Peptide Hydrogels Assembled from Nonionic Alkyl-polypeptide

Communication pubs.acs.org/Biomac

Peptide Hydrogels Assembled from Nonionic Alkyl-polypeptide Amphiphiles Prepared by Ring-Opening Polymerization Chongyi Chen, Decheng Wu, Wenxin Fu,* and Zhibo Li* Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: Three alkyl-polypeptide (AP) amphiphiles were prepared using ring-opening polymerization of α-amino acid Ncarboxyanhydride. The polypeptide segment was composed of diethylene-glycol-monomethyl-ether-functionalized poly-Lglutamate (poly-L-EG2Glu). These AP amphiphiles can spontaneously self-assemble into transparent hydrogels in water. These hydrogels showed shear thinning properties, and their strength can be modulated by hydrophobic alkyl tails. CryoTEM and AFM characterizations suggested that these hydrogels were formed by nanoribbons arising from intermolecular interactions between nonionic poly-L-EG2Glu segments.

P

(AP) amphiphiles using controlled ROP of NCA.27−29 These AP amphiphiles can spontaneously self-assemble into hydrogels possessing shear-thinning and rapid recovery properties suitable for injectable delivery application. These AP hydrogel surfaces possessed active groups subject to biofunctionalization. To our knowledge, this is an unprecedented example of making injectable hydrogel from nonionic AP amphiphiles prepared by ROP. A big advantage of such system is that the materials can be obtained in large quantity using easily available monomer, and these peptide hydrogels have tunable properties and variable functionalities. For the self-assembly PAs, the β-sheet sequence was a vital component for them to form hydrogels because it provided the intermolecular hydrogen bonding interactions that essentially drive the formation of 1D nanostructures.30−32 Moreover, additional charged peptide units were necessary to confer nanostructures’ water dispersibility. Following such design principle, a specific synthetic polypeptide segment that tends to form water-soluble β-sheet would be necessary to make nonionic peptide hydrogels. Hence, we designed and synthesized a water-soluble nonionic polypeptide composed of diethylene glycol monomethyl ether functionalized poly-Lglutamate (poly-L-EG2Glu). The poly-L-EG2Glu displayed

eptide hydrogels, which mimic extracellular matrix, are critical biomaterials.1−3 Their porous nanostructure and high water content made them excellent candidates for 3D scaffolds4−7 and delivery of therapeutic compounds including growth factors, drugs, proteins, and cells.8−12 In general, peptide hydrogels can be prepared via self-assembly of de novo designed oligopeptides or peptide amphiphiles (PAs). For example, Pochan and Schneider groups explored stimuliresponsive hydrogels via self-assembly of β-hairpin peptides.13−15 Yang and coworkers studied the enhanced interactions between protein and peptide hydrogels.16 Peptide hydrogels assembled from PAs, which are composed of a hydrophobic alkyl tail and a functional oligopeptide chain, have been extensively explored for their biomedical applications.17,18 In particular, Stupp and coworkers have made tremendous contributions to design and applications of PA hydrogels.19−21 However, these peptide materials were made by solid-phase peptide synthesizer (SPPS), which involved multistep synthesis and tedious purification with high cost. Also, these peptides usually carried charged groups to confer water solubility. Peptide hydrogels can be prepared using amphiphilic block copolypeptides that were synthesized by controlled ringopening polymerization (ROP) of α-amino acid N-carboxyanhydride (NCA).22−26 Hence, it is important to develop highly efficient methods of preparing peptide hydrogels at low cost but without losing their advanced physical and biofunctional properties. Herein, we report a versatile strategy to make nonionic alkyl-polypeptide © 2013 American Chemical Society

Received: June 6, 2013 Revised: July 3, 2013 Published: July 4, 2013 2494

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transformable secondary conformation from α-helix to β-sheet, which can be tuned by external conditions. 33−35 We demonstrated here that linking poly-L-EG2Glu with alkyl tail can afford a new type of nonionic PA, which assembled into hydrogels with tunable mechanical properties. Three alkyl-poly-L-EG2Glu amphiphiles (AP1-AP3) were synthesized by ROP of γ-(2-methoxyethoxy)esteryl-L-glutamate N-carboxyanhydride (L-EG2Glu NCA) using hexyl/dodecyl/ hexadecyl amine as initiators (Scheme 1). The preparation of L-

