Letter pubs.acs.org/macroletters
Alkyl-poly(L‑threonine)/Cyclodextrin Supramolecular Hydrogels with Different Molecular Assemblies and Gel Properties Sheng-Shu Hou, Yu-Yun Hsu, Jia-Hsien Lin, and Jeng-Shiung Jan* Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan S Supporting Information *
ABSTRACT: We report alkyl-poly(L-threonine)/cyclodextrin (alkyl-PLT/CD) supramolecular hydrogels with different molecular assemblies. Their properties are determined by the interplay between host−guest chemistry and hydrogenbonding interactions. The gelation process was mainly dictated by the formation of alkyl chain/CD inclusion complex and PLT chain conformation. The dodecyl-PLT20/α-CD hydrogel exhibited laminar packing due to the sheet-to-coil conformational change upon forming inclusion complex. The hexadecylPLT20/β-CD hydrogel exhibited ribbon-like assemblies instead, because the peptide adopted mainly sheet conformation. The gel-to-sol transition occurred upon increasing temperature because of the decrease in hydrogen-bonding interactions and partly conformational change.
H
Alkyl-poly(O-benzyl-L-threonine) (alkyl-PBLT), denoted as C6PBLT20, C12PBLT20, and C16PBLT20, were synthesized by ring-opening polymerization (ROP) of NCAs using hexyl, dodecyl, and hexadecyl amines, respectively, as initiators. Alkylpoly(Z-L-lysine) (alkyl-PZLL) and alkyl-poly(γ-benzyl-L-glutamate) (alkyl-PBLG), denoted as C16PZLL20 and C16PBLG20, respectively, were synthesized for comparison by using hexadecyl amine as initiator. 1H NMR spectra of these alkylpeptides revealed that all the chemical shifts can be assigned to the protons on alkyl-peptide (Figures S1 and S2) and the degree of polymerization (DP) was determined by the ratio of the benzyl group (−OCH2C6H5) on peptides and the methylene group (−(CH2)nCH3) on alkyl chain (Tables S1 and S2). The calculated DP was comparable with the feed molar ratio of alkyl amine to NCA (1:20). MALDI-TOF analysis revealed that the derived number-averaged molecular weights (Mn) of C6PBLT20, C12PBLT20, and C16PBLT20 were 3600, 3700, and 4200, respectively (Figure S3 and Table S1). The calculated DPs derived from MALDI-TOF were slightly lower than those from 1H NMR (Table S1), which can be attributed to experimental error. Upon deprotection, the residual protecting groups for most samples were well below 3.0% except for C12PLT20 (3.2%), as determined by 1H NMR (Figures S1 and S2). All alkyl-peptides in the absence of CD cannot form hydrogels in aqueous solution if the polypeptide concentration (Cpeptide) is below 10 wt %. Interestingly, we found that the
ydrogels are three-dimensional networks of hydrophilic polymers with high water content and permeability, that making them ideal material for biomedical applications. Natural proteins and synthetic peptide-based polymers are two important classes of materials that can self-assemble to form supramolecular hydrogels through the induction of ordered conformations.1−3 In contrast to hydrogels based on natural proteins, synthetic peptide-based hydrogels with tunable gel property and functionality can be prepared via varying macromolecular chemistry.4−6 The self-assembly behavior of polypeptide-based copolymers is mainly dictated by amphiphilicity and polypeptide chain conformation. CD-based host− guest chemistry has recently been extensively explored to selfassemble nanoparticles and hydrogels.7−9 However, there are only a few studies on the supramolecular gelation between polypeptide-based copolymers and CDs via host−guest chemistry.10,11 Herein, we report the preparation of alkyl-poly(L-threonine)/cyclodextrin (alkyl-PLT/CD) supramolecular hydrogels with varying alkyl chain length and CD moiety. PLT is known to be a water-soluble peptide and adopts mainly β-sheet conformation.12 Polypeptide chain conformation, gel molecular assembly, and gel properties, including gel-to-sol phase behavior and strength, were investigated. The chain conformation of PLT would be determined by the alkyl chain length and alkyl chain/CD host−guest interactions. The influence of the host−guest and polypeptide inter/intramolecular hydrogen-bonding interactions on the gel molecular assembly and property was investigated. The interplay between the host−guest and hydrogen-bonding interactions may impart the formation of hydrogels with versatile molecular assembly and property. © XXXX American Chemical Society
Received: September 22, 2016 Accepted: September 29, 2016
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DOI: 10.1021/acsmacrolett.6b00716 ACS Macro Lett. 2016, 5, 1201−1205
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ACS Macro Letters alkyl-PLT/CD supramolecular hydrogels can be formed upon mixing C12PLT20 (or C16PLT20) with α-CD (or β-CD) at 1 or 2 of the molar ratio of CD to alkyl-PLT (RCD/peptide) above certain Cpeptide (Figure 1a). It is worth noting that the
Figure 2. (a−d) 1H NMR spectra of C16PLT20/β-CD with RCD/peptide = 0, 1, 2, and 3. In (e) the zoom-in spectra (β-CD region, δ 3.5−4.0) of pure β-CD (blue) and RCD/peptide = 1 (red) are compared. The polypeptide concentration of the samples is 6 mg/mL. The β-CD concentrations for the samples with RCD/peptide = 1, 2, and 3 were 2.66, 5.32, and 7.98 mg/mL, respectively.
