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Gram-Positive Antimicrobial Activity of Amino Acid-Based Hydrogels I. Irwansyah, Yong-Qiang Li, Wenxiong Shi, Dianpeng Qi, Wan Ru Leow, Mark B. Y. Tang, Shuzhou Li,* and Xiaodong Chen* Bacterial infections constitute one of the greatest global challenges facing public healthcare today.[1–4] Extensive efforts have been dedicated to bacterial infection therapy, as reflected by the continuing development of antimicrobial materials, such as antibiotics, silver particles, photosensitizers, antimicrobial peptides, and hydrogels.[5–14] In particular, antimicrobial hydrogels can provide a moist environment and remain in place under physiological conditions while exhibiting antimicrobial activity, making them suitable for medical implants coating, wound healing, and skin infection treatment.[15–17] Several effective antimicrobial hydrogels have been reported mainly based on the supramolecular assembly of synthetic cationic polymers or peptides.[10,15–26] However, the preparation of these cationic polymers and peptides generally requires complicated chemical synthesis and purification processes. Meanwhile, organic solvent and toxic reagents used in such processes would be incorporated in obtained antimicrobial hydrogels, thereby reducing their antimicrobial efficiency and biocompatibility.[27–31] In addition, the antimicrobial hydrogels created by the supramolecular assembly of cationic polymers and peptides frequently result in immunogenicity and inflammation and have high toxicity to mammalian cells.[27,32,33] To address these challenges, it is important and necessary to develop more biocompatible and efficient antimicrobial hydrogels. In order to achieve this, hydrogelator design and antimicrobial ability conferment are two essential points needed to be taken into account. With respect to the former, supramolecular hydrogels derived from 9-fluorenylmethoxycarbonyl-modified oligopeptides (Fmoc-oligopeptides) have recently been of particular interest due to their biocompatibility.[34–43] The hydrogelator of Fmoc-oligopeptides can easily self-assemble in aqueous solution to form fibrous hydrogels by taking advantage of the intermolecular π–π stacking interactions of Fmoc groups and hydrogen bonding of oligopeptides.[34–43] Since oligopeptides are normally synthesized using commercial Fmoc-amino acids as precursor, it would be convenient and cost-effective if the commercially available Fmoc-amino acid can be directly used as gelator for supramolecular hydrogel formation. As the phenyl
I. Irwansyah, Dr. Y.-Q. Li, Dr. W. Shi, Dr. D. Qi, W. R. Leow, Prof. S. Li, Prof. X. Chen School of Materials Science and Engineering Nanyang Technological University 50 Nanyang Avenue, Singapore 639798, Singapore E-mail: [email protected]; [email protected] Dr. M. B. Y. Tang National Skin Centre Singapore 1 Mandalay Road, Singapore 308205, Singapore
DOI: 10.1002/adma.201403339
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group of phenylalanine could provide increased intermolecular π–π stacking interactions to drive gel formation, we hypothesize that Fmoc-phenylalanine (Fmoc-F) could potentially be a simple and cost-effective hydrogelator. Regarding the second point, the conferment of antimicrobial ability, co-assembly is a valuable strategy frequently used in supramolecular systems to endow additional properties for different applications.[44–51] Fmoc-leucine (Fmoc-L) appears to be a promising candidate as an antimicrobial building block in such co-assembly, as leucine has been reported to possess intrinsic anti-inflammatory and antibacterial properties,[52–56] and peptides with leucine-rich residues have been reported to have an important role in gram-positive bacterial recognition.[57–61] Therefore, we hypothesize that by co-assembly of commercial Fmoc-L and Fmoc-F, which act as the antimicrobial agent building block and hydrogelator, respectively, amino acidbased hydrogels with gram-positive antimicrobial activity could be prepared. In this work, we first demonstrate that Fmoc-F can be used as a novel hydrogelator to form supramolecular hydrogels by taking advantage of the intermolecular π–π stacking and hydrogen bonding. Furthermore, the co-assembly of the Fmoc-F and an antimicrobial building block of Fmoc-L can lead to the generation of a supramolecular hydrogel with antimicrobial activity. Finally, we demonstrate that this co-assembled hydrogel exhibits selective gram-positive antimicrobial activity via a mechanism involving cell wall and membrane disruption while being biocompatible with normal mammalian cells. This development may provide a solution to efficiently eliminate the problem of bacterial drug resistance, and it has great potential as antimicrobial coatings for the treatment of clinical skin and wound infections mainly caused by gram-positive bacteria. In typical experiments, Fmoc-amino acids were dissolved in sodium carbonate solution, and the gel phase was transformed using a heating and cooling process. Prior to the preparation of our designed antimicrobial hydrogel, the potential of Fmoc-F as a hydrogelator was first investigated. As expected, in testing six types of commercial hydrophobic Fmoc-amino acids, selfsupporting hydrogel was only formed when Fmoc-F was used as a hydrogelator (Figure 1a). The viscoelastic properties of the formed Fmoc-F hydrogel were then assessed using oscillatory rheology. As shown in Figure S1 (Supporting Information), the storage modulus (G′) of the hydrogel was approximately an order of magnitude greater than the loss modulus (G″), which is indicative of an elastic gel rather than viscous material.[42] To prepare the desired antimicrobial hydrogels, the Fmoc-F hydrogelator was mixed with the antimicrobial building block of Fmoc-L, and co-assembled (F+L) hydrogels were easily obtained (Figure 1a), while no hydrogels were found in the mixtures of
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Figure 1. a) Demonstration of the facile preparation of antimicrobial hydrogel based on the co-assembly of commercial Fmoc-F and Fmoc-L. Transparent self-supporting hydrogel was only formed based on self-assembly of Fmoc-F rather than Fmoc-glycine (Fmoc-G), Fmoc-isoleucine (Fmoc-I), Fmoc-valine (Fmoc-V), Fmoc-alanine (Fmoc-A), and Fmoc-L, thus indicating the potential of Fmoc-F as a hydrogelator. b) Oscillatory rheology of the co-assembled (F+L) hydrogel. c) Field emission scanning electron microscopy (FESEM) images of the co-assembled (F+L) hydrogel indicated the formation of nanofiber structure.
