈ 565 nm (which is attributed to the in-plane dipole resonance of the Ag NPs41) to λ ≈ 716 nm was observed, in addition to an apparent peak broadening and surface plasmon oscillation damping. The red-shift is attributed to the net change in the refractive index of the resulting hollow NP structure formation as reported by Smith et al.42 Moreover, the shoulder peak which corresponds to the in-plane quadrupole resonance41 disappeared. These observations indicate partial loss of the plasmonic nature of the pristine prism-shaped Ag NPs by forming bimetallic NPs with Pt. It is worth noting that our synthesis protocol can also be extended to the synthesis of other AgM NPs (M= Pd and Au) (Figures S12, S14 and S15). However, in this report, further experiments were carried out using AgPt NPs only.


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Figure 1. Synthetic route and characterization of AgPt NPs. (a) Schematic illustration showing the 3-step synthetic route to form porous AgPt NPs: (1) synthesis of silver NPs seeds, (2) synthesis of the sacrificial template in the form of prism-shaped Ag NPs, and (3) galvanic exchange reaction and formation of AgPt NPs. Corresponding photographs of the NP solutions are shown. (b and c) Transmission electron microscopy (TEM) images of the porous AgPt NPs. (d and e) Energy dispersive X-ray spectroscopy (EDS) elemental mapping of Ag and combination of Ag and Pt in AgPt NPs. (f) EDS analysis of one AgPt NP (corresponding to the NP shown in the TEM image in the inset) showing the atomic percentage I of Ag and Pt (table inset). (g) Circular dichroism (CD) spectra of the N- and E-forms of BSA, and of Ag NPs and AgPt NPs with BSA coating. (h) Distribution I of secondary structure elements of BSA within the N- and E-forms of BSA, Ag NPs, and AgPt NPs as estimated from the CD spectra in (g) using an online tool mentioned in the SI. The AgPt NPs showed better colloidal stability in sodium chloride solutions of different ionic strengths as well as in different other media in comparison to the monometallic Ag NPs (Figure



S6). The comparative high colloidal stability of AgPt NPs over Ag NPs in different media may arise due to their porous nature. In case of our AgPt NPs, their hollow nanostructure design offers a higher proportion of surface area, which means higher surface coverage with BSA as compared to the solid nanostructure of Ag NPs. Note the fact that BSA has an intrinsic amphiphilic character43 and coating of the NPs with such amphiphilic moieties enhances the NPs colloidal stability. This is presumably because of the increasing interfacial energy, which leads to the formation of a mechanical barrier that reduces aggregation of the NPs.44 It is also important to mention here that due to the increase of the Pt fraction the bimetallic NPs may confer significant modification of their electrochemical properties in comparison to the Ag NPs. Such change in electronic properties might prevent their oxidative dissolution (as well as Ag ions release in aqueous media) via the formation of internal electron traps within the bimetallic NPs, which in turn means higher chemical stability of AgPt NPs over the monometallic Ag NPs. These results are in agreement with previous reports which showed the excellent chemical stability of Ag NPs upon alloying with Pt and the formation of bimetallic systems.13 The pH of the reaction mixture is very crucial to control the galvanic replacement reaction and the formation of porous NPs. The pH of the reaction medium was varied over a wide range of pH (214) and the results show that characteristic UV-vis absorption spectra of porous AgPt NPs were obtained when the pH was acidic (~4) (Figure S7). At such pH, the galvanic exchange reaction dominates, leading to the formation of porous NPs. Similar results were obtained for other bimetallic and trimetallic NPs.45-46 To address the structural change of the surface capping agent, BSA, during the Ag NP and AgPt NP formation, spectroscopic measurements were carried out using circular dichroism (CD) and fluorescence spectroscopy. The CD spectra of native BSA (N-form, aqueous solution, pH 6.5) shows one spectroscopic band at 190 nm in the positive absorption side and two other bands at ca. 209 and 222 nm in the negative absorption side (Figure 1g). The later peaks are characteristic for the α-helix structure of BSA.47 Deconvolution of the spectra revealed that BSA at nearly neutral pH has 50.8% of α-helix, 3.1% of β-sheet, 14.1% of β-turn, and 32% irregular structures (Figures 1h and S16a), which matches well with reported literature values.39, 48 Since the reaction pH was acidic during the synthesis of the AgPt NPs, the structural analysis of BSA was also carried out in acidic environment. Deconvolution of the CD spectra of acidified BSA revealed that acidified BSA has ca. 25% α-helix, 22% β-sheet, 8% β-turn, and 45% irregular structures (Figures 1h and S16b), which reveals the unfolding of BSA, i.e. subsequently BSA adopts an extended form (E-form) at such acidic environments.49-50 A significant change in the α-helical structure elements in the BSA structure was seen in case of BSA-capped Ag NPs and AgPt NPs when compared to the N-form of BSA (Figures 1h, S16c and S16d). Moreover, there was an additional decrease in the helicity of Ag NPs and AgPt NPs (14% and 20%, respectively) as compared to that exhibited by the Eform of BSA (25%). These results suggest that a strong interaction between the metal ions/atoms and the BSA molecule takes place,51 which results in extra unfolding of the BSA in addition to the unfolding exhibited at low pH which in turn means more loss of the α-helical structure. Similar


