Self-Assembly and Hydrogelation of Coil–Sheet Poly (l-lysine)-block

Oct 3, 2018 - Hierarchical Green-Energy Materials (Hi-GEM) Research Center, ... This study reports the self-assembly and hydrogelation of coil–sheet...
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Self-Assembly and Hydrogelation of Coil−Sheet Poly(L‑lysine)-blockpoly(L‑threonine) Block Copolypeptides Sheng-Shu Hou,†,‡ Nai-Shin Fan,† Yu-Chao Tseng,† and Jeng-Shiung Jan*,†,‡ †

Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan Hierarchical Green-Energy Materials (Hi-GEM) Research Center, National Cheng Kung University, Tainan 70101, Taiwan

Macromolecules Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 10/03/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: This study reports the self-assembly and hydrogelation of coil−sheet poly(L-lysine)-block-poly(L-threonine) (PLLb-PLT) block copolypeptides. Our experiments showed that the PLL-b-PLT block copolypeptides with degrees of polymerization (DPs) between 30 and 60 can self-assemble to form fibrils due to the packing of sheetlike PLT at low polymer concentrations; moreover, further increasing the polymer concentration can result in the formation of transparent hydrogels due to fibril entanglements. The hydrogelation was determined by the orientation and packing of the fibril assemblies, which aremainly dictated by the balance between the intermolecular hydrogen bonding interactions and charge repulsion exerted respectively by sheetlike PLT and positively charged, coil PLL segments. The noncovalent interactions can be manipulated by varying the polypeptide chain length and composition, which would result in these block copolypeptides exhibiting different critical gelation concentrations (CGCs), gel molecular assemblies, and mechanical properties.

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INTRODUCTION Hydrogels exhibiting three-dimensional, water-soluble macromolecular networks have been extensively evaluated as biomaterials for wound dressing, drug delivery, implantable device, and tissue engineering.1−6 Many biomacromolecules including collagen, elastin, silk fibroin, and hyaluronic acid have been utilized to prepare hydrogels for a variety of applications.5,7−10 They are essentially natural proteins, polypeptides, or polysaccharides, which exhibit innate biocompatibility and biodegradability. However, these natural hydrogels exhibit limited structural and functional modification and batch-to-batch variation. Synthetic polymeric hydrogels are advantageous over natural ones due to the ease of tailoring their physical and chemical properties via macromolecular chemistry.11−13 While a variety of synthetic polymers can form hydrogels, it is desirable to prepare hydrogels with biocompatible or biodegradable building blocks such as amino acids or saccharides. One of the important classes of synthetic gel materials is the polypeptide-based hydrogel composed of biocompatible/ biodegradable building blocks, which are amino acids. They possess essential structures and functions of natural proteins, endowing them with biofunctionality and self-assembly versatility. The gelation of polypeptide-based copolymers can be driven by versatile noncovalent interactions including hydrophobic interactions, hydrogen-bonding interactions, ionic attraction, and host−guest complexion.14−25 Moreover, the covalently bonded networks can also facilitate the gelation to form chemically cross-linked hydrogels.21,26−28 For physically cross-linked hydrogels, the polypeptide segments adopting ordered conformations are typically selected so that © XXXX American Chemical Society

the introduction of the hydrogen-bonding interactions trigger the self-assembly and subsequently the gelation of polypeptidebased copolymers. Moreover, the gel properties can be carefully tuned by manipulating the noncovalent interactions exerted by the polypeptides. Although many studies have reported the preparation of physically cross-linked hydrogels based on polypeptides tethered with PEG or Pluronic, there are only a few examples on the gelation of block copolypeptides or amphiphilic polypeptide derivatives.14,15,21,29 The notable example is the gelation of coil− helix poly(L-lysine)-block-poly(L-leucine) block copolypeptides driven by the formation and entanglement of fibril assemblies via the balance between the electrostatic repulsion and hydrophobic interactions exerted by the coil poly(L-lysine) and helical poly(L-leucine) segments, respectively.15,29 Our group is recently interested in preparing polypeptidebased hydrogels and nanogels via a variety of strategies. It is a feasible strategy by using sheetlike polypeptide segments to trigger polymer self-assembly into hydrogel matrix via controlling the balance between different noncovalent interactions. On the basis of this concept, we hypothesize that sheetlike poly(L-threonine) (PLT) is a promising polypeptide candidate to trigger self-assembly and subsequent hydrogelation. To explore whether PLT can be utilized to trigger hydrogel formation, we designed and synthesized a series of poly(L-lysine)-block-poly(L-threonine) (PLL-b-PLT) block copolypeptides with varying polypeptide chain lengths Received: June 13, 2018 Revised: September 12, 2018

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DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

