Self-Assembling Peptide Artificial Enzyme as an Efficient Detection

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Self-Assembling Peptide Artificial Enzyme as Efficient Detection Probers and Inhibitor for Cancer Cells Meiling Lian, Shuo Zhang, Jun Chen, Xuejiao Liu, Xu Chen, and Wensheng Yang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00160 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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

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ACS Applied Bio Materials

Self-Assembling Peptide Artificial Enzyme as Efficient Detection Probers and Inhibitor for Cancer Cells

Meiling Lian, Shuo Zhang, Jun Chen, Xuejiao Liu, Xu Chen*, and Wensheng Yang* State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, P.R. China

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ABSTRACT A new hybrid nanoparticle (NP; fluorenylmethoxycarbonyl- arginine-glycineaspartate and hemin, Fmoc-RGD/hemin NP) was developed for the simultaneous detection and inhibition of breast cancer cells. Hemin can regulate the reactive oxygen species (ROS) while Fmoc-RGD acts as a scaffold for hemin nanocrystallization. Fmoc groups interact with the porphyrin groups of hemin through hydrophobic and π-π interactions to form a hydrophobic core of NPs. The hydrophilic RGD chains surround the core to maintain the stability of the nanoparticles in an aqueous medium. The RGD groups of Fmoc-RGD is also selective for tumor cells. This interaction can be exploited to enhance the selectivity of tumor detection. Based on enhanced peroxidase activity, Fmoc-RGD/hemin NPs were developed as signal transducers in a facile and fast point-of-care cancer diagnosis platform. This platform is sensitive to breast cancer cells and hydrogen peroxide (H2O2), a biomarker for breast cancer. In addition, these Fmoc-RGD/hemin NPs can be used as nano-scavengers for ROS and for regulating the redox status of cancer cells. They also exhibit a targeted inhibitory effect on the epithelialmesenchymal transition (EMT). The peptide-tuned self-assembly of FmocRGD/hemin NPs as functional artificial enzymes boasts simple preparation, biofriendliness, and the versatility required for on-demand therapeutics and diagnostics for metastatic cancer cells. These NPs can therefore be used as 2


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effective tools for potential applications in medicine and biotechnology. KEYWORDS Peptide self-assembly; artificial enzyme; colorimetric detection; ROS scavenging; epithelial-mesenchymal transition

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1. INTRODUCTION Tumor metastasis is an important cause of most cancer-related deaths and presents a major clinical challenge.1 The incidence of metastatic disease is particularly high in breast cancer patients.2 Therefore, the diagnosis and treatment of breast cancer are extremely important and inextricably linked.3 Proper diagnosis informs treatment and is vital to the prevention of tumor growth and metastasis.4 In turn, effective treatments validate diagnoses.5 Therefore, the development of effective strategies for targeted diagnostics and

cancer

treatments

is

of

great

importance

in

nanomedicine.6

Nanomaterials with unique enzyme-like characteristics have been employed in a variety of bionanotechnology applications, from bioimaging and biosensor to therapeutics and tissue engineering.7-10 Despite the existing variety of carbon-based materials, precious metals, and metal oxides that have been used in such systems, the targeting and biocompatibility of materials, as well as new applications, are in need of improvement in this field.11-13 Epithelial-mesenchymal transition (EMT) is a metastasis-related process in which epithelial cells are transformed into mesenchymal phenotype cells by specific procedures, becoming motile and invasive.14 The application of nanomaterials in EMT has only rarely been studied. Most nanomaterials do not show any binding specificity, allowing for non-specific adsorption of 4