The hydrogel strength was characterized using rheology (Figure 1). Frequency sweep measurements showed that the

Scheme 1. Synthetic Routes to AP Amphiphiles

Figure 1. (a) Storage modulus (G′) as a function of concentration for AP1-AP3 hydrogels. (b) G′/G′eq as a function of time for AP2 and AP3 hydrogels, which were sheared at ω = 6 rad/s, γ0 = 50 for 600 S before switching to small strain. The hydrogel strength was monitored through small amplitude oscillations ω = 6 rad/s, γ0 = 0.4. For comparison, G′ was normalized to the equilibrium value (G′eq).

gel strength increased with concentration, as expected (Figure 1a). At the same concentration, the hydrogel strength increased substantially with alkyl chain length (Figure 1a). At 3 wt %, the corresponding modulus increased from 7 to 89 Pa and to 833 Pa for samples AP1−AP3, respectively. Note that the hydrogel strength can be modulated from 1 to 1000 Pa by simply adjusting sample compositions and concentrations to meet specific application requirements. More interestingly, these hydrogels displayed shear-thinning and rapid recovery properties. Strain sweep measurements indicated that AP2 and AP3 hydrogels had clear gel−sol transition under large strain (Figures S7 and S8 in the Supporting Information). In contrast, AP1 was a relative weak and soft hydrogel. It remained as a weak gel even under 100% strain (Figure S6b in the Supporting Information). Both AP2 and AP3 hydrogel showed the capability to recover rapidly from sol to gel. As shown in Figure 1b, both AP2 and AP3 hydrogel underwent a gel−sol transition under large amplitude strain oscillations. Then, the recovery of hydrogels was monitored by measuring the G′ as a function of time. Upon removal of shearing, both samples recovered to gel status within minimum switching time, ca. 10 s. In particular, AP2 recovered 70% of its original strength within 10 s, while AP3 recovered 88% of its original strength upon stopping shearing. Both samples then progressively regained their strength and reached 100% recovery within 1 h. Note that we only explored the effects of alkyl tails and concentrations on AP hydrogel properties. The importance of hydrophilic poly-L-EG2Glu block and its polydispersity on hydrogel properties will be investigated in the near future. It was known that PA can self-assemble into 1-D cylindrical fibril or nanoribbon depending on the nature of β-sheet forming peptide sequence linked to hydrophobic alkyl tail.31,32 We performed cryoTEM and AFM measurements to characterize the corresponding nanostructure of each hydrogel sample (Figure 2). CryoTEM images shown in Figure 2a−c reveal that samples AP1−AP3 form fibril network structures, which accounts for the formation of hydrogel. Careful cryoTEM examinations gave fibril width of about 9, 11, and 11 nm for samples AP1−AP3, respectively. AFM measurements further supported that AP1−AP3 formed a fibril network. All of the elongated fibrils had uniform width and height of approximate 13 and 1 nm (Figures S9−S11 in the Supporting Information).

Table 1. Molecular Parameters of Alkyl-Poly-L-EG2Glu entry

initiator

[M]/[I]

DPa

DPb

PDIb

CGCc

AP1 AP2 AP3

C6H13NH2 C12H25NH2 C16H33NH2

15 15 15

12 12 13

10 11 11

1.06 1.05 1.07

2.0 wt % 2.0 wt % 2.0 wt %

a c

Determined from 1H NMR. bDetermined from MALDI-TOF MS. Determined by inverting tube method.

EG2Glu NCA was reported elsewhere.33 Table 1 summarizes the corresponding molecular parameters. The degree of polymerization (DP) of poly-L-EG2Glu block was determined using 1H NMR by taking the ratio of methoxy peak over the methylene and methyl protons from alkyl chain (Figure S2 in the Supporting Information). The averaged DP was ∼12 for AP1-AP2 and 13 for AP3. These values were slightly lower than the expected number from [M]/[I] ratio, which was fixed at 15. The DP values determined from MALDI-TOF MS were underestimated compared with 1H NMR results possibly due to unequal ionization. The PDI values were estimated from MALDI-TOF MS and were below 1.1. Furthermore, careful analysis of MS spectra revealed that the amino group was the major end group for AP1−AP3, which offered potential active sites for subsequent conjugation with bioactive species (Figures S3−S5 in the Supporting Information).36 It is worth noting that gram-scaled materials can be easily prepared using this method with good control over peptide length and distributions. Samples of AP1-AP3 were obtained as white solid. Interestingly, all three samples spontaneously formed transparent hydrogel when dispersed in water at room temperature (Figure S1 in the Supporting Information). The gelation time ranged from a few minutes to couple of hours depending on the alkyl tail length. The longer the alkyl chains, the shorter the time required for the gelation. An advantage of these nonionic AP amphiphiles was that it was unnecessary to impose heating, pH change, or ions to induce gel formation. Only mechanical vortex was needed to facilitate gelation. For all samples, the critical gelation concentration (CGC) was ∼2 wt % using the inverting tube method. Apparently, the CGC did not show strong dependence on hydrophobic alkyl chain given that the hydrophilic poly-L-EG2Glu blocks had similar length. 2495

