Letter Cite This: ACS Macro Lett. 2018, 7, 944−949
pubs.acs.org/macroletters
Facile Approach to Covalent Copolypeptide Hydrogels and Hybrid Organohydrogels Saltuk B. Hanay,† Joanne O’Dwyer,‡,§ Scott D. Kimmins,† Fernando C. S. de Oliveira,† Matthew G. Haugh,§ Fergal J. O’Brien,§,∥,⊥,# Sally-Ann Cryan,‡,∥,⊥ and Andreas Heise*,†,⊥,# †
Department of Chemistry, Royal College of Surgeons in Ireland, Dublin 2, Ireland Drug Delivery and Advanced Materials Team, School of Pharmacy, RCSI, Dublin 2, Ireland § Tissue Engineering Research Group, Department of Anatomy, RCSI, Dublin 2, Ireland ∥ Trinity Centre for Bioengineering, Trinity College Dublin (TCD), Dublin 2, Ireland ⊥ Centre for Research in Medical Devices (CURAM), RCSI, Dublin 2, and National University of Ireland, Galway, Ireland # Advanced Materials and Bioengineering Research Centre (AMBER) RCSI and TCD, Dublin 2, Ireland
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‡
S Supporting Information *
ABSTRACT: Crosslinking of tryptophan (Trp) containing copolypeptides with varying ratios of benzyl-L-glutamate (BLG) and Nα-(carbobenzyloxy)-L-lysine (Z-Lys) is achieved by the selective reaction with hexamethylene-bis-TAD (bisTAD). Conversion of the resulting organogels into biocompatible hydrogels by full BLG or Z-Lys deprotection is demonstrated. Moreover, diffusion controlled deprotection allows the design of macroscopic hybrid organohydrogels comprising hydrophilic as well as hydrophobic regions at a desired ratio and position. FTIR and SEM analysis confirm the coexistence of both hydrophilic and hydrophobic segments in one copolypeptide piece. Selective loading of hydrogel and organogel segments with hydrophilic and hydrophobic dyes, respectively, is observed on macroscopic amphiphilic gels and films. These materials offer significant potential as dualloaded drug release gels as well as tissue engineering platforms.
H
photocrosslinking,29,30 chemically crosslinked polypeptide hydrogels have not been widely reported to date.31−33 Generally, all crosslinking chemistries necessitate the synthesis of reactive amino acids and the protection of the amino acids to facilitate compatibility with the NCA chemistry followed by deprotection of the polymer to allow the crosslinking reaction to be carried out. The fast and selective reaction of triazolinedione (TAD) with indole groups offers a simpler alternative,34,35 and we have recently shown its feasibility in the reaction with tryptophan (Trp) containing polypeptides omitting protection/ deprotection strategies.36 Here we demonstrate that this reaction is highly suitable for the design of novel polypeptide hydrogels. Moreover, we disclose a strategy for the design of macroscopic organohydrogels (amphiphilic gels, hybrid gels). Organohydrogels comprise regions of organogels and hydrogels with unique mechanical and loading properties.37−40 To date there are only a few reports on macroscopic organohydrogels. In one approach two gels are “glued” together,41 while in another approach, two different viscous (macro)monomers are polymerized in contact with each other.42−44
ydrogels have been broadly explored in many biomedical areas, including drug delivery,1 regenerative medicine,2−6 and wound dressings,7 among others. Hydrogels can be noncovalent, but these often do not meet the minimum mechanical application requirements for specific applications. Covalent hydrogels are generally stronger and can, for example, readily be obtained from hydrophilic acrylates. However, the resulting materials lack biodegradability, which is a desirable feature in applications where hydrogel removal would stipulate surgical intervention. Natural biodegradable hydrophilic