Extremely Stable Supramolecular Hydrogels Assembled from

Jul 11, 2016 - Peptide hydrogels with high stability in different media are of great interest in biomedical applications. In this paper, we report an ...
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Extremely Stable Supramolecular Hydrogels Assembled from Nonionic Peptide Amphiphiles Yaoming Wan, Zuoning Wang, Jing Sun, and Zhibo Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00727 • Publication Date (Web): 11 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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Extremely Stable Supramolecular Hydrogels Assembled from Nonionic Peptide Amphiphiles †





Yaoming Wan, Zuoning Wang, Jing Sun*, and Zhibo Li*,

†,‡



Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. ‡

School of Polymer Science and Engineering, Qingdao University of Science and Technology, Qingdao 266042, China. KEYWORDS: Peptide amphiphile, Hydrogels, Self-assembly ABSTRACT. Peptide hydrogels with high stability in different media are of great interest in biomedical applications. In this paper, we report an easy, fast and scalable method to prepare a family of nonionic peptide amphiphiles (PAs) obtained by direct aminolysis of alkyl-oilgo(γbenzyl-L-glutamate) samples, which were synthesized via alkyl amine initiated sequence ringopening reaction of NCAs. A big advantage of this method is that vast chemical diversity and large-scale yields can be achieved easily using commercially available hydramines. These PA samples can readily form clear hydrogel without any external aid, and show exceptionally enhanced gelation properties with critical gelation concentration (CGC) as low as 0.05wt%. The hydrogels are highly stable against extreme pH of 1 and 14, and high salt concentration of 200 mM NaCl. These properties combined with the shear-thinning properties make these PA hydrogels ideal candidates for new generation of injectable scaffolds.

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INTRODUCTION Hydrogels offer great potential for applications in biomaterials like 3D scaffolds,1, 2, 3, 4 drug delivery carrier5,

6

and surgical dressing.7 In particular, peptide hydrogel is an outstanding

candidate due to its good biocompatibility, biodegradability and versatile biofunctionalization.8, 9, 10

Peptide hydrogel can be easily obtained from natural peptides with specific sequence like

fibrin,11 collagen,12 and de novo designed short peptides13, 14, 15 and peptide amphiphiles (PAs).16, 17, 18

However, natural peptide hydrogels have limitations such as deficient diversity, limited

sources, large batch heterogeneity and inherent presence of water-soluble residuals. In particular, synthetic peptide hydrogels based on PAs offer more advantages in versatility, stability and processing feasibility. Most of the work on synthetic peptide hydrogels involves two components, i.e., hydrophobic segment and hydrophilic moiety. Stupp and coworkers have systematically studied a series of PAs hydrogel with an alkyl tail attached to sequence-specific oligopeptides.19, 20, 21

Whereas, the synthetic approach of PAs is based on solid-phase synthesis, which is time

consuming with low yields. On the other hand, Deming et. al. synthesized charged diblock copolypeptides using controlled ring-opening polymerization (ROP) of α-amino acid Ncarboxyanhydride (NCA). These high molecular weight copolypeptides can easily form hydrogels at low concentration.16 Most previous studies of peptide hydrogels contained charged amino acid residues. However, it is known that the charges, especially positive charges, may cause inevitable side effects such as unfavorable protein absorption in vitro. Hence, it is of critical importance to develop a new and easy method to prepare peptide hydrogels with broad and high tolerance to different media such as pH variation, ions, and charges bioactive molecules. Nonionic PAs offer significant advantages to avoid this issue.22, 23 However, the control over the hydrogel functionality and properties is limited by the diversity of the side-chain 2 ACS Paragon Plus Environment

