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Cite This: ACS Appl. Bio Mater. 2019, 2, 2812−2821
Design and Characterization of Nucleopeptides for Hydrogel SelfAssembly Kiheon Baek,† Alexander D. Noblett,† Pengyu Ren,† and Laura J. Suggs*,† †
Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, United States
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ABSTRACT: Self-assembling peptides can be used in a bottom-up approach to build hydrogels that are similar to the extracellular matrix at both structural and functional levels. In this study, a nucleo-tripeptide library was constructed to identify molecules that form hydrogels under physiological conditions. We used both experimental and computational approaches to study these self-assembled structures. Circular dichroism spectroscopy, transmission electron microscopy, and rheometry were utilized to support and supplement molecular dynamics simulations. Our data demonstrate that nucleo-tripeptides can form nanofibrous hydrogels through Watson−Crick base pairing and π−π stacking interactions. Selfassembly conditions are mediated by nucleo-tripeptide hydrophobicity and amphiphilicity and can therefore be regulated by a rational molecular design. We have found that structures derived from specific peptide and nucleobase conjugations form hydrogels under physiologic conditions, making them promising candidates for biomedical applications. KEYWORDS: self-assembly, nucleopeptides, hydrogel, molecular dynamics simulation, supramolecular chemistry, nanofiber, biocompatibility
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INTRODUCTION Peptide-based biomaterials have numerous biomedical applications due to their biocompatibility, potential for modification, and capacity to self-assemble into hydrogels structurally similar to the extracellular matrix (ECM). The 20 standard proteogenic amino acids have diverse molecular characteristics, and peptide materials are readily made using a bottom-up approach.1,2 The features of self-assembled peptides, which include their biological diversity, chemical versatility, and wealth of studies regarding structure and synthesis, have increased their popularity in biomaterials research. The combination of molecular secondary interactions and peptide secondary structures (e.g., α-helices, β-strands, β-turns, random coils, and coiled coils) gives a myriad of possibilities for controlling self-assembly and developing new structures.3 Further, a peptide sequence can confer specific biologic functionalization to constructs, including targeting and degradation.4,5 Many self-assembling peptide molecules and peptide-based structures have been studied, including peptide amphiphiles, secondary structure peptides, and short modified peptides, leading to a variety of structures, such as micelles, vesicles, fibers, ribbons, and tubes.6−9 These materials are used in multiple applications, such as functional cell scaffolds for tissue engineering, targeted delivery carriers for controlled release, and templates for guiding nanostructure development. Our group has focused on hydrogels composed of short peptide derivatives in order to study self-assembly processes and © 2019 American Chemical Society
develop tissue engineering scaffolds. Short modified peptides have several advantages in the study of self-assembly due to their ease of synthesis and controllable molecular interactions leading to association and supra-structure formation.10,11 Selfassembled peptide-based hydrogels are also well-suited for tissue engineering applications as a result of many ECM-like characteristics, particularly morphology, physical properties, and enzymatic susceptibility.12−16 Our group has reported on the self-assembly of depsipeptides, which have alternating ester and amide bonds along the polymer backbone. We demonstrated experimentally and computationally in short peptides modified by fluorenylmethyloxycarbonyl (Fmoc) that π−π stacking of aromatic groups and hydrophobic considerations are the primary driving forces for self-assembly, while β-sheet interactions were minimal.17 Based on this discovery, a self-assembled, hydrolytically degradable Fmoc-depsipeptide hydrogel was developed containing a cell-attachment peptide moiety, arginine-glycineaspartic acid (RGD). The hydrogel degraded linearly over a period of 60 days and demonstrated increased cell attachment in vitro, but the viability of cells was reduced over time.18 We suggested that this decrease may be caused by chemical toxicity of the degradation products or by a reduction in physical properties during degradation.19−21 Received: March 18, 2019 Accepted: May 14, 2019 Published: May 14, 2019 2812
