CHIRALITY (2016)
Characterization of a Crosslinked Elastomeric-Protein Inspired Polypeptide BRIGIDA BOCHICCHIO,* ANGELO BRACALELLO AND ANTONIETTA PEPE Laboratory of Protein-Inspired Materials, Department of Science, University of Basilicata, Potenza, Italy
ABSTRACT Materials inspired by natural proteins have a great appeal in tissue engineering for their biocompatibility and similarity to extracellular matrix (ECM). Chimeric polypeptides inspired by elastomeric proteins such as silk, elastin, and collagen are of outstanding interest in the field. A recombinant polypeptide constituted of three different blocks, each of them having sequences derived from elastin, resilin, and collagen proteins, was demonstrated to be a good candidate as biomaterial for its self-assembling characteristics and biocompatibility. Herein, taking advantage of the primary amine functionalities present in the linear polypeptide, we crosslinked it with 1,6-hexamethylenediisocyanate (HMDI). The characterization of the obtained polypeptide was realized by CD spectroscopy, AFM, and SEM microscopies. The obtained results, although not conclusive, demonstrate that the crosslinked polypeptide gave rise to porous networks, thin nanowires, and films not observable for the linear polypeptide. Chirality 00:000–000, 2016. © 2016 Wiley Periodicals, Inc. KEY WORDS: AFM; circular dichroism; resilin; elastin; SEM Protein-inspired biomaterials have gained great interest as alternative scaffolds to synthetic polymers derived from petroleum because they offer improved characteristics of biocompatibility.1–5 Elastin-inspired polypeptides are considered particularly suitable for this purpose because elastin is the crosslinked protein responsible for the elasticity of organs and tissues in vertebrates.6–10 Elastomeric proteins such as resilin are equally worthy of attention for their resilience and selfassembling properties together with the presence of crosslinks conferring on them the property of resistance to rupture.11–13 Therefore, synthetic elastin peptides were crosslinked in order to give strength to the polypeptide. The crosslinked polypeptides showed variegated supramolecular structures with twisted-rope fibrils typical of elastin together with branched networks not detectable in linear peptides.14 However, the disadvantage represented by the rather low molecular weights, because they were obtained by chemical synthesis, limited the application of the polymers as efficient biomaterials. Thanks to the versatility of DNA recombinant technologies, we produced a 12kDa chimeric polypeptide inspired by elastin, resilin, and collagen as well (Fig. 1a). The assessment as elastic, self-assembling, and cytocompatible polypeptide suggested it as an interesting candidate in the biomaterial pool.15,16 Herein, taking advantage of primary amine functionalities of lysine residues in elastinand collagen sequences, we crosslinked REC with 1,6hexamethylene-diisocyanate (HMDI) (Fig. 1b,c). Among the requirements that the crosslinker should fulfill, we include the presence of at least two functional groups and the solubility in solvents able to dissolve the linear polypeptide to obtain the exposition of functional groups. Furthermore, the reaction should be carried out under mild conditions with minimal by-products. The choice of HMDI was justified by the solubility of REC in organic solvents at the concentrations necessary for the crosslinking and by the low cytotoxicity of HMDI when compared to other crosslinking agents such as glutaraldehyde.17,18 Furthermore, it was previously demonstrated that polypeptides crosslinked through HMDI were biocompatible after multiple rinses with sterile Milli-Q water.19,20 The crosslinked polypeptide (RECck) was studied at the molecular level by circular dichroism © 2016 Wiley Periodicals, Inc.
(CD) spectroscopy in different solvents as a function of the temperature while, at supramolecular level, it was characterized by scanning electron (SEM) and atomic force microscopies (AFM). CD studies revealed for RECck the presence of a stable type-I beta turn. At the supramolecular level RECck gave rise to porous networks, thin nanowires, and films not observable for the linear polypeptide. MATERIALS AND METHODS Synthesis and Production of Resilin, Elastin, and Collagen Polypeptide (REC) The recursive directional ligation approach (RDL) was used for the gene construction of the chimeric polypeptide REC. The DNA sequences for resilin (R) (flanked at 3′ by Sfi I) and collagen (C) (flanked at 5′ by Bgl I and at 3′ by Sfi I) were synthesized and introduced in pCR 2.1 vector (Invitrogen, MWG Biotech, Germany). The elastin (E) monomer gene was obtained from a recombinant pDrive + ELP plasmid by double digestion with Bgl I and Sfi I restriction endonucleases. The detailed production and expression of the REC monomeric gene, of REC chimeric gene, and of the recombinant REC chimeric polypeptide were performed as previously described16
Crosslinking The hydrogel was obtained by mixing a solution of REC (100 mg/ml) in dimethyl sulfoxide / dimethylformamide (DMSO/DMF) (80:20) with an appropriate solution (25 mg/mL) of the homobifunctional crosslinker 1,6-hexamethylene-di-isocyanate (HMDI) in DMF in a polymer/crosslinker molar ratio of 1:3 at 4 °C. The mixture was stirred overnight and poured into customized Teflon molds (diameter = 13.5 mm; h = 2 mm). Finally, the hydrogel was washed with Milli-Q water to eliminate unreacted reagents and lyophilized.