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findings were obtained using fluorescence spectroscopy measurements (Figure S17) by exciting the tryptophan residues of BSA, which is the significant contributor to the intrinsic fluorescence of BSA52 (details are explained in SI). The nanozyme behavior of our AgPt NPs in different pH environment was studied by evaluating their peroxidase/catalase-like activity. The peroxidase/catalase-like activity was assessed by evaluating the ability to catalyze the oxidation of a typical peroxidase substrate, orthophenylenediamine (OPD) in the presence of H2O2 to produce yellow colored 2,3diaminophenazine (DAP), as well as the ability to catalyze the decomposition of hydrogen peroxide (H2O2) liberating O2 gas, respectively. At acidic environment (pH 4), AgPt NPs showed pronounced peroxidase-like activity as could be revealed from the characteristic absorption peak of DAP (at λ= 425 nm), as well as from the enhanced kinetics of OPD oxidation (Figures S18 and S21, respectively). The peroxidase-like activity of our AgPt NPs showed a composition dependence and increasing the Pt content in the NPs up to a certain level gradually enhanced their catalytic performance (Ag1Pt0.5 showed optimum results) (Figures 2f and S19). In contrast, Ag NPs which are made entirely of Ag showed negligible catalytic activity towards the oxidation of OPD (Figures 2c, 2e, S18 and S21c). The peroxidase-like activity of citrate protected AgPt NPs was also evaluated for the sake of comparison with our BSA protected AgPt NPs and showed almost similar results (Figures S20 and S22). Based on these observations, we hypothesize that this pronounced peroxidase-like activity of AgPt NPs is mainly due to Pt. Pt NPs has been extensively used as catalyst in various electron-transfer reactions (Equation 1) and their peroxidase-like activity could be related to their ability to transfer electrons to H2O2 resulting in H2O2 decomposition.53-54 Nevertheless, our results showed that the bimetallic AgPt NPs exhibit greater peroxidase-like catalytic activity when compared to the monometallic Pt NPs of equivalent Pt content (Figure S23). It has been reported that some metals, metal ions, or metal oxides produce hydroxyl radicals (•OH) during their reaction with H2O2,55-56 Thus we assume that the underlying mechanism of the artificial peroxidase activity of our NPs is a radical chain reaction mechanism during which AgPt NPs (more specifically the Pt atoms in these NPs) react with the adsorbed H2O2 on the NP surfaces, followed by a base-like decomposition of H2O2 in acidic pH, which involves the rapid breaking of the oxygen–oxygen bond of H2O2, finally yielding •OH. The adsorbed •OH on AgPt NP surfaces reacts with OPD (or any other peroxidase substrate) resulting in the generation of OPD radical that interacts with another OPD radical yielding the DAP. The hypothesized mechanism is illustrated in Scheme 1 (cf. the SI). A similar mechanism of H2O2 decomposition on the surfaces of Pt NPs in acidic pH environments takes place in case of absence of the peroxidase substrate leading to the overexpression of highly oxidative •OH (Eq. 2). In contrast, at relatively higher pH values (pH>7), AgPt NPs do not exhibit peroxidase-like activity (Figures 2 and S18), which can also be explained from Eq. 1, as the catalysis reaction needs H+