DOI: 10.1021/acsabm.9b00229 ACS Appl. Bio Mater. 2019, 2, 2812−2821
Article
ACS Applied Bio Materials
The organic layer was back-extracted with saturated NaHCO3 solution (2 times). The combined aqueous layer was placed in an ice bath, and the pH was decreased to 2−3 by adding 1 N HCl. The acidic aqueous phase was then extracted with ethyl acetate (4 times), and the collected organic layer was dried with Na2SO4, filtered, and evaporated to give the product (91%). To remove the methyl protecting group at the acetate linker, the above compound was dissolved in THF and placed in an ice bath. Sodium hydroxide solution (2.2 equiv) was added dropwise, and the reaction proceeded for 1 h at room temperature. The mixture was cooled to 0 °C, and 1 N HCl was added to decrease the pH to 2−3. Ethyl acetate extraction was performed, and the collected organic layer was washed with saturated sodium chloride (NaCl) solution, dried with Na2SO4, filtered, and concentrated to give the guanine acetic acid product (89%). Solid-Phase Synthesis of Nucleo-tripeptides. All nucleotripeptides were made (0.25 mmol scale) by traditional Fmoc solidphase peptide synthesis and coupled on the solid phase using diisopropylcarbodiimide (DIC)/OxymaPure (ethyl 2-cyano-2(hydroxyimino)acetate) amide coupling chemistry. Fmoc-Phe-Wang resin swelled in DCM in a fritted, capped syringe for 20 min and then was rinsed with dimethylformamide (DMF). Five milliliters of 20% piperidine in DMF was used for deprotecting the Fmoc group (20 min × 2). After washing with 5 mL of DMF (3 times) and 5 mL of DCM (2 times), the resin was mixed with a coupling solution (3 equiv of Fmoc-protected amino acids or 2 equiv of nucleobase derivatives, 3 equiv of OxymaPure, and 3 equiv of DIC) for 50 min. The coupling reaction was followed by washing with DMF (10 mL, 4 times) and DCM. (10 mL, 2 times). Fmoc deprotection and coupling were repeated until achieving the desired nucleopeptide. Cleavage from the resin and side group deprotection were simultaneously completed by a 2.5−3 h reaction with 5 mL of 95:2.5:2.5 trifluoroacetic acid/water/triisopropylsilane (TFA/H2O/TIPS). The mixture was then collected in a clean round-bottom flask, and subsequent resin washes (7 × 10 mL DCM) were added to the flask. After concentrating in vacuo on a rotary evaporator, the resulting viscous solution was precipitated with cold diethyl ether. Scavenged protecting groups were removed by washing with cold diethyl ether (×3) and centrifugation, and then the product was dried under a gentle stream of N2. By redissolving in 1:1 water/acetonitrile, the product was purified by reversed phase (RP)-HPLC (5% H2O initially for 5−10 min, followed by a 25−30 min linear gradient of 5−95% acetonitrile in water). Pure product fractions were collected and lyophilized in a low temperature (−100 °C collector) for 2 days. Gelation of Self-Assembling Nucleopeptides. A pH switch method was employed for inducing the self-assembly of nucleotripeptides. Briefly, each nucleopeptide was dissolved in 200 μL of H2O and 40 μL of 0.5 M NaOH, and the solution was gently mixed until clear. An adequate volume of 0.5 M HCl was slowly added, and the mixture was gently mixed until the assembled structure was formed in the solution. The inverted vial method was used to confirm hydrogel formation. In the case of mixture hydrogels, both nucleotripeptide powders were dissolved in 200 μL of H2O and 40 μL of 0.5 M NaOH, and then 0.5 M HCl was gradually added to induce selfassembly. Gel Rheology. Storage and loss moduli of nucleopeptide hydrogels or nucleopeptide mixture hydrogels were measured by an Anton-Paar MCR101 rheometer with a parallel plate geometry (top plate diameter = 8 mm). Hydrogels were prepared by forming in 3 mm deep, 8 mm diameter polydimethylsiloxane (PDMS) molds. Oscillatory shear stress rheometry was performed (1% strain, 0.5−100 Hz). Circular Dichroism (CD) Measurement. Nucleo-tripeptide samples were pipetted into a cylindrical quartz cuvette with a 0.1 mm path length. Spectra between 185 and 400 nm were measured by a Jasco J-810 CD spectrometer. The instrument was operated with a 0.5 nm data pitch, 200 nm/min scan speed, and 1 s response time. Each data point was the average value of three measurements. Transmission Electron Microscopy (TEM) Analysis. FEI Tecnai TEM and negative staining with uranyl acetate were used