water, and the solution pH was adjusted to pH 11−12 using concentrated NaOH solution. The solution was dialyzed against DI water a dialysis tubing cellulose membrane (MWCO 1000 or 3500 g/ mol), and the water was exchanged 2−3 times per day over 3 days. The solution was then lyophilized, and the obtained product was dissolved in DI water. After adjusting the solution pH to pH 2−4 using concentrated HCl solution, the solution was then dialyzed against DI water using a dialysis tubing cellulose membrane (MWCO 1000 or 3500 g/mol) for 3 days and lyophilized to obtain a white spongy material (yield 90−95%). Preparation of Hydrogel and Determination of Gelation Concentration. The hydrogel samples were prepared by dissolving freeze-dried PLL-b-PLT block coolypeptides in DI water at room temperature. To facilitate the dissolution process, the samples were sonicated and vortexed to obtain clear solutions and then equilibrated at room temperature overnight. The gel formation was determined via the vial inverting method.22,34 The 2 mL vials containing hydrogel samples at a given concentration were prepared. After inverting the vial, the gelation concentration was determined at which the hydrogel samples did not flow within 1 min. Instrumentation and Characterization. PZLL-b-PBnT block copolypeptides were analyzed at 35 °C by a Shimadzu gel permeation chromatograph (GPC) equipped with two Shodex HFIP 806 M columns. The eluent was redistilled 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Matrix) with 0.8 mL/min of flow rate, and poly(methyl methacrylate) (molecular weight: 1000−100000 g/mol) was used as the calibration standard. The samples were dissolved in HFIP and filtered through a 0.2 μm PTFE filter (13 mm, Finetech) prior to GPC analysis. The MALDI-TOF spectra of PZLL-b-PBnT block copolypeptides were recorded in reflectron mode on a Bruker MALDI-TOF spectrometer (Autoflex III TOF/TOF). The matrix used in this study was THF (1 mL) with 10 mg of DCTB and 1 wt % NaCl. The samples were prepared by mixing the matrix with the polypeptide dissolved in 100 μL of THF at 1:10 of the volume ratio. 1 H NMR spectra of PZLL-b-PBnT in TFA-d1 and PLL-b-PLT in D2O were recorded on a Mercury 300 Varian spectrometer (600 MHz). Dynamic rheological measurements were measured using a Rheometrics ARES controlled strain rheometer with a cone−plate geometry of 25 mm diameter and 0.1 rad cone angle. The storage modulus (G′) and loss modulus (G″) of hydrogel samples were measured by varying the strain amplitude or angular frequency as the temperature increased from 25 to 60 °C by a heating rate of 1.0 °C/ min. The measurements of hydrogel recovery were performed by the following steps: nonlinear, large-amplitude oscillations at a constant strain of 2% and angular frequency of 6 rad/s for 220 s to break down the gel structure, followed by monitoring hydrogel recovery of mechanical strength at a constant strain of 0.05% and 6 rad/s. Transmission electron microscopy (TEM) images of the polypeptide assemblies were taken with a JEM-1400 microscope (JEOL). The sample solutions were dispensed on a carbon-coated copper grid, negatively stained with uranyl acetate for 30 s, and dried overnight in air. Cryoelectron microscopy (cryoEM) images of hydrogel samples were taken with a JEM-1400 microscope (JEOL). A 5.0 wt % PLL-bPLT hydrogel sample (30 μL) in DI water was dispensed on a lacey carbon grid, and then the EM grid was plunged into liquid nitrogencooled ethane for cryoEM. Field-emission scanning electron microscopy (FE-SEM) images of the lyophilized hydrogel samples were taken with a Hitachi SU8010 microscope. X-ray diffraction (XRD) patterns of hydrogel samples were recorded on a Rigaku Ultima IV-9407F701 X-ray spectrometer using Cu Kα (0.154 nm) radiation (50 kV, 250 mA) and scanned from 2θ = 5° to 40° at a speed of 10°/min. Circular dichroism (CD) spectra of polypeptide solutions in a 0.1 mm quartz cell were recorded on a JASCO J-815 spectrometer (JASCO, Japan). The temperature was controlled between 20 and 60 °C using a PFD-425S/15 Peltier-type temperature controller. The solution was allowed to equilibrate for 10 min at each temperature prior to data collection. Fourier transform infrared (FTIR) spectra of lyophilized hydrogel samples were recorded on a Thermo Nicolet Nexus 670 FTIR. The lyophilized hydrogel samples were pelletized with KBr (Sigma-Aldrich) for FTIR analysis. SAXS

(degrees of polymerization (DPs) between 30 and 60) and block ratios by ring-opening polymerization (ROP) of Ncarboxyanhydrides (NCAs) and investigated their selfassembly and hydrogelation. It is known that PLT is relatively hydrophilic as compared to the sheetlike polypeptides such as ploy(L-alanine), poly(L-phenylalanine), and poly(L-tyrosine).16,21,30 In addition to the formation of stable β-sheet conformation through hydrogen bonds between amide groups, the threonine hydroxyl group might form hydrogen bonds not only with water molecules but also with carbonyl oxygens on the polypeptide main chains. We demonstrate that polypeptide chain conformation, gel molecular assembly, and gel property are dictated by the balance between different noncovalent interactions exerted by PLL and PLT segments, which can be tuned by varying polypeptide chain length and block ratio. Unlike the gelation of poly(L-lysine)-block-poly(L-leucine) block copolypeptides requiring high molecular weight, it is found that the PLL-b-PLT block copolypeptides with relatively low molecular weight can self-assemble to form hydrogels. Moreover, chemical cross-linking on the PLL block can be applied on these hydrogels to meet the requirement for downstream applications. This approach can enhance not only their mechanical properties but also their stability against dilution in aqueous solution.

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EXPERIMENTAL SECTION

Diblock Polypeptides Synthesis. The polypeptide was synthesized by using hexylamine (Sigma-Aldrich) as the initiator to sequentially polymerize Z-L-lysine (ZLL) and O-benzyl-L-threonine (BnT) NCAs with designated feed molar ratios between the initiator and NCAs. ZLL and BnT NCAs were synthesized through reacting triphosgene (98%, Sigma-Aldrich) with Nε-Z-L-lysine (∼99%, SigmaAldrich) and N-Boc-(O-benzyl)-L-threonine (>99%, Bachem), respectively.31−33 The solvents (ACS Reagent) used for polypeptide synthesis, hexane and dichloromethane, were dried using Na metal (99.95%, Sigma-Aldrich) and calcium hydride (95%, Sigma-Aldrich), respectively. The detailed synthesis procedures of a specific block copolypeptide are given below. Synthesis of Poly(Z-L-lysine)30-block-poly(O-benzyl-L-threonine)12 (PZLL30-b-PBnT12). In a glovebox, hexylamine (0.011 g, 0.11 mmol) and ZLL NCA (1 g, 3.27 mmol) were weighted out and dissolved in 16.5 and 13 mL of anhydrous dichloromethane, respectively. The two solutions were mixed well in a 100 mL flask and taken out from the glovebox. The resultant solution was stirred at 30 °C for 2 days, and a 100 μL sample was drawn for analysis. Then, BnT NCA (0.35 g, 1.61 mmol) dissolved in 6.15 mL of anhydrous dichloromethane was transferred to the flask through a cannula under a N2 atomsphere. After stirring at 30 °C for a further 4 days, the polypeptide was precipitated out of the solution by adding 200 mL of anhydrous diethyl ether (95%, Sigma-Aldrich) to the solution. The product was collected via filtration, washed with anhydrous diethyl ether, and then dried under vacuum to yield a white solid (yield 83− 87%). Synthesis of Poly( L -lysine) 30 -block-poly( L -threonine) 12 (PLL30-b-PLT12). PLL30-b-PLT12block copolypeptide was obtained by removing the Z and benzyl protecting groups on PZLL30-b-PBnT12 using HBr (33 wt % in acetic acid, Sigma-Aldrich) and iodotrimethylsilane (TMSiI, Sigma-Aldrich).32,33 In a 100 mL flask, excess HBr with 5-fold of the moles of Z group was added to a solution containing 0.3 g of PZLL30-b-PBnT12 polypeptide dissolved in 30 mL of trifluoroacetic acid (Sigma-Aldrich) via syringe. After stirring for 30 min, TMSiI with the volume based on 150 μL of TMSiI per 1 mmol of benzyl group was added to the flask via syringe, and the resultant solution was stirred for an additional 120 min. The polypeptide was precipitated by adding 100 mL of anhydrous diethyl ether into the solution, collected via centrifugation, and dried under vacuum. The crude product was dissolved in 40 mL of deionized (DI) B

DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules profiles of hydrogel samples in a 1 mm quartz capillary were recorded at 25 °C on a Bruker diffractometer (NanoSTAR U System, Bruker AXS GmbH, Germany), calibrated with a standard sample of silver behenate. The background subtracted data were desmeared against the beam length profile of the source. The scattering wavevector q = 4πλ−1 sin θ was defined by the scattering angle θ and wavelength λ.

