Article pubs.acs.org/Biomac
Mixed α/β-Peptides as a Class of Short Amphipathic Peptide Hydrogelators with Enhanced Proteolytic Stability Jeroen Mangelschots,† Mathieu Bibian,† James Gardiner,‡ Lynne Waddington,‡ Yannick Van Wanseele,§ Ann Van Eeckhaut,§ Maria M. Diaz Acevedo,∥ Bruno Van Mele,∥ Annemieke Madder,*,⊥ Richard Hoogenboom,*,# and Steven Ballet*,† †
Research Group of Organic Chemistry, Departments of Chemistry and Bioengineering Sciences, and ∥Physical Chemistry and Polymer Science, Vrije Universiteit Brussel, Pleinlaan 2, B-1050, Brussels, Belgium ‡ CSIRO Manufacturing Flagship, Bayview Avenue, Clayton, VIC 3169, Australia § Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium ⊥ Organic and Biomimetic Chemistry Research Group, Department of Organic and Macromolecular Chemistry, and # Supramolecular Chemistry Group, Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281, 9000 Ghent, Belgium S Supporting Information *
ABSTRACT: Peptide hydrogels are a highly promising class of materials for biomedical application, albeit facing many challenges with regard to stability and tunability. Here, we report a new class of amphipathic peptide hydrogelators, namely mixed α/β-peptide hydrogelators. These mixed α/βgelators possess good rheological properties (high storage moduli) and form transparent self-supporting gels with shear-thinning behavior. Infrared spectroscopy indicates the presence of β-sheets as the underlying secondary structure. Interestingly, self-assembled nanofibers of the mixed α/βpeptides display unique structural morphologies with alteration of the Cterminus (acid vs amide) playing a key role in the fiber formation and gelation properties of the resulting hydrogels. The incorporation of β3homoamino acid residues within the mixed α/β-peptide gelators led to an increase in proteolytic stability of the peptides under nongelating conditions (in solution) as well as gelating conditions (as hydrogel). Under diluted conditions, degradation of mixed α/βpeptides in the presence of elastase was slowed down 120-fold compared to that of an α-peptide, thereby demonstrating beneficial enzymatic resistance for hydrogel applications in vivo. In addition, increased half-life values were obtained for the mixed α/β-peptides in human blood plasma, as compared to corresponding α-peptides. It was also found that the mixed α/βpeptides were amenable to injection via needles used for subcutaneous administrations. The preformed peptide gels could be sheared upon injection and were found to quickly reform to a state close to that of the original hydrogel. The shown properties of enhanced proteolytic stability and injectability hold great promise for the use of these novel mixed α/β-peptide hydrogels for applications in the areas of tissue engineering and drug delivery.
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genic properties, especially when cross-linking is done in vivo.10 Therefore, in recent years, hydrogels prepared from selfassembling peptides have emerged as an important class of biomaterials in the fields of drug delivery and tissue engineering.11−14 The great diversity of available natural amino acids, coupled with a “natural degradation” pathway via proteolytic enzymes, has led to peptides becoming powerful tools in creating new biocompatible, biodegradable, and functional soft materials.15 The increasing amount of work published on peptide hydrogels in recent years has led to the formulation of crucial design features for self-assembling oligopeptides with predesigned
INTRODUCTION Hydrogels are defined as three-dimensional polymeric networks that are able to absorb a large amount of water while maintaining a semisolid morphology.1−4 Depending on the type of cross-linking, hydrogels can be subdivided into two types: chemical hydrogels (covalently cross-linked materials) and physical hydrogels (noncovalently cross-linked, supramolecular polymers).5 Both of these types of hydrogels have widely been exploited as biomaterials due to their porous structure and high water content, thereby mimicking natural tissue.6 For biomedical applications, such as tissue engineering, medical imaging, or controlled drug delivery, the predominant class under investigation still remains the family of chemical hydrogels.1,7−9 However, for in vivo applications such polymeric cross-linked structures still face challenges with regard to stability in biological fluids, toxicity, and immuno© XXXX American Chemical Society