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helical contents decreased from 65 to 33% and to 7% accompanying β-sheet content increase from 4 to 21% and to 39% for samples AP1−AP3, respectively.38,39 Apparently, the β-sheet content increased with alkyl tail length because elongation of alkyl chain will promote the formation of intermolecular hydrogen bonding.40−42 Then, we used FTIR to characterize the hydrogen-bonding interactions of amide bonds within nanoribbons (Figure 3b). AP1 has two absorption bands with comparable intensity at 1655 and 1620 cm−1 (amide I band), which indicates that the helical content is slightly higher than the β-sheet content.43 When changing the alkyl group from hexyl to dodecyl, the band intensity at 1620 cm−1 is much stronger than that at 1655 cm−1, suggesting the formation of more β-sheet conformation. For AP3, absorbance at 1655 cm−1 almost disappears and the strong absorption at 1620 cm−1 means predominate β-sheet conformation.44 Deconvolution of FTIR spectra showed that the helical content decreased from 39 to 18% and to 5% for samples AP1−AP3, while the β-sheet conformation increased from 38 to 55% and to 72% accordingly (Figure S12 in the Supporting Information). These results were consistent with CD measurements discussed above and suggested that the longer the alkyl tail, the more β-sheet content and the stiffer the hydrogels are. It was also generally accepted that the alkyl unit contributed the hydrophobic interaction for self-assembly, while intermolecular hydrogen bonding, namely, β-sheet conformation, decided the formation of 1D structure.30−32,45 On the basis of this motif, a plausible model was proposed in Scheme 2 to Scheme 2. (a) Molecular Illustration of Dodecyl-poly-LEG2Glu12 Amphiphile and (b) Self-Assembly Mechanism Alkyl-Polypeptide into Nanoribbons Figure 2. (a−c) CryoTEM images of AP1-AP3 hydrogels and (d−f) AFM height images of AP1-AP3 hydrogels with c = 2 wt %.

These results demonstrated that assemblies were indeed nanoribbons instead of cylindrical fibrils. To understand the nanoribbon formation, we applied circular dichroism (CD) and FTIR spectroscopy to determine the role of secondary structure on hydrogel formation (Figure 3).

understand the formation of nanoribbons. It was found that poly-L-EG2Glu segment adopted mixed conformation containing both helical and β-sheet structure (Figure 3). We thus propose that the hydrophobic alkyl chains will form interdigitated hydrophobic core via hydrophobic interactions, while hydrophilic poly-L-EG2Glu adopts parallel packing via partial intermolecular hydrogen bonding among poly-LEG2Glu. The intermolecular hydrogen bonding is parallel to the long axis of nanoribbon, although the exact sequence corresponding to such interaction is uncertain given available information. Increase in alkyl tail length will promote the formation of β-sheet conformation at least for peptide sequence linked to alkyl tail. As a result, elongation of alkyl tail makes hydrogel stiffer and stronger (Figure 1).

Figure 3. (a) CD and (b) FTIR spectra of AP1-AP3 hydrogels.