Fmoc-L with other four Fmoc- amino acids (Figure S2, Supporting Information). The oscillatory rheology confirmed the formation of a solid like gel material (Figure 1b). Moreover, the formation of entangled nanofibers within formed supramolecular hydrogels with width dimensions in the order of tens of nanometers and microscopic lengths was determined based on a variety of microscopy techniques (Figure S3, Supporting Information, and Figure 1c). For Fmoc-oligopeptides-based supramolecular hydrogels, intermolecular interactions of π–π stacking of Fmoc groups and hydrogen bonding were reported to play important roles in gel formation.[34–43] Hence, we first employed fluorescence spectroscopy to investigate the molecular arrangement of Fmoc groups within our hydrogels. As shown in Figure 2a, an emission peak centered at 312 nm wavelength appeared in the complex of Fmoc-F and Fmoc-L solution phase, while the
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maximum emission for co-assembled (F+L) hydrogel appeared at 316 nm with the presence of a shoulder at 368 nm. This indicated the existence of antiparallel intermolecular π–π interactions of Fmoc groups in the gel phase.[42] A redshift of emission peak was also observed for the Fmoc-F hydrogel system (Figure S4, Supporting Information), which can be attributed to the presence of a fluorenyl excimer.[62,63] No redshift of emission peaks was found in other four Fmoc-amino acids systems (Figure S5, Supporting Information). Meanwhile, we used Fourier transform infrared (FTIR) spectroscopy to study the role of intermolecular hydrogen bonding in the formation of the supramolecular hydrogels. As shown in Figure S6 (Supporting Information) and Figure 2b, compared to the FTIR spectrum in solution phase, the characteristic C=O stretching band peaks in Fmoc-F and co-assemble (F+L) hydrogel phases redshifted from 1658 and 1717 cm−1 to 1640 and 1636 cm−1, respectively,
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indicating the presence of intermolecular hydrogen bonding motifs. Information provided by Raman spectroscopy was consistent with this observation; the peaks of C=O stretching band in hydrogel phases exhibited redshift compared to that in solution phases (Figure 2c and Figure S7, Supporting Information). According to these spectroscopic results, it can be concluded that both the intermolecular π–π stacking of Fmoc groups and hydrogen bonding play important roles in the co-assembled (F+L) hydrogel formation. The effect of aromatic phenyl group of Fmoc-F for hydrogel formation was also evaluated, upon which it was discovered that, compared to Fmoc-amino acids without aromatic phenyl group (Fmoc-G, Fmoc-I, Fmoc-V, Fmoc-A, Fmoc-L), only the Fmoc-F could form supramolecular hydrogel in our experiments (Figure 1a). This indicated that the intermolecular π–π stacking of phenyl groups also plays a critical role in our supramolecular hydrogel formation in addition to the intermolecular π–π stacking interactions of Fmoc groups and hydrogen bonding. This conclusion could also be verified by a hydrogel destruction experiment using urea
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as a blocking reagent of π–π stacking and hydrogen bonds (Figure S8, Supporting Information). These three forces work together to drive the co-assembly of Fmoc-F and Fmoc-L and culminate in hydrogel formation (Figure 2d). To further confirm this result, molecular dynamic simulation was performed to study the aggregation of Fmoc-amino acids in water. As illustrated in Figure 2e and Figure S9, Supporting Information, spontaneous formation of cross-linking 3D network structures of nanofibers was observed for Fmoc-F and co-assembled (F+L) systems, while the aggregation of the other five Fmoc-amino acids without aromatic phenyl group merely yielded the separated pillar structure of nanofibers. These simulation results were consistent with the empirical results described above. To evaluate the antimicrobial activity of the co-assembled (F+L) hydrogel, the hydrogel surface was challenged with pathogenic bacteria and the microbial proliferation was assessed by optical density (OD) measurement. Staphylococcus aureus, arguably the most prevalent pathogen to humans,[64–67] was chosen as the model gram-positive bacteria for evaluation.
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Figure 3. a) Time-dependent antimicrobial effects of the co-assembled (F+L) hydrogel, Fmoc-F (F) hydrogel, and the Fmoc-L (L) solution (0.5 mL, 4 mg mL−1) toward S. aureus bacteria. The original concentration of S. aureus used was 1 × 106 CFU mL−1. b) Concentration-dependent antimicrobial effect of the co-assembled (F+L) hydrogel toward S. aureus bacteria. Different concentrations of S. aureus bacteria were incubated with the hydrogel for 48 h before detecting the OD value. c) The difference between antimicrobial effects of the co-assembled (F+L) hydrogel for gram-positive and gram-negative bacteria. The y-axis indicates the increased folds of OD values of the bacteria solution after it has been incubated with the hydrogel for 20 h. Gram-positive bacteria of Bacillus subtilis and S. aureus, and gram-negative bacteria of Pseudomonas aeruginosa and Escherichia coli were tested. d) MTT activities of endothelial cells contacted with the co-assembled (F+L) hydrogel from 1 to 3 d.