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artificial enzymes.15 New strategies for the design and preparation of other types of multifunctional artificial enzyme remain scarce and are challenging to devise.16 Peptides are attractive biomolecules for use as building blocks in the selfassembly of nanomaterials for biomedical applications. Many peptides have beneficial pharmacological properties that are unmatched by inorganic nanomaterials, such as high tissue permeability, non-immunogenicity, and biocompatibility.17-19 Peptides also boast a programmable primary structure and adjustable architecture via self-assembly.20-22 A variety of composites combining hemin groups and peptide chains have been designed to mimic enzyme active sites.23-26 However, designing targetable nanomaterials from these relatively simple

components is challenging. The application of

nanostructures based on peptide self-assembly has not been fully developed due to limited functionality. The development of versatile functional nanomaterials based on simple peptide self-assembly is essential for obtaining materials possessing good catalytic efficiency, ease of preparation, excellent targeting, and versatility in diagnosis and treatment. This work describes the construction of an artificial peroxidase (POD) though self-assembly of hemin and an aromatic short peptide derivative, fluorenylmethoxycarbonyl- arginine-glycine-aspartate (Fmoc-RGD). Domain 1 (RGD) boasts a high binding affinity for cancer cells.27 Domain 2 (Fmoc) 5



allows the formation of nanoparticles NPs) and improves the catalytic activity of hemin by preventing dimerization. The resulting artificial enzyme (FmocRGD/hemin NP) showed excellent POD performance and was used to measure the release of hydrogen peroxide (H2O2) from living cells. The FmocRGD/hemin NP nanozyme was also capable of rapidly detecting cells with excellent sensitivity and specificity. As far as we know, this is the first report of an artificial enzyme resulting from peptide self-assembly being used to colorimetrically detect cellular H2O2 and cancer cells. Our Fmoc-RGD/hemin NP can also act as a therapeutic agent to remove excess reactive oxygen species (ROS) produced by transforming growth factor-β (TGF-β) in cells, thereby suppressing EMT (Scheme 1).

Scheme 1. Schematic illustration of fluorenylmethoxycarbonyl- arginineglycine-aspartate (Fmoc-RGD)/hemin nanoparticles (NPs) for cancer cell

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detection and the inhibition of TGF-β-induced EMT in breast cancer cells.

2. EXPERIMENTAL Materials and instruments Lyophilized Fmoc-RGD peptide was obtained from Bachem (Bubendorf, Switzerland). Hemin (98%) was purchased from Energy Chemical (Shanghai, China). Dimethyl sulfoxide (DMSO) and 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and were purchased from Aladdin Chemicals Co. Ltd. (Shanghai, China). Scanning electron microscopy (SEM) micrographs were recorded with a Supra 55 field emission scanning electron microscope (Zeiss, Oberkochen, Germany). Ultraviolet–visible (UV–vis) absorption spectra were recorded on a UV–vis spectrophotometer (Lambda 35; Perkin-Elmer, Norwalk, CT, USA). Fluorescence images were obtained using a confocal microscope (TCS SP5; Leica, Wetzlar, Germany). Synthesis of Fmoc-RGD/hemin NPs An Fmoc-RGD stock solution was freshly prepared by dissolving the lyophilized Fmoc-RGD in HFIP to a concentration of 15.4 mM, followed by ultrasonicating to obtain a clear solution. A hemin stock solution (3 mM) was prepared in DMSO. Three milliliters of a combined solution containing the two assembly units were prepared with a 1:1 molar ratio (30 μM). Aging this 7



mixed solution for 24 h resulted in Fmoc-RGD/hemin NPs. The aged solution containing Fmoc-RGD/hemin NPs was then dialyzed in 0.01 M PBS (pH 7.4) for 24 hours to remove free hemin. Measurements of peroxidase-like activity and steady-state kinetics The POD-like activity of Fmoc-RGD/hemin NPs was determined using 3,5,3’,5’-tetramethylbenzidine (TMB) as a substrate in a 0.2 M Na2HPO4-citric acid buffer solution (pH 3.0) containing H2O2. The absorbance of the reaction mixture was recorded at 652 nm using a microplate reader. Generally, reaction systems contained 10 μM Fmoc-RGD/hemin NPs, 120 mM H2O2, 1.6 mM TMB, and pH 3.0 buffer. To establish the steady-state kinetics of the Fmoc-RGD/hemin NP enzymatic activity, reactions were performed under optimal conditions at different concentrations of TMB or H2O2. From the Lineweaver–Burk plots of the double reciprocal of the following Michaelis– Menten equation, the key kinetic parameters could be calculated. V= Vmax [S] / (Km + [S]) where Vmax is the maximum rate of conversion, V is the rate of conversion, Km is the Michaelis constant and [S] is the substrate concentration. Cell culture, cell viability, and the flux of cellular H2O2 release Cells were grown in RPMI 1640 medium containing 10% FBS, penicillin (100 U mL−1), and streptomycin (100 mg mL−1) in a 37 °C incubator. Counting 8