polymers such as collagen or chitosan are therefore often favored by biomedical engineers for in vivo applications.8 However, lack of readily accessible functional groups, compositional variabilities depending on source, and possible immune responses are among their drawbacks.9 Polymers consisting of natural building blocks that grant control over composition and functionalization are interesting alternatives.10 Synthetic polypeptides from ring-opening polymerization of amino acid N-carboxyanydrides (NCA) meet these demands and have been studied as hydrogel forming building blocks.11−14 Most reported examples are based on the self-assembly of amphiphilic block copolypeptides into physical hydrogels.15−24 While some strategies for chemical crosslinking of polypeptides were devised, for example, using difunctional NCA monomer,25 disulfide formation,26−28 or © XXXX American Chemical Society
Received: June 6, 2018 Accepted: July 13, 2018
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DOI: 10.1021/acsmacrolett.8b00431 ACS Macro Lett. 2018, 7, 944−949
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ACS Macro Letters Scheme 1. Synthesis of Poly(L-Lysine)-Based Organogels and Hydrogels
A set of tryptophan (Trp) containing copolypeptides with varying ratios of benzyl-L-glutamate (BLG) and Nα-(carbobenzyloxy)-L-lysine (Z-Lys), respectively, was prepared by the copolymerization of the respective NCAs (Scheme 1 and Scheme S1, SI). Size exclusion chromatography (SEC; Figure S1, SI) results highlight good polymerization control and 1H NMR spectra confirm agreement of the copolymer composition with the monomer feed ratio (Figures S2 and S3 and Table S1, SI). Polymer crosslinking was trialed by mixing the copolymers with hexamethylene-bis-TAD (bisTAD) in chloroform. It was found that the TAD-tryptophan reaction occurred so fast in this solvent that it prevented thorough mixing of the reactants before gelation. It was overserved that the gelation rate could be reduced by using a chloroform/acetonitrile 1:1 (v/v) mixture (copolymer concentration: 100 mg/mL). Under these conditions the gelation time was proportional to the amount of Trp in the copolymer as monitored by the vial inversion method (Table 1). The organogels had an opaque to transparent appearance (Figure 1). In rare cases, an orange discoloration could be observed due to unreacted TAD still remaining in the gel, which could be removed in the subsequent reaction and washing steps. Large pieces (∼5 mL) of organogels were readily obtained from all copolypeptides by crosslinking under ambient conditions in a disposable syringe barrel and removing the organogel under moderate pressure (Figure 1, Figure S4, SI, Movie 1). The selectivity of the Trp-TAD reaction allows the gels to be prepared in any mold with the advantage over other crosslinking methods that no prior activation or deprotection is required. For the conversion into hydrogels, the organogel pieces were immersed into a solution containing HBr (in acetic
Table 1. Copolypeptides Used in Hydrogel Synthesis and Their Gelation Times by Reaction with Hexamethylene-BisTAD (bisTAD); Dispersities (Đ) of All Polymers < 1.2 sample
theoretical composition
Mna (g/mol)
molar ratio bisTAD/Trp
gelation timeb (min)
L1 L1b L2 L2b L3 G1 G2 G3
ZLys70Trp30 ZLys70Trp30 ZLys80Trp20 ZLys80Trp20 ZLys90Trp10 BLG70Trp30 BLG80Trp20 BLG90Trp10
11000 11000 13000 13000 12000 10000 12000 8000
1:2 1:4 1:2 1:4 1:2 1:2 1:2 1:2
1−2 2−3 8 60 90 1−2 6 40
a
Measured in DMF against PS standards, theoretical Mn: 22000 g/mol). Determined in chloroform/aceronitrile 1:1 (v/v) at a copolymer concentration of 100 mg/mL and bisTAD of 33 mg/mL by vial inversion method.
