Polyphenol-Binding Amyloid Fibrils Self-Assemble ... - ACS Publications

Polyphenol-Binding Amyloid Fibrils Self-Assemble into Reversible Hydrogels with Antibacterial Activity

Bing Hu,*,†,‡ Yang Shen,§ Jozef Adamcik,‡ Peter Fischer,∥ Mirjam Schneider,⊥ Martin J. Loessner,§ and Raffaele Mezzenga*,‡ Downloaded via UNIV OF NEW ENGLAND on July 12, 2018 at 08:37:32 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing, Jiangsu 210095, People’s Republic of China ‡ Laboratory of Food and Soft Materials, Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland § Laboratory of Food Microbiology, Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland ∥ Laboratory of Food Process Engineering, Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland ⊥ Laboratory of Toxicology, Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland S Supporting Information *

ABSTRACT: Adaptable hydrogel networks with reversible connectivity have emerged as a promising platform for biomedical applications. Synthetic copolymers and lowmolecular-weight gelators (LMWG) have been shown to form reversible hydrogels through self-assembly of the molecules driven by self-complementary hydrophobic interaction and hydrogen bonding. Here, inspired by the adhesive proteins secreted by mussels, we found that simply adding natural polyphenols, such as epigallocatechin gallate (EGCG) to amyloid fibrils present in the nematic phase, successfully drives the formation of hydrogels through self-assembly of the hybrid supramolecules. The hydrogels show birefringence under polarized light, indicating that the nematic orientation is preserved in the gel phase. Gel stiffness enhances with incubation time and with an increase in molecular ratios between polyphenol and fibrils, fibril concentration, and pH. The hydrogels are shear thinning and thermostable from 25 to 90 °C without any phase transition. The integrity of the trihydroxyl groups, the gallate ester moiety in EGCG, and the hydrophobicity of the polyphenols govern the interactions with the amyloid fibrils and thus the properties of the ensuing hydrogels. The EGCG-binding amyloid fibrils, produced from lysozyme and peptidoglycans, retain the main binding functions of the enzyme, inducing bacterial agglomeration and immobilization on both Gram-positive and Gram-negative bacteria. Furthermore, the antibacterial mechanism of the lysozyme amyloid fibril hydrogels is initiated by membrane disintegration. In combination with the lack of cytotoxicity to human colonic epithelial cells demonstrated for these hybrid supramolecules, a potential role in combating multidrugresistant bacteria in biomedical applications is suggested, such as in targeting diseases related to infection of the small intestine. KEYWORDS: polyphenols, amyloid fibrils, self-assembly, reversible hydrogels, antibacterial activity of inorganic and organic surfaces.1,2 The polyphenolic motifs play essential roles in enhancing the strength of the adhesive protein and bulk solidification.3 With respect to the specific

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he notion of mimicking natural structures provides inspiration not only to design functional artificial materials but also to further expand fundamental research. Dihydroxy-L-phenylalanine (DOPA) identified from mussel byssus is a phenolic molecule that acts as an essential part of the adhesive, associating with the collagen fibrous filler to generate cohesive strength to attach the animal to virtually all types © 2018 American Chemical Society

Received: December 19, 2017 Accepted: March 19, 2018 Published: March 19, 2018 3385

DOI: 10.1021/acsnano.7b08969 ACS Nano 2018, 12, 3385−3396

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Cite This: ACS Nano 2018, 12, 3385−3396

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Figure 1. Schematic of polyphenol-binding amyloid fibrils self-assembly into reversible hydrogels with antibacterial activity.

Driving forces responsible for β-sheet interactions include hydrogen bonding, π−π stacking of aromatic groups, and hydrophobic sequestering in the interior gap between β-sheets.24 Until now, amyloid fibrils based hydrogels are mainly prepared through electrostatic interactions, inducing physical entanglement by shielding surface charge with addition of salts or electrostatically crosslinking with oppositely charged molecules or nanomaterials.25−28 Polyphenols, primarily categorized into flavonoids and nonflavonoids, are a group of naturally occurring phytochemicals that are present in high amounts in fruits, vegetables, green tea, and other natural products. With a basic skeleton of C6−C3−C6, flavonoids have various subclasses such as flavonols, flavones, and flavan-3-ols. In recent years, polyphenols have gained increasing interest in the prevention of degenerative diseases, particularly cancers, cardiovascular diseases, and neurodegenerative disorders.29,30 Polyphenols have demonstrated capacities in directing the progression of amyloid peptides toward disordered and amorphous aggregates,4,31 and thus provide protective mechanisms for the nervous system.5,31 Among them, (−)-epigallocatechin gallate (EGCG), the most abundant and bioactive flavan-3-ol in green tea,32 is well known due to its high water solubility (33.3−100 g/L) and capability in remodeling mature protein amyloids.5,6 Here, we show that simply adding polyphenols to amyloid fibrils (produced from globular peptidoglycans, lysozyme, and lysostaphin M23 endopeptidase) present in the nematic phase induces the formation of hydrogels through exogenous complementary hydrophobic interactions, π−π stacking, and hydrogen bonding (Figure 1). The hydrogels are reversible and thermal resistant. Furthermore, the polyphenol-binding amyloid fibrils and the amyloid fibrils alone show effective antibacterial activity by agglomerating both Gram-negative and Gram-positive bacteria, with the antibacterial mechanism initiated by membrane disintegration. Recently, agglomeration and clumping of bacteria have been demonstrated to effectively remove bacteria, accounting for protection against microbial infection in the brain by amyloid fibrils33 and in the intestine by IgA.34 Importantly, the polyphenol-binding amyloid fibrils have no influence on the viability of human colonic epithelial cells. The observed antibacterial activity without direct cytotoxicity endows the hybrid biomaterials with high biomedical application potential in targeting drug-resistant virulent bacteria and diseases related to infection of the small intestine (∼pH 6.0).