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RESULTS AND DISCUSSION Polypeptide Synthesis and Characterization. The PZLL-b-PBnT diblock copolypeptides with varying chain lengths and block ratios were synthesized by sequential ROP of ZLL and BnT NCAs using hexylamine as the initiator (Scheme 1). Based on GPC and 1H NMR analyses, the Scheme 1. Preparation of the PLL-b-PLT Block Copolypeptides and the Formation of Polypeptide Hydrogels Due to Polypeptide Self-Assembly

Figure 1. 1H NMR spectra of (a) PZLL11 in TFA-d1, (b) PZLL11-bPBnT17 in TFA-d1, and (c) PLL11-b-PLT17 in D2O.

(Table S2). PLL-b-PLT diblock copolypeptides were then obtained by removing the Z and benzyl groups using HBr and Me3SiI, respectively. Upon deprotection, 1H NMR analysis of the as-synthesized polypeptides reveals that the percentages of the residual Z and benzyl groups are well below 5% (for example, Figure 1c). Hydrogelation, Gel Morphology, and Mechanical Properties. PLL-b-PLT diblock copolypeptides with targeted DPs (30−60) and block ratios of PLL to PLT (2:1, 1:1, and 1:2) can spontaneously form transparent hydrogel upon dispersing in DI water at room temperature, and their critical gelation concentrations (CGCs), which were determined by using the inverting tube method, are ranged between 3.0 and 7.0 wt % (Scheme 1 and Table 1). It is worth noting that the solution pH values are ranged between 6.5 and 7.0, which is much lower than the pKa value of the amino group.22,35 Hence, it would not significantly affect the charge state of the amino group. To understand the hydrogelation of these relatively lowmolecular-weight polypeptides, we first compare the CGCs of

number-averaged molecular weight (Mn), molecular weight distribution (Mw/Mn), and composition ratio were determined and are summarized in Table S1. 1H NMR analysis confirms the successful synthesis of the diblock copolypeptides, evidenced by the presence of the chemical shifts for all the protons on the polypeptides and initiator (Figure 1). The degree of polymerization (DP) for the PZLL block was determined based on the integrals of the benzyl group (−NHCOOCH2C6H5) on the PZLL block and the methyl group (CH3CH2−) on the initiator (for example, Figure 1a). It is worth to note that 1H NMR analysis was performed by drawing a small portion of the reaction solution when the PLL block was prepared. With the known DP of the PZLL block, the block ratio of PZLL-b-PBnT can then be determined based on the intergrals of the benzyl groups (−COOCH2C6H5 and −CHOCH2C6H5) on the both blocks and the methyl group (CH3CH2−) on the initiator (for example, Figure 1b). The calculated DPs and block ratio are found to be reasonably comparable with the feed molar ratio of the initiator and NCAs (Table S1). The molecular weights of PZLL-b-PBnT diblock copolypeptides determined from GPC analysis are in good agreement with those calculated based on the obtained DPs (Table S1). MALDI-TOF analysis was also performed on the selective polypeptides to determine the molecular weights of PBnT blocks. The calculated DPs based on MALDI-TOF are reasonably comparable with those based on 1H NMR analysis

Table 1. Critical Gelation Concentration (CGC), Degree of Polymerization (DP), Block Ratio, and Percentages of Different Secondary Conformations Adopted by PLL-b-PLT Block Copolypeptides Computed by Using the Software CD-fit 4

C

polypeptide

CGC (wt %)

DP

block ratio

secondary conformation coil/ α-helix/β-sheet (%)

PLL20 PLT20 PLL11-b-PLT17 PLL20-b-PLT8 PLL22-b-PLT16 PLL20-b-PLT41 PLL30-b-PLT12 PLL30-b-PLT30 PLL41-b-PLT21

----7.0 5.0 4.5 3.5 4.0 3.0 7.0

20 20 28 28 38 61 42 60 62

----11:17 5:2 5:4 1:2 5:2 1:1 2:1

88.6/0/11.4 19.8/0/80.2 48.1/1.6/50.3 53.3/1.4/45.3 53.4/0/46.6 41.7/3.5/54.8 64.9/2.5/32.6 48.2/5.2/46.6 62.7/10.4/26.9 DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX

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Figure 2. SEM images of freeze-dried (a) PLL20-b-PLT8, (b) PLL22-b-PLT16, (c) PLL30-b-PLT12, and (d) PLL30-b-PLT30 hydrogel samples prepared at 5.0 wt % of polypeptide concentration.

Figure 3. SEM images of freeze-dried (a) PLL20-b-PLT8, (b) PLL22-b-PLT16, (c) PLL30-b-PLT12, and (d) PLL30-b-PLT30 sol samples prepared at 1.0 wt % of polypeptide concentration.

PLT chain length. The same trend is also seen for PLL30-bPLT12 and PLL30-b-PLT30. Based on the above observations, PLL30-b-PLT30 showed the lowest CGC value among all the studied samples. These results reveal that only the PLL-b-PLT diblock copolypeptides with optimal polypeptide chain length and block ratio would exhibit good gelation ability. SEM analysis was performed to characterize the morphology of the gel matrix exhibited in the freeze-dried hydrogel samples. Previous studies have demonstrated that the native morphology of hydrogels can be preserved by the freeze-drying procedure.25,36,37 The SEM images of the freeze-dried polypeptide hydrogels (5.0 wt % of polypeptide concentration) indicate the gel matrix exhibited two-dimensional, interconnected membranes (Figure 2). For comparison, the freeze-

diblock copolypeptides with comparable PLT chain length (PLL11-b-PLT17, PLL22-b-PLT16, and PLL41-b-PLT21), which are 7.0, 4.5, and 7.0 wt %, respectively. It should be noted that PLT20 cannot form hydrogel at the polypeptide concentration below 10 wt %. PLL22-b-PLT16 exhibits the lowest CGC among them. For the ones with comparable block ratio of PLL to PLT (PLL20-b-PLT8, PLL30-b-PLT12, and PLL41-b-PLT21), PLL30-b-PLT12 exhibits the lowest CGC among them. Therefore, tethering with PLL blocks on PLT and an appropriate block ratio of PLL to PLT are crucial for promoting hydrogelation. Then we compare the samples with fixed chain length of PLL (for example, PLL20-b-PLT8, PLL22-b-PLT16, and PLL20-b-PLT41), and the CGC of the block copolypeptide is found to decrease with increasing the D

DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX

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molecular weight) with a given PLT to PLL block ratio. Compared to PLL20-b-PLT8 (or PLL30-b-PLT12), the gel structure of PLL22-b-PLT16 (or PLL30-b-PLT30) hydrogel is disrupted at a smaller strain (Figure 4b), and the gel structure of PLL30-b-PLT30 hydrogel is disrupted at a smaller strain than that of PLL22-b-PLT16 one. The increment of PLT to PLL block ratio and/or chain length with a given PLT to PLL block ratio results in the increase of the stiffness of the gel network, which consequently required smaller strain to disrupt the gel network. The gel recovery experiments were conducted on PLL20-b-PLT8 and PLL22-b-PLT16 hydrogels to investigate their ability to recover after large-amplitude strain oscillations. Figure S2 shows that both samples recover ∼91% of its original strength upon removal of shearing and then progressively regain their strength toward 100% recovery. The results demonstrate that these hydrogels are capable to recover rapidly from sol to gel upon stopping shearing. The influence of temperature on the mechanical strengths of PLL30-b-PLT12 and PLL30-b-PLT30 was also investigated (Figure S3a,b). The mechanical strengths of PLL30-b-PLT12 and PLL30-b-PLT30 hydrogels are found to slightly increase as the temperature increases. The storage modulus G′ gradually increases with the temperature increase from 30 to 60 °C. On the other hand, the loss modulus G″ does not exhibit apparent increase between 30 and 50 °C except at 60 °C. CD analysis reveals that the percentages of β-sheet conformation adopted by PLL30-bPLT12 and PLL30-b-PLT30 slightly increase with the increase of temperature from 30 to 60 °C (Figure S3c,d and Table S3). Polypeptides Self-Assembly and Gel Network. The chain conformations adopted by these PLL-b-PLT diblock copolypeptides at neutral condition were characterized by CD and FTIR analyses. Their chain conformations were computed by fitting the CD spectra using the software CD-fit 4.35,38,39 For comparison, the chain conformations adopted by PLL20 and PLT20 were also characterized by CD analysis. PLL20 and PLT20 adopt mainly random coil (88.6%) and β-sheet (80.2%) conformations at neutral condition, respectively (Table 1 and Figure 5a). The results are consistent with the previous studies,22,30,35,40 showing that PLL and PLT homopolypeptides adopt mainly random coil and β-sheet conformations at neutral condition, respectively. In this study, CD analysis reveals that PLL-b-PLT diblock copolypeptides adopt mainly a mixture of random coil and β-sheet conformations at neutral condition (Table 1, Figure 5a,b, and Figure S4a). Consistent with the results from CD analysis, FTIR spectra of these PLLb-PLT diblock copolypeptides exhibit the amide I peak at 1650 and 1623 cm−1, which are the characteristics of random coil and β-sheet conformations, respectively (Figure 5c,d and Figure S4b). For the polypeptides with a given PLL chain length (for example, PLL20-b-PLT8, PLL22-b-PLT16, and PLL20-b-PLT41), CD and FTIR analyses show that the percentage of β-sheet conformation adopted by the polypeptides increases with the increment of the block ratio of PLT to PLL. For the polypeptides with roughly 2:1 block ratio of PLL to PLT, the percentage of β-sheet conformation adopted by PLL20-b-PLT8 is higher than that adopted by PLL30-bPLT12 and PLL41-b-PLT21. It is worth noting that PLL41-bPLT21 also exhibites about 10−12% of α-helical conformation. However, the polypeptides with roughly 1:1 block ratio of PLL to PLT (i.e., PLL22-b-PLT16, and PLL30-b-PLT30) exhibit comparable percentage of β-sheet conformation (∼47%). Previous studies have shown that the conjugation of a hydrophobic polymer block (or alkyl chain) on the chain

dried polypeptide sol solutions (1.0 wt % of polypeptide concentration) were also characterized by SEM analysis and found they exhibit more like one-dimensional network (Figure 3). To understand the effect of chain length and block ratio on the mechanical strengths of these PLL-b-PLT polypeptide hydrogels, the hydrogels formed by PLL20-b-PLT8, PLL22-bPLT16, PLL30-b-PLT12, and PLL30-b-PLT30 were selected and investigated by using an oscillatory shear rheometer. The dynamic strain and frequency sweeps were conducted on the selected PLL-b-PLT polypeptide hydrogels at 5.0 wt % of polypeptide concentration (Figure 4). At 25 °C, the formation

Figure 4. (a) Storage modulus G′ (solid symbols) and loss modulus G″ (open symbols) of (■) PLL20-b-PLT8, (▲) PLL22-b-PLT16, (●) PLL30-b-PLT12, and (▼) PLL30-b-PLT30 hydrogels as a function of angular frequency at strain = 1.0%. (b) Strain sweeps for the same samples at frequency = 1.0 rad/s. The polypeptide concentration of the samples is 5.0 wt %.

of hydrogels is evident since the storage modulus G′ is found to be larger than the loss modulus G″. Strain sweep measurements show that the storage modulus G′ decreases with the increment of strain and the gel network is disrupted under large strain, revealing the shear-thinning properties and the disruption of the gel structure at high strain (Figure 4). PLL22-b-PLT16 and PLL30-b-PLT30 hydrogels exhibit higher mechanical strengths compared to PLL20-b-PLT8 and PLL30-bPLT12, respectively (Figure 4a). The mechanical strength of PLL30-b-PLT30 hydrogel is slightly higher than that of PLL22-bPLT16 one. The results show that the gel mechanical strength increases with the increment of PLT to PLL block ratio (that is, the percentage of β-sheet) and chain length (that is, E

DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX

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Figure 5. (a, b) CD spectra of the block copolypeptides. The polypeptide concentration is 0.125 mg/mL. (c, d) FTIR spectra of freeze-dried polypeptide hydrogel samples prepared at 5 wt % of polypeptide concentration.