*Correspondence to: B. Bochicchio, Laboratory of Protein-Inspired Materials, Department of Science, University of Basilicata, Via Ateneo lucano, 10, 85100 Potenza, Italy. E-mail:
[email protected] Received for publication 5 May 2016; Accepted 7 June 2016 DOI: 10.1002/chir.22619 Published online in Wiley Online Library (wileyonlinelibrary.com).
BOCHICCHIO ET AL.
Fig. 1. Overview of REC polypeptide. REC polypeptide is constituted of three blocks inspired by resilin, elastin, and collagen proteins, respectively. MAHHHHHHVDDDD K sequence contains a 6 Histidine tag helpful in order to purify the polypeptide through affinity chromatography Ni-NTA. VDDDDK sequence is a recognition site for an enterokinase enzyme able to cleave 6-Histine tag portion. (a) The three different blocks and the tag sequence MAHHHHHHVDDDDK; (b) Lysine residues in elastin-like and collagen-like motifs can react through amine groups with the homobifunctional crosslinker HMDI; (c) Full amino acid sequence of the chimeric polypeptide.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and with the nominal resonance frequency and force constant of 330 kHz and 42 N/m, respectively
The polyacrylamide gel electrophoresis in the presence of SDS-PAGE was used in order to show the presence of the polypeptides having different molecular weight as crosslinking reaction products. The sample was dissolved in loading buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 1% betamercaptoethanol, 10% glycerol) and heated for 2 min at 100 °C. 10–30 μL of each sample solution were loaded onto 4–15% SDS PAGE. 50, 100, 150, and 300 μg of RECck were loaded in lanes 2, 3, 4, 5, respectively. Protein bands were visualized after 45 min staining (40.0% [vol./vol.] methanol, 10% [vol./ vol.] acetic acid, 0.1% [wt./vol.] Coomassie Blue) and 60 min in destaining solution (40.0% [vol./vol.] methanol, 10% [vol./vol.] acetic acid). The molecular weight of resulting bands was approximated by measuring the relative mobility of the standard protein molecular weight markers
The internal structure of the polypeptides both before and after crosslinking reaction were observed using a scanning electron microscope (Philips-Fei ESEM XL30-LaB6). Prior to imaging the freeze-dried REC and RECck were freeze-fractured after immersion in liquid nitrogen and then mounted using carbon tape on aluminum SEM stubs and sputtered with a thin gold layer. Fifty different pores were randomly selected, measured, and the mean pore diameters calculated by ImageJ software (NIH, Bethesda, MD) on the RECck sample
Circular Dichroism Spectroscopy Circular dichroism spectra were recorded at 0, 25, 37, and 70 °C using a Jasco (Tokyo, Japan) model J-815 spectropolarimeter equipped with a Haake thermostat (Thermo Fisher Scientific, Waltham, MA), averaging 16 scans. Baselines were corrected by subtracting the solvent contribution. Cylindrical, fused quartz cells of 0.1 cm path length (Hellma, Mullheim, Germany) were employed. The data are expressed in terms 1 of [θ] MRW, the mean residue ellipticity value (deg cm2 dmol ). 2,2,2Trifluoroethanol (TFE) was purchased from Romil (Waterbeach, Cambridge, UK). Deionized water was further purified using a Milli-Q reagent grade water system from Millipore (Beford, MA)
AFM Microscopy The sample was solubilized in ultrapure water at a final concentration of 1 mg/mL and an aliquot of the solution was sonicated. Two different aliquots of the initial solution, sonicated and not sonicated, were deposited as drops on Si (100) wafer substrates. The silicon wafers were cleaned by using a two-solvent method consisting of an immersion of the Si wafer in warm acetone bath for 10 min and in a successive methanol bath for 5 min immediately followed by a final deionized water rinse. After water evaporation, the AFM images were carried out by using the XE-120 microscope (Park Systems, Suwon, Korea) in air and at room temperature. Data acquisition was carried out in intermittent contact mode at scan rates between 0.4 and 0.7 Hz, using rectangular Si cantilevers (NCHR, Park Systems) having the radius of curvature less than 10 nm Chirality DOI 10.1002/chir
SEM Microscopy
RESULTS AND DISCUSSION Crosslinking Reaction
SDS-PAGE of RECck obtained through covalent crosslinks between the homobifunctional crosslinker HMDI and the εamino groups of lysine residues is shown in Figure 2a. Roughly, band sizes in lanes 2–5 at about 12, 36, 60 kDa are visible and are ascribed to unreacted REC and (RECck)3, (RECck)5, respectively. Bands at higher molecular weights are also present, suggesting the existence of higher molecular weight polymers. Macroscopically, the final product looked like a transparent hydrogel (Fig. 2b). Circular Dichroism Spectroscopy