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functionalities. Herein, we report a convenient approach by post-modification of well-defined alkyl-oligopeptide precursors to prepare nonionic PAs, which offer unique opportunities to expand the versatility of PA hydrogels. A series of PA samples were firstly synthesized by sequence ring-opening of γ-benzyl-Lglutamate-N-carboxyanhydride (BLG-NCA) using alkyl amine as initiator, followed by aminolysis of different hydramines in one-pot reaction.24 The chemistry applied here is robust, versatile and easily scalable. The chemical diversity is therefore vast because there are literally numbers of commercially available hydramines, which can offer distinct functionalities and chemical identities. The hydrogel forming process is entirely spontaneous and the as-prepared hydrogel behaves highly stable to external stimuli such as pH and ions in solution. Moreover, the critical gel concentration (CGC) of these PAs is highly tunable to an extremely low level with respect to reported examples. In addition, the PA hydrogels display shear-thinning and rapid recovery properties. All of these advantages make this system an ideal candidate for next generation of injectable hydrogel materials. EXPERIMENTAL SECTION Materials and methods. Tetrahydrofuran (THF) was deoxygenated and dried by purging with nitrogen and passage through activated alumina columns prior to use. Deionized water (18 MΩcm ) was obtained from a Millipore Milli-Q purification unit. Super dry N,N-dimethylformamide (DMF) was purchased from J&K Scientific Ltd. γ-Benzyl-L-glutamate 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. 1

H NMR spectra were acquired on Bruker AV400 FT-NMR spectrometer. Matrix-assisted laser

desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) measurements were carried out on Bruker BIFLEX III equipped with a 337 nm nitrogen laser. All Fourier transform 3 ACS Paragon Plus Environment

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infrared (FTIR) spectra were performed using a Nicolet Avatar 330 FT-IR spectrometer. The solid samples was milled with potassium bromide (Aldrich) at mass ratio of 1:100 and pressed into disk before FTIR measurements. 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 TEM grids and stained with 1% uranyl acetate. Atomic force microscopy (AFM) was performed in tapping mode (Multimode 8, Bruker, Inc.) with silicon cantilever probes. The scanning rate was usually 1 Hz. AFM samples were prepared by tapping hydrogels on freshly cleaved mica disk (diameter 1.0 cm). Rheology measurements were performed on the AR2000ex rheometer using the 40mm diameter and 1° cone angle cone-plate with 31um gap. Synthesis of PAs. Peptide amphiphiles were synthesized by sequence ring-opening of γ-benzylL-glutamate-N-carboxyanhydride (BLG-NCA) using dodecylamine or 1-hexadecylamine as the initiator in anhydrous DMF at room temperature, followed by aminolysis of benzyl ester group using ethanolamine(AE), 3-amino-1-propanol(AP), 2-(2-aminoethoxy) ethanol (AE2), 1- amino2-propanol (iAP) or 3-amino-1,2-propanediol (AP2) respectively. Taking C12-(G-AE) as the example, we firstly prepared BLG-NCA following reported procedure.25 370mg (2 mmol) dodecylamine dissolved in 3 ml dry THF was mixed with 1.578 g (6 mmol) BLG-NCA in 15 ml dry DMF. The mixture was stirred in N2 atmosphere for 48 hours at room temperature. Then the reaction solution was poured into excess diethyl ether to give white precipitate, and the white

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precipitate was vacuum dried to give solid product C12-PBLG. For the deprotection, 900 mg (1 mmol) C12-PBLG and 1.83 g ethanolamine (30mmol, 10 molar equivalent of benzyl group) was dissolved into dry DMF followed by addition of 1.42 g 2-hydroxypyridine to catalyze the aminolysis. The aminolysis reaction was preceded under a N2 atmosphere at 50 °C for 48 hours. After that, the solution was concentrated using rotary evaporator to remove most DMF. Then the crude product was dissolved in 5 ml MeOH, and subsequently poured into excess diethyl ether. The white precipitates were collected by centrifugation. The crude product was dissolved in DIwater, and the solution was dialyzed (1000Da molecular weight cutoff) against deionized water for 48 hours with water change every 12 hours. White powder (120mg, 80%) was obtained after lyophilization. The hydrogel was prepared by dispersing PA samples in water or other solutions. The obtained solutions were gently heated for a few minutes to ensure complete dissolution. The solutions were then cooled down to room temperature to allow the gelation. The gelation time was different ranging from several minutes to hours for PAs with different chemical structure. RESULTS AND DISCUSSION It is known that ROP of NCA has been demonstrated a powerful method to make well-defined (co)polypeptides in large quantity, albeit lacking specific sequence control.26, 27 In this work, we firstly synthesize a series of alkyl-oligo-γ-benzyl-L-glutamate precursors by sequence ringopening of NCA. The synthetic route of post-modified PAs is shown in Scheme 1. By varying the amine initiator/NCA ratio, we can easily control the peptide length from a few residuals to hundreds. Then, different PA samples were obtained by direct aminolysis of benzyl ester group, which is quite efficient to achieve high-yields in polar solvent and mild reaction condition.28 It is found that the aminolysis is highly efficient as all the benzyl ester bonds can be converted into amide bonds as verified by 1H NMR spectra (Figure. S1). Two hydrophobic alkyl segment with 5 ACS Paragon Plus Environment

