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Uncharged Helical Modular Polypeptide Hydrogels for Cellular Scaffolds Caroline Chopko Ahrens, M. Elizabeth Welch, Linda G. Griffith, and Paula T. Hammond Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01076 • Publication Date (Web): 13 Oct 2015 Downloaded from http://pubs.acs.org on October 17, 2015
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Uncharged Helical Modular Polypeptide Hydrogels for Cellular Scaffolds Caroline C. Ahrens,|| M. Elizabeth Welch,|| Linda G. Griffith, †¶ and Paula T. Hammond||δ*
||
Department of Chemical Engineering, δKoch Institute for Integrative Cancer Research,
†
Department of Biological Engineering, and ¶Center for Gynepathology Research, Massachusetts
Institute of Technology, Cambridge, MA, USA KEYWORDS Step growth hydrogels; synthetic polypeptide; N-carboxyanhydride; poly(γpropargyl-L-glutamate) (PPLG); click-functionalized hydrogels; synthetic extracellular matrix
ABSTRACT Grafted synthetic polypeptides hold appeal for extending the range of biophysical properties achievable in synthetic extracellular matrix (ECM) hydrogels. Here, Ncarboxyanhydride polypeptide, poly(γ-propargyl-L-glutamate) (PPLG)
macromers were
generated by fully grafting the “clickable” sidechains with mixtures of short polyethylene glycol (PEG) chains terminated with inert (-OH ) or reactive (maleimide and/or norbornene) groups, then reacting a fraction of these groups with an RGD cell attachment motif. A panel of synthetic hydrogels was then created by crosslinking the PPLG macromers with a 4-arm PEG star molecule. Compared to well-established PEG-only hydrogels, gels containing PPLG exhibited dramatically less dependence on swelling as a function of crosslink density. Further, PPLG-containing gels, which retain an alpha-helical chain conformation, were more effective
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than standard PEG gels in fostering attachment of a human mesenchymal stem cell (hMSC) line for a given concentration of RGD in the gel. These favourable properties of PPLG-containing PEG hydrogels suggest they may find broad use in synthetic ECM.
Poly(ethylene glycol) (PEG) crosslinked hydrogels have been extensively explored as a platform for creating synthetic extracellular matrix (ECM) for tissue engineering applications, through modification of multifunctional PEG macromers with crosslinking and adhesion peptides.1,2 The field has expanded from initial studies of gels made from free radical crosslinked PEG macromers, in which crosslinked PEG chains extend from radical-polymerized hydrocarbon backbones,3 to include gels made from step-growth crosslinking approaches, in which multi-arm PEG macromers with complementary endgroups react to form a crosslinked network.4,5 Despite proven utility and continual advances in the field of step-growth PEG hydrogels, established gels only partly capture all the desirable features of the native ECM in terms of eliciting desired cellular responses, due in part to the difficulty of presenting sufficient densities of functional groups from the polyether backbone. To address these limitations, we introduce here a new hydrogel platform in which standard 4-arm PEG macromers are combined with a novel macromer comprising an α-helical polypeptide grafted with a dense brush containing a mixture of inert and reactive short ethylene glycol chains, such that the macromer can be modified with adhesion ligands while retaining crosslinking functionalities. These hybrid PEG-polypeptide gels, crosslinked with macromers possessing both defined secondary structure and readily-functionalized side chains, offer at least three key enhancements to PEG-only hydrogel systems used for synthetic ECM: (i) potential for systematic presentation of highly clustered adhesion ligands on an α-helical scaffold via modification of reactive side chains; (ii) a more diverse spectrum of nano- and meso-scale mechanical properties than standard PEG gels,
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including values in a range more closely mimicking those of the native ECM and (iii) minimal swelling of crosslinked gels. Despite the continued interest in both grafted synthetic polypeptides,
6–12
and in step-growth
PEG hydrogels formed from -functional and star macromers and even proteins,13 the literature offers limited precedence for step-growth hydrogels in which a synthetic polypeptide acts as the primary polymer backbone in the network. Established polypeptide gels rely almost exclusively on crosslinking charged polypeptides from native amino acids such as poly(glutamic acid),14,15 poly(lysine),16 and poly(aspartic acid),17 with only recent work introducing nanogels crosslinked through photodimerization of polypeptide-side chain cinnamyloxy groups18 or horseradish peroxidase mediated enzymatic crosslinking.19 Hydrogels formed from neutral synthetic polypeptides have consisted almost exclusively of physical self-assembled gels,6,20,21 rather than covalent crosslinking which is introduced here. One specific N-carboxyanhydride polymerized polypeptide, poly(γ-propargyl
L-glutamate)
(PPLG), was previously established in the Hammond group22 and forms the foundation of this work. As outlined in a recent review,23 this polypeptide’s pendant alkynes allow grafting of a wide variety of azide functionalized side chains via copper catalyzed 1,3-cyclo addition. Examples of reported grafting groups include long PEG chains,22 short sugar molecules,24 amines,25 sulfonate ions,26 and thermoresponsive ethylene glycol grafting groups.27 The geometry associated with the robust α-helix of the polypeptide both before and after grafting of the polypeptide allows for almost perfect grafting efficiency, largely insensitive to the properties of the side chains azide groups.22 We demonstrated that step-growth hydrogels crosslinked through grafted α-helical polypeptides exhibit an increased elastic modulus compared to gels from polypeptides having random coil secondary structure.28 Gel crosslinking was demonstrated
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through anhydrous activation of ethylene oxide grafted PPLG by a non-specific coupling agent in organic solvent, and, without purification, crosslinking through 4-arm PEG-thiol (10K).28 In this report, a new PPLG crosslinked hydrogel platform is developed by introducing a waterbased modular, biocompatible, macromer synthetic strategy allowing peptide modification and cell encapsulation.