In the current study, we aimed to design a small selfassembling nucleopeptide library. These molecules have a nucleobase, which we postulate may have reduced toxicity relative to Fmoc, at the N-terminus of the peptide chain. We also hypothesize that nucleobases provide an opportunity to enhance molecular self-assembly. Both of these features would be advantageous in the development of a biocompatible hydrogel that assembles in a physiological environment. In particular, we are interested in molecules that assemble at low concentrations and a neutral pH in order to maintain a high cell viability in a culture. The candidate molecules were derived from a previously reported template of Nuc-FF (Nuc, nucleobase, including Ade = adenine, Thy = thymine, Cyt = cytosine, Gua = guanine, F = phenylalanine), which form nanofibrous structures above 2 wt % and below a physiological pH (∼5).22−24
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EXPERIMENTAL SECTION
Materials. All chemical compounds were purchased from Milipore Sigma (Milwaukee, WI), and Fmoc-Phe-Wang was purchased from Bachem (Torrance, CA). Synthesis of Nucleotide Derivatives. Nucleobase acetic acids, except thymine acetic acid, were prepared in a 15 mmol scale based on the synthetic route (Figure S1) from Porcheddu et al. and Mercurio et al.25,26 Adenine or cytosine (1 equiv) and 4-dimethylaminopyridine (DMAP, 0.1 equiv) were dissolved in dry tetrahydrofuran (THF), and di-tert-butyl decarbonate (Boc2O, 4 equiv) was added to the reaction batch. The reaction mixture was stirred overnight at room temperature, and excess THF was evaporated the following day. The product was dissolved in methanol (MeOH), and saturated sodium bicarbonate (NaHCO3 (aq)) was added. The reaction mixture was stirred for 1 h at 50 °C; then water was added to the suspension after evaporating MeOH. The product was extracted with dichloromethane (DCM, 3 times) and the collected organic layer was dried with sodium sulfate (Na2SO4), filtered, and evaporated to give a Bocprotected nucleobase (98%). In order to attach a methyl acetate linker, a Boc-protected nucleobase (adenine or cytosine) was dissolved in THF and sodium hydride (NaH, 1.2 equiv) was added. The solution was placed in an ice bath to decrease the reaction temperature to 0 °C. Methyl bromoacetate (1 equiv) was added dropwise; then the ice bath was removed after 1 h to react overnight at room temperature. Water was added to quench the reaction, and THF was evaporated. The residue was extracted with DCM (4 times), and the collected organic layer was dried with Na2SO4, filtered, and evaporated. The product was then purified by flash chromatography on a silica gel column (ethyl acetate/hexane 5%−95% gradient) to obtain pure a Boc-protected nucleobase with a methyl acetate linker (93%). To remove the methyl protecting group, the above compound was dissolved in H2O/THF (1:1) and cooled to 0 °C in an ice bath. Sodium hydroxide (NaOH, 1.2 equiv) was added dropwise, and the reaction mixture was stirred for 30 min. Hydrochloric acid (1 N HCl) was added to decrease the pH to 2−3, and the solution was extracted with ethyl acetate (4 times). The product was then dried with Na2SO4, filtered, and evaporated to give a Boc-protected nucleobase acetic acid (82%). For guanine acetic acid synthesis, 2-amino-6-chloro purine was used as a starting material because guanine has a very low solubility in organic solvents. The same Boc protection and methyl-protected linker attachment procedures were performed first. To replace chlorine at the purine ring with an oxo group, the compound was dissolved in 1,4-dioxane and potassium acetate (CH3COOK, 3 equiv) and triethylamine (TEA, 3 equiv) were added. 1,4Diazabicyclo[2.2.2]octane (DABCO, 0.1 equiv) was then added portion-wise, while stirring, and the reaction batch was stirred overnight at room temperature. Saturated NaHCO3 solution was added, and after 30 min, the solution was washed with ethyl acetate. 2813