CD spectroscopy is a useful tool to determine the secondary structure of polypeptides. To detect temperature-induced conformational transitions, the peptides were monitored by CD spectroscopy at different temperatures. CD spectra were analyzed by comparing the curves with CD spectra of peptides with known secondary structures or with peptides that adopt a mixture of known structures. The microenvironment that determines the conformation of an amino acid sequence is not known a priori and can be different from the bulk macroscopic solution conditions (i.e., physiological conditions). Predicting the functional solvent environment for insoluble elastin is particularly difficult because the protein’s hydrophobicity and
ELASTOMERIC-PROTEINS INSPIRED CHIMERA
Fig. 2. characterization. (a) SDS-PAGE of RECck polypeptide. Lane M: electrophoresis markers (8–220 kDa); Lane 1: REC; Lane 2–5: RECck at different concentrations. 50, 100, 150, and 300 μg of RECck were loaded in lanes 2, 3, 4, 5, respectively. Bands at molecular weight of 12, 36, 60 kDa corresponding to REC, (RECck)3, (RECck)5 are visible together with bands at higher molecular weights; (b) Photograph of the RECck hydrogel. Bar corresponds to 1 cm.
highly crosslinked nature suggest a less polar internal environment than the surrounding solvent. For this reason, the experiments in this study were performed in both water and 2,2,2 trifluoroethanol (TFE). TFE is a significantly less polar solvent than water and is usually considered a structure-inducing solvent because it favors intramolecular hydrogen bonding, thus promoting folded conformations such as helices and β-turns.21–23 While the real local solution conditions for insoluble elastin are unknown, it is likely to be intermediate between the two solvent
extremes of water and TFE. The spectra of RECck were recorded in aqueous solution and TFE. They were compared to those carried out for REC polypeptide (Fig. 3). In aqueous solution the spectra of REC and RECck are very similar between them. The CD spectra of REC are shown in Figure 3a. They suggest the presence of β-turn and unordered conformations, as elsewhere discussed.15 The CD spectrum of RECck, shown in Figure 3b, at 0 °C is characterized by a strong negative band at ~196 nm. As the temperature increases from 0 to 70 °C the band is slightly red-shifted and reduced in intensity. The strongly negative π–π* band at ~196 nm is usually attributed either to unordered or PPII conformations. However, CD spectrum at 0 °C shows a negative band at [θ]200 ~ 4000, too weak to be assigned either to PPII or unordered conformations.24 That finding together with the reduction in intensity of the band with increasing temperature could be indicative of the growing contribution from conformations with positive ellipticity at 200 nm such as β-turn, β-strand, and (less likely) α-helix.25,26 The hypothesis of the co-presence of multiple conformations is strengthened by the lack in the CD spectrum at 0 °C of the positive, essentially n–π*, band at ~216 nm expected for peptide with high amounts of PPII structure very stable at low temperatures27–29 and by the absence of an isodichroic point.24 Summarizing, RECck in aqueous solution is able to adopt flexible and folded structures in conformational equilibrium among them. The CD spectra of RECck recorded in TFE are shown in Figure 3b. They show a positive band at 190 nm and two negative bands at 203 and 221 nm. These spectral findings are diagnostic of type I β-turn conformation. Furthermore, at first glance it is evident that they remain substantially unchanged on increasing the temperature. That finding suggests a stable conformation as a function of the temperature. When compared between them, the spectral features of REC and RECck are very similar, even if with the presence of a slightly more stable beta-turn in the case of the crosslinked polypeptide. AFM Microscopy
Fig. 3. Dichroism spectra in aqueous solution (filled symbols) and in TFE (empty symbols) at 0 (circles), 25 (squares), 37 (diamonds), 70 (triangles) °C of REC (a) and RECck (b).
AFM images of RECck polypeptide are shown in Figure 4. RECck self-aggregate in branched and long fibers (Fig. 4a). The enlarged zone in Figure 4b shows the unraveling branched fiber. Interestingly, if sonicated the sample is able to give rise to long and slender filaments appearing as nanowires interacting side-by-side (Fig. 4c). For the sake of clarity, REC polypeptide gave rise to bundles of fibers only Chirality DOI 10.1002/chir
BOCHICCHIO ET AL.
