Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release † ‡ § ∥ ⋈ † ⋈ † † † Lindsay K

Article Cite This: Biomacromolecules 2019, 20, 3340−3351 pubs.acs.org/Biomac Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release † ‡ § ∥ ⋈ † ⋈ † † † Lindsay K. Hill, , , , , Michael Meleties, , Priya Katyal, Xuan Xie, Erika Delgado-Fukushima, † † ⊥ ⊥ # Teeba Jihad, Che-Fu Liu, Sean O’Neill, Raymond S. Tu, P. Douglas Renfrew, # ∇ ○ § ∥ † ∥ ◆ ¶ Richard Bonneau, , , Youssef Z. Wadghiri, , and Jin Kim Montclare*, , , , † Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, Brooklyn, New York 11201, United States ‡ Department of Biomedical Engineering, SUNY Downstate Medical Center, Brooklyn, New York 11203, United States § ∥ Center for Advanced Imaging Innovation and Research (CAI2R) and Bernard and Irene Schwartz Center for Biomedical Imaging, Department of Radiology, New York University School of Medicine, New York, New York 10016, United States ⊥ Chemical Engineering Department, The City College of New York, 160 Convent Avenue, New York, New York 10031, United States (UTC). # Center for Computational Biology, Flatiron Institute, Simons Foundation, New York, New York 10010, United States ∇ Center for Genomics and Systems Biology, New York University, New York, New York 10003, United States ○ Computer Science Department, Courant Institute of Mathematical Sciences, New York University, New York, New York 10009, United States ◆ Department of Chemistry, New York University, New York, New York 10012, United States o legitimately share published articles. ¶ Department of Biomaterials, New York University College of Dentistry, New York, New York 10010, United States *S Supporting Information Downloaded via NEW YORK UNIV on September 13, 2019 at 16:05:26 ABSTRACT: Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution See https://pubs.acs.org/sharingguidelines for options on how t temperature-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here, we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled- coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein-phase behavior and that of synthetic polymer systems. ■ INTRODUCTION protein and peptide engineering have resulted in the design of fi Hydrogels are three-dimensional (3D) crosslinked polymer hydrogels possessing speci c primary sequences and secondary matrices with a high retention of water that are used in applications including tissue engineering, biomaterial implants, Received: January 23, 2019 and drug delivery.1 Although traditional examples of hydrogels Revised: July 21, 2019 have been comprised of synthetic polymers,2 advances in Published: July 29, 2019 © 2019 American Chemical Society 3340 DOI: 10.1021/acs.biomac.9b00107 Biomacromolecules 2019, 20, 3340−3351 Biomacromolecules Article structures ideal for drug binding, stimuli responsiveness, cell sulfate, calcium chloride (CaCl2), manganese chloride tetrahydrate 3 · · adhesion, and mechanical integrity. Though substantial work (MnCl2 4H2O), cobaltous chloride hexahydrate (CoCl2 6H2O), zinc · β 4 α sulfate heptahydrate (ZnSO4 7H2O), boric acid (H3BO3), isopropyl has focused on -structured systems, -helical coiled-coil β − − domains composed of two or more α-helices that form a -D-1-thiogalactopyranoside (IPTG), tris hydrochloride (Tris 5 HCl), Pierce bicinchoninic acid (BCA) assay kit, Pierce snakeskin supercoil have also been employed to generate hydrogels. dialysis tubing 3.5 kDa molecular weight cut off (MWCO), sodium Helical hydrogels include hybrids of the coiled-coil motif and 6−8 dodecyl sulfate (SDS), and Molecular Probes FluoSpheres poly- synthetic polymers, engineered protein-block co-polymers styrene microspheres were acquired from Thermo Fisher Scientific. bearing coiled-coils fused to conformationally distinct All 20 naturally occurring amino acids, nickel(II) chloride 9−18 19−22 · domains, and coiled-coil peptides. These systems hexahydrate (NiCl2 6H2O), sodium molybdate dihydrate 23−29 · have demonstrated evidence of either porous networks or (Na2MoO4 2H2O), iron(III) chloride (FeCl3), and thiamine hydro- interconnected nanofibers, 2.519−20 nm20 in diameter. Such chloride (vitamin B), were purchased from Sigma-Aldrich. Copper(II) matrices are physically crosslinked hydrogels, where physical chloride anhydrous (CuCl2), sodium selenite (Na2SeO3), imidazole, crosslinks are mediated through