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12 and 16 carbon atoms were used here. Five different functional side chains with hydroxyl groups were employed. The molecular structures and average molecular weights are further verified by Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Figure. S2). Apparently, all PA samples are not mono-disperse as expected. It is also revealed by MALDI analysis that the terminal of the molecule is a cyclic ring instead of amine group due to the cyclization reaction between the terminal amine and adjacent benzyl ester group.29 The average number of peptide unit was optimized at 3 for most PAs studied here. The molecular characteristics of all molecules are given along with their abbreviations in Table 1. A big advantage of this method is that we can make tens grams of PA samples with different side chains that can be easily introduced within two days. O

C12/16-(G-AE)3

O

H N

5/7 N

N H

3

H

5/7 N H

O

NH

O

OH

O

OH

OH

O

OH

O O NH

NH2 5/7

O

H2N

H2N

O

C12/16-(G-AE2)3

O

N H

3

O

NH

O

H N

O

+

H2N

H N

ROP 5/7 N

N H

3

H

O

OH

O

H N

5/7 N

N H

3

H

O

O

O O O

Alkyl-PBLG

O

C12/16-(G-AP)3

O

BLG-NCA OH

OH

H2N

H2N

OH OH O

H N

5/7 N H

O

C12/16-(G-iAP)3

NH

O

N H

3

H

O O

O

C12/16-(G-AP2)3 O

O

O

H N

5/7 N

N H

3

NH OH

NH OH OH

Scheme 1. The synthetic routes towards PAs.

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Most nonionic PAs spontaneously form self-supported hydrogels at concentration of 0.5% or below when dispersed in water. A typical photo of clear hydrogel formed from sample C12-(GAE)3 at a concentration of 0.5 wt% is shown in Figure 1. Interestingly, all these PA hydrogels display great stability against pH and ionic strength variation. As shown in Figure 1, C12-(G-AE)3 self-assembles into hydrogels at extreme pH of 1 and 14, and high salt concentration up to 200 mM NaCl. At 0.5 wt%, the sample remains as hydrogels in cell culture medium Dulbecco's Modified Eagle Medium (DMEM) containing diverse salts and biomolecules. For all the other PA samples listed in Table 1, similar gelation behaviors in different media and stability are also observed regardless of the structure of side chains except that the CGC might be slightly different depending on side chain structures (Figure. S4). Apparently, all of the side chains of L-glutamide contain a terminal hydroxyl group, which contributes the hydrophilicity to PA molecules. We attribute such unusual pH and salt stability to the absence of ionic groups in PA samples. Table 1. Molecular parameters and critical gelation concentration (CGC) of different PAs. Sample

Alkyl Lengtha

DPb

CGCc(wt)

C12-(G-AE)3

12

3

0.1%

C12-(G-AE2)3

12

3

0.5%

C12-(G-AP)3

12

3

0.05%

C12-(G-AP2)3

12

3

0.3%

C12-(G-iAP)3

12

3

Precipitated

C16-(G-AE)3

16

3

0.08%

C16-(G-AE2)3

16

3

0.3%

C16-(G-AP)3

16

3

0.05%

C16-(G-AP2)3

16

3

0.1%

C16-(G-iAP)3

16

3

Precipitated

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a)

number of carbon atom on the alkyl chain; b) average degree of polymerization of peptide segment determined by 1H-NMR; c) CGC was tested using inverting tube method at room temperature.