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Kit-8 (CCK-8) was used to performed the cell viability assay. Briefly, cells were seeded at a density of 11 × 104 cells well-1 into 96 well cell culture plates. After 24 hours, different concentrations of Fmoc-RGD/hemin NPs was added to the cell cultures. The cells were grown for an additional 24 h at 37°C prior to adding CCK-8 solution (10 μL) to the cultures. After 24 hours of cell regrowth, a CCK-8 solution (10 μL) was added to the culture. The absorbance (A) of each well was measured at 450 nm. As for the measurement of cellular release of H2O2, 100 μL of phorbol 12myristate-13-acetate (PMA) (200 ng mL−1) was added to the well containing 1 × 108 MCF-7 cells. After 10 s of incubation, TMB (1.6 mM) and FmocRGD/hemin NPs (10 μM) were added to each well and incubated in 0.2 M Na2HPO4-citric acid solution (pH 3.0) for 5 min to allow color development. The absorbance of each well at 652 nm, corresponding to the release of cellular H2O2, was recorded with a microplate reader. Colorimetric detection For colorimetric assays, different numbers of cells were seeded into individual wells of a 96-well plate and allowed to grow for 1 day. Each well was then washed with PBS (10 mM, pH 7.4) and the cells were fixed with 4% paraformaldehyde at room temperature for 10 min to disrupt endocytosis. After removing formaldehyde by washing, the cells were incubated with Fmoc-RGD/hemin NPs (10 μM) for 2 h. Then the unattached Fmoc9



RGD/hemin NPs are removed, 0.2 M Na2HPO4-citric acid buffer solution (pH 5.0, 250 μL) containing TMB (1.6 mM) and H2O2 (120 mM) was added to the well. Finally, the absorbance was recorded at 652 nm. Intracellular ROS assays Intracellular ROS levels were monitored using non-fluorescent 2’,7’dichlorfuorescein diacetate (DCFH-DA), which penetrates the cell membrane and is hydrolyzed to DCFH in the cytosol by an intracellular esterase. Intracellular free radicals can react with DCFH to form DCF with fluorescent properties. DCFH then reacts with intracellular free radicals to produce fluorescent DCF, which exhibits excitation and emission wavelengths of 488 and 520 nm, respectively. The specific steps are as follows. After treated with TGF-β1, cells were incubated with or without hemin. The cells were then incubated with DCFDA (0.02 M) at 37˚C in the dark. After 30 minutes, DCFDA fluorescence was measured at excitation and emission wavelengths of 488 and 520 nm, respectively. . Migration of MCF-7 breast cancer cells Migratory ability of MCF-7 cells was determined by awound healing assay. Briefly, MCF-7 cells were first incubated with hemin or FmocRGD/hemin NPs (10 μM) for 72 hours. It was then seeded into 6-well plates at 10


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60,000 cell well-1. When the cells were grown to almost full confluence, the cell monolayer was injured using a 200-μL disposable pipette tip. Finally, TGF-β1 (10 ng mL-1) was added to each well. Scratched wounds are visualized under the microscope. In vivo anti-inflammation Inflammation models were generated in Kunming mice (20 g). A 50-μLPMA (100 μg mL-1) solution was injected into the right ear of the mouse to induce local inflammation. The mice were then injected with Fmoc-RGD/hemin NPs (10 μM kg-1) after 6 h induction. Similarly, DCFH-DA (50 μL, 1 mM) was injected after a 30-min incubation. Finally, an in vivo Imaging System was used to record whole body fluorescence images at excitation and emission wavelengths of 465 nm and 520 nm, respectively.