Figure 6. SAXS patterns of (a) PLL20-b-PLT8, (b) PLL22-b-PLT16, (c) PLL30-b-PLT12, and (d) PLL30-b-PLT30 solutions and hydrogels prepared at different polypeptide concentrations.

the contrary, the conjugation of a PLT block on the chain end of a short PLL do not facilitate the conformational transition of PLL, evidenced by the percentage of ordered conformation

end (or side chain) of a short PLL can facilitate the conformational transition of PLL due to the confinement of polypeptide chains in the self-assembled structures.35,39−42 On F

DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX

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width of PLL20-b-PLT8 assemblies is calculated to be about 15.0 nm. The widths of the PLL20-b-PLT8 fibrils determined from the TEM image are found to be slightly smaller than the calculated ones, which can be attributed to the shrinkage of coil PLL segments upon drying. The SAXS patterns with characteristic I(q) ∝ q−n exhibit the n exponent increases with the increase of the polypeptide concentration at intermediate scattering vectors (0.03 Å−1 < q < 0.08 Å−1). Moreover, the SAXS patterns exhibit a plateau at low scattering vectors (q < 0.03 Å−1) for the samples with the polypeptide concentration higher than their CGCs. Previous studies have shown that the scattering intensity for hydrogels composed of flexible polymer coils can be described by the following equation:51,52

comparable with or slightly lower than the mole fraction of PLT block except PLL20-b-PLT8. It can be attributed that the sheetlike PLT is not as hydrophobic as other sheetlike polypeptides such as poly(L-alanine), poly(L-phenylalanine), and poly(L-cysteine).22,30 Hence, the confinement effect exerts only by the intermolecular hydrogen-bonding interactions between the PLT block cannot facilitate the conformational transition of PLL. To understand the self-assembly of PLL-b-PLT, SAXS analysis was performed on both PLL-b-PLT sol solutions and hydrogels at different polypeptide concentrations. SAXS analysis of the PLL-b-PLT sol solutions with 0.5 wt % of polypeptide concentration indicates the scattering intensity (I) with characteristic I(q) ∝ q−1 at the scattering vectors (q) lower than 0.08 Å−1 (Figure 6), revealing the formation of onedimensional (1D) fibril assemblies.43 TEM analysis of the selected PLL-b-PLT polypeptide assemblies negatively stained with uranyl acetate, which were prepared from a PLL20-b-PLT8 sol solution (0.1 wt %), exhibits 1D fibril structures (Figure 7).

I(q) =

I1 2 2

1+qξ

+

I2 (1 + q2 Ξ2)2

(1)

The equation is composed of Ornstein−Zernike and Debye− Bueche equations describing the polymer concentration fluctuation and excess scattering from a spatial inhomogeneity, respectively. ξ is the thermal correlation length or mesh size of the polymer network, whereas Ξ is the characteristic length of the network inhomogeneity. The I1 and I2 terms, that correspond to the respective static and dynamic contributions, can be obtained by extrapolating the curves to q = 0. For the Ornstein−Zernike equation, the SAXS intensity profiles would exhibit t respectively I(q) ∝ q−2 and I(q) ∝ q0 if the q2ξ2 values are much larger and smaller than 1. For the hydrogel samples, their SAXS patterns exhibit the n exponents to be −2 and 0 at two different ranges of scattering vectors, which are 0.03 Å−1 < q < 0.08 Å−1 and q < 0.03 Å−1. It suggests that the spatial inhomogeneity of the samples, which is described by Debye−Bueche function, exhibits much larger dimension than mesh size of the polymer network. Hence, the SAXS intensity profiles of the hydrogel samples can be fitted by the Ornstein− Zernike equation. Based on the given available information, the thermal correlation lengths (or mesh sizes) for PLL20-b-PLT8, PLL22-b-PLT16, PLL30-b-PLT12, and PLL30-b-PLT30 hydrogels (6.0 wt %) are about 5.7, 4.8, 7.6, and 5.4 nm, respectively. The mesh sizes of the polypeptide hydrogels with roughly 1:1 block ratio of PLL to PLT are larger than those with roughly 2:1 block ratio. At a given block ratio, the mesh size of the polypeptide hydrogels slightly increases with the increase of molecular weight. The results indicate that both the hydrogenbonding interactions between PLT segments and charge repulsion between PLL segments would contribute to the self-assembly of polypeptides. The large mesh size for PLL20-bPLT8 and PLL30-b-PLT12 hydrogels is caused by the fact that they self-assemble to form loose and wide spatial distribution of the fibrils, which are due to the relatively weak hydrogenbonding interactions and strong charge repulsion. Self-Assembly and Gelation Mechanism. The above results suggest that the hydrogelation of PLL-b-PLT diblock copolypeptides is mainly governed by a delicate balance between the charge repulsion and hydrogen-bonding interactions exerted by PLL and PLT blocks, respectively. Moreover, the hydrogen-bonding interactions between the partly polar hydroxyl group on PLT and water molecules would also promote the molecular interaction in the hydrogels. The PLL-b-PLT diblock copolypeptides would self-assemble to form fibril assemblies with the interdigitated packing of the sheetlike PLT block in water (Figures 3 and 7). Upon

Figure 7. TEM image of PLL20-b-PLT8 assemblies. The concentration of polypeptide solution was 0.1 wt %, and the TEM sample was negatively stained with uranyl acetate.

CryoEM analysis of the vitrified PLL20-b-PLT8 hydrogel (5.0 wt %) without staining showed interconnected nanostripes with widths smaller than 5 nm, resembling the sheetlike PLT assemblies (Figure S5). XRD patterns of freeze-dried hydrogel samples exhibit one major peak at 2θ = 18.9°, giving the d spacing to be 0.46 nm (Figure S6). The d spacing of 0.46 nm can be attributed to the formation of antiparallel β-sheet conformation.44−46 The much broader peak at 2θ > 20° suggests the presence of amorphous phase, which is from the coil PLL domain. Previous studies have shown that coil−sheet peptide-based block copolymers would form fibril assemblies via intermolecular hydrogen bonding interactions (i.e., β-sheet conformation).20,47,48 In this study, the PLL-b-PLT polypeptide would possibly form fibril assemblies with the interdigitated packing of the sheetlike PLT block via intermolecular hydrogen-bonding interactions, which is parallel to the long axis of fibril assemblies. In other words, the width of the fibril assemblies should be the length of sheetlike PLT domain plus the length of the two coil PLL ones. Based on the contour lengths of the coil PLL and sheetlike PLT,32,49,50 the G

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PLT block copolypeptides. The PLL30-b-PLT12 and PLL30-bPLT30 hydrogels exhibit slightly higher mechanical strengths upon increasing the temperature. It can be attributed to the dehydration of polypeptides and the slight increase in the intermolecular hydrogen-bonding interactions (i.e., higher percentage of β-sheet conformation) at elevated temperatures. The increase of temperature would result in the dehydration of polypeptides and the increase in the molecular motion of the PLT domain, which consequently would offset the amphiphilic balance in the block copolypeptides and promote the efficient self-assembly of polypeptides and packing of the fibrils.