interaction between polyphenols and proteins, one of the most impressive properties of polyphenols is the inhibition of amyloid protein fibril formation.4 In addition, they remodel or cut mature amyloid fibrils under low protein concentrations,5,6 which is used to design therapies to slow down the progress of amyloid-related diseases. Inspired by the adhesive polyphenolic proteins secreted by mussels, we focus here on how polyphenols can interact with mature amyloid fibrils in high protein concentration to form complex hierarchical structures. Hydrogels are three-dimensional polymer networks formed through cross-linking of hydrophilic macromolecular chains within an aqueous microenvironment.7 Due to their water-rich nature, hydrogels are widely used materials in a multitude of applications, including biomedicine,8,9 soft electronics,10 sensors,11,12 and scaffold for catalysis.13,14 Recently, adaptable hydrogel networks with reversible linkages have emerged as a promising and attractive platform in biomedicine.15,16 In previous studies, selfassembly of synthetic amphiphilic block copolymers and polymer blocks (peptides) conjugated with ureidopyrimidinone (UPy) or nucleobases resulted in hydrogels driven by self-complementary hydrophobic interaction and hydrogen bonding, respectively.16 An alternative strategy to prepare hydrogels is via the self-assembly of chemically modified low-molecular-weight gelators (LMWG) into one-dimensional structures, which entangle to form the 3D matrix.17,18 Hydrophobic interaction, π−π stacking, and hydrogen bonding constitute the driving force directing the molecular self-assembly.17 However, the chemical synthesis process is usually labor-intensive, and toxic chemical reagents and solvents are typically used in the modification reactions. Furthermore, the biocompatibility or toxicity of the synthetic polymers and LMWG is an issue raising further concerns. Self-assembled protein nanofibrils, also called amyloid fibrils, are insoluble aggregates formed in vivo from misfolded proteins19 and are known for their implications in severe neurodegenerative diseases, type II diabetes, and other protein-aggregation-prone disorders.20 In recent years, interest in the applications of amyloid fibrils as templates or building blocks in ordered nanomaterials21 is steadily increasing due to the high aspect ratio and mechanical properties of the fibrils.22 Under appropriate conditions of temperature and pH, nontoxic and monomeric globular proteins undergo hydrolysis and can self-assemble into charged amyloid fibrils capable of forming entangled networks and nematic liquid crystals.23 Amyloid fibrils share the remarkably similar structural features at the atomistic length scale, with amino acids arranged into β-strands running orthogonal to the fibril axis and closely packed into β-sheets running parallel to the fibril axis.21

RESULTS AND DISCUSSION Amyloid fibrils prepared by heating a 2 wt % solution of lysozyme monomers at pH 2 for 8 h possess the typical morphology of 3386


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Figure 2. Formation of lysozyme amyloid fibrils and the polyphenol-induced fibril hydrogels. AFM images of the morphology of lysozyme fibrils in pH 2 water (A) and 10 mM pH 6.8 Bis-Tris buffer (B) at a fibril concentration of 0.005 wt %. Linear charge density of lysozyme fibrils after incubation with (−)-epigallocatechin-3-gallate (EGCG) for different time periods (C). (D) Upturn tests of EGCG alone, incubation of EGCG with native lysozyme protein, lysozyme fibrils alone, and incubation of EGCG with lysozyme fibrils [lysozyme fibril (native protein) concentration is 0.5 wt %, molecular ratio between EGCG and lysozyme fibril (native protein) is 4:1, incubation time is 12 h under pH 5.8]. (E) Upturn tests of incubation of lysozyme fibrils (0.5 wt %) with EGCG, (−)-epicatechin gallate (ECG), and (−)-epigallocatechin (EGC) for 12 h with molecular ratio between polyphenol and lysozyme fibril of 4:1 (polyphenol concentration of 1.4 mM) at pH 5.8. (F) Upturn tests of incubation of lysozyme fibrils (0.5 wt %) with 14 μM and 1.4 mM of EGCG and 14 μM morin for 12 h at pH 5.8. (G) Upturn tests and birefringence of the samples of lysozyme fibrils with polyphenols incubated for 96 h under polarized light (fibril concentration of 0.5 wt % and molecular ratio between EGCG, ECG, or EGC and lysozyme fibril of 4:1, molecular ratio between morin and lysozyme fibril of 0.04:1). (H) Frequency dependence of the storage modulus (G′, filled symbols) and loss modulus (G″, open symbols) for the hydrogels induced by incubation of lysozyme fibrils with EGCG, ECG, EGC, and morin for 96 h with a fixed fibril concentration of 0.5 wt % and molecular ratio between EGCG, ECG, or EGC and lysozyme fibril of 4:1, with a molecular ratio between morin and lysozyme fibril of 0.3:1.