3. RESULTS AND DISCUSSION Characterization of Fmoc-RGD/hemin NPs Our peptide-based, bioactive NPs were formed through the molecular self-assembly of building blocks consisting of a simple, short peptide, FmocRGD, and hemin, a ferrous porphyrin (Figure 1a). The SEM images show uniform Fmoc-RGD/hemin NPs with an average diameter of about 220 nm (Figure 1b). The uniform distribution of Fe (found only in the hemin moiety), 11



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N, and C further illustrates the successful complexation of hemin with FmocRGD (Figure 1c). As with the SEM-based measurements, dynamic light scattering (DLS) measurements yielded a mean hydrodynamic diameter of about 220 nm (Figure 1d). The formation of these self-assembled NPs is based on multiple weak intermolecular interactions, such as hydrophobic interactions, π-π stacking, and electrostatic forces.21 To explore the versatility of this assembly method, we evaluated hemin assembly with other peptide monomers.

As

expected,

the

co-assembly

of

hemin

with

fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) or FF yielded different sizes of NP (Figure S1). These results show that different peptide monomers exhibit different stacking geometries with hemin, thereby affecting the overall diameter of the resulting NP. Next, the structure of hemin was studied after its assembly with FmocRGD. From the UV-visible spectrum (Figure 1e), it can be seen that the free hemin has a Soret band at 385 nm, a shoulder peak of 365 nm, and a low intensity band at 630 nm. These bands indicate the presence of a mixture of monomeric hemin hydroxide (haematin) and μ-oxo bihemin (the dimer of hemin).28 The presence of μ-oxo dimer is detrimental to the stability and catalytic efficiency of hemin in water. Fmoc-RGD/hemin NPs have a Soret band at 400 nm which is similar to the hemin in aqueous micelles or artificial proteins, suggesting the presence of monomeric hemin chloride.29 In addition, 12


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Fmoc-FF/hemin NPs and FF/hemin NPs can also present as structural defects that reduce the packing efficiency of hemin and limit their nanoscale aggregation (Figure S2). To study the stability of Fmoc-FF/hemin NPs in aqueous phase and in cell culture media, the UV-Vis spectra of the FmocRGD/hemin NPs after 10-day aging was measured. No peaks of monomer and dimer of hemin were observed from Figure S3, indicating that the nanoparticles were stable and there was no leakage of hemin. Furthermore, it can be seen from the microscope image that the nanoparticles retain their original granular form, and the morphology in the aqueous phase and the medium is almost identical. The above data indicates that the nanoparticles are stable.

13



Figure

1.

(a)

Schematic

fluorenylmethoxycarbonyl-

diagram

of

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the

arginine-glycine-aspartate

preparation

of

(Fmoc-RGD)/hemin

nanoparticles (NPs). (b) Typical scanning electron microscopy (SEM) micrographs are shown with (c) mapping and (d) the hydrodynamic diameter distribution of Fmoc-RGD/hemin NPs. (e) Ultraviolet-visible (UV−vis) spectra of free hemin in aqueous solution and hemin in the peptide.

Peroxidase activity of Fmoc-RGD/hemin NPs After the successful synthesis and characterization of Fmoc-RGD/hemin NPs, the POD-like activity of Fmoc-RGD/hemin NPs was studied, using TMB and H2O2 as substrates. The UV-vis absorbance spectrum of oxidized TMB contains a characteristic peak at 652 nm, corresponding to a blue solution.30 The inset of Figure 2a shows photographs of different reaction systems. In the presence of H2O2, Fmoc-RGD/hemin NPs accelerate the oxidation of TMB, resulting in a distinct color change. Conversely, incubating TMB with either Fmoc-RGD/hemin NPs or H2O2 alone failed to yield an absorption band. These data indicate that the reaction of H2O2with TMB was promoted by the presence of Fmoc-RGD/hemin NPs. The data in Figure 2a also show that the catalytic activity of Fmoc-RGD/hemin NPs was much higher than that of hemin alone, revealing that our supramolecular self-assembly method enhances the POD-like activity of hemin. In fact, the catalytic activity of 14


