pH-Responsive Peptide Supramolecular Hydrogels with Antibacterial

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pH-Responsive peptide supramolecular hydrogels with antibacterial activity Yaoming Wan, Libing Liu, Shuaishuai Yuan, Jing Sun, and Zhibo Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03986 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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pH-Responsive peptide supramolecular hydrogels with antibacterial activity Yaoming Wan,a,b Libing Liu,b Shuaishuai Yuana, Jing Sun*,a and Zhibo Li*,a,b a

School of Polymer Science and Engineering, Qingdao University of Science and Technology,

Qingdao, 266042, China b

Institute of Chemistry, Chinese Academy of Science, Beijing, 100190, China

KEYWORDS pH responsive, hydrogel, peptide, antibacterial

ABSTRACT Smart hydrogels have received increasing attention for many applications. Here, we synthesized a class of cationic peptide amphiphiles that can self-assemble into hydrogels by ring-opening polymerization (ROP) and post-modification strategy. The incorporation of cationic lysine residues suppress the formation of fibril-like structure and further the gelation ability of the samples. Sodium alginate (SA) is used to enhance the rheology performance of the hydrogels. The hydrogels exhibit pH-dependent self-assembling and the gelation behaviour that enables them ideal smart hydrogel system for biomedical applications. Furthermore, the as-prepared hybrid peptide hydrogel show antibacterial activity.

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Introduction

Hydrogels are a promising class of materials for biomedical applications,1-3 e.g. 3D scaffolds,4 drug delivery carriers5 and wound care.6 A wide variety of compounds have been successfully used to prepare hydrogels in suitable condition. These compounds generally include both natural polymers like alginate,7 chitosan8 and proteins4, and synthetic molecules, such as polyesters,9 polypeptides,10-12 peptide amphiphiles (PAs).13,14 To meet various requirements for application, hybrid hydrogels combining the advantages of both natural and synthetic compounds have been attracting increasing attention.15-17 Particularly, synthetic PA molecule, consisting of a hydrophobic segment and a hydrophilic peptide moiety, receive extensive interests due to its great biocompatibility, biodegradability and versatile biofunctionalization.14 The hydrophilic peptide moieties contain ionic residues, e.g. amines and carboxylic acids that enable the water solubility and meanwhile the pH-responsive property.

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The pH-stimuli can therefore change the

secondary conformation and self-assembling behaviour of the PA molecule, which causes the resultant influence on its hydrogel properties.21-23 The hydrogels exhibiting antibacterial activity is of particular interests for biomedical applications, e.g. wound dressing.24,25 The antibacterial property can be achieved by physical mixing antibacterial agents into bulk hydrogels.26 In this case, however, the antibacterial activity of the hydrogels is generally short-term. Alternatively, chemical modification with antibacterial agents through covalent linkage offers many advantages to achieve long-term antibacterial activity. Antimicrobial peptides are a class of natural antimicrobial agents with broad-spectrum antimicrobial activity. These peptides generally contain rich cationic residues such as lysine or arginine.27,28 Cationic group are known to


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act against bacteria by disrupting the bacterial membrane via a direct-contact mechanism.29,30 However, the natural sources of the antimicrobial peptide are limited, and the extraction and purification are costly and time consuming. Solid-phase peptide synthesis (SPPS) is used to prepare the antimicrobial peptides; however, it is hampered by the high cost.28 We have previously reported extremely stable supramolecular hydrogels assembled from a series of nonionic peptide amphiphiles (PAs).31 In this work, we obtained a class of cationic PA supramolecular hydrogels by ring-opening polymerization (ROP) and post-modification strategy. The designing motif is the incorporation of lysine residues into the PA molecules, which act as a dual-functional unit, i.e., pH-responsive and antibacterial. The obtained PA hydrogels therefore exhibit both pH-responsive behaviour and antibacterial activity. Sodium alginate (SA) was used to strengthen the gel.