semiflexible amyloid fibrils (Figure 2A). The morphology of lysozyme amyloid fibrils is not changed by dilution in 10 mM pH 6.8 Bis-Tris buffer, as shown in Figure 2B. Mixing the fibril solution with the Bis-Tris buffer (pH 6.8) is an effective way to adjust the pH value of the fibrils. The fibrils show slightly decreased linear charge density after exposure to less acidic environments (Figure S1A). Without obvious change in their linear charge density (Figure 2C), incubating lysozyme fibrils (0.5 wt %, equal to ∼350 μM protein monomers, pH 5.8) with EGCG (1.4 mM) for 12 h results in hydrogels (Figure 2D) that do not flow and can withstand an upturn test. Here, the slightly acidic environment was used for maintaining the stability of the polyphenols, and the molecular weight of the native lysozyme protein was used to approximately calculate the molar concentration of the lysozyme fibrils. Under the same experimental conditions, the control systems without either lysozyme fibrils (Figure 2D) or EGCG (Figure 2D) could not form hydrogels. In addition, incubation of EGCG with native lysozyme protein under the same experimental

conditions fails to form hydrogels (Figure 2D). After 2 weeks, the upturn test results still show that only the mixture of EGCG and lysozyme fibrils is a hydrogel (data not shown). Therefore, formation of the hydrogels can unambiguously be attributed to the interaction between EGCG and lysozyme fibrils. The hydrogels can form with different preparation parameters and show translucence as well as birefringence under polarized light, indicating that the nematic orientation is preserved in the gel phase (Supporting Information, Figure S1B−F). The translucent behavior is induced by the nematic grains in the fibril suspensions and disappears when going back through the nematic− isotropic transition via dilution (Figure S2). The gel stiffness increases with incubation time and with an increase in molecular ratios between polyphenol and fibrils, fibril concentration, and pH (Supporting Information, Figure S3A−E). In addition, the hydrogels are shear thinning and thermostable from 25 to 90 °C without any phase transition (Supporting Information, Figure S3F−H). 3387


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morin induces hydrogels with gel strengths similar to that of the hydrogels induced by the more concentrated EGCG (Figure 2H). Six more polyphenols, including two flavonoids, three nonflavonoid phenolic acids, and resveratrol, are also found to induce the lysozyme fibril hydrogels (Figures S7 and S8). Generally, the more hydrophobic the polyphenol is, the less amount of polyphenol is required to form the hydrogels. The binding between the polyphenol molecules and lysozyme fibrils was directly measured by surface plasmon resonance (SPR). Quantitative SPR analysis demonstrates that lysozyme fibrils indeed possess discriminative binding specificity for EGCG, ECG, EGC, and morin (Figure 4). Further, we demonstrate that lysozyme fibrils interact with the polyphenols in a dose-dependent manner (Figure 4A−D). The binding constants of the watersoluble polyphenols EGCG, ECG, and EGC to lysozyme fibrils generally lie in a subnanomolar range, which increases significantly (p < 0.01) to the nanomolar level for the hydrophobic morin (Figure 4E). The binding kinetics of ECG (Figure 4B) to lysozyme fibrils is similar to that of EGCG (Figure 4A), and the binding constant of ECG to lysozyme fibrils decreases slightly compared with that of EGCG (Figure 4E). Interestingly, the binding kinetics of EGC (Figure 4C) to lysozyme fibrils seems to be very different from those of EGCG and ECG, and the affinity also decreases significantly (p < 0.01) (Figure 4E). For the hydrophobic polyphenol, morin shows binding affinity to lysozyme fibrils at a concentrations hundreds of times lower compared with those of the water-soluble ones. Compared to removal of one of the trihydroxyl groups (ECG), removal of the gallate group (EGC) influences the interaction between polyphenols and lysozyme fibrils much more significantly. These data are consistent with the results of formation and strength of the hydrogels. The more hydrophobic the polyphenol molecule is, the higher the affinity shown to the fibrils and the less the polyphenol concentration required to form the hydrogels. In previous studies, the integrity of the trihydroxyl groups and the gallate ester moiety in EGCG were also found to be important for EGCG’s efficiency on inhibiting genesis and remodeling of amyloid fibrils with protein concentrations less than 32 μM.38 In addition, it was found that EGCG inhibits amyloid formation less efficiently and cannot disaggregate existing amyloid fibrils at phospholipid interfaces, which was attributed to decreased π−π stacking interactions between EGCG and the hydrophobic parts in amyloid peptides (fibrils) that had been buried in the phospholipid phase.47 In a model of two complementary pentapeptides modified by connecting pyrene, aromatic−aromatic interactions were found to enable α-helix to β-sheet transition of the short peptides, leading to supramolecular hydrogels.48 Furthermore, hydrophobic morin shows significantly stronger interaction with lysozyme fibrils compared to water-soluble polyphenols (EGCG, ECG, and EGC). Morin has been reported to be more effective in inhibiting amyloid formation and disaggregating preformed amyloid fibrils at low protein concentrations compared with its derivatives.31 Therefore, hydrophobic interactions, π−π stacking interactions, and hydrogen bonding are inferred to provide the driving forces for the interactions between polyphenols and lysozyme fibrils, and thus the formation of the hydrogels. Long fibrils (0.5 wt %, equal to ∼350 μM protein monomers) are homogeneously distributed in the atomic force microscopy (AFM) image of the control fibril sample in the absence of polyphenols (Figure 5A) and in the nematic phase. Conversely, the EGCG-induced lysozyme fibril hydrogels show domains of fibril assemblies in the AFM image (Figure 5B). For the polyphenols

To identify the driving forces for hydrogel formation, two naturally occurring derivatives of EGCG, (−)-epicatechin gallate (ECG) and (−)-epigallocatechin (EGC) (Figure 3), as well as a

Figure 3. Chemical structures of (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin-3-gallate (EGCG), and morin.