Figure 1. (a) Gel-to-sol phase transition temperature of peptide amphiphile/cyclodextrin hydrogels with RCD/peptide = 1 (open symbols) and 2 (solid symbols) as a function of peptide concentration and (b) schematic illustration of the molecular assembly of C12PLT20/α-CD and C16PLT20/β-CD hydrogels and the photographs of C12PLT20/αCD and C16PLT20/β-CD hydrogels (6 wt % of peptide concentration, RCD/peptide = 2). The α-CD and β-CD concentrations were 59.1 and 56.6 mg/mL in C12PLT20/α-CD and C16PLT20/β-CD hydrogels, respectively.
CD (RCD/peptide = 3) solution (spectrum a; Figure 2). Obviously, in the presence of β-CD, a new resonance peak for the terminal methyl protons of the C16PLT20 was observed upon inclusion of the alkyl part of C16PLT20, and this peak was downfield shifted from δ = 0.818 ppm (C16PLT20 alone) to 0.857 ppm (C16PLT20/β-CD). In addition, the shift became more pronounced with increasing amount of β-CD. On the other hand, the large chemical shift changes of β-CD protons (protons 2, 3, 5, and 6 in Figure 2e) also confirmed the formation of inclusion complexes. The peaks of the protons 2, 3, and 5 on β-CD became broader and were not well resolved after adding β-CD to C16PLT20. Such broad peak pattern suggested that the mobility of the protons 2, 3, and 5 was restricted by the included alkyl chain of the guest C16PLT20. The chemical shifts and peak patterns of the protons α and β of C16PLT20, however, were hardly affected by the addition of βCD. Therefore, the peptide chain of C16PLT20 was not encapsulated into the cavity of β-CD. The PLT chain conformation upon the formation of inclusion complex was studied at Cpeptide = 0.25 mg/mL. CD spectrum of C12PLT20 solution that exhibited two minima at 198 and 222 nm (Figure 3a) suggested that C12PLT20 adopted mainly a mixture of random coil (43.4%) and β-sheet (40.9%) conformations (Table S3). Rather, the CD spectrum of C16PLT20 solution exhibited a maximum at 198 nm and a minimum at 222 nm (Figure 3b), suggesting that C16PLT20 adopted mainly β-sheet (67.1%) conformation (Table S3). Consistent with previous studies,15−17 the conjugation of the alkyl chain can promote the formation of intermolecular hydrogen bonding on the short PLT segments. Upon forming
calculation of RCD/peptide was based on the average molecular weight of alkyl-PLT (Table S2). Rather, mixing CD with C6PLT20, alkyl-poly(L-lysine) (C16PLL20), or alkyl-poly(Lglutamic acid) (C16PGA20) at Cpeptide ≤ 10 wt %, this process did not result in the formation of supramolecular hydrogel. The gel-to-sol phase transition temperatures (Tgel) of the hydrogels determined by test tube inversion method, that typically ranged between 5 and 55 °C, were found to be a function of Cpeptide and RCD/peptide. The gelling ability of C 12PLT20/α-CD (C16PLT20/β-CD) was better than that of C12PLT20/β-CD (C16PLT20/α-CD). At Tgel = 30 °C, the gelling concentration (Cgel) of C12PLT20/α-CD and C16PLT20/β-CD with RCD/peptide = 2 were determined to be about 5.2 and 4.0 wt %, respectively. Rather, C12PLT20/β-CD and C16PLT20/α-CD with the same Cpeptide did not form supramolecular hydrogel at the same temperature. At fixed Cpeptide, decreasing RCD/peptide would result in the decrement of the Tgel. For example, at Cpeptide = 6 wt %, the Tgels of C12PLT20/α-CD and C16PLT20/β-CD with RCD/peptide = 2 were determined to be about 35 and 47 °C, respectively. The Tgel of these hydrogels decreased to 10−13 °C upon decreasing RCD/peptide to 1. The host−guest interactions between alkyl chains and CDs were first investigated. Consistent with previous studies,13,14 the formation of channel-type inclusion complex between C16PLT20 and β-CD was evident by comparing the C16PLT20 alone solution (spectrum d; Figure 2) with that of C16PLT20/β1202
DOI: 10.1021/acsmacrolett.6b00716 ACS Macro Lett. 2016, 5, 1201−1205
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ACS Macro Letters
Figure 3. CD spectra of (a) C12PLT20/α-CD and (b) C16PLT20/β-CD with RCD/peptide = 0, 1, 2, and 4; CD spectra of (c) C12PLT20/α-CD and (d) C16PLT20/β-CD with RCD/peptide = 2 at varying temperatures. The polypeptide concentration of the samples is 0.25 mg/mL. The α-CD concentrations for C12PLT20/α-CD sample with RCD/peptide = 1, 2, and 4 were 0.116, 0.232, and 0.463 mg/mL, respectively. The β-CD concentrations for C16PLT20/β-CD sample with RCD/peptide = 1, 2, and 4 were 0.111, 0.222, and 0.443 mg/mL, respectively.