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ions. 𝐻2𝑂2 + 2 𝐻 + + 2 𝑒 ―

𝑨𝒈𝑷𝒕 𝑵𝑷𝒔

2 𝐻2𝑂

𝐸𝑞.1

This intrinsic nanozyme capability and the ability to produce •OH exhibited by AgPt NPs at acidic pH environment (which would be the case if the NPs are trapped within the acidic endosomal/lysosomal compartments of cells) might be of interest in various biomedical applications aimed at eliciting targeted cytotoxic effects such as potential cancer therapy.

Figure 2. Peroxidase-like activity of AgPt NPs and steady-state kinetics of the catalytic reaction. Photographs of the peroxidase-like activity of (a) control, (b) horseradish peroxidase (HRP), (c) Ag NPs, and (d) AgPt NPs at different pH (1, 2 and 3 represents pH 4, pH 7 and pH 11, respectively). Gas bubbles were clearly observed in case of Ag NPs and AgPt NPs due to their apparent catalase-like activity at basic pH (c and d, 3). (e) Time-dependent absorbance changes at wavelength λ= 425 nm upon oxidation of OPD to DAP in absence of catalyst, or in presence of HRP, Ag NPs, or AgPt NPs. (f) Intrinsic peroxidase-like activity of AgPt NPs as a function of the elemental composition of the AgPt NPs. The inset shows the corresponding photographs. (g) Steady-state kinetics of the catalytic reaction where the rate of reaction (v) was plotted vs. different concentrations of H2O2 (cH O ) while keeping the OPD concentration fixed. (h) Steady-state 2 2

kinetics of the catalytic reaction where v was plotted vs. different concentrations of OPD (cOPD) while keeping H2O2 concentration fixed. The inset shows the schematic illustration of the


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peroxidase-like activity of AgPt NPs. The error bars represent the standard deviation of three measurements. The data points in g and h were fitted via non-linear regression to the MichaelisMenten model and the vmax, Km and Kcat values were calculated thereof. R2 indicates how close the data points fit to the non-linear regression line. The steady-state kinetics of the peroxidase-like activity of AgPt NPs with OPD and H2O2 as the substrates were evaluated. A series of kinetics experiments were performed to determine the rates of the catalytic reaction by varying the concentration of OPD while keeping the concentration of H2O2 constant (Figure S24), or with changing the concentration of H2O2 while keeping the concentration of OPD constant (Figure S25). Typical Michaelis–Menten curves were obtained (Figure 2). The data points were fitted thereafter via non-linear regression to the Michaelis-Menten model and the kinetic parameters, i.e. the Michaelis constant (Km), maximum reaction velocity (vmax) and turnover number (Kcat, where Kcat= vmax/ccat) were calculated (ccat is the concentration of catalyst (details of the NP concentration calculations are mentioned in the SI). The kinetics data were compared with the ones of the well-studied horseradish peroxidase (HRP) enzyme, see the summary in Table 1. The Km values reflect the degree of affinity of an enzyme to its substrate and the smaller the Km values are, the higher the affinity is. vmax indicates the maximum catalytic activity when an enzyme is saturated with its substrate. The apparent Km value of the AgPt NPs with OPD as the substrate is 0.129 mM, which is about 5 times smaller than that of HRP (Table 1). These results reflect the higher affinity of our AgPt NPs for the OPD substrate as compared to the native HRP. This may be due to the abundance of exposed binding sites on the larger surface area provided by the AgPt NPs in comparison with HRP. This phenomenon has been reported for many NPs.57 However, the Km value with H2O2 as the substrate was much higher than that of HRP (Table 1), which indicates that higher concentrations of H2O2 are required to observe maximal catalytic activity of the AgPt NPs. The same findings were observed in studies on iron oxide nanozymes.29 In addition, the Kcat values of AgPt NPs with OPD and H2O2 substrates were 751 s1 and 1075 s-1, respectively which means that at the same molar concentrations, the AgPt NP nanozyme showed peroxidase-like activity 13 times and 9 times higher than HRP with OPD and H2O2 as the substrates, respectively, suggesting higher catalytic capability of our AgPt NPs as compared to the native enzyme. Still, for fair comparison one also needs to consider that the surface area of one AgPt NPs is much higher than the one of one HRP molecule. The nanozyme behavior of AgPt NPs was also evaluated in neutral and basic environments (pH 7.1 and 11, respectively). However, the catalytic reaction in neutral and basic pH environment (Figure 3) was accompanied by the liberation of oxygen (gas bubbles), inferring that AgPt NPs might act similarly as the natural catalases which decompose H2O2 into water (H2O) and molecular oxygen (O2). The hypothesized catalase activity of AgPt NPs was monitored by evaluating the dissolved oxygen levels in the reaction solutions containing H2O2 and AgPt NPs in different pH environment (pH 4, 7 and 11) (Figure 3). Our results confirmed the catalase-like activity of AgPt NPs only in neutral