significantly more hydrophobic flavonoid, morin (Figure 3), were incubated with lysozyme fibrils. Compared to EGCG, ECG has a similar molecular structure with retention of the gallate ester, but without one of the trihydroxyl groups (Figure 3). EGC has a chemical structure similar to EGCG but lessened by the gallate ester group (Figure 3). The hydroxyl group is prone to form hydrogen bonds in an aqueous environment. The gallate ester moiety is widely reported to endow gallate-type catechins with stronger interactions with proteins,35,36 amyloid peptides,37,38 polysaccharides,39,40 synthetic molecules,41,42 and natural small molecules,43−46 due to multiple intermolecular π−π stacking interactions. Morin is a hydrophobic flavonoid with a water solubility of 0.25 g/L. After incubation with lysozyme fibrils for 12 h with the same molecular ratio between polyphenol and the fibrils, both EGCG and ECG induce formation of the hydrogels, but EGC does not (Figure 2E). With prolonging of the incubation time, the mixture of lysozyme fibrils and EGC also forms hydrogels (Figure 2G). It is interesting that morin induces lysozyme fibril hydrogels with a molecular concentration (14 μM) 100 times lower compared with that of EGCG (1.4 mM) (Figure 2F). Morin induces lysozyme fibril hydrogels at a very low molecule concentration range of 14 to 105 μM (Figure S5), beyond which it causes precipitates rather than hydrogels. The mechanical strength of the hydrogels also increases with the increase of the molecular ratio between morin and lysozyme fibrils (Figure S6). All the hydrogels induced by the polyphenols show birefringence under polarized light (Figure 2G). Removal of one of the trihydroxyl groups from EGCG (ECG) decreases the gel strength compared to EGCG-induced fibril hydrogels, which is further weakened in the case of removal of the gallate ester group (EGC, Figure 2H). Even at a concentration more than 10 times lower than EGCG, 3388


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Figure 4. Binding kinetics of the interaction of EGCG (A), ECG (B), EGC (C), and morin (D) with lysozyme fibrils. The baseline response obtained with no (the 0 nM control) polyphenol has been subtracted from the binding curves and is not shown. For all curves, the “1:1 Langumir” model gave the best fit and was therefore used for calculation of the equilibrium association constants (KA). The KA of EGCG, ECG, EGC, and morin to lysozyme fibrils is shown in E.

surface, cluster with each other, and interact directly with the fibrils,49 with the interaction occurring in surface grooves along the amyloid axis.50 Changing the type of amyloid does not change significantly the conclusions of the structure and properties of the gel. Incubation of EGCG with the amyloid fibrils produced from lysostaphin M23 endopeptidase (Figure S10A) also leads to hydrogels (Figure S10B), with domains of the fibril assemblies (Figure 5F) similar to those observed in the lysozyme case (Figure 5B,E). This infers that the effect of EGCG and other polyphenols in controlling gelation is not proteindependent. Lysostaphin is a Staphylococcus simulans metalloendopeptidase. For investigating the effects of the EGCG-binding lysozyme fibrils on bacteria, Gram-negative Escherichia coli (E. coli) and Grampositive Listeria monocytogenes (L. m.) were used as the representative microorganisms. First, the dynamic effect was evaluated by colony-forming unit (CFU) counting for the EGCG-binding lysozyme fibrils in the concentration range of 0−50 μg/mL in terms of lysozyme fibrils, as well as for EGCG alone and the fibrils alone at various time points (0, 2, 6, and 24 h) (Figure 6, Figures S11 and S12). The results in Figure 6(B and D) show that the EGCG

with removal of one of the trihydroxyl groups (ECG) and the gallate ester group (EGC) from EGCG, morphologies of mixtures of the polyphenols with the fibrils (Figure 5C and D) change from the control fibrils (Figure 5A). The different behavior of the amyloid fibrils induced by EGCG, ECG, and EGC on the microscopic level is at the origin of their different viscoelasticity properties (Figure 2H), consistent with their binding features (Figure 4). With a molecular concentration 10 times lower than that of EGCG, morin also causes domains of fibril assemblies (Figure 5E), which could account for the comparable rigidity of the hydrogels induced by EGCG and morin (Figure 2H). The impact of the lysozyme amyloid fibrils’ concentration on formation of the hydrogels is shown in Figure S9. At the same molecular ratio between EGCG and the fibrils, 0.1 and 0.5 wt % lysozyme fibrils form hydrogels, but 0.05 wt % remains liquid (data not shown). For the hydrogel samples, incubation with EGCG leads to domains of fibril assemblies (Figure S9), which become more intensive upon increase of fibril concentration. This could be the reason for higher gel strength at higher fibril concentrations (Figure S3D). Previous simulation studies found that EGCG molecules could expel water from the amyloid 3389