Table 2. Summary of the Characteristic Parameters Deduced from Fitting the SAXS Profiles for Different Polypeptide Hydrogels Using the Ornstein−Zernike Equation, Where ξ Is the Thermal Correlation Length or Mesh Size of the Polymer Network and I1 Is the Scattering Intensities at q = 0, Corresponding to the Static Contribution polypeptide

concn (%)

I1

ξ (Å)

r2

PLL20-b-PLT8

6 5 6 5 6 5 7 6

215.0 240.0 248.0 201.0 410.5 370.0 530.0 232

56.9 56.7 48.5 48.4 76.6 76.0 53.9 53.7

0.980 0.984 0.994 0.995 0.985 0.988 0.987 0.994

PLL22-b-PLT16 PLL30-b-PLT12 PLL30-b-PLT30

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CONCLUSION We have demonstrated that coil−sheet PLL-b-PLT block copolypeptides can self-assemble to form transparent hydrogels with the CGCs between 3.0 and 7.0 wt % due to the formation and entanglement of fibril assemblies via the intermolecular hydrogen-bonding interactions and charge repulsion exerted by sheetlike PLT and positively charged PLL blocks, respectively. The CGC, gel molecular assembly, and gel mechanical property are governed by the polypeptide chain length and composition. Increasing the percentage of β-sheet conformation adopted by the polypeptides would exhibit the increase in the gel mechanical properties and decrease in CGC and mesh size in the gel network. This study illustrates that the sheetlike PLT is a promising polypeptide candidate to trigger self-assembly and hydrogelation. Moreover, these block copolypeptides may lead to new insights into the mechanisms of hydrogelation and could serve as biomimetic templates for the synthesis of nanomaterials.

increasing the polypeptide concentration, the propagation of the length of fibril assemblies results in the entanglement of the fibril assemblies and the formation of gel network, consistent with previously reported peptide hydrogels.20,48 Contrary to fibril network in the micro- and macroscales shown in the previous studies,20,48 the PLL-b-PLT fibril assemblies prepared in this study can aggregate to form interconnected, membraneous network at relatively high polypeptide concentration, evidenced by the SEM analysis (Figure 2). It can be attributed that these positively charged fibril assemblies tend to elongate and aggregate in two-dimensional space, in which consequently the interconnected, membraneous network would form in the micro- and macroscales. The polypeptide chain length and block ratio are the two major parameters to manipulate the noncovalent interactions and amphiphilic nature exhibited by these block copolypeptides. The experimental data show the CGC for the hydrogels is in the following order: PLL30-b-PLT30 < PLL20-b-PLT41 < PLL30-b-PLT12 < PLL22-b-PLT16 < PLL20-b-PLT8 < PLL11-bPLT17 ∼ PLL41-b-PLT21. The results reveal that PLL-bPLT block copolypeptides with appropriate polypeptide chain length and block ratio would exhibit better gelation ability. It can be attributed that the hydrogel formation is determined by the molecular orientation and packing of the fibril assemblies, which are dictated by the balance between the charge repulsion and hydrogen bonding interactions as well as the amphiphilic nature of the block copolypeptides. Increasing the block ratio of PLT to PLL increases the percentage of βsheet conformation adopted by the polypeptides and subsequently facilitates their hydrogelation as a result of the enhancement of the intermolecular hydrogen-bonding interactions between polypeptide chains. Consequently, this leads to the increase in the gel mechanical properties and decrease in CGC and mesh size in the gel network. However, it is not the case for PLL11-b-PLT17 because it has a relatively short PLL block. This would offset the balance between the charge repulsion and hydrogen-bonding interactions, which would lead to poor gelation ability. The CGC for PLL30-b-PLT12 is slightly lower than that for PLL22-b-PLT16 even though PLL30b-PLT12 adopts less percentage of β-sheet conformation than PLL22-b-PLT16. The results suggest that PLL30-b-PLT12 chains can pack to form fibril assemblies and subsequent interconnected network more efficient than PLL22-b-PLT16 ones. It highlights the importance of the molecular orientation and packing of the fibril assemblies for hydrogelation of PLL-b-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b01265. Experimental details and characterization data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Fax +886-6-2344496 (J.-S.J.). ORCID

Jeng-Shiung Jan: 0000-0002-8379-404X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the Ministry of Science and Technology, Taiwan (MOST 1052221-E-006-248 and 106-2221-E-006-206). The authors acknowledge S.S.-S. Wang for access to circular dichroism. This work was financially supported by the Hierarchical GreenEnergy Materials (Hi-GEM) Research Center, from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.

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REFERENCES

(1) Nuttelman, C. R.; Rice, M. A.; Rydholm, A. E.; Salinas, C. N.; Shah, D. N.; Anseth, K. S. Macromolecular Monomers for the Synthesis of Hydrogel Niches and Their Application in Cell