Received: October 1, 2015 Revised: December 23, 2015
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DOI: 10.1021/acs.biomac.5b01319 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
modified by substitution of one or two α-amino acids with the corresponding β-amino acids (Fmoc-βAla-Xxx-OH; Xxx = Phe, Val, His) and naphthalene-βPhe-βPhe-OH) to give stronger self-assembling hydrogels in similar gelation conditions.31−33 However, due to the low pH required to initiate gelation and the lack of toxicity studies on such aromatic group-capped peptide gelators, the potential usefulness for such compounds in vivo is still unknown.34,35 Yang and co-workers also used a similar β-amino acid-containing tripeptide as a substrate for the enzyme-triggered formation of a stable hydrogel.35,36 A phosphatase was used to cleave a phosphate group from a tyrosine side chain of the nongelating precursor peptide, Napβ3-hPhg-β3-hPhg-P-Tyr-OH to give Nap-β3-hPhg-β3-hPhg-TyrOH, which subsequently self-assembled to form a hydrogel. Common to all these examples however is the use of an additional external stimulus, either pH or an enzyme, to induce formation of the hydrogel. Therefore, as part of our design we envisaged the development of a class of β-amino acidcontaining self-assembling peptides that could form dynamic hydrogels after simple dissolution in water or buffer. Herein, we report on the design, synthesis, and gelating properties of a set of linear amphipathic mixed α/βoctapeptides. These peptides readily form stable self-assembled hydrogels after dissolution in water or phosphate buffer. The introduction of two hydrophobic dyads, composed of β3homophenylalanine and Phe or Tyr, into an amphipathic hexapeptide, gave a set of short linear amphipathic α/β-peptide hydrogelators (2, 3, and 4, Table 1). These novel mixed α/β-
supramolecular architectures (including β-sheet, helical, or βhairpin-based fibrils and nanotubes).16−20 It has been stated that a perfect balance between hydrophobicity and hydrophilicity needs to be maintained when designing oligopeptide gelators in order to obtain a self-assembled system under suitable conditions.19 The self-association of two peptide strands can be aided by using aromatic−aromatic interactions (specific π−π interactions).20 However, the use of oligopeptide hydrogels as injectable controlled-delivery systems continues to raise challenges with respect to drug release rate, mechanism of release, determination of toxicity, fine-tuning of viscoelastic and release properties, and assessment of and control over a correct stability/degradability balance in biological environments. After the discovery of the hydrogelating peptide EAK16-II (H-AEAEAKAKAEAEAKAK-OH) by Zhang et al., various amphipathic peptides have been designed for drug delivery applications. These include VEK (H-VEVKVEVK-OH) and FEFEFKFK octapeptide analogues, the self-assembling series KXE12 (H-XKXEXKXEXKXE-OH; X = F, I, or V) from Caplan et al., and our own hexapeptide MBG-1 alphahydrogelator 1 (H-FEFQFK-OH).16,21−23 In addition, a novel subclass of β-hairpin self-assembling peptide, MAX1 (HVKVKVKVKVDPPTKVKVKVKV-NH2), emerged from work performed by Pochan and Schneider.24 An important criterion for drug delivery from peptide hydrogels is that of enzymatic stability in order to maintain sufficient control over the release of active components from a hydrogel matrix. If the peptide fibers are amenable to fast proteolytic degradation in vivo, or peptides in solution on the periphery of the fibers are degraded before they can reself-assemble, then the equilibrium of the selfassembled hydrogel may be altered and degradation of the matrix may be enhanced. In this context, α-peptide hydrogelators that are easily cleaved by endo- and exopeptidases could possess insufficient enzymatic stability leading to in vivo burst effects and destabilization of the supramolecular system. In addition, the previously mentioned amphipathic αoligopeptides are reported to exhibit hours-to-days release profiles of various encapsulated compounds.25,26 In this context, sustained delivery of pharmaceuticals is necessary, preferably with a duration of multiple weeks. This can increase patient compliance by decreasing the amount of injections