Figure 3a shows the time-dependent antimicrobial test of the co-assembled (F+L) hydrogel. It was found that the presence of the co-assembled (F+L) hydrogel completely suppressed bacterial growth, while bacteria showed significant growth during incubation in sample with Fmoc-F gel (F gel) or without gel (control), indicating that the co-assembled (F+L) hydrogel possessed efficient antimicrobial effect. In addition, the Fmoc-L (L) solution was discovered to inhibit bacterial growth moderately and show better antimicrobial activity than the pure leucine solution (Figure 3a and Figure S10, Supporting Information). For the reason of this phenomenon, we hypothesize that the Fmoc modification may have a potential role in bacteria killing through its hydrophobic interaction with the hydrophobic sections of bacterial cell membrane, as employed by AMPs. The synergistic antimicrobial effect of Fmoc modification and leucine on bacterial growth leads to a relative lower OD value in Fmoc-L solution, compared to the control experiment. The co-assembled (F+L) hydrogel was found to effectively inhibit ≈95% of S. aureus bacteria proliferation after 20 h incubation (Figure S11, Supporting Information). To assess the antimicrobial capacity of the co-assembled (F+L) hydrogel, the hydrogel was subjected to various concentrations of S. aureus and allowed to incubate for 48 h. As shown in Figure 3b, the co-assembled (F+L) hydrogel was capable of inhibiting up to 1 × 108 colony-forming units (CFU) per milliliter of S. aureus 4
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proliferation, but lost its antimicrobial ability when subjected to 1 × 109 CFU mL−1 bacteria. Furthermore, the difference between antimicrobial activity of the co-assembled (F+L) hydrogel for gram-positive and gram-negative bacteria was investigated systematically to demonstrate its selective grampositive bactericidal ability. As we expected, the co-assembled (F+L) hydrogel indeed exhibited preferential antimicrobial effect for gram-positive bacteria (Figure 3c and Figure S12, Supporting Information), while the co-assembled (F+I) hydrogel showed no antimicrobial activity and gram-positive bacteria selectivity (Figure S13, Supporting Information), indicating that Fmoc-L has a critical role in the selective antimicrobial effect of the co-assembled (F+L) hydrogel for gram-positive bacteria. The selective antimicrobial activity of our co-assembled (F+L) hydrogel indicates that it is a good candidate for the clinical treatment of gram-positive bacterial infections, such as the clinical skin and wound infections mainly caused by gram-positive bacteria of S. aureus.[68] Moreover, the in vitro biocompatibility of the co-assembled (F+L) hydrogel was investigated. Endothelial cells were contacted with the co-assembled (F+L) hydrogel for 3 d with the tissue culture polystyrene dish as control, and cell viability was tested by methyl tetrazolium (MTT) assay. As shown in Figure 3d, the MTT absorbance increased with culture time and was statistically similar to that of the control from 1 to 3 d, indicating that the cell proliferated well in the presence
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was found in bacteria before contact with the gel, further confirming the disruption of the bacterial cell wall and membrane after contact with the hydrogel, since redfluorescent propidium iodide is well known to penetrate only bacteria with destroyed cell walls and membranes.[69–71] The timedependent live/dead bacterial staining assay also revealed that the S. aureus bacteria were killed rapidly by the co-assembled (F+L) hydrogel within 30 min (Figure S16, Supporting Information). For our antimicrobial hydrogel system, contact killing and Fmoc-L release are possible mechanisms, but there is a time sequential process of bacteria contact and Fmoc-L release. Fmoc-L release may only occur after the bacterial contact, which is the reason that Fmoc-L release was found in bacterial LB medium (Figure S17, Supporting Information) rather than pure LB medium (Figure S18, Supporting Information). When bacteria contact the surface of hydrogel, Fmoc-L would insert into bacteria and release in which the hydrophobic interactions between hydrogels (Fmoc and phenyl group) and the hydrophobic sections of bacterial cell membrane may play a critical role, and finally kill bacteria. It is noteworthy that Figure 4. a) Representative SEM images and b) overlapping fluorescence images for live/dead the co-assembled (F+L) hydrogel has no posibacterial staining assay of S. aureus before and after contact with the co-assembled (F+L) tive charge in its structure, indicating that hydrogel for 2 h. Two fluorescent dyes were used in live/dead staining in which SYTO 9 with the binding interaction between the hydrogel green color labeled both live and dead bacteria while propidium iodide with red color stained and S. aureus is unlikely to be caused by eleconly dead bacteria. trostatic interactions, as seen in AMPs. It is hypothesized that the interactions between the S. aureus cell wall and the co-assembled (F+L) hydrogels of the co-assembled (F+L) hydrogel. In addition, endothelial are related to the Fmoc-L used, since it has been found that cells contacted with the co-assembled (F+L) hydrogel were leucine-rich peptides can bind to S. aureus cell wall rapidly and found to show healthy spread morphologies (Figure S14, Supeffectively.[57–61] porting Information). These preliminary data suggest that the co-assembled (F+L) hydrogel is in vitro biocompatible toward In conclusion, we report the preparation of biocompatible normal mammalian cells. Furthermore, the co-assembled hydrogel with gram-positive antimicrobial activity based on (F+L) hydrogel was found to remain stable in two nutrientthe co-assembly of commercial Fmoc-amino acids. By taking rich mediums (lysogeny broth (LB) bacteria culture medium advantage of the intermolecular π–π stacking interactions of and Dulbecco’s modified eagle cell culture medium (DMEM)) Fmoc and phenyl group, a novel supramolecular hydrogel platduring 2 d at 37 °C, indicating its stability for long-term antiform derived from Fmoc-F can be easily established. Moreover, bacterial applications (Figure S15, Supporting Information). antimicrobial ability can be conferred on this supramolecular hydrogel platform via a co-assembly mechanism by combining To gain insight into the antimicrobial mechanism of the the Fmoc-F hydrogelator with another commercial Fmoc-L as co-assembled (F+L) hydrogel, the morphological changes of antimicrobial building block. The co-assembled (F+L) supramobacteria after contact with the hydrogel were investigated. As lecular hydrogel exhibited selective gram-positive bactericidal shown in Figure 4a, S. aureus bacteria showed clear edges and activity via a mechanism involving cell wall and membrane dissmooth bodies prior to contact with the hydrogel. In sharp conruption while is biocompatible with normal mammalian cells. trast, cellular deformation and surface collapse were clearly Therefore, it may efficiently eliminate the problem of bacterial found after contact with hydrogel for 2 h. In addition, lysed drug resistance. In addition, it shows great potential as antimibacteria and debris were found, suggesting that the antimicrobial coatings in clinical devices, wound dressings, or topical crobial mechanism of the co-assembled (F+L) hydrogel should agents for the treatment of clinical skin and wound infections be similar to that of AMPs which involved bacterial cell wall mainly caused by gram-positive bacteria of S. aureus. The prepand membrane disruption. This conclusion was further suparation of this antimicrobial hydrogel relies on the co-assembly ported by the live/dead bacterial staining assay. As shown in of commercially available Fmoc-amino acids, which enable us Figure 4b, S. aureus bacteria were stained red after contact to fabricate biocompatible antimicrobial hydrogel easily and with the co-assembled (F+L) hydrogel while little red color
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cost-effectively without involving complicated organic synthesis and purification processes, as compared to the commonly used antimicrobial hydrogels based on cationic materials.