Figure 1. Photos of C12-(G-AE)3 hydrogel (0.5wt%) formed in different aqueous medium, including deionized water, 200mM NaCl, pH=1, pH=14 and DMEM aqueous solution.

C12-PAs

0.5

C16-PAs

p :precipitated

0.4

CGC(wt%)

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0.3 0.2 0.1 p

0.0 AE

AE2

AP

AP2

p

iAP

Figure 2. Critical gelation concentration (CGC) of PAs with different side-chains. The CGC of the hydrogels was determined using inverting tube method, as summarized in Table 1 and shown in Figure 2. The CGC of all PA samples listed in Table 1 is below 0.5wt %.

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Given the same oligopeptide segment, the samples with longer hydrophobic alkyl chains, i.e. C16PAs, generally have lower CGC than that of C12-PAs. When the chain length of alkyl tail is shortened to 6, no hydrogel is formed in all cases. The lowest CGC of 0.05wt% is achieved by the samples containing 3-propanol side chain, including C12-(G-AP)3 and C16-(G-AP)3), is more than 10 folds lower than that of hydrogels from previous work,16 indicating enhanced gelation ability of these nonionic PA hydrogels. C12-(G-AE)3 and C12-(G-AP2)3, which contain less hydrophobic side chains than that of C12-(G-AP)3, show higher CGCs. Similar results are observed for C16-Glu serial samples. The samples containing more hydrophobic 2-propanol side chains, both C12-(G-iAP)3 and C16-(G-iAP)3 precipitate from solution in the experimental window, instead of showing gelation behavior. Apparently, the branched methyl groups increase steric hindrance of the hydroxyl groups, resulting in the decreasing hydrophilicity of the oligopeptide moieties. C12-(G-AE2)3 and C16-(G-AE2)3 show highest CGC due to the presence of more hydrophilic diethylene glycol units. All above results suggest that the hydrophilichydrophobic balance of side chains plays a critical role on the formation of hydrogel and dominates their CGCs. Moreover, the samples with more hydrophobic side-chains show shorter gelation time, consistent with the CGC results. 22, 30 The mechanical properties of the PA hydrogels are investigated by rheology measurement (Figure 3A and Figure S5). As expected, the storage modulus (G’) increases with increasing concentration. Note that the C12-(G-AE2)3 hydrogel shows much less strength than hydrogels of C12-(G-AE)3 and C12-(G-AP)3 given the same concentration. This is possibly due to the increased hydrophilicity arising from diethylene glycol units. The obtained hydrogel is highly stable over 6 months. As shown in Figure 3B, the hydrogel undergo a gel-sol transition under large amplitude strain oscillations and rapid recover to gel status upon removal of shearing, which represents

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typical shear-thinning and rapid recovery properties. These advantageous properties of PA hydrogels enable potential applications as injectable scaffolds.31

A

4

100

B

10

C12-(G-AP)3 C12-(G-AP2)3 2

10

60

eq

C12-(G-AE2)3

G'/G'

C12-(G-AE)3

3

10

(%)

80

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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40 1200s

20

0.5% C12-(G-AE)3 0

0.2

0.4

0.6

0.8

Concentration (wt%)

1.0

0

1000

2000

3000

4000

5000

Time (s)

Figure 3. (A) Storage modulus (G`) as a function of concentration for C12-(G-AE)3, C12-(GAE2)3, C12-(G-AP)3, C12-(G-AP2)3 hydrogels. (B) G`/G`eq as a function of time for C12-(G-AE)3 (0.5wt%) hydrogel, which were sheared at ω = 6 rad/s, γ0 = 1000% for 1200s before switching to small strain (γ0 = 0.4%).