DOI: 10.1021/acsabm.9b00229 ACS Appl. Bio Mater. 2019, 2, 2812−2821
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ACS Applied Bio Materials for supra-structure analysis. The surface of a carbon-coated, 300 mesh copper specimen support grid (Electron Microscopy Services, Hatfield, PA) was initially ionized using a glow discharge system. Approximately, 10 μL of each nucleopeptide hydrogel was placed on the grid and was allowed to adsorb for 5 min. Excess sample was removed by using a Kimwipe, and the grid was air-dried for 5 min. Two microliters of 2% uranyl acetate was placed on the dried grid for 5 min and then removed and dried before mounting in the TEM. Molecular Dynamics (MD) Simulations. All simulations and calculations were performed using AMBER 1427 with AMBERff12SB force field,28 with periodic boundary conditions and explicit water molecules. Nonstandard residues, nucleobase acetic acid (ADL, THL, CYL, and GUL) and C-terminal protonated phenylalanine (AHP), were parametrized for building nucleo-tripeptide molecules. The charge and force field parameter of individual atoms in the residue were assigned by AM1-BCC29 and Amber atom types, respectively. AMBER GAFF force field30 was used to assign the missing parameters. Nuc-FFF molecules were built in AMBER with the parametrized nonstandard residues. Four peptide systems, Ade-FFF (120 molecules of Ade-FFF), (Ade-,Thy-)FFF (60 Ade-FFF molecules and 60 Thy-FFF molecules), Cyt-FFF (Cyt-FFF 120 molecules), and (Cyt-,Gua-)FFF (60 molecules of Cyt-FFF and 60 molecules of Gua-FFF), were selected for simulations. To build an initial simulation system, Packmol31 was used for arranging 120 molecules randomly in the simulation box (100 Å cube), which was then solvated with TIP3P water molecules using the SOLVATEBOX in AMBER. The proper number of ions (Na+ and Cl−) were added to represent the ionic strength (0.1 M). Before the productive simulated annealing (SA) MD, energy minimization and equilibration simulations were applied. Initially, starting structures were subjected to two steps of energy minimization: minimization with solute restrain (500 cycles by the steepest descent algorithm and 500 cycles by the conjugate gradient algorithm) and full system minimization (1000 cycles by the steepest descent algorithm and 1500 cycles by the conjugate gradient algorithm) to remove high-energy contacts. Next, 100 ps NVT with solute restrained and 1 ns NPT simulations without restraints were performed to equilibrate the system. Then, 50 ns SA MD simulations were then performed to simulate the assembly. The temperature of the system was increased to 500 K during the initial 5 ns and then gradually decreased to 300 K in 45 ns followed by 5 ns equilibration at 300 K. After SA MD, an additional 20 ns NPT equilibration was performed to analyze ther assembled structures. Hydrogen mass repartitioning was adopted in the NPT equilibration in order to use a time step to 3 fs.32 The SHAKE algorithm was used to constrain covalent bonds with hydrogen atoms, allowing a 2 fs time step for MD simulations.33 Constant temperature and pressure were maintained by a Langevin thermostat34 and a Berendsen barostat,35 respectively. In Vitro Biocompatibility Assay. In order to evaluate the cytotoxicity of our synthesized nucleopeptides, an MTS [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, CellTiter 96, Promega] assay was performed to measure the proliferation 3T3 fibroblasts in a culture with media containing various concentrations of four nucleopeptides. Briefly, 104 cells/cm2 of 3T3 fibroblasts (after at least 2 passages) were seeded on 48-well microplates. After 24 h of culture with complete media, the medium was replaced by nucleopeptidecontaining media (0.6−0.075 wt %), and fibroblasts were incubated for 16 h. Because the chosen concentrations were below their critical gelation concentration, they reduced the media pH more than that in the gelled form. As a result, the pH of the media was neutralized by adding 0.1 M NaOH prior to incubation with cells. The proliferation of fibroblasts after 16 h incubation was measured by MTS according to the manufacturer’s protocol.