Experimental Section Preparation of Supramolecular Hydrogels: All the Fmoc-amino acids were purchased from Merck and used as received. The gelation test was performed by dissolved 10 mg of Fmoc-amino acid in a vial with an internal diameter of 10 mm containing an equivalent amount of sodium carbonate solution (0.1 M, 130 µL). The volume of solution was adjusted to 500 µL by adding deionized (DI) water followed by slowly heating (2 °C min−1) until all solid disappears. The hot solutions (about 90 °C) were allowed to cool slowly (undisturbed) to achieve room temperature. After 20–30 min, supramolecular hydrogel formation was verified by the vial inversion method. The co-assembled supramolecular hydrogels were prepared in an identical manner but contain 10 mg of Fmoc-F and 2 mg of Fmoc-L or Fmoc-I so as to keep the ratio of Fmocamino acids and sodium carbonate almost constant (molar ratio (F+L) or (F+I):Na2CO3 ≈ 2:1). In addition, the antimicrobial effect of Fmoc-L solution (0.5 mL, 4 mg mL−1) was tested as a control. Spectrum Characterization of Hydrogels: FTIR spectra were collected on an FTIR system (IFS 66/S, Bruker) with the gel pressed between crystal zinc selenium plates and scanned between 1800 and 1550 cm−1 with an interval of 1 cm−1. Fluorescence emission spectra were measured on a fluorescence spectrometer (RF-5301, PC) with excitation at 280 nm and emission data range between 300 and 450 nm. The Raman spectra were obtained with a spectrophotometer (WITec, alpha300R) with an operating wavelength of 532 nm. SEM Study of Hydrogels: Field-emission scanning electron microscopy (FESEM) was used to study the morphology of hydrogels obtained. For such measurement, the hydrogel samples were first air-dried at room temperature and then placed on a copper tape. The copper tape was mounted onto an aluminum stud and vacuum coated with platinum before SEM (JSM-7600F, JEOL) examination. Oscillatory Rheology of Hydrogels: Rheological measurements were conducted on a rheometer (AR-G2, TA Instruments) operating in oscillatory mode. Hydrogel samples were placed on the rheometer stage, and a dynamic frequency sweep was performed from 0.1 to 100 Hz with 0.2% strain at 25 °C. All reported values of storage and loss modulus are the averages of three runs. Bacteria Culture: Bacteria of S. aureus (ATCC 6538), Bacillus subtilis (ATCC 6051), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 9027) were used in our experiments. Prior to the experiments, the bacteria were grown overnight in Luria–Bertani broth medium (LB) (Sigma-Aldrich) and harvested at the exponential growth phase via centrifugation. The supernatant was then discarded and the cell pellet was resuspended in phosphate-buffered saline (PBS). The bacteria concentration could be monitored photometrically by measuring the OD at a wavelength of 600 nm. Before performing the antibacterial experiments with the hydrogels, the OD600 values of bacteria stock solutions were readjusted to 0.1, which corresponded to the concentrations, obtained based on the colony-counting method, of 4 × 108, 2 × 108, 1 × 108, and 2 × 108 CFU mL−1 for S. aureus, B. subtilis, E. coli, and P. aeruginosa, respectively. Time-Dependent and Concentration-Dependent Antibacterial Experiments of Supramolecular Hydrogels: Supramolecular hydrogels for the antibacterial assays were prepared in 24-well tissue culture plates (NUNC). For time-dependent antibacterial assay, 1 mL of bacteria LB solution was introduced onto the surface of the 2% (w/v) supramolecular hydrogel (F, (F+I) or (F+L) gel). The 24-well tissue culture plates containing gels and bacteria cultures were placed on a shaker at 37 °C, and the OD readings of bacteria solutions were monitored every 2 h by measuring the OD600. The bacteria solution without hydrogel or with L solution was used as control. For concentration-dependent antibacterial assay, 1 mL of S. aureus LB solution with different bacteria concentration
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(1 × 104 to 1 × 109 CFU mL–1) was introduced onto the surface of the co-assembled (F+L) supramolecular hydrogel (2% (w/v)) for 48 h, and the OD readings of bacteria solutions were monitored by measuring the OD600. The bacteria solution without hydrogel was used as control. Live/Dead Bacterial Assay: Live/dead bacterial assay was carried out to examine the viability of bacteria before and after supramolecular hydrogel treatment. After incubation with or without hydrogel for different times, S. aureus bacteria solution was mixed with 0.5 mL of dye solution containing 1 µM SYTO 9 and 5 µM propidium iodide (Invitrogen) for 20 min at room temperature. The green-colored SYTO 9 dye enters both intact and membrane-compromised bacterial cells, while the red-colored propidium iodide dye can only enter cells with damaged membranes. The bacteria were then imaged using a confocal microscopy (TCS SP5, Leica). Morphology Study of Bacteria: The morphology of bacteria before or after supramolecular hydrogel treatment was examined by FESEM. Firstly, the S. aureus bacteria solution before or after hydrogel treatment was dropped onto silicon wafer, and the silicon wafer was then treated with 2% glutaraldehyde (Sigma-Aldrich) fixation for 2 h at room temperature and gradient dehydration by a series of ethanol solutions (50%, 70%, 90%, 95%, and 100%) for 10 min with each step. Next, the silicon wafer was subjected to nitrogen drying to maintain the morphologies of the bacteria captured on substrate. After complete drying, the silicon wafer was sputter coated with platinum and imaged with FESEM (JSM-7600F, JEOL).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements I.I. and Y.-Q. Li contributed equally to this work. This work was supported by the Singapore National Research Foundation (CREATE Programme of Nanomaterials for Energy and Water Management and NRF-RF2009-04) and NTU-Northwestern Institute for Nanomedicine. Received: July 24, 2014 Revised: September 22, 2014 Published online:
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Gram-Positive Antimicrobial Activity of Amino Acid-Based Hydrogels I. Irwansyah, Yong-Qiang Li, Wenxiong Shi, Dianpeng Qi, Wan Ru Leow, Mark B. Y. Tang, Shuzhou Li,* and Xiaodong Chen* Bacterial infections constitute one of the greatest global challenges facing public healthcare today.[1–4] Extensive efforts have been dedicated to bacterial infection therapy, as reflected by the continuing development of antimicrobial materials, such as antibiotics, silver particles, photosensitizers, antimicrobial peptides, and hydrogels.[5–14] In particular, antimicrobial hydrogels can provide a moist environment and remain in place under physiological conditions while exhibiting antimicrobial activity, making them suitable for medical implants coating, wound healing, and skin infection treatment.[15–17] Several effective antimicrobial hydrogels have been reported mainly based on the supramolecular assembly of synthetic cationic polymers or peptides.[10,15–26] However, the preparation of these cationic polymers and peptides generally requires complicated chemical synthesis and purification processes. Meanwhile, organic solvent and toxic reagents used in such processes would be incorporated in obtained antimicrobial hydrogels, thereby reducing their antimicrobial efficiency and biocompatibility.[27–31] In addition, the antimicrobial hydrogels created by the supramolecular assembly of cationic polymers and peptides frequently result in immunogenicity and inflammation and have high toxicity to mammalian cells.[27,32,33] To address these challenges, it is important and necessary to develop more biocompatible and efficient antimicrobial hydrogels. In order to achieve this, hydrogelator design and antimicrobial ability conferment are two essential points needed to be taken into account. With respect to the former, supramolecular hydrogels derived from 9-fluorenylmethoxycarbonyl-modified oligopeptides (Fmoc-oligopeptides) have recently been of particular interest due to their biocompatibility.[34–43] The hydrogelator of Fmoc-oligopeptides can easily self-assemble in aqueous solution to form fibrous hydrogels by taking advantage of the intermolecular π–π stacking interactions of Fmoc groups and hydrogen bonding of oligopeptides.[34–43] Since oligopeptides are normally synthesized using commercial Fmoc-amino acids as precursor, it would be convenient and cost-effective if the commercially available Fmoc-amino acid can be directly used as gelator for supramolecular hydrogel formation. As the phenyl
I. Irwansyah, Dr. Y.-Q. Li, Dr. W. Shi, Dr. D. Qi, W. R. Leow, Prof. S. Li, Prof. X. Chen School of Materials Science and Engineering Nanyang Technological University 50 Nanyang Avenue, Singapore 639798, Singapore E-mail: [email protected]; [email protected] Dr. M. B. Y. Tang National Skin Centre Singapore 1 Mandalay Road, Singapore 308205, Singapore
DOI: 10.1002/adma.201403339
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group of phenylalanine could provide increased intermolecular π–π stacking interactions to drive gel formation, we hypothesize that Fmoc-phenylalanine (Fmoc-F) could potentially be a simple and cost-effective hydrogelator. Regarding the second point, the conferment of antimicrobial ability, co-assembly is a valuable strategy frequently used in supramolecular systems to endow additional properties for different applications.[44–51] Fmoc-leucine (Fmoc-L) appears to be a promising candidate as an antimicrobial building block in such co-assembly, as leucine has been reported to possess intrinsic anti-inflammatory and antibacterial properties,[52–56] and peptides with leucine-rich residues have been reported to have an important role in gram-positive bacterial recognition.[57–61] Therefore, we hypothesize that by co-assembly of commercial Fmoc-L and Fmoc-F, which act as the antimicrobial agent building block and hydrogelator, respectively, amino acidbased hydrogels with gram-positive antimicrobial activity could be prepared. In this work, we first demonstrate that Fmoc-F can be used as a novel hydrogelator to form supramolecular hydrogels by taking advantage of the intermolecular π–π stacking and hydrogen bonding. Furthermore, the co-assembly of the Fmoc-F and an antimicrobial building block of Fmoc-L can lead to the generation of a supramolecular hydrogel with antimicrobial activity. Finally, we demonstrate that this co-assembled hydrogel exhibits selective gram-positive antimicrobial activity via a mechanism involving cell wall and membrane disruption while being biocompatible with normal mammalian cells. This development may provide a solution to efficiently eliminate the problem of bacterial drug resistance, and it has great potential as antimicrobial coatings for the treatment of clinical skin and wound infections mainly caused by gram-positive bacteria. In typical experiments, Fmoc-amino acids were dissolved in sodium carbonate solution, and the gel phase was transformed using a heating and cooling process. Prior to the preparation of our designed antimicrobial hydrogel, the potential of Fmoc-F as a hydrogelator was first investigated. As expected, in testing six types of commercial hydrophobic Fmoc-amino acids, selfsupporting hydrogel was only formed when Fmoc-F was used as a hydrogelator (Figure 1a). The viscoelastic properties of the formed Fmoc-F hydrogel were then assessed using oscillatory rheology. As shown in Figure S1 (Supporting Information), the storage modulus (G′) of the hydrogel was approximately an order of magnitude greater than the loss modulus (G″), which is indicative of an elastic gel rather than viscous material.[42] To prepare the desired antimicrobial hydrogels, the Fmoc-F hydrogelator was mixed with the antimicrobial building block of Fmoc-L, and co-assembled (F+L) hydrogels were easily obtained (Figure 1a), while no hydrogels were found in the mixtures of
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Figure 1. a) Demonstration of the facile preparation of antimicrobial hydrogel based on the co-assembly of commercial Fmoc-F and Fmoc-L. Transparent self-supporting hydrogel was only formed based on self-assembly of Fmoc-F rather than Fmoc-glycine (Fmoc-G), Fmoc-isoleucine (Fmoc-I), Fmoc-valine (Fmoc-V), Fmoc-alanine (Fmoc-A), and Fmoc-L, thus indicating the potential of Fmoc-F as a hydrogelator. b) Oscillatory rheology of the co-assembled (F+L) hydrogel. c) Field emission scanning electron microscopy (FESEM) images of the co-assembled (F+L) hydrogel indicated the formation of nanofiber structure.