Experimental section Materials Tetrahydrofuran (THF) was deoxygenated and dried by purging with nitrogen and passage through activated alumina columns prior to use. Deionized water (18 MΩ-cm) was obtained from a Millipore Milli-Q purification unit. Super dry N, N-dimethylformamide (DMF) was purchased from J&K Scientific Ltd. Benzyl-γ-L-glutamate, Nε-Trifluoroacetyl-L-lysine and triphosgene were purchased from GL Biochem (Shanghai) Ltd. All other chemicals were purchased from commercial suppliers and used as received without further purification unless otherwise stated. BLG-NCA, and Lys(tfa)-NCA were synthesized as reported previously.32,33 Synthesis of peptide amphiphiles (PAs)


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PAs were synthesized by ring-opening polymerization of γ-benzyl-L-glutamate Ncarboxyanhydride (BLG-NCA) and ε-Trifluoroacetyl-L-lysine N-carboxyanhydride (Lys(tfa)NCA) using 1-hexadecylamine as the initiator in anhydrous DMF/THF mixed solution at room temperature, followed by aminolysis of benzyl ester group at each Glu unit using 3-amino-1propanol and deprotection of lys unit. The PA sample was abbreviated as PA-Kn, where n represents the number of lysine unit in the peptide segment. \PA-K0 , without lysine units in the molecule, is prepared as a control sample. Taking PA-K1 as an example, 482mg (2mmol) 1hexadecylamine was dissolved in 3 ml dry THF and 2.104g (8mmol) BLG-NCA and 0.536g (2mmol) Lys(tfa)-NCA in 25 ml dry DMF was added, the mixture was allowed for 48 hours stirring under a N2 atmosphere at room temperature. Then the reaction solution was poured into excess diethyl ether to give white precipitate, which was then vacuum dried to give solid product C16-P(BLG4-Lys(tfa)1). Then, 1.341g (1mmol) C16-P(BLG4-Lys(tfa)1) and 3g (40mmol, 10 mole equivalent of benzyl group) 3-amino-1-propanol was dissolved into dry DMF and 1.42g (5 mole equivalent of benzyl group) 2-hydroxypyridine was added to catalyze the aminolysis. The aminolysis reaction proceeded under a N2 atmosphere at 50°C for 48 hours. After that, most of the solution DMF was removed using rotary evaporator to give a crude concentrated product solution. Then the crude product was dissolved in 5 ml MeOH, and subsequently poured into excess diethyl ether. The white precipitates were collected by centrifugation. Re-dissolve the crude product into MeOH/H2O (20/1) solution and the solution was heated to reflux. 276 mg (2 mmol) K2CO3 was added to the reaction mixture. After 4 h, the reaction mixture was cooled to room temperature and solvents were removed under vacuum. The residue was dissolved into Milli-Q water and dialysis (1000Da molecule weight cutoff) against deionized water for 48 hours, white powder product was obtained after lyophilization.


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Preparation of hydrogel All PA-Kn hydrogel were prepared at an appropriate concentration in Milli-Q water. Firstly, a certain amount of PA-Kn powder was dissolved in water with the help of gentle heat to ensure totally dissolved. Then the clear solution was kept overnight in a sealed bottle at room temperature to form hydrogel. PA-Kn/SA (with low viscosity) hybrid hydrogels were prepared in a similar way.

Antibacterial assays Escherichia coli were used in our experiments. Prior to the experiments, the bacterial on a solid LB/Amp agar plate was transferred to 10 mL of liquid LB/Amp culture medium and grown at 37°C and 180 rpm overnight. Then the bacterials were harvested at the exponential growth phase via centrifugation (8000 rpm for 3 min). The supernatant was discarded and E. coli was resuspended in liquid LB culture medium, and diluted to an optical density of 1 at 600 nm (OD600 = 1.0). According to the test requirement, the bacterial concentration was adjusted to a target value. For antibacterial property test, in diluted PA-Kn solution, a certain amount of PA-Kn solution was added into 2 ml 104 CFU/ml bacterial culture in vial to make the final PA concentration 0.05 wt%. The culture was incubated at 37 °C and 180 rpm for 8h. Then 10µl bacterial solution was pipetted onto carbon coated TEM grids to observe the bacterial morphology after interacting with PAs. Hybrid hydrogels in the antibacterial assays were prepared in separate wells of 48-well tissue cultured-treated polystyrene plate (TCTP). To each well, 0.3 mL of a PA-Kn/SA (1 wt% /