Figure 1. Molecular structure and library of nucleo-tripeptides.
diphenylalanine molecules were shown to form nanofibers leading to gelation, but the self-assembly conditions via pH change, namely, high concentration (2 wt %) and low pH (∼5) of the resulting hydrogel, are not ideal for cell culture applications. In order to raise the pH of gelation, increase potential self-assembly interactions, and control the amphiphilicity, we decided to make a small library of nucleo-peptides. We inserted an additional amino acid between the nucleobase and diphenylalanine. Four amino acids having different side chain characters were chosen: bulky, hydrophobic phenylalanine (F), small, hydrophobic alanine (A), small, flexible glycine (G), and charged, hydrophilic lysine (K). Each nucleobase acetic acid (except thymine acetic acid) was produced by following a similar synthetic route (Figure S1), and these were used for the solid-phase peptide synthesis of nucleo-tripeptides. Sixteen nucleo-tripeptides were successfully synthesized, purified, and lyophilized for further analysis. Self-Assembly Properties of Nucleo-tripeptides. In order to study the self-assembly of our nucleo-tripeptides, we developed a protocol for inducing gelation in our small molecules via pH change. All of 16 nucleo-tripeptides were well dissolved in a basic aqueous solution. A solution of 0.5 M hydrochloric acid was gradually added to nucleo-tripeptide solutions, leading to neutralization of the carboxylic acid group of the C-terminus and formation of the structure. The gelled supra-structures were investigated by the inverted-vial test (Table 1). We found that all of the nucleo-tripeptides except Gua-KFF were able to form hydrogels. Next, TEM was used to analyze the self-assembled structure at the nanoscale (Figure 2). All nucleo-tripeptide hydrogels with FFF and AFF formed nanofibers, in which the diameter was in the range 6−18 nm. The Ade-GFF hydrogel also had a network structure of nanofibers, but other nucleo-tripeptides with the GFF sequence did not. In the case of Thy-GFF and Gua-GFF, microscale crystals were observed. Interestingly, Cyt-GFF was initially assembled into a nanofibrous structure, but this structure was lost over time and changed to ∼100 nm connected spherical aggregates. Nucleo-tripeptides having KFF generally showed larger nanofibers (20 nm) than other hydrogels. In comparing the formed hydrogels based on the hydrophobicity of the additional peptide insertion (FFF, AFF, GFF), the more hydrophobic nucleo-tripeptides tended to form selfassembled structures at lower concentrations and higher pH’s. This result confirms that hydrophobicity and π−π interactions are the primary driving force for assembly.36 Furthermore, a hydrophobic environment constructed by hydrophobic peptides increased the pKa of the C-terminal carboxylic acid group, thus raising the pH for gelation.37 On the other hand, nucleotripeptides having glycine between the nucleobase and diphenylalanine required a higher concentration and resulted
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RESULTS AND DISCUSSION Design and Synthesis of Nucleo-tripeptides. Sixteen nucleo-tripeptides were designed based on the nucleodiphenylalanine previously reported (Figure 1).24 Nucleo2814
DOI: 10.1021/acsabm.9b00229 ACS Appl. Bio Mater. 2019, 2, 2812−2821
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ACS Applied Bio Materials Table 1. Gelation Conditions and Properties of Nucleo-Tripeptide Hydrogelsa
a
1 wt % of Ade and Thy hydrogels and 1.5 wt % of Cyt and Gua hydrogels were used for measuring the storage (G′) and loss (G′′) modulus.