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E. coli has a cell membrane that can act as a barrier hindering the diffusion of EGCG into the cell. The amyloid fibrils produced from lysostaphin M23 endopeptidase also show the effect on reducing CFUs of E. coli and L. m. (data not shown). To investigate the behavior of bacteria after incubation with the hydrogels, as well as to detect whether the bacterial membrane is damaged or not, a LIVE/DEAD BacLight staining assay was performed under a confocal microscope. This assay is known to discriminate intact (green) and damaged (orange-red) bacterial membranes, respectively.52 Both the EGCG-induced fibril hydrogels and the corresponding lysozyme fibrils alone induce the agglomeration of the bacterial cells, which has been previously demonstrated as the major mechanism for protection against microbial infection in the brain by amyloid-β peptide fibrils.33 Figure 6E-d and F-d show that a portion of E. coli and L. m. display a compromised membrane after incubation with the hydrogels just for 30 min, which is mainly attributed to lysozyme fibrils (Figure 6E-c and F-c) rather than EGCG (Figure 6E-b and F-b). Similar results were also found for control studies incubating E. coli with lysozyme amyloid fibril hydrogels induced by added salt, without the presence of polyphenols (Figure S15), indicating that the antibacterial mechanism is not polyphenolrelated. No bacteria clumps were observed under microscopy after 24 h of treatment with the polyphenol-binding fibrils (data not shown), suggesting that vast majority of bacteria should be killed or underwent lysis, which is in agreement with antibacterial activity shown in previous CFU counting studies. Lysozyme is known as muramidase, an antibacterial enzyme produced by mammals that forms the innate immune system and acts against bacteria mainly by hydrolyzing peptidoglycan of bacterial cell walls. The enzyme itself, in native monomeric format, has been demonstrated in the literature as a promising antibacterial agent.53 However, Gram-negative bacteria are normally insensitive to lysozyme by virtue of their outer membrane, which acts as a physical barrier preventing access of the enzyme. Coupling lysozyme with nanomaterials exhibits pronounced antibacterial activity with desired features.54−57 Here, the amyloid fibrils prepared through unfolding, hydrolyzing, and self-assembling of lysozyme monomers themselves, without addition of any other nanomaterials, show a broad antibacterial spectrum by the agglomeration effect, not only against Gram-positive bacteria, including multidrug-resistant Staphylococcus aureus, but also against Gramnegative ones (Figure 6, Figure S16). Lysozyme is well known to catalyze the hydrolysis of 1,4-βlinkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan, which is the major component of the Gram-positive bacterial cell wall. We indeed took further steps on investigating whether the amyloid fibrils still exhibit lysozyme-like activity using fluorogenic substrates. Surprisingly, no lysozyme activity is detected using the fibrils compared to the monomer control (data not shown). For Gram-negative bacteria, taking E. coli K12 for instance, lysozyme fibrils show significantly stronger antibacterial activity than the native lysozyme protein monomers (Figure S17). It has been known that the disease-related amyloid peptide oligomers always show significantly higher cytotoxicity than their corresponding monomers or mature fibrils.33 In the present study, it is interesting that the short fibrils prepared through homogenization of the mature lysozyme fibrils (Figure S18) do not show a stronger effect on reducing CFU compared with the original mature fibrils (Figure S17). Since homogenization leads to a larger number of free peptides during the cutting process, the lack of obvious difference in the antibacterial activity between the

Figure 5. Behavior of amyloid fibrils induced by different polyphenols on the microscopic scale. The AFM images of the morphology of lysozyme fibrils (A) and the mixtures of lysozyme fibrils with EGCG (B), ECG (C), EGC (D), and morin (E) with the same fibril concentration of 0.5 wt % and incubation time of 24 h at pH 5.8. (The molecular ratio between EGCG and its derivatives (EGC, ECG) and lysozyme fibrils is 4:1; that between morin and lysozyme fibrils is 0.3:1.) (F) AFM image of the morphology of the mixture of lysostaphin M23 endopeptidase amyloid fibrils and EGCG with a fibril concentration of 0.5 wt % and incubation time of 24 h at pH 5.8. (The molecular ratio between EGCG and the lysostaphin M23 endopeptidase amyloid fibrils is 4:1.)

binding lysozyme fibrils are able to reduce E. coli K12 and L. m. WSLC 104251 CFUs by more than 4 orders of magnitude after 24 and 2 h, corresponding to a more than 99.99% reducing efficiency. No CFUs could be found in the Petri dishes (Figures S13 and S14), the amount of which has already been shown to be below the detection limit. For E. coli, the effect of EGCG binding lysozyme fibrils on reducing CFUs is attributed to the combined effects of lysozyme fibrils (50 μg/mL) and EGCG (14 μM) (Figure 6B). For L. m., the EGCG-binding lysozyme fibrils show much faster effect on reducing CFUs, 2 h versus 24 h, compared to E. coli. Lysozyme fibrils alone show a stronger effect on reducing CFUs compared to the hybrid ones, which is shown in the case of 10 μg/mL of the fibrils in Figure S12B and C. After incubation with L. m. for only for 2 h, EGCG alone shows a stronger effect on reducing bacterial CFUs compared to incubation with E. coli for 24 h (Figure 6B and D). The Gram-negative 3390


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Figure 6. Antibacterial activity by the agglomeration effect of the polyphenol-binding lysozyme amyloid fibrils (hydrogels). (A) Time and concentration dependency for reducing colony-forming units (CFUs) of E. coli K12 by EGCG-binding fibrils with the concentrations in terms of fibril concentrations. (B) Effect on reducing CFUs of E. coli after 24 h of exposure to EGCG-binding fibrils (50 μg/mL lysozyme fibrils and 14 μM EGCG), lysozyme fibirls (50 μg/mL), EGCG (14 μM), blank working solution (WS), and Bis-Tris buffer (control). (C) Time and concentration dependency for reducing CFUs of L. monocytogenes WSLC1042 by EGCG-binding fibrils with the concentrations in terms of fibril concentrations. (D) Effect on reducing CFU of L. m. after 2 h of exposure to EGCG-binding fibrils (50 μg/mL lysozyme fibrils and 14 μM EGCG), lysozyme fibrils (50 μg/mL), EGCG (14 μM), blank WS, and Bis-Tris buffer (control). (A−D) Values are means ± SD for n = 6; significant differences among different treatments on reducing CFUs determined with one-way ANOVA followed by Tukey’s test, p ≤ 0.05, are indicated by different lowercase letters in A−D. CLSM images of E. coli incubated with blank WS (E-a), EGCG (1.4 mM) (E-b), lysozyme fibrils (0.5 wt %) (E-c), and the EGCG-induced lysozyme fibril hydrogels (E-d), respectively, for 30 min by LIVE/DEAD BacLight bacterial viability assay. CLSM images of L. m. incubated with blank WS (F-a), EGCG (1.4 mM) (F-b), lysozyme fibrils (0.5 wt %) (F-c), and the EGCG-induced lysozyme fibril hydrogels (F-d), respectively, for 30 min by LIVE/DEAD BacLight bacterial viability assay. Bacterial cells with intact membrane are stained green, and bacteria with compromised membrane are stained orange or red.