Fmoc-L with other four Fmoc- amino acids (Figure S2, Supporting Information). The oscillatory rheology confirmed the formation of a solid like gel material (Figure 1b). Moreover, the formation of entangled nanofibers within formed supramolecular hydrogels with width dimensions in the order of tens of nanometers and microscopic lengths was determined based on a variety of microscopy techniques (Figure S3, Supporting Information, and Figure 1c). For Fmoc-oligopeptides-based supramolecular hydrogels, intermolecular interactions of π–π stacking of Fmoc groups and hydrogen bonding were reported to play important roles in gel formation.[34–43] Hence, we first employed fluorescence spectroscopy to investigate the molecular arrangement of Fmoc groups within our hydrogels. As shown in Figure 2a, an emission peak centered at 312 nm wavelength appeared in the complex of Fmoc-F and Fmoc-L solution phase, while the
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maximum emission for co-assembled (F+L) hydrogel appeared at 316 nm with the presence of a shoulder at 368 nm. This indicated the existence of antiparallel intermolecular π–π interactions of Fmoc groups in the gel phase.[42] A redshift of emission peak was also observed for the Fmoc-F hydrogel system (Figure S4, Supporting Information), which can be attributed to the presence of a fluorenyl excimer.[62,63] No redshift of emission peaks was found in other four Fmoc-amino acids systems (Figure S5, Supporting Information). Meanwhile, we used Fourier transform infrared (FTIR) spectroscopy to study the role of intermolecular hydrogen bonding in the formation of the supramolecular hydrogels. As shown in Figure S6 (Supporting Information) and Figure 2b, compared to the FTIR spectrum in solution phase, the characteristic C=O stretching band peaks in Fmoc-F and co-assemble (F+L) hydrogel phases redshifted from 1658 and 1717 cm−1 to 1640 and 1636 cm−1, respectively,
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indicating the presence of intermolecular hydrogen bonding motifs. Information provided by Raman spectroscopy was consistent with this observation; the peaks of C=O stretching band in hydrogel phases exhibited redshift compared to that in solution phases (Figure 2c and Figure S7, Supporting Information). According to these spectroscopic results, it can be concluded that both the intermolecular π–π stacking of Fmoc groups and hydrogen bonding play important roles in the co-assembled (F+L) hydrogel formation. The effect of aromatic phenyl group of Fmoc-F for hydrogel formation was also evaluated, upon which it was discovered that, compared to Fmoc-amino acids without aromatic phenyl group (Fmoc-G, Fmoc-I, Fmoc-V, Fmoc-A, Fmoc-L), only the Fmoc-F could form supramolecular hydrogel in our experiments (Figure 1a). This indicated that the intermolecular π–π stacking of phenyl groups also plays a critical role in our supramolecular hydrogel formation in addition to the intermolecular π–π stacking interactions of Fmoc groups and hydrogen bonding. This conclusion could also be verified by a hydrogel destruction experiment using urea
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as a blocking reagent of π–π stacking and hydrogen bonds (Figure S8, Supporting Information). These three forces work together to drive the co-assembly of Fmoc-F and Fmoc-L and culminate in hydrogel formation (Figure 2d). To further confirm this result, molecular dynamic simulation was performed to study the aggregation of Fmoc-amino acids in water. As illustrated in Figure 2e and Figure S9, Supporting Information, spontaneous formation of cross-linking 3D network structures of nanofibers was observed for Fmoc-F and co-assembled (F+L) systems, while the aggregation of the other five Fmoc-amino acids without aromatic phenyl group merely yielded the separated pillar structure of nanofibers. These simulation results were consistent with the empirical results described above. To evaluate the antimicrobial activity of the co-assembled (F+L) hydrogel, the hydrogel surface was challenged with pathogenic bacteria and the microbial proliferation was assessed by optical density (OD) measurement. Staphylococcus aureus, arguably the most prevalent pathogen to humans,[64–67] was chosen as the model gram-positive bacteria for evaluation.
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Figure 3. a) Time-dependent antimicrobial effects of the co-assembled (F+L) hydrogel, Fmoc-F (F) hydrogel, and the Fmoc-L (L) solution (0.5 mL, 4 mg mL−1) toward S. aureus bacteria. The original concentration of S. aureus used was 1 × 106 CFU mL−1. b) Concentration-dependent antimicrobial effect of the co-assembled (F+L) hydrogel toward S. aureus bacteria. Different concentrations of S. aureus bacteria were incubated with the hydrogel for 48 h before detecting the OD value. c) The difference between antimicrobial effects of the co-assembled (F+L) hydrogel for gram-positive and gram-negative bacteria. The y-axis indicates the increased folds of OD values of the bacteria solution after it has been incubated with the hydrogel for 20 h. Gram-positive bacteria of Bacillus subtilis and S. aureus, and gram-negative bacteria of Pseudomonas aeruginosa and Escherichia coli were tested. d) MTT activities of endothelial cells contacted with the co-assembled (F+L) hydrogel from 1 to 3 d.