with the gelation tests, showing that C16PLT20/β-CD hydrogel exhibited better gelling ability than C12PLT20/α-CD hydrogel. The molecular assembly of C12PLT20/α-CD and C16PLT20/ β-CD hydrogels was further characterized by X-ray diffraction (XRD) and small-angle X-ray scattering (SAXS) analyses. XRD patterns of C12PLT20 and C16PLT20 powders exhibited two major peaks at 2θ = 9.4° and 18.9°, giving the d spacing to be 0.94 and 0.46 nm, respectively (Figure 4). The d spacing of 0.46 nm can be attributed to the formation of antiparallel βsheet conformation.18−20 The d spacing of 0.94 nm was from the alternative packing of alkyl chains. The much broader peak at 2θ > 20° for C12PLT20 than that for C16PLT20 suggested that C12PLT20 adopted more coil conformation than C16PLT20 did. XRD patterns of α-CD and β-CD powders showed a major peak at 11.8° and 12.5°, respectively, with many other diffraction peaks, suggesting a high degree of crystallinity. Upon the formation of hydrogels with CD, C12PLT20/α-CD and C16PLT20/β-CD hydrogels exhibited two major peaks at 2θ = 11.5° and 28.2°. The peak at 2θ = 11.5°, corresponding to the d spacing of 0.77 nm, was the height of CD, suggesting the crystallization of CD upon forming channel-type inclusion complex. The hydrogels exhibited low degree of crystallinity, evidenced by the broad peak at 2θ = 28.2°. The peak at 2θ = 11.5° for C12PLT20/α-CD hydrogel was broader than that for C16PLT20/β-CD hydrogel, suggesting C12PLT20/α-CD hydrogel exhibited lower degree of crystallinity than C16PLT20/β-CD hydrogel. Two humps at 2θ = 18.9° and 21.8° can be clearly observed for C16PLT20/β-CD hydrogel (Cpeptide = 5.0 wt % and RCD/peptide = 2), but they are not clear for C12PLT20/α-CD hydrogel, revealing the presence of ordered conformation for C16PLT20. SAXS pattern of C12PLT20/α-CD hydrogel showed a set of Bragg peaks with scattering wave vector ratio of q1/2q1/ 3q1 (Figure 5a), suggesting the lamellar packing of C12PLT20/
inclusion complex, C16PLT20 remained adopting mainly sheet conformation (Figure 3b and Table S3). Rather, the addition of α-CD (RCD/peptide = 1) to C12PLT20 solution triggered sheet-tocoil conformational transition and C12PLT20 adopted mainly random coil (69.2%) conformation (Table S3). Further increasing RCD/peptide to 4 resulted in a slight increase of sheet conformation. For C12PLT20/α-CD and C16PLT20/β-CD inclusion complexes with RCD/peptide = 2, C12PLT20 and C16PLT20 adopted mainly coil (61.2%) and β-sheet (61.8%) conformations, respectively. Upon increasing temperature from 25 to 60 °C, C16PLT20 remained adopting mainly sheet conformation, with an increase in helical conformation (∼12%), and C12PLT20 rather adopted coil conformation with lower percentage (∼54%; Figure 3d and Table S4). The influence of the host−guest and polypeptide hydrogenbonding interactions on the gel molecular assembly and property was then investigated. The mechanical properties of the selected C12PLT20/α-CD and C16PLT20/β-CD hydrogels at Cpeptide = 5.0 wt % and RCD/peptide = 2 were measured at varying temperatures by using an oscillatory shear rheometer (Figures S4 and S5). At 25 °C, the storage modulus G′ was larger than the loss modulus G″, indicating the formation of alkyl-PLT/CD hydrogels via the formation of inclusion complex and hydrogen bond. The small magnitude of G′ for C12PLT20/α-CD showed that it was a relatively weak gel at 5.0 wt %, which is just above its gelation threshold. Upon increasing the temperature, the magnitude of G′ (or G″) and the G′/G″ ratio decreased, suggesting the decrease in the gel strength. The mechanical properties of C16PLT20/β-CD hydrogel were superior than that of C12PLT20/α-CD hydrogel, evidenced by the higher magnitude of G′ (or G″) for C16PLT20/β-CD hydrogel than that of C12PLT20/α-CD hydrogel. These results are consistent 1203
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Figure 4. XRD patterns of (a) α-CD powder (1), C12PLT20 powder (2), and C12PLT20/α-CD hydrogels (3−5) with and (b) β-CD powder (1), C16PLT20 powder (2), and C16PLT20/β-CD hydrogels (3−5). The samples 3 and 5 were prepared at 9 wt % of peptide concentration with RCD/peptide = 2 and 1, respectively. The sample 4 was at 6 wt % of peptide concentration with RCD/peptide = 2. The α-CD concentrations for samples 3−5 (a) were 91.6, 59.1, and 45.8 mg/mL, respectively. The β-CD concentrations for samples 3−5 (b) were 87.7, 56.6, and 43.8 mg/mL, respectively.