Apparently, AP1 exhibits a positive peak near 195 nm and two negative peaks at 208 and 222 nm, suggesting α-helical conformation.37 For AP2, CD showed a notable decrease in helical content compared with AP1. CD spectrum of AP3 displayed the negative peak near 218 nm, indicating predominate β-sheet conformation.37 Quantitative analysis of conformation using Contin-LL on DICHROWEB revealed that 2496

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Also, the dimensions of nanoribbon determined by cryoTEM and AFM were consistent with the above model. For perfect βsheet, the distance between peptide backbone is ∼0.47 nm, and the length per amino acid residue is ∼0.35 nm.37,46 From the average DP of poly-L-EG2Glu block, we can estimate that their contour length will be about 4.2, 4.2, and 4.6 nm for AP1-AP3, respectively. In contrast, the contour length of alkyl unit for AP1-AP3 will be about 0.7, 1.4, and 1.9 nm, respectively. According to the model in Scheme 2, the maximum width of nanoribbons will be about 9.1, 9.8, and 10.3 nm for AP1-AP3. CryoTEM examination revealed that the average width for each nanoribbon was about 9, 11, and 11 nm for AP1-AP3. These values agreed rather well with molecular dimensions considering sample polydispersity and experimental deviation. Regarding nanoribbon thickness, the maximum thickness of nanoribbons (h) is about 2.3 nm for double contour length of side chain. AFM measurements gave thickness of AP1−AP3 nanoribbons of about 1 nm. Apparently, the theoretical thickness is larger than that obtained from AFM. We believe the reason is due to collapse of side chains on substrate during sample preparation. For AP amphiphiles, the alkyl tail acts as hydrophobic domain while poly-L-EG2Glu segment plays dual roles in hydrogel formation. First, the partial β-sheet conformation of poly-L-EG2Glu segment accounts for 1D nanoribbons, which grow into 3D networks. Second, the polar ethylene glycol side chains contribute to the water solubility of resulting nanoribbons. These two complementary factors are realized by the unique poly-L-EG2Glu block. It would be expected that any changes on poly-L-EG2Glu might break the self-assembly equilibrium. For example, we made racemic poly-rac-EG2Glu segment by mixing D-EG2Glu NCA with L-EG2Glu NCA and found the obtained hexadecyl-poly-rac-EG2Glu16 amphiphile could not form hydrogel under identical conditions. Moreover, the length of side chain on poly-L-glutamate was critical in terms of gel formation. For example, hexyl-poly-L-EG1Glu10 and dodecyl-poly-L-EG1Glu10 were insoluble in water because the poly-L-EG1Glu block was more hydrophobic than poly-LEG2Glu and forms dominant β-sheet structure.33 In contrast, dodecyl-poly-L-EG3Glu18 and hexadecyl-poly-L-EG3Glu11 did not form hydrogel as well. The reason was that poly-L-EG3Glu was more hydrophilic than poly-L-EG2Glu and predominately adopted α-helical structure.34 It was known the decorating of nanoribbons’ surface with bioactive species such as epitope or growth factors was important for practical biomedical applications. We already demonstrated that the AP was made by ROP-bearing amino end groups, which were subject to bioconjugation. As proof of concept, we labeled AP2 with fluorescein isothiocyanate (FITC). The FITC-labeled AP2 amphiphile was then coassembled with pristine AP2 at molar ratio of 5/95. The mixture formed hydrogel at 2.0 wt %, as expected. Laser scanning confocal microscopy (LSCM) characterization revealed that a porous 3D network structure was obtained for the partially labeled AP2 amphiphile (Figure 4a). TEM characterization of this fluorescent hydrogel confirmed nanoribbon structure consistent with pristine AP2 amphiphile hydrogel (Figure 4b). From the above discussion, we can draw an important conclusion that these AP hydrogels’ surfaces can be functionalized with bioactive compounds without sacrificing their gelation capabilities, which offered the platform for specific bioconjugation.

Figure 4. (a) Confocal image and (b) TEM image of FITC-labeled AP2 hydrogel with c = 2 wt %. The sample was negatively stained using 0.5 wt % uranyl acetate solution.

In summary, we demonstrated a convenient and low-cost method to prepare nonionic polypeptide hydrogels via selfassembly of AP amphiphiles. These AP amphiphiles were made through ROP of L-EG2Glu NCA. The peptide hydrogels were composed of nanoribbons directed by the intermolecular hydrogel bonding of poly-L-EG2Glu segment. These AP hydrogels showed shear-thinning and rapid recover properties, which made them great candidates as injectable hydrogel for biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

General materials section and a detailed experimental describing synthesis of Alkyl-PolyEG2Glu, 1HNMR, MALDITOF MS, rheology test, FTIR spectroscopy, and X-ray diffraction. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-010-62565612. E-mail: [email protected]; zbli@ iccas.ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (20974112, 50821062, 91027043), the Chinese Academy of Sciences (XDA01030302).

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REFERENCES

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