Two different crosslinking moieties are used to create systems that can be
singly as well as doubly crosslinked in a subsequent step. Specifically, a 4-arm PEG-thiol is crosslinked with PPLG macromonomers pre-grafted with a short PEG brush presenting a mixture of two orthogonal crosslinking chemistries: maleimides and norbornenes. Maleimides react extremely efficiently in a pH dependent addition reaction with thiols29–31 while norbornenes react with thiols in UV activated thiol-ene reactions.32 This design, which allows double crosslinking, builds on recent advances in fabricating cell culture systems engineered with temporal and spatial modulation of both mechanical properties33–35 and bio-active grafted groups.35,36 Fabricating hydrogels from polypeptide macromers having orthogonal groups leverages the robust, efficient, and tractable PPLG grafting to enable synthetic controls of a wide range of well-characterized and highly versatile hydrogel systems. The strategies presented here for grafting norbornene and maleimide functionality onto PPLG can be readily extended to introduce a variety of crosslinking chemistries,37,38 crosslinker lengths,39 and solubilizing brushes, creating a versatile system for biomolecular scaffolds and tunable 3D cellular environments. Finally, the modular synthesis of crosslinker-grafted PPLG polypeptides enables foundational theoretical and experimental characterization of how introducing polypeptide α-helical crosslinkers can direct bulk gel properties. Materials and Methods
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L-(+)-Glutamic acid 99% minimum was purchased from EMD Chemicals (Gibbstown, NJ). 3Maleimidopropionic acid N-hydroxysuccinimide ester was purchased from Alfa Aesar (Ward Hill, MA). 2-(2-azidoethoxy)ethanol (EO2) was synthesized as previously reported (Chopko et al. 2012). Maleimide functionalized polymers 8-arm PEG-mal (10K) and 8-arm PEG-mal (40K) were purchased from JenKemTechnologyUSA (Plano, TX). 4-arm PEG-thiol (10K) thiol was purchased from Laysan Bio (Arab, Alabama). Stock solutions of multiarm PEG were dissolved at 10 wt% in ultrapure water, pH 5, and stored at -80 °C until use. Gels were UV crosslinked with a PK50 Omnicure series 2000 lamp with a 365 nm filter at reported intensities as measured by Dymax Corp Accu-Cal-50 Smart UV intensity meter from Dymax Corperation (Torrington, Connecticut) which measures UV-A (320-390nm) intensity. Adhesive peptides, GCRE-RGDSPNH2(RGD), negative control peptide, GCRE-RGESP-NH2(RGE), and heparin binding peptide, GCRE-RKRLQVQLSIRT-NH2 (AG73) were synthesized by Boston Open Labs (Fall River, MA). Human telomerase reverse transcriptase (hTERT) immortalized human mesenchymal stem cells (hMSC) were a gift from Dr. Junya Toguchida (Kyoto University, Kyoto, Japan) and were used passage 10 (Okamoto et al. 2002). A µ-Plate Angiogenesis 96 well was purchased from Ibidi, LLC (Verona, WI). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. 10x stock solutions diluted to 1x in the crosslinking gels were made at pH 7.4, pH 6.0, pH 5.3 and pH 5.2. All stock buffer solutions contained 10x phosphate buffered saline (PBS). Additional 200 mM MES was added to stock solutions below pH 7.4 and buffers were titrated with concentrated HCl or NaOH to the designated pH. Synthesis and Characterization.