Figure 5. SAXS patterns of (a) C12PLT20/α-CD and (b) C16PLT20/βCD hydrogels with RCD/peptide = 2 at varying temperatures. The polypeptide concentration of the samples is 6.0 wt %. The α-CD and β-CD concentrations were 59.1 and 56.6 mg/mL in C12PLT20/α-CD and C16PLT20/β-CD hydrogels, respectively.
C 12 PLT 20 /α-CD lamellar thickness and dimension of C16PLT20/β-CD assemblies based on CD and SAXS results. In addition, the hydrogen-bonding interactions decreased upon increasing solution temperature. Consequently, the combined effects led to the decrease in gel strength and eventually the collapse of the gel network. In conclusion, we demonstrated the preparation of alkylpoly(L-threonine)/cyclodextrin (alkyl-PLT/CD) supra-molecular hydrogels with molecular assembly and property determined by the host−guest chemistry and hydrogenbonding interactions. For dodecyl-PLT20/α-CD hydrogel, the formation of inclusion complex triggered the chain conformational change of PLT and dodecyl-PLT20 adopted mainly coil conformation and, for hexadecyl-PLT 20/β-CD hydrogel, hexadecyl-PLT20 remained adopting mainly sheet conformation. Consequently, these hydrogels exhibited different molecular packings and gel properties including strength and gel-to-sol phase behavior. The results demonstrated that this is a viable approach to prepare hydrogels with versatile molecular assembly and property.
α-CD.21 It can be seen that the scattering wave vector q1 increased with the increase of temperature. The low intensity at the diffraction angles of 2q1 and 3q1 suggested the lamellar packing did not exhibited long-range order. For C12PLT20/αCD, the scattering wave vector, q1, at 25 °C was 0.075 Å−1, corresponding to the d spacing of 8.38 nm. Upon increasing the temperature to 35 and 55 °C, the scattering wave vector shifted to 0.077 and 0.083 Å−1, respectively, corresponding to the d spacing of 8.14 and 7.59 nm, respectively. SAXS patterns of C16PLT20/β-CD hydrogel did not exhibit observable peak (Figure 5b), suggesting that C16PLT20/β-CD did not form ordered molecular packing. TEM analysis showed that C16PLT20/β-CD self-assembled to form nanoribbons (Figure S6). The molecular assemblies of C 12 PLT 20 /α-CD and C16PLT20/β-CD are proposed as shown in Figure 1b. C16PLT20/β-CD hydrogel exhibited nanoribbon-like structure given that C16PLT20 adopted mainly sheet conformation. For C12PLT20/α-CD hydrogel, the PLT20 adopted parallel packing via partial intermolecular hydrogen bonding, while the inclusion complexes crystallized to align together via hydrogen bonding. The contour lengths of the coil PLT20 and alkyl chain (C12) were calculated to be about 6.0 and 1.4 nm, respectively.22,23 The d spacing of 8.38 nm, corresponding to the thickness of one C12PLT20/α-CD lamellar at 25 °C, is in agreement with the length of PLT20 domain plus the length of the two adjacent inclusion complexes. Upon increasing solution temperature, the PLT20 contour length decreased due to the partially peptide conformational change, resulting in the decrease in both the
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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/acsmacrolett.6b00716. Experimental details and characterization data (PDF).
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AUTHOR INFORMATION
Corresponding Author
*Fax: +886-6-2344496. E-mail:
[email protected]. 1204
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ACS Macro Letters Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the financial support from the Ministry of Science and Technology Taiwan grant MOST 104−2811-E006−243 and Headquarters of University Advancement at National Cheng Kung University, which is sponsored by the Ministry of Education, Taiwan, ROC. The authors acknowledge S.S.-S. Wang for access to circular dichroism.
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