Figure 4. (A) TEM image of multiple 1D nanostructures formed by C12-(G-AE)3 in water at room temperature. Enlarged image of typical twisted nanoribbon (B) and helical nanoribbon (C) of dotted area from panel (A)

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Scheme 2. The possible self-assembly mechanism of PA in water. To systematically study the hydrogel forming mechanism, TEM and AFM are employed to characterize the assembly nanostructures. It is well known that PAs containing hydrophobic alkyl tail and hydrophilic peptide block can self-assemble into 1D nanofiber or nanoribbon, driven by both collapse of hydrophobic alkyl tails and hydrogen bonding between β-sheets of peptide blocks along the long axis.20, 32 For these nonionic PA hydrogels, TEM characterization reveals that sample C12-(G-AE)3 hydrogels assembles into ultra-long 1D fibril structures (Figure. 4). Hydrogels from all the other PAs also exhibit fibril-like structures (Figure. S3). The fibril-like structures include complex morphologies such as cylindrical nanofibers, helical ribbons, twisted ribbons and grooved nanobelts. For C12-(G-AE)3 hydrogels, TEM images show that the twisted ribbons are composed of several nanofibers with a diameter of 4.5 nm arranged side by side (Figure 4). The pitch of twisted ribbons and helical ribbons varies from one hundred to several hundreds of nanometers. Based on these, a possible self-assembly mechanism is proposed in Scheme 2. Firstly, the cylindrical nanofiber forms as a primary assembly unit, driven by hydrophilic-hydrophobic interactions and β-sheet hydrogen bonding along the long axis. Several of proto filaments are then adhered to each other and pack side by side through the hydrogen bonding between the peripheral hydroxyl groups, which result in the formation of nanoribbons or 11 ACS Paragon Plus Environment

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nanobelts. It has been reported that the nanoribbons are inclined to stepwise curve.33 Two ways to curve are reported, mean curvature and Gaussian curvature.21 When the nanoribbons adopt Gaussian curvature, twisted ribbons are formed, as shown in Figure 4B. Helical ribbons are formed when mean curvature is adopted. Interwound helical ribbons are shown in Figure 4C. The AFM results further confirm the structures shown in TEM images. The regular height fluctuations along the long axis of fibrils indicate the helical or twisted structures matching very well with the structures from TEM images (Figure 5A). The phase diagram clearly shows twisted ribbon structure (fibril structure with the red line) and helical ribbon structure (fibril structure with the black line) in Figure 5B. The height profile of Figure 5A clearly shows that the diameter of twisted structure (27 nm) is distinct from that of helical structure (20 nm), which is possibly due to the difference in bundled numbers of the nanofibers (Figure 5D). The mean and Gaussian curvature of nanoribbons seems to be random in the TEM images. But we believe that the twisted ribbon is an intermediate state as in other PA self-asembly reported elsewhere.34 However, both structure have no influence on the gelaiton process and gel properties.

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Figure 5. The height (A), phase (B) and 3D (C) AFM images of C12-(G-AE)3. (D), (E), (F) are corresponding height profiles of blue curve, red curve and black curve from panel (A).

Interestingly, C12-(G-iAP)3 can still self-assemble into 1D fibril-like structure without forming hydrogels. This may be attributed to its side-chain structures. The periphery of nanofibers is full of the hydrophilic peptide sidechains with hydroxyl groups and terminal cyclic rings that hold two adjacent nanofibers together through hydrogen bonding. The transition from nanofibers to nanoribbons or nanobelts is attributed to hydrophobic and hydrophilic balance. In the case of C12(G-iAP)3, the presence of extra hydrophobic methyl groups adjacent to hydroxyl groups make the formed nanofibers unstable in hydrophilic environment. Nanofibers are inclined to aggregate together which eventually results in precipitation from solution. Further, it is evident that the nanofibers formed by PAs with relatively more hydrophobic side-chains, i.e. C12-(G-AP)3 and C12-(G-iAP)3, intend adhere and fuse into ribbons and nanobelts at the edge. The boundary between these nanofibers is barely seen due to the adhesion. C12-(G-iAP)3, which has the most hydrophobic compositions, mostly self-assembles into twisted ribbons and grooved nanobelts, as shown in Figure S3. The surface of the twisted ribbon seems smooth, and the boundary between nanofibers completely disappears. However, the grooves in nearby nanobelts indicate that nanofibers are basic assembly unit. Instead, C12-(G-AP)3, can form hydrogels with lowest CGC due to the presence of large fraction of 1D nanofibril structures. In addition, we also try to determine PA solution`s surface tension to get information about their surface activity and their critical aggregation concentration (CAC). However, the aggregation behavior of these PA samples is quite different from traditional surfactants. The surface tension of sample C12-(G-AE)3 keeps decreasing with concentration increase without showing the turning point. There are two possible reasons for such phenomena. One is the polydisperse nature of these PAs samples. 13 ACS Paragon Plus Environment