Figure 2. Nanofiber structures of self-assembled nucleo-tripeptide hydrogels were observed via transmission electron microscopy (TEM) images (scale bar = 200 nm).
Figure 3. Mechanical properties of Ade-FFF with a concurrent strain profile demonstrated the self-healing ability of Ade-FFF (A). An AdeFFF hydrogel injection through a needle was evidence of self-healing (B).
in a lower pH for gelation. Except for Ade-GFF, other GFFcontaining molecules either formed crystals or, in the case of Gua-GFF, lost their gelled form rapidly, after 10 min. This more transient gelation was evidence of the negative effect of increased flexibility and the decreased molecular interaction conferred by glycine.38 Interestingly, unlike the other peptide sequences, nucleotripeptides containing lysine formed gels at a similar pH to those with FFF but took a longer time for gelation. (Both Ade-
KFF and Thy-KFF showed a change in optical transparency after approximately 20 min, and sonication was required to promote gelation for Thy-KFF.) Presumably, protonated lysine side chains lead to higher pH values, while the increased overall hydrophilicity of the molecule may limit the driving force for gelation and increase the time for self-assembly. 2815
DOI: 10.1021/acsabm.9b00229 ACS Appl. Bio Mater. 2019, 2, 2812−2821
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ACS Applied Bio Materials
Figure 4. Circular dichroism spectrum of various concentrations of nucleo-triphenylalanine indicated the peptide secondary helical structure and DNA-like stacking structure (A−D). Ramachandran plots of Ade-FFF and Cyt-FFF models also supported the existence of peptides having φ and ψ angles in a helical structure (E, F).
Mechanical Properties of Nucleo-tripeptide Hydrogels. Rheological characterization of nucleo-tripeptide hydrogels was performed in order to compare their mechanical properties (Table 1). A higher storage than loss moduli of nucleo-tripeptide self-assembled structures demonstrated that elastic hydrogels were formed. The formed hydrogels achieved storage moduli on the order of 10 Pa to 1 kPa. This modulus is appropriate for the engineering of soft tissues having a similar stiffness to endothelial tissue.39 Intriguingly, nucleo-tripeptide hydrogels having a KFF sequence showed much higher storage and loss moduli than other relevant nucleo-tripeptides. These unusual mechanical properties may have been derived from the charge interaction via lysine’s protonated amine group. One of the important features of this family of selfassembled hydrogels is the ability to self-heal, recovering its fiber network formed by secondary interactions. We evaluated
this property by conducting a dynamic strain step test on the Ade-FFF hydrogel (Figure 3). The storage modulus immediately dropped from approximately 1800 to 10 Pa when subjected to a 100% strain, but both storage and loss moduli were immediately recovered when the strain returned to 0.1%. The recovered storage modulus maintained its initial storage modulus, indicating that Ade-FFF may restore the secondary interactions leading to hydrogel formation in spite of repeated strain stress. This self-healing ability makes the Ade-FFF hydrogel suitable as an injectable material, which will be useful for biomedical applications. Molecular Dynamics Simulations of Nucleo-tripeptide Self-Assembly. We carried out MD simulations for the detailed analysis of nucleo-triphenylalanines’ self-assembled structure. Simulated annealing was performed to predict the assembled structures from initial random configurations. Four 2816
DOI: 10.1021/acsabm.9b00229 ACS Appl. Bio Mater. 2019, 2, 2812−2821
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ACS Applied Bio Materials
Figure 5. MD simulation results of Ade-FFF and Cyt-FFF models supported their experimentally determined structure and properties. Stabilized SASA and RMSD of both models showed equilibrated assembled structures (A, B). Cyt-FFF had more hydrogen bonds between molecules and nucleobases (C), but a higher number of π−π stacking of nucleobases was seen in the Ade-FFF model (D, E). Concurrently, the Ade-FFF hydrogel demonstrated a higher storage modulus (F).
was examined based on three conditions, minimum distance between two bases’ heavy atom (