required for efficient treatment. It is well-known that the proteolytic stability of oligopeptides can be enhanced by incorporation of β-amino acids or 27−29 D-amino acids into the sequence. In the field of amphipathic peptide hydrogelators, the use of all D-amino acids has been explored and reported to be beneficial for in vivo stability of the hydrogel and hydrogel rigidity.27 However, inversion of the chirality of a single amino acid residue (L- to Damino acid) in a β-sheet forming peptide motif has been shown to result in loss of the gelation properties due to inappropriate stacking of β-sheets.27,30 In light of this, we hypothesized that the incorporation of commercial β3-homoamino acids possessing proteinogenic side chains would have the potential to form stable hydrogels without significant interference of the β-sheet structure, and in addition possess the added property of enhanced enzymatic stability. However, introducing more flexible β3-homoamino acid residues may obstruct hydrogel formation by destabilization of β-sheet structures, posing an important challenge. The use of β-amino acids has, to the best of our knowledge, only been reported for the design of low molecular weight gelators (LMWG). Four self-assembling Fmoc-dipeptides were
Table 1. Hydrogelator Peptide Sequences, Minimum Gelation Concentration (MGC), Gelation Conditions, HPLC Retention Times (As a Measure of Peptide Polarity) sequence 2 3 4
H-FEβ3hFFQβ3hFFK-OH H-FEβ3hFFQβ3hFFK-NH2 H-FEβ3hFYQβ3hFYK-NH2
MGCa (mM)
gelation conditionsb (v/v)
trc (min)
17 17 21
mQ mQ/PBS (1:1) PBS
13.4 13.1 11.6
a
Minimum gelation concentration (MGC), expressed in mM, corresponds to 2, 2, and 3% w/v for peptides 2, 3, and 4, respectively. b Formation of a stiff transparent hydrogel at the above-mentioned gelation conditions in an aqueous solution of mQ, a mixture of mQ/ PBS (10 mM PBS) at a 1:1 volume ratio, or PBS for 2, 3, and 4, respectively. cThe HPLC retention times of the peptide TFA salts (tr) in minutes under identical mobile phase conditions.
gelators will be shown to form transparent self-supporting gels with shear-thinning behavior and relatively high storage moduli. Moreover, biodegradation experiments were performed in order to assess the biostability toward elastase, a prominent enzyme present in the subcutaneous compartment.
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MATERIALS AND METHODS
Synthesis and Purification of Peptides. The synthesis of peptide 1 was realized as previously reported.23 All other peptides used were prepared using standard 9-fluorenylmethoxycarbonyl (Fmoc) strategy. While the synthesis of mixed α/β-peptide 2 was carried out on Fmoc-Lys(Boc)-loaded Wang resin (FluoroChem, 100−200 mesh, 0.35 mmol g−1), mixed α/β-peptides 3 and 4 were assembled on Fmoc-Rink Amide resin (Chem-Impex, 200−400 mesh, 0.60 mmol g−1). Nα-Fmoc-L-β3-homophenylalanine (2 equiv) was preactivated by HATU (2 equiv), DIPEA (4 equiv) in DMF for 1 min at room temperature and the α-amino acids (4 equiv) were preactivated by TBTU (4 equiv) and DIPEA (8 equiv). The peptides were cleaved manually from the solid support and the side chains were deprotected B
DOI: 10.1021/acs.biomac.5b01319 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Å−2 for all imaging. Images were recorded using a FEI Eagle 4k × 4k CCD camera (FEI, Eindhoven, The Netherlands). Negative Staining TEM Analysis. Carbon-coated 300-mesh copper grids were glow-discharged in nitrogen to render the carbon film hydrophilic. Hydrogels were prepared as described above, diluted to allow handling, and a 4 μL aliquot of the sample was pipetted onto each grid. After 30 s adsorption time, the excess was drawn off using Whatman 541 filter paper, followed by staining with 2% aqueous potassium phosphotungstate at pH 7.2, for 10 s. Grids were air-dried until needed. The samples were examined using a Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. Images were recorded using a Megaview III CCD camera and AnalySIS camera control software (Olympus.) Each grid was systematically examined and imaged to reflect a representative view of the sample. Enzymatic Degradation Study of Peptides in Nongelating Conditions (i.e., in Solution). The following buffer was used to assay the proteolytic stability of our peptides toward elastase: 10 mM PBS at pH 7.2. First, a stock solution of elastase (60 μM in 10 mM PBS) was prepared. Peptides 1−4 were subjected to the degradation study and prepared as a stock solution of 350 μM in mQ water (with a resistivity of 18.2 mΩ·cm). Enzymatic degradation was carried out by incubation of peptide (900 μL, 350 μM) with the enzyme (150 μL, 60 μM) at 37 °C for 6 days. The enzyme concentration was selected such that the reference α-peptide 1 was completely degraded after 1 h. In the case of α-peptide 1, aliquots (70 μL) were periodically taken at 0, 5, 10, 15, 20, and 30 min, while for the mixed α/β-peptides aliquots (70 μL) were taken at 0, 20, and 30 min, 1, 2, and 5 h, and 1, 2, 3, 4, 5, and 6 days. A 5 μL aliquot of 25% AcOH in water (v/v) was added and degradation was monitored by HPLC analysis. Stability Study of Peptides in Human Blood Plasma. Human plasma was obtained from the Belgian Red Cross (Vlaams-Brabant, Leuven). Prior to the stability test, selectivity, stability of the compound in the injection solvent, the effect of various incubation times at 4 °C, linearity, accuracy and precision of the method were investigated. Frozen (−20 °C) human plasma samples were thawed and thermostated to 37 ± 2 °C. Dissolution of the lyophilized peptide and consecutive dilutions were performed in water. The resulting aqueous solutions of peptides 1−4 were spiked in human plasma (10:90 v/v), with final plasma concentrations of 34, 33, 33, and 33 μM, respectively. During the stability study of mixed α/β-peptides, samples were taken after 0, 5, 10, 15, 30, 60, 90, 120, 240, and 1500 min. For αpeptide 1, degradation was monitored after 0, 1, 2, 3, 4, 5, and 10 min. On every time point, 100 μL of spiked plasma was transferred to a 500 μL Eppendorf tube, and a protein crash was performed using 300 μL of methanol + 0.1% TFA (4 °C). Suspensions were vortexed for 15 s and placed at 4 °C for 30−45 min. After centrifugation at 18625 g for 20 min, 100 μL of supernatant was diluted with 100 μL of water in the injection vial. Injection samples were vortexed for 5 s and placed in the autosampler. For the calculation of the peptide half-life, only points with an area under the curve (AUC) higher than the AUC of the lowest standard were used. Concentrations were calculated by use of the calibration curve and transferred to a semilog chart presenting the log concentrations as a function of time. The optimum curve was used to calculate the peptide-half-life. Calculations were performed using Microsoft Office Professional 2010 Excel. Graphs representing the % recovery as a function of time were made using Graphpad Prism 4. Enzymatic and Erosion Degradation Study of Hydrogels. The proteolytic and erosion stability tests comprise four consecutive steps (A−D): (A) The peptide hydrogel (1 mL) was prepared in a 10 mL glass tube using the appropriate gelation conditions (see Peptide Self-Assembly). After leaving the hydrogel to rest overnight the degradation evaluation was started. First, general stability (without the incubation of an enzyme) was evaluated for α-peptide 1: 3 mL of mQ water was gently placed on top of the hydrogel and erosion experiments were performed by placing 3 mL PBS solution (10 mM) on top of the hydrogel to assess the hydrogels macroscopic decay in time. The proteolytic stability of the hydrogels was analyzed by placing an elastase solution (60 μM in 10 mM PBS) on top of the hydrogel. In all the stability experiments a total volume of 4 mL (gel +
using a trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/water (95:2.5:2.5, v/v/v) mixture at room temperature for 1.5 h. Subsequently, ∼90% of the cleavage cocktail was evaporated. The crude peptides were precipitated in cold diethyl ether and washed three times with cold Et2O. Next, the crude peptides were dissolved in water and lyophilized before purification. Preparative reverse-phase high-performance liquid chromatography (HPLC) was used for the purification of all crude peptides. During the purification of peptides 1−4, solvent A consisted of 0.1% TFA in water and solvent B of 0.1% TFA in acetonitrile. A linear gradient of 3−100% B in 20 min was applied. The purity of each peptide was verified by analytical reversephase liquid chromatography methods based on the same solvent system and gradient. The resulting pure peptides (>95%) were obtained after lyophilization of the collected fractions. Peptide Self-Assembly. Peptide