Figure 3a shows the time-dependent antimicrobial test of the co-assembled (F+L) hydrogel. It was found that the presence of the co-assembled (F+L) hydrogel completely suppressed bacterial growth, while bacteria showed significant growth during incubation in sample with Fmoc-F gel (F gel) or without gel (control), indicating that the co-assembled (F+L) hydrogel possessed efficient antimicrobial effect. In addition, the Fmoc-L (L) solution was discovered to inhibit bacterial growth moderately and show better antimicrobial activity than the pure leucine solution (Figure 3a and Figure S10, Supporting Information). For the reason of this phenomenon, we hypothesize that the Fmoc modification may have a potential role in bacteria killing through its hydrophobic interaction with the hydrophobic sections of bacterial cell membrane, as employed by AMPs. The synergistic antimicrobial effect of Fmoc modification and leucine on bacterial growth leads to a relative lower OD value in Fmoc-L solution, compared to the control experiment. The co-assembled (F+L) hydrogel was found to effectively inhibit ≈95% of S. aureus bacteria proliferation after 20 h incubation (Figure S11, Supporting Information). To assess the antimicrobial capacity of the co-assembled (F+L) hydrogel, the hydrogel was subjected to various concentrations of S. aureus and allowed to incubate for 48 h. As shown in Figure 3b, the co-assembled (F+L) hydrogel was capable of inhibiting up to 1 × 108 colony-forming units (CFU) per milliliter of S. aureus 4
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proliferation, but lost its antimicrobial ability when subjected to 1 × 109 CFU mL−1 bacteria. Furthermore, the difference between antimicrobial activity of the co-assembled (F+L) hydrogel for gram-positive and gram-negative bacteria was investigated systematically to demonstrate its selective grampositive bactericidal ability. As we expected, the co-assembled (F+L) hydrogel indeed exhibited preferential antimicrobial effect for gram-positive bacteria (Figure 3c and Figure S12, Supporting Information), while the co-assembled (F+I) hydrogel showed no antimicrobial activity and gram-positive bacteria selectivity (Figure S13, Supporting Information), indicating that Fmoc-L has a critical role in the selective antimicrobial effect of the co-assembled (F+L) hydrogel for gram-positive bacteria. The selective antimicrobial activity of our co-assembled (F+L) hydrogel indicates that it is a good candidate for the clinical treatment of gram-positive bacterial infections, such as the clinical skin and wound infections mainly caused by gram-positive bacteria of S. aureus.[68] Moreover, the in vitro biocompatibility of the co-assembled (F+L) hydrogel was investigated. Endothelial cells were contacted with the co-assembled (F+L) hydrogel for 3 d with the tissue culture polystyrene dish as control, and cell viability was tested by methyl tetrazolium (MTT) assay. As shown in Figure 3d, the MTT absorbance increased with culture time and was statistically similar to that of the control from 1 to 3 d, indicating that the cell proliferated well in the presence
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was found in bacteria before contact with the gel, further confirming the disruption of the bacterial cell wall and membrane after contact with the hydrogel, since redfluorescent propidium iodide is well known to penetrate only bacteria with destroyed cell walls and membranes.[69–71] The timedependent live/dead bacterial staining assay also revealed that the S. aureus bacteria were killed rapidly by the co-assembled (F+L) hydrogel within 30 min (Figure S16, Supporting Information). For our antimicrobial hydrogel system, contact killing and Fmoc-L release are possible mechanisms, but there is a time sequential process of bacteria contact and Fmoc-L release. Fmoc-L release may only occur after the bacterial contact, which is the reason that Fmoc-L release was found in bacterial LB medium (Figure S17, Supporting Information) rather than pure LB medium (Figure S18, Supporting Information). When bacteria contact the surface of hydrogel, Fmoc-L would insert into bacteria and release in which the hydrophobic interactions between hydrogels (Fmoc and phenyl group) and the hydrophobic sections of bacterial cell membrane may play a critical role, and finally kill bacteria. It is noteworthy that Figure 4. a) Representative SEM images and b) overlapping fluorescence images for live/dead the co-assembled (F+L) hydrogel has no posibacterial staining assay of S. aureus before and after contact with the co-assembled (F+L) tive charge in its structure, indicating that hydrogel for 2 h. Two fluorescent dyes were used in live/dead staining in which SYTO 9 with the binding interaction between the hydrogel green color labeled both live and dead bacteria while propidium iodide with red color stained and S. aureus is unlikely to be caused by eleconly dead bacteria. trostatic interactions, as seen in AMPs. It is hypothesized that the interactions between the S. aureus cell wall and the co-assembled (F+L) hydrogels of the co-assembled (F+L) hydrogel. In addition, endothelial are related to the Fmoc-L used, since it has been found that cells contacted with the co-assembled (F+L) hydrogel were leucine-rich peptides can bind to S. aureus cell wall rapidly and found to show healthy spread morphologies (Figure S14, Supeffectively.[57–61] porting Information). These preliminary data suggest that the co-assembled (F+L) hydrogel is in vitro biocompatible toward In conclusion, we report the preparation of biocompatible normal mammalian cells. Furthermore, the co-assembled hydrogel with gram-positive antimicrobial activity based on (F+L) hydrogel was found to remain stable in two nutrientthe co-assembly of commercial Fmoc-amino acids. By taking rich mediums (lysogeny broth (LB) bacteria culture medium advantage of the intermolecular π–π stacking interactions of and Dulbecco’s modified eagle cell culture medium (DMEM)) Fmoc and phenyl group, a novel supramolecular hydrogel platduring 2 d at 37 °C, indicating its stability for long-term antiform derived from Fmoc-F can be easily established. Moreover, bacterial applications (Figure S15, Supporting Information). antimicrobial ability can be conferred on this supramolecular hydrogel platform via a co-assembly mechanism by combining To gain insight into the antimicrobial mechanism of the the Fmoc-F hydrogelator with another commercial Fmoc-L as co-assembled (F+L) hydrogel, the morphological changes of antimicrobial building block. The co-assembled (F+L) supramobacteria after contact with the hydrogel were investigated. As lecular hydrogel exhibited selective gram-positive bactericidal shown in Figure 4a, S. aureus bacteria showed clear edges and activity via a mechanism involving cell wall and membrane dissmooth bodies prior to contact with the hydrogel. In sharp conruption while is biocompatible with normal mammalian cells. trast, cellular deformation and surface collapse were clearly Therefore, it may efficiently eliminate the problem of bacterial found after contact with hydrogel for 2 h. In addition, lysed drug resistance. In addition, it shows great potential as antimibacteria and debris were found, suggesting that the antimicrobial coatings in clinical devices, wound dressings, or topical crobial mechanism of the co-assembled (F+L) hydrogel should agents for the treatment of clinical skin and wound infections be similar to that of AMPs which involved bacterial cell wall mainly caused by gram-positive bacteria of S. aureus. The prepand membrane disruption. This conclusion was further suparation of this antimicrobial hydrogel relies on the co-assembly ported by the live/dead bacterial staining assay. As shown in of commercially available Fmoc-amino acids, which enable us Figure 4b, S. aureus bacteria were stained red after contact to fabricate biocompatible antimicrobial hydrogel easily and with the co-assembled (F+L) hydrogel while little red color
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cost-effectively without involving complicated organic synthesis and purification processes, as compared to the commonly used antimicrobial hydrogels based on cationic materials.