b
acid) in chloroform for a defined period of time followed by soaking the hydrogels in DMF and water to remove all unreacted acid residues (Scheme 1 and Scheme S1, SI). Glutamic acid hydrogels were further treated with a K2CO3 solution to ionize the carboxylic acid groups and increase their hydrophilicity. It was observed that if the deprotection agents were not thoroughly removed by washing they caused hydrolysis of the hydrogel, while properly washed hydrogels were stable for at least 12 months in deionized water. Hydrogels are denoted HL# and HG# when obtained from the Lys L-series and glutamic acid G-series, respectively (for example, HL1: hydrogel obtained from sample L1 in Table 1). 945
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ACS Macro Letters
hydrogels relative to the cells alone. This effect was significant for the HG1 hydrogel at a metabolic activity of 65% after 168 h, while HL1 retained a metabolic activity similar to the control group, suggesting no cytotoxic effect. The reason for this difference has not been further investigated. The conversion of the organogels into hydrogels by Z-Lys or BLG deprotection is governed by the diffusion of acid through the solid hydrophobic organogel network. The hydrogel formation is relatively slow and occurs from the outer surface toward the core following the diffusion of the deprotecting agent. The slow diffusion is probably caused by shrinking of the deprotected networks as the deprotection medium is a poor solvent for the hydrogel. It was hypothesized that novel materials combining hydrophobic organogel and hydrophilic hydrogel sections could be obtained from the crosslinked organogel precursors by a time controlled deprotection reaction. In initial trails smooth cylindrical organogels were prepared and deprotected under gentle conditions (nonstirring). The first test was done with a low molecular weight copolypeptide poly(Z-Lys10-st-Trp5), which is soluble in DMF in its protected and deprotected form. After crosslinking with bisTAD (Figure S7a, SI), the organogel was placed into a TFA/CHCl3 solvent mixture and treated with HBr (Figure S7b, SI). After 1.5 h, the material was taken from the solution and washed with diethyl ether. At this stage the material had shrunken and became opaque. Finally, it was placed in DMF and left until fully swollen. Unreacted hydrophobic reddish TAD acts as a convenient reporter to visualize the hydrophobic region of the organohydrogel. In Figure 3 (top image) two distinct regions can be observed, with a reddish hydrophobic organogel core surrounded by a clear hydrogel shell. The experiment was repeated with higher molecular weight copolypeptides L1, poly(Lys70-st-Trp30) and the same core−shell structure was obtained after optimization of deprotection time and acid amount. It was possible to remove excess bisTAD by solvent washing. Initially, diethyl ether was used and while the reddish color could be removed it resulted in cracked organohydrogels presumably due to the high vapor pressure of diethyl ether (Figure S6, SI). Washing with excess DMF instead produced clear, intact organohydrogels. FTIR signature peaks of the Z-protecting group at 1686 (CO) and 1247 cm−1 (C−O) confirm the fully protected core polypeptide, while these peaks are absent in the deprotected shell (Figure S8, SI). The different regions of the organohydrogel are also clearly distinguishable in SEM images (Figure 3a−c). The hydrogel shell is characterized by a porous structure similar to that of the full hydrogels depicted in Figure 1, while the hydrophobic core network appears as a collapsed solid material. SEM images taken of the interface display both morphologies with a sharp border between the hydrogel and the organogel region. In order to obtain images of hydrated organohydrogels, Environmental SEM (ESEM) images were taken of water swollen samples. In agreement with the SEM images, the porous hydrated hydrogel and the collapsed organogel regions can clearly been recognized in the ESEM images (Figure 3d−f), separated by a sharp interface. Notably, both regions of the organohydrogel comprise the same polymeric material and a continuous network structure unlike previously reported organohydrogels. They can be made in any shape and offer new opportunities as biocompatible and degradable platform materials. To demonstrate their potential, dual loading was trialed with model dyes. In the first instance, a piece of organohydrogel was placed in an aqueous solution of Rhodamine B. Visual inspection confirmed
Figure 1. Organo- (O) and hydrogels (H) obtained from tryptophan containing lysine (L) and glutamic acid (G) copolypeptides and SEM images of the freeze-dried hydrogels (scale bars 2 μm).