H

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Article

Macromolecules Encapsulation and Tissue Engineering. Prog. Polym. Sci. 2008, 33, 167−179. (2) Bhattarai, N.; Gunn, J.; Zhang, M. Chitosan-Based Hydrogels for Controlled, Localized Drug Delivery. Adv. Drug Delivery Rev. 2010, 62, 83−99. (3) Li, Y.; Rodrigues, J.; Tomás, H. Injectable and Biodegradable Hydrogels: Gelation, Biodegradation and Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2193−2221. (4) Lee, K. Y.; Mooney, D. J. Alginate: Properties and Biomedical Applications. Prog. Polym. Sci. 2012, 37, 106−126. (5) Jonker, A. M.; Löwik, D W. P. M.; van Hest, J. C. M. Peptideand Protein-Based Hydrogels. Chem. Mater. 2012, 24, 759−773. (6) Gyles, D. A.; Castro, L. D.; Silva, J. O. C., Jr.; Ribeiro-Costa, R. M. A Review of the Designs and Prominent Biomedical Advances of Natural and Synthetic Hydrogel Formulations. Eur. Polym. J. 2017, 88, 373−392. (7) Malda, J.; Visser, J.; Melchels, F. P.; Jüngst, T.; Hennink, W. E.; Dhert, W. J. A.; Groll, J.; Hutmacher, D. W. 25th Anniversary Article: Engineering Hydrogels for Biofabrication. Adv. Mater. 2013, 25, 5011−5028. (8) Van Vlierberghe, S.; Dubruel, P.; Schacht, E. Biopolymer-Based Hydrogels as Scaffolds for Tissue Engineering Applications: A Review. Biomacromolecules 2011, 12, 1387−1408. (9) Kapoor, S.; Kundu, S. C. Silk Protein-Based Hydrogels: Promising Advanced Materials for Biomedical Applications. Acta Biomater. 2016, 31, 17−32. (10) Radhakrishnan, J.; Subramanian, A.; Krishnan, U. M.; Sethuraman, S. Injectable and 3D Bioprinted Polysaccharide Hydrogels: From Cartilage to Osteochondral Tissue Engineering. Biomacromolecules 2017, 18, 1−26. (11) Park, M. H.; Joo, M. K.; Choi, B. G.; Jeong, B. Biodegradable Thermogels. Acc. Chem. Res. 2012, 45, 424−433. (12) He, X.; Fan, J.; Wooley, K. L. Stimuli-Triggered Sol−Gel Transitions of Polypeptides Derived from α-Amino Acid NCarboxyanhydride (NCA) Polymerizations. Chem. - Asian J. 2016, 11, 437. (13) Liu, W.; Zhang, W.; Yu, X.; Zhang, G.; Su, Z. Synthesis and Biomedical Applications of Fluorescent Nanogels. Polym. Chem. 2016, 7, 5749−5762. (14) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J. Rapidly Recovering Hydrogel Scaffolds from Self-Assembling Diblock Copolypeptide Amphiphiles. Nature 2002, 417, 424−428. (15) Breedveld, V.; Nowak, A. P.; Sato, J.; Deming, T. J.; Pine, D. J. Rheology of Block Copolypeptide Solutions: Hydrogels with Tunable Properties. Macromolecules 2004, 37, 3943−3953. (16) Choi, Y. Y.; Jang, J. H.; Park, M. H.; Choi, B. G.; Chi, B.; Jeong, B. Block Length Affects Secondary Structure, Nanoassembly and Thermosensitivity of Poly(Ethylene Glycol)-poly(L-Alanine) Block Copolymers. J. Mater. Chem. 2010, 20, 3416−3421. (17) Chen, Y.; Pang, X. H.; Dong, C. M. Dual Stimuli-Responsive Supramolecular Polypeptide-Based Hydrogel and Reverse Micellar Hydrogel Mediated by Host−Guest Chemistry. Adv. Funct. Mater. 2010, 20, 579−586. (18) Cheng, Y.; He, C.; Xiao, C.; Ding, J.; Zhuang, X.; Huang, Y.; Chen, X. Decisive Role of Hydrophobic Side Groups of Polypeptides in Thermosensitive Gelation. Biomacromolecules 2012, 13, 2053− 2059. (19) Cheng, Y.; He, C.; Xiao, C.; Ding, J.; Cui, H.; Zhuang, X.; Chen, X. Versatile Biofunctionalization of Polypeptide-Based Thermosensitive Hydrogels via Click Chemistry. Biomacromolecules 2013, 14, 468−475. (20) Zhang, S.; Fu, W.; Li, Z. Supramolecular Hydrogels Assembled from Nonionic Poly(Ethylene Glycol)-b-Polypeptide Diblocks Containing OEGylated Poly-L-Glutamate. Polym. Chem. 2014, 5, 3346− 3351. (21) Zhang, S.; Alvarez, D. J.; Sofroniew, M. V.; Deming, T. J. Design and Synthesis of Nonionic Copolypeptide Hydrogels with