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0.2 wt%) stock solution in sterile filtered H2O (1 wt%) was introduced and was allowed to form hydrogel overnight at 37 °C. Then 0.6 ml LB culture was added on the hydrogels, and the hydrogel was equilibrated overnight at 37 °C. After that the culture was removed and 0.2 ml bacterial solution of different concentration (104, 105, 106, 107 CFU/ml) were introduced to the surface of hydrogel, as well as the TCTP surface, and allowed for incubating for 24h at 37 °C. The next day, 0.2 ml LB culture was added to wash gently and then transferred the bacterial solution for measurement. Bacterial growth was monitored by measuring the OD600 of the solution above the gel. Each assay was performed in triplicate. Antibacterial activity was calculated as non-viable bacterial percentage, where non-viability (%) = [1 - (OD600 hydrogel surface / OD600 TCTP)] * 100.

Characterization 1

H NMR spectra were acquired on Bruker AV400 FT-NMR spectrometer. All Fourier

transform infrared (FTIR) spectra were performed using a Nicolet Avatar 330 FT-IR spectrometer. The samples were dissolved into deuterated water at 0.5 wt% concentration, and then were transferred to a self-made calcium fluoride cell with 50 µm thickness to achieve the FTIR measurement. Circular dichroism (CD) spectra were recorded on a Jasco J-815 CD spectropolarimeter. Deionized water was used as a reference for baseline correction before measurement. The solution was placed into a quartz cell with a path length of 1 mm at a sample concentration of 0.5 mg/mL. The ellipticity (θ in deg cm2/dmol) was calculated as (millidegrees*mean residue weight) / (path length in milli-metres*concentration of peptide in mg/mL). Transmission electron microscopy (TEM) samples were examined with a JEOM 2200FS TEM (200 keV). TEM samples were prepared by pipetting hydrogels on carbon coated


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TEM grids and stained with 1% uranyl acetate. Scanning electron microscopy (SEM) samples were examined with a Hitachi S-4300 field-emission SEM. SEM samples were prepared by pipetting solution on clean silica wafer which was adhered on a double-sided sticky carbon tape mounted on aluminum sample holder and dry in the air, then sputter-coated with platinum. Rheology measurements were performed on the AR2000ex rheometer using the 40mm diameter and 1° cone angle cone-plate with 31um gap.

Results and Discussion A series of PA samples with different lysine contents were synthesized (Scheme 1). These samples are designated as PA-Kn, where n represents the average number of lysine residues per molecule. The polymerization degree was fixed at 5 for all PAs studied and n varied from 0 to 3. The structures and compositions of PA-Kn were confirmed by 1HNMR (Figure S1). Table 1 summarizes the gelation ability of the PA molecules in water. PAK0 without lysine residues displays the best gelation ability with lowest critical gelation concentration (CGC) of 0.1 wt% and can spontaneously form clear hydrogels within a few minutes. In contrast, the other three PA molecules containing lysine units (n = 1~3) show weaker gelation ability at pH=7. PA-K1 and PA-K2 form hydrogel with CGC of 0.5 wt% and 2 wt%, respectively. Their gelation time takes several to tens hours. PA-K3 remains viscous solution with concentrations up to 10 wt% at pH=7. In general, the CGCs of PA-Kn increase with increasing of number of lysine units. We therefore demonstrate that the incorporation of cationic lysine residues suppress the gelation ability of the PA-Kn samples due to the disrupted hydrophilicity and hydrophobicity balance.22