mature fibrils and the short fibrils points to the cationic, hydrophobic, and conformational properties of the amyloid fibrils as main sources for the observed antibacterial activity, via the control of bacterial agglomeration. AFM analysis of E. coli after antibacterial treatment further reveals not only the agglomeration of the bacterial cells but also significant surface morphology changes (Figure 7D−F) upon interaction with the polyphenolinduced amyloid fibril hydrogels, whereas bacteria not in contact with the hydrogels maintain their regular surface morphology (Figure 7A−C). Hence, we deduce that the antibacterial mechanism of the lysozyme amyloid fibril hydrogels is initiated by membrane disintegration. Lysozyme amyloid fibril gels are already well known to be noncytotoxic, and for these reasons they are used as efficient cellular scaffolds in tissue engineering and bone regeneration.58−60 However, in the present work we wanted to check that the observed antimicrobial activity of the gels does not come at the expenses of induced cytotoxicity; therefore a human colonic epithelial cell line was selected to run ad-hoc studies. The cell viability of the EGCG binding lysozyme fibrils to human colonic epithelial cells (HCEC) issued from noncancerous tissues that retain characteristics of

normal epithelial cells61 was further investigated to establish whether these fibril gels are also cytotoxic. Different concentrations of EGCG-binding lysozyme fibrils were exposed to HCEC cells, and the metabolic activity was assessed. With concentrations up to 125 μg/mL of lysozyme fibrils, EGCG-binding lysozyme fibrils do not show any influence on the cell viability after 48 h of exposure (Figure 8). Additionally, the corresponding EGCG alone (maximum 35 μM) and the lysozyme fibrils alone (maximum 125 μg/mL) also have no impact on the cell viability (Figure 8), confirming that despite the antibacterial activity, the fibrils and their polyphenol-induced gels show no cytotoxic behavior. Many serious chronic diseases of the small intestine, such as ulcers and even carcinogenesis, have been demonstrated to be related to epidermal infection.62 The small intestine is comparatively difficult for drugs or pharmaceutics to reach and more importantly to retain for long periods. The pH environment in the human small intestine is around pH 6,63 conditions at which the polyphenol-induced reversible fibril hydrogels could coat the epidermal surface and exert an antibacterial activity, along with other bioactivities associated with polyphenols, such as 3391


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Figure 7. AFM images of samples observed by confocal microscope displayed in Figure 6. AFM images of E. coli incubated for 30 min with blank working solution (upper part) and the EGCG-induced lysozyme fibril hydrogels (lower part).

Figure 8. Metabolic activity of HCEC cells upon exposure to increasing concentrations of EGCG-binding lysozyme fibrils and corresponding EGCG alone and lysozyme fibrils alone for 48 h. Results are expressed as mean ± SD % metabolic activity relative to untreated cells (control). Data are from three different experiments with six technical replicates. Significant differences from untreated control cells determined with oneway ANOVA, p ≤ 0.05, are indicated by *.

amyloid fibrils in the nematic phase induce controlled fibril aggregation without phase separation. The integrity of the trihydroxyl groups and the gallate ester moiety in the polyphenol EGCG and the hydrophobic property of the polyphenols play important roles in their interaction with amyloid fibrils, pointing to exogenous complementary hydrogen bonding, π−π stacking, and hydrophobic interactions as the main driving forces for hydrogel assembly. The hydrogels are shear thinning, reversible, and thermal resistant, providing a highly desirable palette of physical properties. The ensuing hydrogels exhibit strong antibacterial activities against both Gram-negative and Gram-positive bacteria, controlled by bacterial agglomeration, without any notable cytotoxicity to human cells. The antibacterial mechanism of the lysozyme amyloid fibril hydrogels is initiated by membrane disintegration. Although a deeper understanding of the antibacterial

antioxidant and anticancer activities. Furthermore, all the materials used here are food grade, which are safer compared to drugs for preventing or treating chronic gastrointestinal diseases. Therefore, these amyloid fibrils prepared from the globular proteins and assembled by polyphenols into hydrogels are expected to target diseases in the small intestine, by exhibiting a strong antibacterial activity against both Gram-negative and Gram-positive bacteria, without any notable cytotoxicity to human colonic epithelial cells.

CONCLUSIONS A novel and effective strategy to prepare reversible hydrogels is achieved through nonelectrostatic physical interactions between small molecular polyphenols and amyloid fibrils. Polyphenol-binding 3392