self-assembly was analyzed in (i) a PBS solution (10 mM), (ii) a PBS/mQ mixture 1:1 (v/v; mQ water with a resistivity of 18.2 mΩ·cm) or mQ water (with a resistivity of 18.2 mΩ·cm). The TFA salt of 1 (1 mg) was dissolved in 50 μL mQ water, followed by the addition of 50 μL of PBS solution. This mixture was left to rest overnight, resulting in a 1% w/v hydrogel. The TFA salts of mixed α/β-peptides 2 and 3 (2 mg) were dissolved in mQ (100 μL) and PBS/mQ mixture (100 μL), respectively. The TFA salt of mixed α/β-peptide 4 (3 mg) was dissolved in PBS (100 μL) to induce gelation. Solubilization of the peptide salts was assisted by vortexing for 2−3 s and gentle heating, followed by hydrogelation of the clear sample. All self-assembled gels were analyzed by circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy, as well as cryogenic transmission electron microscopy (Cryo-TEM). FT-IR Analysis. FT-IR spectra were collected on a Nicolet 6700 FT-IR spectrometer in attenuated total reflectance (ATR) mode, using a diamond ATR sample holder. An aliquot of the gel was transferred on the diamond. Scans were performed between 4000 and 600 cm−1 with 64 accumulations at a resolution of 0.4 cm−1. CD Spectroscopy Analysis. The secondary structure of the peptides was analyzed using a 0.1 cm quartz cell on a Jasco J815 Spectropolarimeter, with 1 s integrations, 1 accumulation, and a step size of 1 nm, with a bandwidth of 1 nm over a range of wavelengths from 200 to 270 nm. Peptide hydrogels were freshly prepared in their corresponding gelation conditions (see above Peptide Self-Assembly) directly in the CD cell and spectra were recorded after 2 h. Measurements were repeated at least three times, and their average was plotted. Spectral data with HT values below 700 V were used to generate figures. Data with HT values above 700 V (below 190 nm) were not used. Rheological Analysis of Mixed α/β-Peptide Gelators. Dynamic rheometry measurements were carried out on a TA Instruments AR-G2 rheometer equipped with Electrically Heated Plates and aluminum plate−plate geometry with a diameter of 10 mm. A ring shaped reservoir filled with a saturated NH4Cl solution was placed around the measuring plates for humidity control. Rheological properties of the hydrogels were studied by oscillatory frequency sweeps performed at 37 °C in the range between 0.01 and 10 Hz using a strain control of 0.05% to ensure linear viscoelastic response. Cryo-TEM Analysis. A laboratory-built humidity-controlled vitrification system was used to prepare the hydrogels for imaging in a thin layer of vitrified ice using cryo-TEM. Humidity was kept close to 80% for all experiments, and ambient temperature was 22 °C. 200 Mesh copper grids coated with perforated carbon film (Lacey carbon film: ProSci- Tech, Qld, Australia) were used for all experiments. Grids were glow discharged in nitrogen for 5 s immediately before use. The hydrogels were prepared as described above and analyzed after 24 h. Approximately 4 μL aliquots of sample were pipetted onto each grid prior to plunging. After 30 s adsorption time, the grid was blotted manually using Whatman 541 filter paper. The grid was then plunged into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until required. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, U.S.A.) and Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. At all times, low dose procedures were followed, using an electron dose of 8−10 electrons C
DOI: 10.1021/acs.biomac.5b01319 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 1. Structure of mixed α/β-peptide 2. Left: Two β3-homophenylalanine residues are incorporated in reference α-peptide 1, forming an amphipathic octapeptide. Right: Picture of the vial inversion test revealing the self-supporting nature of our hydrogel prepared by dissolving mixed α/β-peptide 2 in mQ water.
with hydrophilic amino acids (α-Glu, α-Gln, and α-Lys), two hydrophobic dyads (β3-hPhe-α-Phe), and an N-terminalcapped hydrophobic α-Phe residue. Hydrogelation of 2 was readily accomplished at 17 mM (corresponding to 2% w/v) by simply adding mQ water to the peptide salt of 2. Vortexing for 2−3 s and gentle heating resulted in complete dissolution of the peptide after which formation of a stable, self-supporting hydrogel was observed (Figure 1, right; see also Table 1). Lowering the molar concentration (