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and basic environments, and showed the ability of AgPt NPs to decompose H2O2 liberating O2. In contrast, AgPt NPs did not show any catalase-like activity in acidic pH environments. Furthermore, the decomposition rate of H2O2 was highly dependent on the Ag:Pt ratio of the NPs, and higher Ag:Pt ratio (Ag96Pt4 and Ag93Pt7) results generally in diminished catalase-like catalytic activity (Figure 3f). Our studies showed that the bimetallic AgPt NPs exhibit greater catalase-like catalytic activity when compared to the monometallic Pt NPs of equivalent Pt content (Figure S27). Hence, the catalytic activity of AgPt NPs could be tailored and optimized by tuning the Ag:Pt ratio which brings some changes in the electronic structure of the resulted bimetallic system. Moreover, BSA-coated AgPt NPs showed greater catalase-like activity (Figure 3) in comparison to sodium citrate-stabilized AgPt NPs (Figure S26). H2O2 + H + H2O2 + OH

_

H2O + •OH

Eq. 2

H2O + HO2

Eq. 3

_ H2O + OH + O2 H2O2 + HO2 --------------------------------------------------2 H2O + O2 2 H2O2

Eq. 4

(Base-like decomposition)

(Acid-like decomposition)

Eq. 5

We assume that such catalytic activity of AgPt NPs in alkaline pH is due to the preferential H2O2 adsorption on the NPs, which will then react with the preadsorbed OH-. This enhances the decomposition of H2O2 on the NP surface (acid-like decomposition) during which H2O2 passes H+ to the pre-adsorbed OH- yielding OH that interacts with another molecule of H O , giving rise to 2

2

2

H2O and O2 liberation (Eq. 3-5). The apparent steady-state kinetics of catalase-like activity of AgPt NPs were further investigated by plotting the rate of oxygen generation as a function of H2O2 concentration (Figure S28). The experimental data were fitted thereafter to the Michaelis-Menten model and the kinetic parameters were compared to those of catalase (summarized in Table 1). The apparent Km value of the AgPt NPs with H2O2 as the substrate is 63.0 mM, which is almost similar to that of catalase (54.3 mM). whereas, the Kcat values of AgPt NPs with H2O2 as the substrate was 1.84×105 s-1 in comparison to only 6.1×104 s-1 for catalase. This means that at the same molar concentration, AgPt NP nanozymes show catalase-like activity 3 times higher than that of catalase. Such higher turnover number of our AgPt NPs over the native enzyme might render them useful for applications. These findings suggest the ability of AgPt NPs to catalyze the decomposition of H2O2 in acidic as well as in neutral and basic environment, but with two different catalytic pathways: i) H2O2 decomposition in acidic pH in a reaction that does not involve the liberation of gas bubbles suggesting a peroxidase-like catalytic activity accompanied by •OH formation (Eq. 2), and ii) H2O2


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decomposition in neutral and basic environment accompanied by the formation of gas bubbles possibly through a reaction that yields H2O and O2 (Eq. 3-5), a direct evidence of a catalase-like activity. A similar overall dual enzymatic-like trend was reported for iron oxide NPs,58 Pt NPs,59 and Au@Pt NPs.60