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It was reported that PA-K0 can easily self-assemble into extremely long fibrils that results in formation of networks and hydrogels.31 To determine the correlation between the hydrogel nanostructure and the number of lysine units, the PA-Kn solutions were first examined by TEM. It was observed that the sample PA-Kn (n = 1~3) mainly form fibril structures at pH=7 (Figure S2). Note that the vesicular structure appeared in negatively stained TEM images of PA-K may be artificial, as it is absent in unstained TEM results (Figure S2). In comparison to PA-K0, PA-K1 with one lysine unit is less prone to form long fibrils. Furthermore, the proportion of the fibrillike structure reduces with the number of the lysine units increasing, which explains the remained sol state of the sample PA-K3.

Scheme 1. Synthetic route of PA-Kn. (m and n represent the average number of glutamate and lysine residues, respectively. m + n = 5. Table 1. Chemical parameters and gelation ability of different PAs


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SAMPLE M

N

GELATION PH CGC

GLU UNITS LYS UNITS @1 WT%

@PH=7*

PA-K0

5

0

1-14

0.1%

PA-K1

4

1

1-14

0.5%

PA-K2

3

2

≥8

2%

PA-K3

2

3

≥11

sol

*determined by inverting tube method. In order to determine the hydrogen bonding interactions, we performed FTIR and CD spectroscopy on the samples. FTIR is an efficient tool to track the intra/ inner hydrogen bonding interactions.34 To exclude the impact of H2O on the amide I band absorption in FTIR spectra, D2O is used as the solvent to prepare the PA hydrogel or solution at a concentration of 1 wt%.35 The PA hydrogels or solutions are then transferred into a home-made calcium fluoride cell for the FTIR characterization. The FTIR spectra of the four PA molecules in solution are shown in Figure 1. The amide I band of PA-K0 locates at 1618 cm-1 and 1629 cm-1, indicating dominant β-sheet conformation.36 PA-K1 shows similar absorption peak at 1628 cm-1 but with a broad shoulder at higher wavenumbers. In the case of PA-K2, an obvious absorbance at 1649 cm-1 is shown in addition to the strong absorbance at 1629 cm-1, indicative of the coexistence of β-sheet and non-hydrogen bonded carbonyls.35 Sample PA-K3 shows a single amide I band at 1649 cm1

, suggesting that the secondary conformation is dominated by non-hydrogen bonded

carbonyls.12 It is evident that the fraction of β-sheet conformation in PA-Kn samples decreases with increasing number of lysine units. This is possibly due to the increased repulsive interaction of charged groups that decrease the intermolecular hydrogen bonding interaction. It has been reported that the formation of extended fibril structures is dependent on the stability and fraction of β-sheet structure, which results in the hydrogel formation.37 Hence, we


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demonstrate that the β-sheet structure of PA-Kn dominates the formation of the fibril structure and further the gelation ability, confirmed by the TEM results. CD spectrum is further used to determine the conformation of PA-Kn. Apart from PA-K3, all the other three PAs show a positive peak at ~196 nm and a negative peak at ~220 nm in neutral and alkaline solution (Figure S3 and Figure 2D), indicative of a typical β-sheet conformation, consistent with FTIR results. In the case of PA-K0 and PA-K1, β-sheet conformation persists over the entire pH measurement window. We will address this shortly.