SEM images taken of freeze-dried hydrogels (Figure 1) revealed porous structures for all Lys hydrogels. Notably, glutamic acid derived hydrogels HG1 and HG2 did not show any noticeable porous microstructure under the applied conditions. This reflects in the higher Young’s moduli of the HG samples (∼11 kPa, Figure S5). Lysine hydrogels obtained from copolypeptides with lower amounts of Try or lower amounts of added bisTAD resulted in Young’s moduli of around 4 kPa. The highest modulus was measured for HL1 (12 kPa), which correlate to the copolypeptide with the highest Trp content. Preliminary biocompatibility data was obtained using HL1 and HG1 hydrogels using human mesenchymal stem cells (hMSCs). Fluorescence microscope images of cells exposed to the hydrogel media (Figure 2) qualitatively confirmed cell
Figure 2. Top: Metabolic activity at different time points of human mesenchymal stem cells (hMSCs) exposed to glutamic acid (HG1) and lysine (HL1) hydrogels. Bottom: Live/dead analysis of hMSCs after 72 h exposure to said hydrogels (green cells, alive; red cells, dead); **p < 0.01.
viability for both samples after 72 h with significant numbers of viable cells visible after exposure to both hydrogels. This was quantitatively corroborated by examining hMSC metabolic activity using an MTS assay. No statistically significant differences in metabolic activity existed between the hydrogel-treated cells and untreated cells (cells alone) up to 72 h. At longer exposure time, a slight drop of the metabolic activity was observed for both 946
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Figure 3. Top: Partially deprotected organohydrogel swollen in DMF. Bottom: SEM images (a−c) of freeze-dried samples and ESEM images (d−f) in water taken at different regions of the organohydrogel. From left to right: hydrogel, interface, and organogel regions.
Figure 4. (a) Organohydrogel soak-loaded with Rhodamine B; (b) cut through Rhodamine B loaded organohydrogel. (c) Organohydrogel dyed with Reichardt’s dye in THF followed by reswelling in water. (d) Organogel film from copolypeptide L2; (e) water-soaked film after HBr solution spotted onto film by TLC capillary tube; (f) patterned film exposed to Rhodamine B solution.
that the hydrophilic dye only penetrated into the hydrophilic hydrogel shell, while the hydrophobic core remained dye free
(Figure 4a,b). The reverse effect was observed when hydrophobic Reichardt’s dye was used in THF solution accumulating 947
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(6) Lin, C. C.; Anseth, K. S. PEG Hydrogels for the Controlled Release of Biomolecules in Regenerative Medicine. Pharm. Res. 2009, 26, 631−643. (7) Kamoun, E. A.; Kenawy, E-RS; Chen, X. A Review on Polymeric Hydrogel Membranes for Wound Dressing Applications: PVA-based Hydrogel Dressings. J. Adv. Res. 2017, 8, 217−233. (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) Hersel, U.; Dahmen, C.; Kessler, H. RGD Modified Polymers: Biomaterials for Stimulated Cell Adhesion and Beyond. Biomaterials 2003, 24, 4385−4415. (10) Benoit, D. S. W.; Anseth, K. S. The Effect on Osteoblast Function of Colocalized RGD and PHSRN Epitopes on PEG Surfaces. Biomaterials 2005, 26, 5209−5220. (11) Tian, H.; Tang, Z.; Zhuang, X.; Chen, X.; Jing, X. Biodegradable Synthetic Polymers: Preparation, Functionalization and Biomedical Application. Prog. Polym. Sci. 2012, 37, 237−280. (12) Shirbin, S. J.; Karimi, F.; Chan, N. J. A.; Heath, D. E.; Qiao, G. G. Macroporous Hydrogels Composed Entirely of Synthetic Polypeptides: Biocompatible and Enzyme Biodegradable 3D Cellular Scaffolds. Biomacromolecules 2016, 17, 2981−2991. (13) Ahrens, C. C.; Welch, M. E.; Griffith, L. G.; Hammond, P. T. Uncharged Helical Modular Polypeptide Hydrogels for Cellular Scaffolds. Biomacromolecules 2015, 16, 3774−3783. (14) Cui, H.; Zhuang, X.; He, C.; Wei, Y.; Chen, X. High Performance and Reversible Ionic Polypeptide Hydrogel Based on Charge-Driven Assembly for Biomedical Applications. Acta Biomater. 