Figure 3. Catalase-like activity of AgPt NPs and steady-state kinetics of the catalytic reaction. Photographs of the catalase-like activity of (a) control, (b) catalase, (c) Ag NPs, and (d) AgPt NPs at different pH (1, 2 and 3 represents pH 4, pH 7 and pH 11, respectively) and (e) the corresponding kinetics of catalytic activity of (a), (b), (c) and (d) at pH 11. (f) Intrinsic catalaselike activity of AgPt NPs as a function of the elemental composition of the AgPt NPs. (g) Steadystate kinetics of the catalytic reaction where the rate of reaction (v) is plotted vs. different concentrations of H2O2 (cH O ). The inset is the schematic illustration of the catalase-like activity 2 2

of AgPt NPs. The error bars represent the standard deviation of three measurements. The data points in (g) were fitted via non-linear regression to the Michaelis-Menten model and the vmax, Km and Kcat values were calculated thereof. CO2 refers to the concentration of dissolved oxygen in the solution. R2, indicates how close the data points match with the fitted non-linear regression line. This phenomenon means that AgPt NPs exhibit pH-switchable peroxidase/catalase-mimicking activity. Whether AgPt NPs will exhibit peroxidase-mimicking or catalase-mimicking behavior



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depends mainly on the surrounding pH. This interesting dual enzymatic property could be beneficial for eliciting oxidative stress (cytotoxicity) in acidic pH compartments such as in tumor tissues while reducing the H2O2 levels (artificial antioxidant) in neutral pH as in normal tissues (when not entrapped within lysosomes) (Figure S33). Table 1. Steady state kinetic parameters of peroxidase- and catalase-like reactions catalyzed by AgPt NPs. ccat is the concentration of AgPt NPs, HRP or catalase, Km is the Michaelis-Menten constant, vmax is the maximum reaction velocity, and Kcat is the turnover number, where Kcat = vmax/ccat. Details of the determination of the molar concentration of NP are mentioned in SI. Type of Enzyme Catalysis

Catalyst

ccat [nM]

AgPt NPs

0.0332

HRP

82.5

Peroxidase mimicking

Catalase mimicking

Substrate

Km [mM]

vmax [µM s-1]

KCat [103 s-1]

OPD

0.129

89.71

0.751

H2O2

76.05

128.49

1.075

OPD

0.59

16740

0.564

H2O2

0.34

34128

0.115

Reference

Present work

61

AgPt NPs

0.0332

H2O2

62.98

6.1

183.735

Catalase

0.266

H2O2

54.30

16.2

60.902

Present work 62

The porous structure, protein-capping nature, as well as the subsequent reaction possibility with H2O2 of our AgPt NPs, had lead us to evaluate also their dye/drug-loading and -controlled release capability, potentially suitable as drug carrier vehicle. Hydrophilic fluorescent calcein dye was used as a model cargo for this experiment. First, a standard curve of different calcein concentrations was generated (Figure S29). Then Ag NPs and AgPt NPs were allowed to react with a definite concentration of calcein solution and the absorption values of free calcein dye in the supernatants were recorded thereafter. AgPt NPs entrapped three times more calcein dye (~60 µM) as compared to the amount of calcein entrapped by Ag NP (̴ 20 µM), giving the same concentration of both NPs (Figures 4b and S30), due to the porous structure of the former one. Then the loading efficiency (% LE) and loading content (LC) of calcein into AgPt NPs were evaluated (Figures 4c and S31). By increasing the amount of calcein used during the incubation with AgPt NPs, the LC of calcein increased significantly and reached about 50 mg calcein/mg AgPt NPs when only about 1.3 mg of calcein was incubated with 0.1 mg of AgPt NPs. Whereas the loading efficiency of calcein into AgPt NPs reduced from 65% to 15% when the added calcein increased from ca. 0.01 mg to 1.3 mg.