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Fmoc-RGD/hemin NPs was similar to that of natural enzymes. That is, the POD-like activity of Fmoc-RGD/hemin NPs was dependent on both concentration and pH.31 The data in Figure 2b show that the catalytic activity of Fmoc-RGD/hemin NPs increased with increasing concentration from 2 to 15 μM. In addition, the POD-like activity of Fmoc-RGD / hemin NPs is much lower in neutral solutions than in weakly acidic solutions (Figure S4). This behavior is similar to horseradish peroxidase (HRP) and several previously reported nanomaterials-based POD-like mimics.32 The steady-state kinetics of Fmoc-RGD/hemin NPs were studied and the curves obtained were consistent with the Michaelis−Menten equation (Figures 2c and 2d).33 The key parameters, such as the maximum initial rate (Vmax) and Michaelis-Menten constant (Km), were calculated in the light of a LineweaverBurk plot (inset of Figures 2c and 2d). The Vmax and Km of previously reported systems listed in Table S1. Note that the Km values obtained for FmocRGD/hemin NPs with TMB and H2O2 as substrates were lower than those of natural HRP and other nanozymes. These results indicate the remarkable affinity of Fmoc-RGD/hemin NPs for TMB and the attainment of maximum activity at a relatively low H2O2 concentration. This provides indirect evidence for the POD-like activity of our prepared Fmoc-RGD/hemin NPs. Given the POD-like properties of our Fmoc-RGD/hemin NPs, we explored their potential application as the active component for sensing H2O2 15



released by cells. Sensors based on Fmoc-RGD/hemin NPs exhibited a wide linear dynamic range (1–1,000 μM) and a low detection limit (0.33 μM) than many other sensors based on POD mimics (Figure S5 and Table S2). Seven times 100 μM H2O2 was repeatedly measured and the relative standard deviation (RSD) obtained was 2.5%, demonstrating a high degree of reproducibility. In addition, H2O2 detection using Fmoc-RGD/hemin NPs was very specific (Figure S6). The release of H2O2 from cancer cells is induced by PMA as a stimulator, which is consistent with a chemotactic response (Figure S7a).34 Figure S6b shows the colorimetric response of a Fmoc-RGD/hemin NPs sensor system to H2O2 released from cancer cells. An average absorbance of 0.148 (n = 3) was observed at 652 nm, corresponding to 0.702 μM of H2O2 based on the calibration curve in Figure 3b. Thus, the amount of H2O2 released from a single cell can be calculated as ∼7.02 × 10−15 mol. This value agrees with published values in a previous report ([6.3−7.1] × 10−15 mol cell1),30

indicating that our detection method may be useful in practical

applications.

16


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Figure 2. (a) Typical absorption spectra of different Fmoc-RGD/hemin NPs solutions. Images in the inset show the color of the corresponding solution: (i) Fmoc-RGD/hemin NPs + TMB + H2O2; (ii) Hemin + TMB + H2O2; (iii) mocRGD/hemin NPs + TMB; (iv) TMB + H2O2. (b) The absorbance changes of a H2O2 (13 mM) solution containing TMB (0.1 mM) are shown at different concentrations of Fmoc-RGD/hemin NPs. Steady-state kinetic analyses yielded Michaelis−Menten and Lineweaver−Burk models (insets) for FmocRGD/hemin NPs at (c) different concentration of H2O2 with a fixed amount of TMB (1.6 mM) and (d) different concentration of TMB with a fixed amount of H2O2 (120 mM).