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Different samples containing different amino acid residues can display distinct assembly behaviors and surface activity although it is difficult to quantify surface activity of each species. The other is that the strong intermolecular hydrogen bonding tendency to result in β-sheet formation, which promote the solution self-assembly of these samples instead of at interface. It is worth noting that we also prepared monodispersed PA via solution coupling method. These welldefined pure PAs also show similar gelation properties as those polydispersed ones.35 We thus demonstrate that the key factors for hydrogel forming are the formation of 1D nano-fibril structures with large quantity and high stability. In other words, the formation of hydrogels is dominated by the formation of 1D self-assembled structure that is quite dependent on hydrophilic-hydrophobic balance of side chains of PAs. To further understand the mechanism and different gelation ability of the hydrogels, solidFTIR was conducted to determine secondary structure and molecule arrangement of the PA hydrogels (Table S1). Two stretching vibration absorption bands of methylene groups in these PAs lay at 2922 cm-1 and 2851 cm-1, which indicates that the alkyl chains are packed in an extended highly ordered conformation (all-trans zig-zag conformation).36 All PA molecules have absorption band ranging from 1634cm-1 to 1640cm-1 (amide I band), which suggests β–sheet secondary conformation.37 CD spectra results reveal that most PA adopt β–sheet secondary conformation with a maximum at around 197nm and a minimum at around 218nm (Figure S6). C12-(G-AE2)3, and C12-(G-AP2)3 showed a CD pattern like random coil, which may due to their relative more hydrophilic property. Both β–sheet and random coil conformation exist in the two PA solutions. The spectrum of C16-(G-iAP)3 may due to strong light scattering of large aggregates. According to the FTIR and CD results, we therefore propose that the β–sheet secondary structure dominates the formation of 1D nanofiber and hydrogels in our system. While 14 ACS Paragon Plus Environment

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considerable work remains to be done, our work offers a convenient and rapid way to prepare a family of highly designable pH-resistant peptide hydrogels in our system.

CONCLUSION We reported a convenient method to prepare a family of nonionic PA amphiphiles hydrogels via post-modification. The PA amphiphiles were synthesized by sequence ring-opening of γbenzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) using alkyl amine as initiator, followed by aminolysis with a series of hydramines. These PAs self-assemble into 1D fibril nanostructures that further enable the formation of 3D network hydrogels, which are surprisingly stable against pH variation, ions and biomolecules in solution due to the nonionic properties. Furthermore, the hydrogels are injectable due to shear-thinning and rapid recovery properties. The CGC of the PA amphiphiles is highly tunable. The lowest CGC is 0.05wt%, achieved by the sample containing 3-propanol side chains, C-(G-AP)3. These investigations show that this novel and facile method provide a unique platform for injectable peptide hydrogel preparation. We meanwhile present a systematic study of the relationship between chemical structure and property of PA amphiphile hydrogels and demonstrate that the hydrophilic-hydrophobic balance of side chains of PA molecules play a vital role on self-assembling and the hydrogel forming process. ASSOCIATED CONTENT Supporting Information. Details of synthesis, additional TEM images, AFM images, rheology data, MALDI-TOF spectrum, FTIR data and CD spectra. This material is available free of charge via the Internet at http:// pubs.acs.org. AUTHOR INFORMATION

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Corresponding Author * E-mail: [email protected]; [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. 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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