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Differential Scanning Calorimetry (DSC). DSC measurements were performed with a DSC 1 STARe System differential scanning calorimeter (Mettler Toledo, Greifensee, Switzerland) from 25 to 90 °C at a heating rate of dT/dt = 1 K min−1 and using 40 μL aluminum crucibles under a N2 atmosphere. Surface Plasmon Resonance Analysis. Sensorgrams of binding of the polyphenols to immobilized lysozyme fibrils were obtained using surface plasmon resonance (Biacore X, GE Healthcare, Glattbrugg, Switzerland) as previously described65 with slight modifications. First, the carboxymethylated surface of a CMD500L chip (Xantec Bioanalytics GmbH, Duesseldorf, Germany) was coated with 0.2 mg/mL lysozyme fibrils at a flow rate of 5 μL/min (10 mM sodium acetate pH 4.2), using the amine coupling procedure according to the manufacturer’s manual. To control nonspecific binding, a second flow cell was treated in the same manner, but without immobilization of lysozyme fibrils. For kinetics studies, the polyphenol samples with various concentrations were flowed through both cells in running working solution (7.5 mM Bis-Tris, 2.5 mM HCl, 1.25% v/v DMSO, pH 5.8) at 10 μL/min at 25 °C. For each concentration, association was measured for 180 s, and dissociation was monitored for 720 s. The surface was then regenerated with a 5 s injection of regeneration buffer (10 mM glycine, pH 2) at a flow rate of 10 μL/min prior to the next measurement. This cycle was repeated after each measurement. For all curves, the “1:1 Langmir” model gave the best fit and was therefore used for the calculation of binding constants KA. Atomic Force Microscopy. Fibril solutions (2 wt %, pH 2.0) were diluted into a final concentration of 0.005 wt % by pH 2 H2O and Bis-Tris buffer (10 mM, pH 6.8), respectively, and then 20 μL of solution was deposited onto freshly cleaved mica, incubated for 2 min, rinsed with Milli-Q water, and dried with compressed air flow. For the samples of a mixture of lysozyme fibrils with different polyphenols, the samples were slightly shaken three times to change the solid reversible hydrogels to a flowing viscous liquid, and then 20 μL of solution was deposited onto freshly cleaved mica, incubated for 2 min, rinsed with Milli-Q water, and dried with compressed air flow. AFM experiments were performed by using a MultiMode VIII scanning probe microscope (Bruker, USA) covered with an acoustic hood to minimize noise. AFM images were acquired in tapping mode under ambient conditions. Bacterial Reducing Assay. The Gram-negative bacteria Escherichia coli K12, Klebsiella pneumoniae DSM789, and Pseudomonas aeruginosa PAO1 strain cells were inoculated and grown in lysogeny broth (LB) media overnight under aerobic conditions. The Gram-positive bacteria Listeria monocytogenes WSLC 1042, clinically isolated methicillinresistant Staphylococcus aureus ZH124, and Streptococcus oralis 580 were grown in brain heart infusion (BHI) media overnight prior to bacterial killing tests. Bacterial density was determined by OD 600 measurements and adjusted to ∼106 bacterial CFU mL−1 for E. coli and L. m. with Bis-Tris buffer (pH 6.8), respectively. The EGCG-binding lysozyme fibril solution after incubation of EGCG and the fibrils for 30 min was used as the testing sample. Time and concentration dependency of the effect on reducing CFUs of the EGCG binding fibrils was studied by mixing bacterial suspensions with the samples with different concentrations of the EGCG binding fibrils (0−50 μg/mL in terms of lysozyme fibrils). Corresponding to the EGCG binding fibrils at different concentrations, the blank working solution, lysozyme fibrils alone, and EGCG alone were mixed with bacterial suspensions, respectively. Then aliquots of the testing bacteria samples were taken at each time point and used for serial dilution on agar plates. Quantification of the viability was conducted after 24 h of incubation at 37 °C by CFU counting. The experiment was performed independently three times with six technical replicates. Statistical information was obtained from one-way analysis of variance (ANOVA). LIVE/DEAD BacLight Viability Assay. LIVE/DEAD BacLight viability assay (Life Technology) was applied to evaluate the behavior of bacteria after incubation with the hydrogels and the antibacterial potentials of the EGCG-induced fibril hydrogels. The bacteria suspensions of E. coli and L. m. respectively were preliminarily incubated for 20 min with propidium iodide and SYTO9 according to the manufacturer’s instructions. Then, the bacterial suspension (20 μL) was mixed with 200 μL of hydrogels, EGCG solution (1.4 mM), fibril solution

mechanisms against Gram-negative and Gram-positive bacteria is still needed, these polyphenol-binding amyloid fibrils promise to become outstanding hybrid biomaterials in biomedical and microbiological applications, especially for targeting diseases related to infection of the small intestine.