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Colorimetric detection of cancer cells Colorimetric assays are ideal method for the detection of cancer as they require simple instrument, and yet provide excellent accuracy and sensitivity. Our Fmoc-RGD/hemin NPs were designed in this way which they selectively target cancer cells by an RGD peptide sequence. MCF-7 human breast cancer cells were selected as targets, while human umbilical vein endothelial cells (HUVECs) were chosen as a control system (Figure 3a). Selectivity was evaluated by monitoring the generation of blue color in a TMB-H2O2 solution catalyzed by Fmoc-RGD/hemin NPs using these two types of cell in a buffered medium. Figure 3b shows that the absorbance of a mixture containing Fmoc-RGD/hemin NPs and target cells (MCF-7) was significantly higher than that of wells containing HUVEC cells and buffer medium, confirming the specificity of our system to cancer cells. To further validate our Fmoc-RGD/hemin

NPs

in

colorimetric

assays

for

the

quantitative

determination of cancer cells, various concentrations of MCF-7 cells were incubated with Fmoc-RGD/hemin NPs. The data in Figure 3c show increasing absorbance with increasing cell counts from 50 to 100,000 cells. This limit of detection is comparable to those of many electrochemical or fluorescent assays and other colorimetric methods, and much lower than those obtained using other detection strategies (Table S3). To determine the cytotoxicity of Fmoc-RGD/hemin NPs, cell viability was 18


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assessed using CCK-8 assays. The data in Figure S8 show no obvious inhibitory effect on cell proliferation after incubating Fmoc-RGD/hemin NPs with MCF-7 cells for 24 hours. These data show that the cytotoxicity of FmocRGD/hemin NPs can be ignored. Thus, Fmoc-RGD/hemin NPs are effective signal transducers for sensitive, accurate, and rapid detection of cancer cells.

Figure 3. (a) A schematic shows the detection of cancer cells using FmocRGD/hemin NPs. (b) The absorbance of the sensing system is shown in response to different types of cell. (c) A linear relationship was obtained between the log of the MCF-7 concentration and the solution absorbance at 652 nm.

19



Therapeutic treatment of cancer cells Previous reports have shown that ROS is very important in TGF-βinduced metastasis.35 Intracellular ROS levels increase significantly after TGFβ stimulation, while hemin was shown to inhibit ROS production and the migration of MCF-7 cells.36 The pharmacological effects of active molecules are limited by their residence time, solubility, stability, and in vivo selectivity. Many of the applications of nanomedicine aim to overcome these limitations.37 Therefore, one goal of the current study was to determine if hemin continues to suppress EMT after its incorporation into a peptide assembly. This would provide a new type of targeted nanotherapeutic agent for the treatment of tumor metastasis. EMT is typically accompanied by morphological changes. Figure 4a shows morphological changes and wider intercellular spacing following stimulation with TGF-β1. These changes were reversed by sequential treatment with hemin or Fmoc-RGD/hemin NPs. Next, we measured the generation of ROS using the fluorescence of DCFH-DA as an indicator.38 Figure 4b shows the fluorescence micrographs of cells under various conditions. When the cells were pretreated with Fmoc-RGD/hemin NPs, the observed fluorescence was negligible. This result confirms the low cytotoxicity of Fmoc-RGD/hemin NPs. In contrast, pre-incubation of cells with TGF-β1 resulted in strong green fluorescence, indicating high levels of intracellular ROS. This fluorescence was significantly reduced in the presence 20


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of hemin and Fmoc-RGD/hemin NPs. Quantitative analyses show that 35.8% of the ROS in Rosup-stimulated cells could be scavenged by hemin compared to 67.2% ROS scavenged by Fmoc-RGD/hemin NPs (Figure 4c). Wound-healing assays were performed to assess the effects of FmocRGD/hemin NPs on the migration of MCF-7 cells. Compared to the TGF-β1treated group, hemin and Fmoc-RGD/hemin NPs obviously inhibited the migration of MCF-7 cells (Figure 4d). The inhibitory effects of FmocRGD/hemin NPs on EMT were obvious (Figure 4e), and were due primarily to the scavenging activity of NPs. In addition, the RGD sequence recognizes cancer cells, facilitating the specific elimination of ROS in targeted cancer cells, thereby providing a new nano-therapeutic agent for tumor treatment and metastasis prevention. Fmoc-RGD/hemin NPs were shown herein to inhibit cellular EMT by scavenging ROS. As an extension of this work, a ROS-induced ear inflammation model was constructed to assess the in vivo anti-inflammatory capability ability of Fmoc-RGD/hemin NPs. Figure S9 shows inflammation after 6 hours of PMA injection into the right ear of the mouse. To evaluate the ROS scavenging ability of Fmoc-RGD/hemin NPs in vivo, inflamed mouse ears were subcutaneously treated with Fmoc-RGD/hemin NPs. The data in Figures S10 showed no obvious fluorescence in the DCFH-DA, PMA or DCFH-DA/Fmoc-RGD/hemin