molecular entanglement or and curcumin were purchased from Acros Organics. Hydrochloric acid (HCl) and Coomassie Brilliant Blue G-250 were purchased from noncovalent interactions including hydrogen bonds, van der ffi 30,31 VWR. HiTrap immobilized metal a nity chromatography (IMAC) Waals, electrostatic, or hydrophobic interactions. The fast flow (FF) 5 mL column for protein purification and Whatman physical interactions eliminate the need for potentially toxic filter paper for transmission electron microscopy (TEM) sample chemical crosslinkers and often permit stimuli-responsive preparation were purchased from GE Healthcare Life Sciences. behavior.32 In the case of coiled-coil fusions that form Macrosep and Microsep Advance centrifugal devices 3 kDa MWCO hydrogels, physical crosslinks are formed via reversible and 0.2 μm syringe filters were purchased from Pall Corporation. intermolecular associations between coiled-coil domains.32 Acrylamide/bis solution (30%) 29:1 and natural polypeptide SDS- Thermoresponsive hydrogels, demonstrating temperature- polyacrylamide gel electrophoresis (SDS-PAGE) standard were sensitive solution-to-gel (sol−gel) transitions, are the most purchased from Bio-Rad. Formvar/carbon-coated copper grids 33 (FCF400-Cu) and 1% uranyl acetate for TEM were purchased from widely studied stimuli-responsive gels. Thermoresponsive Electron Microscopy Sciences. Silicon wafers for scanning electron hydrogels are traditionally described as lower critical solution microscopy (SEM) were purchased from Ted Pella, Inc. temperature (LCST)-type hydrogels, which are miscible at Protein Expression. Chemically competent M15MA E. coli lower temperatures and gel when heated above their critical cells,37 100 μL in volume, carrying the kanamycin-resistant pREP4 transition temperature (LCST), or upper critical solution plasmid38 (Qiagen) were transformed via heat shock with 250 ng of temperature (UCST)-type hydrogels that gel when cooled ampicillin-resistant pQE30/Q plasmid35 maintaining an N-terminal below their critical temperature (UCST).30 Resilin-based 6× histidine tag (6× His-tag). Transformed cells were allowed to μ ° protein-engineered materials are, thus far, the only examples recover in 700 L lysogeny broth and grown at 37 C and 300 rpm for 45 min. Transformed cells were then plated onto tryptic soy agar of single-protein-based UCST-type hydrogels; however, these −1 34 plates containing ampicillin (0.2 mg mL ) and kanamycin (0.35 mg polypeptides require chemical or photochemical crosslinking. mL−1) and allowed to grow overnight at 37 °C for approximately 16 Although coiled-coil peptides have been utilized for hydrogel 19−22 h. Ampicillin and kanamycin were used to positively select for assembly, all are comprised of synthetic peptides that do M15MA cells containing the plasmid vector. Starter cultures were not fit UCST-type hydrogel criteria due to their lack of prepared from single colonies in modified M9 medium (0.5 M 19,20 thermoresponsiveness or their dual-peptide composi- Na2HPO4, 0.22 M KH2PO4, 0.08 M NaCl, and 0.18 M NH4Cl) 21,22 containing all 20 natural amino acids (100 μgmL−1), ampicillin (0.2 tion. To date, there are no described thermoresponsive − − − hydrogels, including those with UCST-type behavior, mg mL 1), kanamycin (0.35 mg mL 1), vitamin B (0.034 mg mL 1), −1 −1 D-glucose (0.1 mg mL ), magnesium sulfate (0.22 mg mL ), comprised solely of a single coiled-coil protein. −1 The study described herein investigates the ability of a calcium chloride (0.01 mg mL ), and trace metals (0.02% (v/v), see the Supporting Information). Starter cultures were incubated at 37 °C rationally designed coiled-coil protein, Q,35,36 to self-assemble fi fi and 350 rpm for 16 h. Single starter cultures were added to expression into a brous network-based hydrogel. This is the rst report flasks at 4% (v/v) in 400 mL of M9 media, supplemented as described of a thermoresponsive hydrogel solely comprised of a single α- above, and incubated at 37 °C and 350 rpm for approximately 3 h helical coiled-coil domain. The physically crosslinked Q gel until the optical density at 600 nm was 0.7−0.9. Protein Q expression demonstrates a porous network composed of interconnected was induced with IPTG (200 μgmL−1) and returned to 37 °C and nanofibers with reversible thermoresponsiveness

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