Reversible Thermoresponsive and Tunable Physical Properties. Biomacromolecules 2015, 16, 1331−1340. (22) Hou, S.-S.; Hsu, Y.-Y.; Lin, J.-H.; Jan, J.-S. Alkyl-poly(Lthreonine)/Cyclodextrin Supramolecular Hydrogels with Different Molecular Assemblies and Gel Properties. ACS Macro Lett. 2016, 5, 1201−1205. (23) Lin, J.-Y.; Lai, P.-L.; Lin, Y.-K.; Peng, S.; Lee, L.-Y.; Chen, C.N.; Chu, I.-M. A Poloxamer-Polypeptide Thermosensitive Hydrogel as A Cell Scaffold and Sustained Release Depot. Polym. Chem. 2016, 7, 2976−2985. (24) Sun, Y.; Wollenberg, A. L.; O’Shea, T. M.; Cui, Y.; Zhou, Z. H.; Sofroniew, M. V.; Deming, T. J. Conformation-Directed Formation of Self-Healing Diblock Copolypeptide Hydrogels via Polyion Complexation. J. Am. Chem. Soc. 2017, 139, 15114−15121. (25) Shen, X.-Y.; Tang, C.-C.; Jan, J.-S. Synthesis and Hydrogelation of Star-Shaped Poly(L-Lysine) Polypeptides Modified with Different Functional Groups. Polymer 2018, 151, 108−116. (26) Inomata, K.; Terahama, T.; Sekoguchi, R.; Ito, T.; Sugimoto, H.; Nakanishi, E. Shape Memory Properties of Polypeptide Hydrogels Having Hydrophobic Alkyl Side Chains. Polymer 2012, 53, 3281− 3286. (27) Wang, S. S.; Hsieh, P.-L.; Chen, P.-S.; Chen, Y.-T.; Jan, J.-S. Genipin-Cross-Linked Poly(L-Lysine)-Based Hydrogels: Synthesis, Characterization, and Drug Encapsulation. Colloids Surf., B 2013, 111, 423−431. (28) Zhang, Y.-X.; Chen, Y.-F.; Shen, X.-Y.; Hu, J.-J.; Jan, J.-S. Reduction-and pH-Sensitive Lipoic Acid-Modified Poly (L-Lysine) and Polypeptide/Silica Hybrid Hydrogels/Nanogels. Polymer 2016, 86, 32−41. (29) Li, Z.; Deming, T. J. Tunable Hydrogel Morphology via SelfAssembly of Amphiphilic Pentablock Copolypeptides. Soft Matter 2010, 6, 2546−2551. (30) Kubota, S.; Fasman, G. D. The β Conformation of Polypeptides of Valine, Isoleucine, and Threonine in Solution and Solid-State: Optical and Infrared Studies. Biopolymers 1975, 14, 605−631. (31) Gibson, M. I.; Cameron, N. R. Experimentally Facile Controlled Polymerization of N-Carboxyanhydrides (NCAs), Including O-Benzyl-L-Threonine NCA. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 2882−2891. (32) Chen, Y.-F.; Shiau, A.-L.; Chang, S.-J.; Fan, N.-S.; Wang, C.-T.; Wu, C.-L.; Jan, J.-S. One-Dimensional Poly(L-Lysine)-block-Poly(LThreonine) Assemblies Exhibit Potent Anticancer Activity by Enhancing Membranolysis. Acta Biomater. 2017, 55, 283−295. (33) Huang, Y. F.; Lu, S. C.; Huang, Y. C.; Jan, J. S. Cross-Linked, Self-Fluorescent Gold Nanoparticle/Polypeptide Nanocapsules Comprising Dityrosine for Protein Encapsulation and Label-Free Imaging. Small 2014, 10, 1939−1944. (34) Yu, L.; Zhang, H.; Ding, J. A Subtle End-Group Effect on Macroscopic Physical Gelation of Triblock Copolymer Aqueous Solutions. Angew. Chem., Int. Ed. 2006, 45, 2232−2235. (35) Huang, Y.-C.; Arham, M.; Jan, J.-S. Alkyl Chain Grafted Poly(L-Lysine): Self-Assembly and Biomedical Application as Carriers. Soft Matter 2011, 7, 3975−3983. (36) Hiemstra, C.; Zhou, W.; Zhong, Z.; Wouters, M.; Feijen, J. Rapidly in situ Forming Biodegradable Robust Hydrogels by Combining Stereocomplexation and Photopolymerization. J. Am. Chem. Soc. 2007, 129, 9918−9926. (37) Khan, F.; Tare, R. S.; Oreffo, R.; Bradley, M. Versatile Biocompatible Polymer Hydrogels: Scaffolds for Cell Growth. Angew. Chem., Int. Ed. 2009, 48, 978−982. (38) Bradbury, E.; Crane-Robinson, C.; Goldman, H.; Rattle, H. Proton Magnetic Resonance and Optical Spectroscopic Studies of Water-Soluble Polypeptides: Poly-L-Lysine HBr, Poly(L-Glutamic Acid), and Copoly(L-Glutamic Acid42, L-Lysine HBr28, L-Alanine30). Biopolymers 1968, 6, 851−862. (39) Chen, B.-Y.; Huang, Y.-C.; Jan, J.-S. Molecular Assembly of Alkyl Chain-Grafted Poly(L-Lysine) Tuned by Backbone Chain Length and Grafted Alkyl Chain. RSC Adv. 2015, 5, 22783−22791. I

DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules (40) Huang, Y.-C.; Arham, M.; Jan, J.-S. Bioactive Vesicles from Saccharide-and Hexanoyl-Modified Poly(L-Lysine) Copolypeptides and Evaluation of The Cross-Linked Vesicles as Carriers of Doxorubicin for Controlled Drug Release. Eur. Polym. J. 2013, 49, 726−737. (41) Harada, A.; Cammas, S.; Kataoka, K. Stabilized α-Helix Structure of Poly(L-Lysine)-block-Poly(Ethylene Glycol) in Aqueous Medium Through Supramolecular Assembly. Macromolecules 1996, 29, 6183−6188. (42) Chen, B.-Y.; Huang, Y.-F.; Huang, Y.-C.; Wen, T.-C.; Jan, J.-S. Alkyl Chain-Grafted Poly(L-Lysine) Vesicles with Tunable Molecular Assembly and Membrane Permeability. ACS Macro Lett. 2014, 3, 220−223. (43) Chu, B. Laser Light Scattering: Basic Principles and Practice; Courier Corp.: 2007. (44) Rösler, A.; Klok, H.-A.; Hamley, I. W.; Castelletto, V.; Mykhaylyk, O. O. Nanoscale Structure of Poly(Ethylene Glycol) Hybrid Block Copolymers Containing Amphiphilic β-Strand Peptide Sequences. Biomacromolecules 2003, 4, 859−863. (45) Gibson, M. I.; Cameron, N. R. Organogelation of Sheet−Helix Diblock Copolypeptides. Angew. Chem., Int. Ed. 2008, 47, 5160− 5162. (46) Huang, Y.-C.; Jan, J.-S. Carboxylmethyl Chitosan-graft-Poly(γBenzyl-L-Glutamate) Glycopeptides: Synthesis and Particle Formation as Encapsulants. Polymer 2014, 55, 540−549. (47) Missirlis, D.; Chworos, A.; Fu, C. J.; Khant, H. A.; Krogstad, D. V.; Tirrell, M. Effect of The Peptide Secondary Structure on The Peptide Amphiphile Supramolecular Structure and Interactions. Langmuir 2011, 27, 6163−6170. (48) Huang, J.; Hastings, C. L.; Duffy, G. P.; Kelly, H. M.; Raeburn, J.; Adams, D. J.; Heise, A. Supramolecular Hydrogels with Reverse Thermal Gelation Properties from (Oligo)tyrosine Containing Block Copolymers. Biomacromolecules 2013, 14, 200−206. (49) Wong, M. S.; Cha, J. N.; Choi, K.-S.; Deming, T. J.; Stucky, G. D. Assembly of Nanoparticles into Hollow Spheres using Block Copolypeptides. Nano Lett. 2002, 2, 583−587. (50) Jan, J.-S.; Lee, S.; Carr, C. S.; Shantz, D. F. Biomimetic Synthesis of Inorganic Nanospheres. Chem. Mater. 2005, 17, 4310− 4317. (51) Shibayama, M.; Isono, K.; Okabe, S.; Karino, T.; Nagao, M. SANS Study on Pressure-Induced Phase Separation of Poly(NIsopropylacrylamide) Aqueous Solutions and Gels. Macromolecules 2004, 37, 2909−2918. (52) Hirokawa, Y.; Okamoto, T.; Kimishima, K.; Jinnai, H.; Koizumi, S.; Aizawa, K.; Hashimoto, T. Sponge-Like Heterogeneous Gels: Hierarchical Structures in Poly(N-Isopropylacrylamide) Chemical Gels as Observed by Combined Scattering and Confocal Microscopy Method. Macromolecules 2008, 41, 8210−8219.

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DOI: 10.1021/acs.macromol.8b01265 Macromolecules XXXX, XXX, XXX−XXX