2015, 11, 183−190. (15) 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. (16) Pakstis, L. M.; Ozbas, B.; Hales, K. D.; Nowak, A. P.; Deming, T. J.; Pochan, D. Effect of Chemistry and Morphology on the Biofunctionality of Self-Assembling Diblock Copolypeptide Hydrogels. Biomacromolecules 2004, 5, 312−318. (17) Pochan, D. J.; Pakstis, L.; Ozbas, B.; Nowak, A. P.; Deming, T. J. SANS and Cryo-TEM Study of Self-Assembled Diblock Copolypeptide Hydrogels with Rich Nano- through Microscale Morphology. Macromolecules 2002, 35, 5358−5360. (18) 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. (19) 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. (20) Fan, J.; Li, R.; Wang, H.; He, X.; Nguyen, T. P.; Letteri, R. A.; Zou, J.; Wooley, K. L. Multi-Responsive Polypeptide Hydrogels Derived from N-Carboxyanhydride Terpolymerizations for Delivery of Nonsteroidal Anti-Inflammatory Drugs. Org. Biomol. Chem. 2017, 15, 5145−5154. (21) Shinde, U. P.; Joo, M. K.; Moon, H. J.; Jeong, B. J. Sol−Gel Transition of PEG−PAF Aqueous Solution and its Application for hGH Sustained Release. J. Mater. Chem. 2012, 22, 6072−6079. (22) Murphy, R.; Borase, T.; Payne, C.; O’Dwyer, J.; Cryan, S.-A.; Heise, A. Hydrogels from Amphiphilic Star Block Copolypeptides. RSC Adv. 2016, 6, 23370−23376. (23) 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. (24) Oh, H. J.; Joo, M. K.; Sohn, Y. S.; Jeong, B. Secondary Structure Effect of Polypeptide on Reverse Thermal Gelation and Degradation of L/DL-Poly(Alanine)−Poloxamer−L/DL-Poly(Alanine) Copolymers. Macromolecules 2008, 41, 8204−8209.
in the core of the organohydrogel (Figure 4c). As a second example, a thin layer of copolypeptide organogel was prepared in glass Petri dishes (Figure 4d). Subsequently, drops of deprotecting HBr in chloroform were placed on the organogel surface followed by washing. Exposing of the gel layer to water reveals the hydrophilic pattern as evidenced by the hydrogel spots on the hydrophobic film (Figure 4e). When the gel was then covered with an aqueous solution of hydrophilic Rhodamine B (20 mg/mL overnight), they dye accumulated in the hydrophilic spots (Figure 4f). While just a pilot experiment, it demonstrates that surface patterning can be achieved with reasonable resolution governed by spatially confined deprotection of the crosslinked copolypeptide. In conclusion, we have demonstrated a novel approach to preparing biocompatible organo- and hydrogels utilizing the selective crosslinking reaction between bisTAD and tryptophan. The robustness of the crosslinking reaction puts no limitations on the size and shape of the gel mold. Selective deprotection allows the design of hybrid gels comprising hydrophilic as well as hydrophobic regions at a desired ratio and position, which can be selectively loaded with small molecules of opposite polarity. These materials offer significant potential as dual drug release gels as well as tissue engineering platforms.
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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.8b00431. Experimental procedures, SEC plots, mechanical analysis, FTIR analysis, and additional images (PDF). Supporting movie, Movie S1 (MOV).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Fergal J. O’Brien: 0000-0003-2030-8005 Sally-Ann Cryan: 0000-0002-3941-496X Andreas Heise: 0000-0001-5916-8500 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was undertaken as part of the Translational Research in Nanomedical Devices (TREND) research project facilitated via a Science Foundation Ireland Investigators Program 13/IA/1840.
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REFERENCES
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