NPs

treated

ear,

21


indicating

that

the


nanoparticles have good biocompatibility and will not destroy the redox state in the body. In contrast, strong fluorescence was observed after treatment with PMA/DCFH-DA, indicating elevated levels of ROS because of PMAinduced inflammation. The fluorescence in the inflamed ear decreased significantly after treatment with Fmoc-RGD/hemin NPs. These results indicate that Fmoc-RGD/hemin NPs were able to efficiently scavenge ROS in the inflamed ears of live mice. The intracellular ROS scavenging activity of Fmoc-FF/hemin NPs and FF/hemin NPs was also investigated. It can be seen from Figure S11 that treatment with Rosup resulted in significant fluorescence that decreased with the addition of Fmoc-FF/hemin NPs or FF/hemin NPs. Therefore, our hemin/Fmoc NPs inhibit inflammation and may be suitable for treating diseases such as Alzheimer's and Parkinson's disease.

Figure 4. (a) Representative photomicrograph and (b) fluorescence images of 22


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MCF-7 cells following various treatments. (c) Corresponding fluorescence intensity of 2’,7’-dichlorfuorescein diacetate (DCFH-DA) from panel (b). (d, e) The wound-healing abilities of various treatments are shown for MCF-7 cells.

4. CONCLUSIONS In summary, Fmoc-RGD/hemin NP nanozymes were fabricated from the self-assembly of simple peptides, Fmoc, and hemin. Fmoc-RGD is an ideal hemin carrier that provides an outer hydrophilic scaffold and an inner hydrophobic environment, inhibiting its dimerization and stabilizing hemin in aqueous media at the same time. The resulting nanozyme exhibited a POD activity greater than that of hemin alone. Furthermore, RGD acts as a recognition element for tumor cells, enabling the selective detection of cancer cells by Fmoc-RGD/hemin NPs. In this study, Fmoc-RGD/hemin NPs were used in the quantitative and selective detection of H2O2 and cancer cells, and as a therapy for inhibiting EMT. Compared to other methods, this approach provides rapid, direct, and real-time measurements. The developed hybrid material is a potential candidate for the development of cancer diagnostics and treatments. In addition, this proposed approach, based on the peptideregulated

self-assembly

of

therapeutic

agents

into

well-defined

nanostructures, represents a universal strategy. That is, peptide monomers can be selected to fabricate unique NPs with tailored size and functionality, 23



providing new opportunities for the design and functionalization of composite materials.

ASSOCIATED CONTENT Supporting Information SEM images of Fmoc-FF/hemin NPs and FF/hemin NPs，UV−vis spectra of hemin, the absorbance of Fmoc-RGD/hemin NPs in different pH, detection of H2O2, selectivity assay, detection of cellular H2O2, relative viability of MFC-7 cells, in vivo fluorescence images.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected] ORCID Xu Chen: 0000-0001-6187-3890; Wensheng Yang: 0000-0001-8921-3966 Author Contributions X. C. conceived and directed the research. M. L. performed the experiments. S. Z. helped to do the living experiments. 24


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Funding Sources The National Natural Science Foundation of China (21521005, 201874005), the Fundamental Research Funds for the Central Universities (PYBZ1821, XK1901) support the research of the manuscript Notes Any additional relevant notes should be placed here. ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation of China (21521005, 201874005), the Fundamental Research Funds for the Central Universities (PYBZ1821, XK1901).

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Self-Assembling Peptide Artificial Enzyme as an Efficient Detection

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