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Poly(γ-propargyl-L-glutamate) (PPLG). PPLG was synthesized as previously reported.
22,27
The polymer had a degree of polymerization (DOP) of 160 by proton NMR (1H NMR), as determined by comparing heptylamine initiator proteins to those on the PPLG backbone, and a polydispersity index (PDI) of 1.18 as determined by gel permeation chromatography (GPC) against poly(methyl methacrylate) standards.22 5-Norbornene-2-carboxylic acid N-hydroxysuccinimideester. Reaction was adapted from published synthesis.40 During a typical procedure 5-norbornene-2-carboxylic acid, 97% exo isomer, (1g, 7.23 mmol), N-hydroxysuccinimide (1g, 8.7 mmol), and dicyclohexylcarbodiimide (1.79g, 8.7 mmol) were dissolved in 10mL anhydrous tetrahydrofuran (THF) and stirred under N2 for 3 hrs. The reaction was concentrated and purified on a silica column (CH2Cl2) to yield a white solid (95% yield). 1H NMR (400 MHz, CDCl3) δ 6.21 (dd, J = 5.7, 3.0 Hz, 1H), 6.15 (dd, J = 5.7, 3.0 Hz, 1H), 3.35 – 3.23 (m, 1H), 3.00 (s, 1H), 2.84 (d, J = 2.3 Hz, 4H), 2.51 (ddd, J = 9.0, 4.5, 1.4 Hz, 1H), 2.11 – 1.99 (m, 1H), 1.57 – 1.55 (m, 1H), 1.54 – 1.53 (m, 1H), 1.48 – 1.43 (m, 1H). In-situ crosslinker-azide conjugation. During a typical procedure O-(2-aminoethyl)-O′-(2azidoethyl)nonaethylene glycol or N3-PEG10-NH2 (0.032 g, 0.061 mmol), N,N,N′,N′,N′′pentamethyldiethylenetriamine
(PMDETA)
(12.7
μL,
0.061
mmol)
and
N-(3-
maleimidopropionyloxy) succinimide (0.018g, 0.067 mmol) were all dissolved in dry dimethylformamide (DMF) (1.95 mL, amine at 0.03 mmol/mL). After 20 minutes, consumption of the amine was verified by thin layer chromotagrophy. Specifically, lanes were spotted with a 0.75 μL reaction mixture or a standard curve of N3-PEG10-NH2 and PMDETA diluted in DMF from 1/8 to 1/128 of the reaction amine starting concentration and run in a solvent system of methanol:dichloromethane at a volume ratio of 20:1. The plate was stained with ninhydrin (0.04
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g ninhydrin, 40 mL acetone, and 200 μL acetic acid) and resolved with heat. The reaction was assumed complete when the reaction spot for O-(2-aminoethyl)-O′-(2-azidoethyl)nonaethylene glycol, (Rf=0.05) was less intensely pink than the spot of the 1/128 dilution, representing greater than 99.2% amine conversion. Synthesis of N3-PEG10-nobornene is as above, substituting 5norbornene-2-carboxylic acid N-hydroxysuccinimide ester for the activated maleimide. This procedure
was
adopted
from
the
protocols
recommended
by
the
vendor
at
ClickChemistryTools.com. Crosslinker-functionalized PPLG. A typical procedure includes grafting onto the PPLG backbone the azide functionalized maleimide and norbornene crosslinker molecules as side chains
in
a
two-stage
reaction
at
an
overall
target
molar
feed
ratio
of
alkyne/azide/CuBr/PMDETA equal to 1/1.2/0.1/0.1. To target 5% grafting of maleimide functionality, PPLG (0.05 g, 0.30 mmol alkyne repeat units), crude N3-PEG10-maleimide (0.5 ml at 0.03 mmol/mL with 0.0063 μL/mL PMDETA, 0.015 mmol azide) and neat PMDETA (3.1 μL, 0.015 mmol) were all dissolved in DMF (1.25 mL). The copper bromide catalyst (0.002 g, 0.015 mmol) was added to the degassed solution, and the reaction solution was stirred at room temperature. After 1 hour, 2-(2-azidoethoxy)ethanol (0.047 g, 0.359 mmol) was added quickly under a blanket of argon. After another 1 hour, the reaction solution was precipitated in 40 mL cold diethylether, dissolved in 10 mL distilled water, and incubated for 30 min with 5 mg Dowex® M4195 sulfate copper chelating resin. The beads were removed by filtration and the polymer solution was dialyzed against water acidified by HCl (pH