Experimental Section Preparation of Supramolecular Hydrogels: All the Fmoc-amino acids were purchased from Merck and used as received. The gelation test was performed by dissolved 10 mg of Fmoc-amino acid in a vial with an internal diameter of 10 mm containing an equivalent amount of sodium carbonate solution (0.1 M, 130 µL). The volume of solution was adjusted to 500 µL by adding deionized (DI) water followed by slowly heating (2 °C min−1) until all solid disappears. The hot solutions (about 90 °C) were allowed to cool slowly (undisturbed) to achieve room temperature. After 20–30 min, supramolecular hydrogel formation was verified by the vial inversion method. The co-assembled supramolecular hydrogels were prepared in an identical manner but contain 10 mg of Fmoc-F and 2 mg of Fmoc-L or Fmoc-I so as to keep the ratio of Fmocamino acids and sodium carbonate almost constant (molar ratio (F+L) or (F+I):Na2CO3 ≈ 2:1). In addition, the antimicrobial effect of Fmoc-L solution (0.5 mL, 4 mg mL−1) was tested as a control. Spectrum Characterization of Hydrogels: FTIR spectra were collected on an FTIR system (IFS 66/S, Bruker) with the gel pressed between crystal zinc selenium plates and scanned between 1800 and 1550 cm−1 with an interval of 1 cm−1. Fluorescence emission spectra were measured on a fluorescence spectrometer (RF-5301, PC) with excitation at 280 nm and emission data range between 300 and 450 nm. The Raman spectra were obtained with a spectrophotometer (WITec, alpha300R) with an operating wavelength of 532 nm. SEM Study of Hydrogels: Field-emission scanning electron microscopy (FESEM) was used to study the morphology of hydrogels obtained. For such measurement, the hydrogel samples were first air-dried at room temperature and then placed on a copper tape. The copper tape was mounted onto an aluminum stud and vacuum coated with platinum before SEM (JSM-7600F, JEOL) examination. Oscillatory Rheology of Hydrogels: Rheological measurements were conducted on a rheometer (AR-G2, TA Instruments) operating in oscillatory mode. Hydrogel samples were placed on the rheometer stage, and a dynamic frequency sweep was performed from 0.1 to 100 Hz with 0.2% strain at 25 °C. All reported values of storage and loss modulus are the averages of three runs. Bacteria Culture: Bacteria of S. aureus (ATCC 6538), Bacillus subtilis (ATCC 6051), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 9027) were used in our experiments. Prior to the experiments, the bacteria were grown overnight in Luria–Bertani broth medium (LB) (Sigma-Aldrich) and harvested at the exponential growth phase via centrifugation. The supernatant was then discarded and the cell pellet was resuspended in phosphate-buffered saline (PBS). The bacteria concentration could be monitored photometrically by measuring the OD at a wavelength of 600 nm. Before performing the antibacterial experiments with the hydrogels, the OD600 values of bacteria stock solutions were readjusted to 0.1, which corresponded to the concentrations, obtained based on the colony-counting method, of 4 × 108, 2 × 108, 1 × 108, and 2 × 108 CFU mL−1 for S. aureus, B. subtilis, E. coli, and P. aeruginosa, respectively. Time-Dependent and Concentration-Dependent Antibacterial Experiments of Supramolecular Hydrogels: Supramolecular hydrogels for the antibacterial assays were prepared in 24-well tissue culture plates (NUNC). For time-dependent antibacterial assay, 1 mL of bacteria LB solution was introduced onto the surface of the 2% (w/v) supramolecular hydrogel (F, (F+I) or (F+L) gel). The 24-well tissue culture plates containing gels and bacteria cultures were placed on a shaker at 37 °C, and the OD readings of bacteria solutions were monitored every 2 h by measuring the OD600. The bacteria solution without hydrogel or with L solution was used as control. For concentration-dependent antibacterial assay, 1 mL of S. aureus LB solution with different bacteria concentration
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(1 × 104 to 1 × 109 CFU mL–1) was introduced onto the surface of the co-assembled (F+L) supramolecular hydrogel (2% (w/v)) for 48 h, and the OD readings of bacteria solutions were monitored by measuring the OD600. The bacteria solution without hydrogel was used as control. Live/Dead Bacterial Assay: Live/dead bacterial assay was carried out to examine the viability of bacteria before and after supramolecular hydrogel treatment. After incubation with or without hydrogel for different times, S. aureus bacteria solution was mixed with 0.5 mL of dye solution containing 1 µM SYTO 9 and 5 µM propidium iodide (Invitrogen) for 20 min at room temperature. The green-colored SYTO 9 dye enters both intact and membrane-compromised bacterial cells, while the red-colored propidium iodide dye can only enter cells with damaged membranes. The bacteria were then imaged using a confocal microscopy (TCS SP5, Leica). Morphology Study of Bacteria: The morphology of bacteria before or after supramolecular hydrogel treatment was examined by FESEM. Firstly, the S. aureus bacteria solution before or after hydrogel treatment was dropped onto silicon wafer, and the silicon wafer was then treated with 2% glutaraldehyde (Sigma-Aldrich) fixation for 2 h at room temperature and gradient dehydration by a series of ethanol solutions (50%, 70%, 90%, 95%, and 100%) for 10 min with each step. Next, the silicon wafer was subjected to nitrogen drying to maintain the morphologies of the bacteria captured on substrate. After complete drying, the silicon wafer was sputter coated with platinum and imaged with FESEM (JSM-7600F, JEOL).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements I.I. and Y.-Q. Li contributed equally to this work. This work was supported by the Singapore National Research Foundation (CREATE Programme of Nanomaterials for Energy and Water Management and NRF-RF2009-04) and NTU-Northwestern Institute for Nanomedicine. Received: July 24, 2014 Revised: September 22, 2014 Published online:
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Adv. Mater. 2014, DOI: 10.1002/adma.201403339
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