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Figure 4. Cargo-loading into porous AgPt NPs and their oxidative stress-triggered release. (a) Schematic illustration of calcein-loading into AgPt NPs and its controlled release upon the addition of triggering agent, H2O2. (b) Quantitative assessment of calcein in the supernatants after reaction of Ag NPs and AgPt NPs with calcein using a calibration curve (Figure S29). (c) Calcein loading content (LC) and loading efficiency (LE) into the AgPt NPs after reacting the hollow AgPt NPs with different concentrations of calcein (Figure S31). (d) Controlled release of calcein upon the addition of H2O2. The inset show photographs of the H2O2-triggered calcein release from Ag NPs and AgPt NP. (e) Column representation of (d) for the fluorescence intensities before and after triggered calcein release. The triggered release of the calcein from the AgPt NPs upon specific stimulus is benefiting from their intrinsic peroxidase-like activity and their Ag NP scaffold which is susceptible to etching upon increased H2O2 levels. Both, calcein-loaded Ag NPs and AgPt NPs exhibited very low



fluorescece intensity (1040 a.u. and 1100 a.u., respectively, Figure 4 d and e). In contrast, the intesity of calcein increased significantly upon addition of H2O2 (16590 a.u. and 30440 a.u., respectively). AgPt NPs showed two-fold enhanced fluorescence intensity than corresponding Ag NPs. The same experiment was carried out using different calcein concentrations and showed the same trend (Figure S32). We propose that H2O2 interacts with calcein-loaded NPs with two mechanisms: first, H2O2 is decomposed into •OH on the surfaces of AgPt NPs owing to their peroxidase-like activity, and this mechanism contributes majorly; second, the powerful oxidizing etchant, H2O2, interacts with the Ag0 scaffold and elicits an oxidative dissolution process which oxidizes the Ag scaffold and releases Ag+ ions, followed by gradual disruption of the NP structure and the ultimate release of calcein dye (Figure 4a). It is worth noting that the coexistence of Pt in the AgPt NPs decreases the release kinetics of calcein when compared to Ag NP. This should be linked to the higher colloidal stability as well as chemical stability of AgPt NPs against oxidative dissolution as compared to the pristine Ag NPs as mentioned earlier.13 This mechanism of release may be of interest for sustainable triggered drug release stratigies. The combined properties could be advantageous for treating diseased tissues with overexpressed H2O2 such as tumor tissues. AgPt NP could be a potential candidate for a combined cancer treatment modality based on their synergistic release of Ag+ ions, hydroxyl radicals, and the liberation of loaded anticancer drugs in tumor tissues (acidic environments), eliciting deleterious effects and subsequent tumor tissue damage. In summary, hollow, BSA-protected AgPt NPs were synthesized through an environmentally benign approach using a controlled galvanic replacement reaction between a preformed sacrificial template of Ag NPs and Pt precursor in aqueous solution. The AgPt NPs showed pH-switchable peroxidase/catalase-like activities. These facile and tunable AgPt NPs exhibit high colloidal stability in harsh conditions as well as over different pH range. Thus, they can overcome the drawbacks of natural enzymes, which often deteriorate under such conditions.27 Furthermore, the catalytic activities of AgPt NPs could be easily tuned by controlling their elemental composition via adjusting the molar ratios of the metal precursors. In addition, the steady-state kinetics of AgPt NPs revealed their higher turnover numbers during their peroxidase/catalase-like catalytic reactions. Our findings suggest that AgPt NPs are efficient catalase and peroxidase mimics. Moreover, AgPt NPs showed good cargo-loading capacity owing to their porous structure and triggered cargo-release upon H2O2 etching stimulus of their Ag backbone. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at https://pubs.acs.org. Details of synthesis and characterization of seed NPs, AgNPs and AgPt NPs, concentration determination of AgPt NPs, colloidal stability, structural characterization of proteins, control experiments, enzymatic activities and cargo loading.


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ACKNOWLEDGEMENTS This work was supported by the German Research Foundation (grant DFG PA 794/28-1). I.C. was supported by an Alexander von Humboldt fellowship. M.G. acknowledges the Ministry of Higher Education and Scientific Research (MHESR) of Egypt and the Deutscher Akademischer Austausdienst (DAAD) for his fellowship. The authors are grateful to Mrs. Marta Gallego (CIC Biomagune) for part of the TEM images, EDS-elemental mapping and EDS-spot analysis. We acknowledge CD instrumental support by the SPC facility at EMBL Hamburg.

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Protein-Protected Porous Bimetallic AgPt Nanoparticles with pH

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