-1

Absorption (%)

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1650cm

-1

1629cm

PA-K3 PA-K2 PA-K1 -1

1618cm

PA-K0 1800

1700

1600

1500

1400

1300

-1

Wavenumbers(cm ) Figure 1. FTIR spectra of PA-Kn samples in D2O at a concentration of 1%. The PA molecule is expected to show pH-dependent self-assembly and gelation behaviour due to the presence of charged lysine units. The pH dependence on the gelation behaviour is summarized in Table 1. The sample PA-K1 can form gels over the entire pH window possibly due to the low amounts of lysine units. In the case of PA-K2 with two lysine units at a concentration of 1 wt%, Newtonian solution is formed at pH 1 without forming fibril-like structures by TEM (Figure 2A). As pH is increased to 7, the sample solution becomes sticky, and long fibril-like structures are observed in the TEM (Figure 2B). Slight increase


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of the solution pH to 8 results in the formation of the hydrogel, showing a highly pHdependent gelation behavior. As pH is further increased to 14, the hydrogel remains with a dense fibril network shown in the TEM images (Figure 2C). CD spectroscopy was used to determine the secondary structure of PA-K2 at different pHs. The related CD spectra are shown in Figure 2D. PA-K2 mainly adopts β-sheet structure in neutral and alkaline media, and no specific conformation is shown in acidic solution. This result confirms that the β-sheet content dominates the formation of long fibril structures and further the gelation behaviour. In acidic solution (pH=1), the amine groups of PA-K2 are fully protonated (pKa = 9.2 in lysine). The charged groups increase the hydrophilicity of the system and suppress the formation of βsheet conformation due to electrostatic repulsion. In the case of alkaline condition, the amine groups are deprotonated and hydrophobic, which facilitates the intermolecular hydrogen bonding interactions and formation of β-sheet conformation. In neutral solution, the amine groups are partially protonated based on the CD results. Note that the fraction of protonated lysine units is not clear as its pKa may decrease when lysine units are conjugated onto a long alkyl tail. Further, the self-assembling process may also change the pKa.38 The deprotonated PA-K2 lead to the formation of β-sheet conformation that results in fibril-like structures. The sample PA-K3 remains sol until the pH reaches 11 at a concentration of 1 wt%, showing week gelation ability due to the high amount of charged units.


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Figure 2. pH-dependent self-assembling behavior of PA-K2. TEM images of assemblies formed by PA-K2 at pH=1(A), pH=7(B), pH=14(C). (D) Circular dichroism spectra of PA-K2 at related pHs. The inset photos are related PA-K2 (1 wt%) sol or gel. 5

10

PA-Kn 4

PA-Kn/SA

10

3

10

G'(Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2

10

1

10

0

10

-1

10

PA-K0

PA-K1

PA-K2


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Figure 3. Rheology properties of PA-Kn hydrogels before and after mixed with sodium alginate at a concentration of 1 wt%. We further investigated the mechanical properties of the hydrogels by rheology measurement. The physical cross-linked cationic PA hydrogel is generally weak, shown in Figure S4. In order to enhance its strength, we mixed negatively charged sodium alginate (SA) with PA-Kn (1 wt%) to prepare a hybrid hydrogel. It is observed that the addition of SA can significantly increase gel modulus at the same concentration of PA-Kn, particularly in the case of n = 1 and 2 (Figure 3). The gel strength of PA-K0, in the absence of amine groups, increases slightly upon addition of SA, possibly due to the entanglement between PA-Kn and SA. Therefore, the possible reinforcing mechanism can be considered as an “electrostatic interaction” mechanism, presented in Scheme 2. The SA molecules are negatively charged and thus interact with positively charged lysine residues in PA-Kn molecules. The binding site acts as physical cross-links between SA and PA-Kn nanofibers through electrostatic interaction and therefore increases the mechanical property of hybrid hydrogel.

Scheme 2. Schematic illustration of hybrid hydrogels (n =1).