EXPERIMENTAL SECTION Materials. Hen egg white lysozyme (HEWL, L-6876), EGCG (E4143), EGC (E3768), morin hydrate (M4008), baicalein (465119), myricetin (70050), chlorogenic acid (C3878), ferulic acid (128708), rosmarinic acid (536954), resveratrol (R5010), dimethyl sulfoxide (DMSO, 41640), and sodium acetate (71183) were purchased from Sigma-Aldrich. ECG (CFN99570, ≥98%) was purchased from Wuhan ChemFaces Biochemical Co., Ltd. (Wuhan, China). Lysostaphin M23 endopeptidase was prepared according to our previously published method.64 EDC, NHS, and ethanolamine used in SPR analysis were premade by the manufacture (Biacore X, GE Healthcare, Glattbrugg, Switzerland). Bis-Tris (14880) was purchased from Fluka. HCl was purchased from VWR. Fibril Formation. A 10 g amount of HEWL was dissolved in 90 mL of deionized water (Milli-Q purification system, Millipore) to obtain 10 wt % HEWL solution. In order to remove all extraneous materials completely, the HEWL solution was dialyzed (6000−8000 MWCO, Spectrum Laboratories) against Milli-Q water for 3 days with daily bath changes in a 4 °C cold room. The purified protein was lyophilized for further study. The lysozyme monomer solution was incubated at 2 wt % monomer concentration, pH 2, and 90 °C for 8 h with magnetic agitation. During the incubation the protein monomer unfolded, hydrolyzed, and self-assembled into amyloid fibrils. A lysozyme fibril solution (2 wt %, pH 2) was blended with Bis-Tris buffer (10 mM, pH 6.8) with different volume ratios, to obtain working fibril solutions with different concentrations and different pH values. At the same volume ratio of the lysozyme fibril solution (2 wt %, pH 2) and Bis-Tris buffer, pH 2 Milli-Q water without fibrils was mixed with the Bis-Tris buffer to prepare the corresponding blank working solution. The working fibril solution was diluted with its corresponding blank working solution to prepare different concentration samples with the same pH value. The pH value of each sample was measured before further experiment. Purified lysostaphin M23 endopeptidase (2 wt %) was converted into amyloid fibrils in the same way as above. Hydrogel Formation. Polyphenols were dissolved in DMSO. Polyphenol solution was added to the lysozyme (lysostaphin M23 endopeptidase) amyloid fibril solution at pH 5.8 with magnetic agitation for 30 s. The highest polyphenol concentration in the final mixture system was 1.4 mM. The systems without polyphenol and without lysozyme fibrils and the system replacing fibrils with native lysozyme protein monomers were used as the controls. The formation of hydrogel was preliminary checked by an upturn test. The effects of different preparation parameters, including different incubation times, molecular ratios between polyphenol and lysozyme fibrils, lysozyme fibril concentrations, pH values, and heating temperatures, on the formation and viscoelastic properties of the hydrogels were investigated by rheological measurements. The molecular weight of native lysozyme protein was applied to approximately calculate the molar concentration of the lysozyme fibrils. The samples were also investigated under polarized light for birefringence. Rheological Measurements. For oscillatory rheological experiments, a shear rheometer (Physica MCR702, Anton Paar, Austria) with a cone−plate geometry (CP25, Anton Paar, Austria) was used. Frequency sweeps were performed at a fixed strain amplitude of γ = 1%. Amplitude sweeps were carried out at a fixed angular frequency of ω = 1 rad/s to determine the linear viscoelastic regime. For each sample, a frequency sweep was carried out at 25 °C. The viscoelastic properties of the hydrogel samples with a fibril concentration of 0.5 wt % at different temperatures of 37, 45, and 50 °C were measured. The measuring cell was sealed by covers with a wet sponge to make a moist environment to prevent water evaporation. The shear viscosity was studied by measuring flow curves recorded at shear rates from 10 to 500 s−1 and back from 500 to 10 s−1. 3393


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ACS Nano (0.5 wt %), and the blank working solution, respectively, for 30 min. The viability of the bacteria cells was assessed by a Leica TCS SPE confocal microscope at wavelengths of 480/500 nm (SYTO9) and 490/635 nm (PI) (Leica Microsystems GmbH, Wetzlar, Germany), operated by a Leica LAS AF interface. After confocal microscopy measurements, the samples (E. coli suspension with EGCG hydrogels and E. coli suspension in blank working solution) were dried, and AFM scanning in air was performed in order to observe the same samples. Cytotoxicity Assay. The HCEC (Clone 1CT) cells were obtained from Prof. Jerry Shay (University of Texas Southwestern Medical Center, Dallas, TX, USA) and cultured under previously reported conditions.61 The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 and were routinely tested to be mycoplasma free using the MycoAlert mycoplasma detection kit from Lonza. Metabolic activity was assessed with the CellTiter-Glo luminescent cell viability assay from Promega. Cells were seeded in 96-well plates (12 500 cells/well). After 24 h the cell culture medium was replaced by medium containing the EGCG-binding fibrils with concentrations from 1.95 to 125.00 μg mL−1 in terms of the fibrils, the corresponding fibrils alone (1.95−125.00 μg mL−1), and EGCG alone (0.55−35.00 μM). After 48 h the cell viability assay was performed according to the manufacturer’s protocol. Triton X-100 (2%, v/v) and untreated cells were used as positive and negative control, respectively. The experiment was performed independently three times with six technical replicates. Statistical information was obtained from one-way analysis of variance (ANOVA).

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b08969. Additional information (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: raff[email protected]. ORCID

Peter Fischer: 0000-0002-2992-5037 Raffaele Mezzenga: 0000-0002-5739-2610 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Dr. Antoni Sánchez-Ferrer for DSC measurement and discussion and Prof. Shana Sturla for granting access to facilities for cytotoxicity assay. This work is supported by the National Natural Science Foundation of China (No. 31501488), the Natural Science Foundation of Jiangsu Province−Outstanding Youth Foundation (BK20160075), the National Social Science Foundation Major Project (15ZDB168), and the National Key Research and Development Program of China (2017YFD0400800). REFERENCES (1) Waite, J. H.; Tanzer, M. L. Polyphenolic Substance of Mytilus Edulis: Novel Adhesive Containing L-Dopa and Hydroxyproline. Science 1981, 212, 1038−1040. (2) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. MusselInspired Surface Chemistry for Multifunctional Coatings. Science 2007, 318, 426−430. (3) Lee, H.; Scherer, N. F.; Messersmith, P. B. Single-Molecule Mechanics of Mussel Adhesion. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 12999−13003. (4) Bieschke, J.; Herbst, M.; Wiglenda, T.; Friedrich, R. P.; Boeddrich, A.; Schiele, F.; Kleckers, D.; Lopez del Amo, J. M.; Gruning, B. A.; Wang, Q.; Schmidt, M. R.; Lurz, R.; Anwyl, R.; Schnoegl, S.; Fandrich, M.; 3394


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