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Figure 4. TEM pictures of bacterial morphology incubated at 37 °C for 8h at a concentration of 0.05wt% (B) PA-K0, (C) PA-K1 and (D) PA-K2. (A) pictures of bacterial culture solutions of PAKn. The presence of cationic groups further makes the PA-Kn hydrogels antibacterial materials. We therefore used Gram-negative E. coli to evaluate the preliminary antibacterial activity of the materials. Figure 4A shows a photo of the bacterial culture solution after incubated with different PA-Kn solutions (n = 0, 1, 2). In comparison with the control PA-K0 solution, the PA-K1 and PA-K2 solution incubated with the E. coli culture show distinct aggregation behaviour, indicating that lysine-containing PA-Kn solutions exhibit higher antibacterial activity. TEM was used to determine the morphology of bacterial cell after incubated in culture with PA-Kn (Figure 4). The bacterial cells incubated with PA-K1 and PA-K2 are coated and entangled by PA-Kn nanofibers. The bacterial cells are fractured and the cytoderm is fused (Figure 4C and 4D). It is therefore reasonable to believe that the antibacterial mechanism is the cell wall lysis. While incubated with the control sample PA-K0, the bacterial grow very


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well with integrated cell structure (Figure 4B). As expected, the presence of lysine residues indeed increases the antibacterial activity of the PA molecules. The bacterial viability assay was then carried out on PA-K1/SA hybrid hydrogel with different concentrations of E.coli bacteria for 24h. The concentration of PA-K1 and SA are 1wt% and 0.2wt %, respectively. In spite of the nearly equal ratio of amine groups and carboxyl groups, some of the amine groups are free without interacting with carboxyl groups due to the spatial confinement. The free amine groups can provide the effective antibacterial activity. The bacterial proliferation was assessed by optical density measurement. The non-viability of bacterial incubated on PA-K1/SA hydrogel is maximized over 70% (Figure 5) with different concentrations of E.coli bacteria. In contrast, the bacteria incubated with both PA-K0/SA hydrogel and a control well of tissue cultured-treated polystyrene plate (TCTP without gel) proliferate well. SEM images show the bacterial morphology after incubated with different hydrogels (Figure 6). Lesions and holes are observed in E. coli incubated with the PA-K1/SA hybrid hydrogel. In the case of the control (TCTP and PA-K0/SA hydrogel) substrates, the bacteria remain integrated cell structure. Although considerable work remains to be done, we demonstrate that the hydrogels prepared by PA molecules containing the lysine residues possess antibacterial activity. 100

80

Non-viablity (%)

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60

40

20

0 4

10

5

10

6

10

10

7

Concentration (CFU/ml)


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Figure 5. Antibacterial activity of 1 wt % PA-K1/SA hydrogel surfaces with different concentration of E.coil bacteria for 24h.

Figure 6. SEM pictures of bacterial morphology incubated on different surfaces at 37 °C for 24h. (A) TCTP, (B) PA-K0/SA hydrogel, (C) and (D) PA-K1/SA hydrogel. Conclusion In conclusion, a series of lysine-containing PA-Kn molecules were synthesized via NCA-ROP and subsequent modification. The PAs display pH-dependent self-assembling and gelation behaviour. Increasing the amounts of lysine residues result in the decreased self-assembling ability into nanofibers, which further reduces the gelation ability of the hydrogels. SA is used to enhance the rheology performance of PA-Kn hydrogels via the electrostatic interaction between negatively charged carboxyl groups of SA and positively charged lysine residues of PA. The as-prepared hybrid peptide hydrogel show antibacterial activity which may find application for biomedicine.

ASSOCIATED CONTENT


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Supporting Information. Details of synthesis, additional TEM images, CD Spectra, rheology data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Prof. Zhibo Li, email: [email protected] Prof. Jing Sun, email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources National Natural Science Foundation of China (21434008 and 51225306). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21434008 and 51225306).

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Langmuir

TOC

pH-Responsive peptide supramolecular hydrogels with antibacterial activity Yaoming Wan,a,b Libing Liu,b Shuaishuai Yuana, Jing Sun*,a and Zhibo Li*,a,b a

School of Polymer Science and Engineering, Qingdao University of Science and Technology,

Qingdao, 266042, China b

Institute of Chemistry, Chinese Academy of Science, Beijing, 100190, China


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pH-Responsive Peptide Supramolecular Hydrogels with Antibacterial

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