Fig. 4. AFM images of RECck polypeptide (a,b) RECck sonicated polypeptide (c). Right: 3-D reconstruction of AFM images. Bars represent: (a) 30, (b) 5, (c) 2 microns.
after a variable period of incubation at 37 and 50 °C ranging from 3–6 days, respectively.15 Scanning Electron Microscopy
SEM analysis was used to investigate the structure of the hydrogel, paying particular attention to the porosity and the pore size of the scaffolds influencing the adhesion, penetration, and growth of the cells within 3D structures of scaffolds in tissue engineering. SEM was carried out on REC and RECck and the results are shown in Figure 5. REC showed an amorphous arrangement of aggregates (Fig. 5a). The enlarged image shown in Figure 5b shows sheets and filaments, sometimes twisted-rope (arrows). At higher magnification, a fiber having a diameter of about 20 micrometers is shown (Fig. 5c). RECck is shown in Figure 5d,e. The first micrograph indicates a different morphological pattern. Increased homogeneity is evident in the 3D structure (Fig. 5d) of RECck when compared to REC (Fig. 5a). The presence of pores is evident. The mean pore diameter was roughly determined to be in the range of 100–200 micrometers. Sporadic globules together with thin fibrils and numerous flat films appear in Figure 5e,f. Summarizing, the data demonstrate that the REC was crosslinked in a transparent hydrogel. CD spectroscopy substantially supports the invariance of the secondary structure of RECck in comparison to REC, even if with slight Chirality DOI 10.1002/chir
modifications. In other words, the constraints induced by the crosslinking bonds triggered the adoption of a more stable type I-beta turn as suggested by the invariance of CD spectra carried out at different temperatures in TFE. On the other hand, more pronounced differences were observed at the supramolecular level by AFM and SEM micrography. Interestingly, AFM studies demonstrated for RECck the tendency to self-aggregate in long fibers without the intervention of any other external variable triggering the aggregation such as, for example, the time or the temperature. Furthermore, RECck gave thin fibrils after sonication. That finding suggests that the manipulation of the sample could be used for tuning its final properties in order to obtain smart materials with applications in bioelectronics. Such properties were assessed for an elastin-inspired polypeptide whose nanometric fibrils were sectioned by the AFM tip realizing biomolecular gaps in the range of nanometers.30 Furthermore, SEM micrography revealed for RECck peculiar morphologies such as films and networks interconnected through pores not observed for REC. Even if some caution is used in interpreting AFM and SEM data, because we are dealing with dried samples susceptible to artifacts due to sample drying and preparation, it is undoubtedly demonstrated that elastin-derived peptides, even when very small, are able to give rise to fibrils and filaments similar to those exhibited by the entire protein.14,31–33 Nevertheless, all data were
ELASTOMERIC-PROTEINS INSPIRED CHIMERA
Fig. 5. SEM images of freeze-dried samples; REC polypeptide (a–c); RECck polypeptide (d–f). Bars represent: (a,d) 500 microns; (b,e) 100 microns; (c,f) 10 microns.
equally collected on lyophilized materials both for REC and for RECck despite the different supramolecular structures observed. On the basis of the collected data and regarding the potential mechanism of assembly, it may be argued, even if in a speculative way, that the capacity to self-assemble more readily by RECck could be related to the increase in molecular weight of the soluble RECck oligomers obtained during crosslinking reaction rather than to the crosslinking process itself. However, further studies are in progress in our laboratory in order to gain more insights into the general mechanism of assembly of these recombinant polypeptides.
CONCLUSION
This report describes the crosslinking of a recombinant polypeptide inspired by repeated sequences of elastomeric proteins and its preliminary characterization. The data show that the crosslinking reaction improved the self-assembly properties of the polypeptide when compared to REC. The full physico-chemical characterization carried out at the molecular and supramolecular level makes RECck a good candidate as an electronic device and nanostructured biomaterial. As future perspectives, biological tests to define biocompatibility and cell adhesion properties will be defined.
ACKNOWLEDGMENTS
We Thank Dr. Neluta Ibris and Mr. Alessandro Laurita (ex-CIGAS, University of Basilicata) for AFM and SEM images. Financial support from the Italian Ministry of University and Research (MIUR) (PRIN 2010-Project 2010 L(SH3K)) is gratefully acknowledged. LITERATURE CITED 1. Gosline J, Lillie M, Carrington E, Guerette P, Ortlepp C, Savage K. Elastic proteins: biological roles and mechanical properties. Philos Trans R Soc Lond B Biol Sci 2002; 357: 121–32. 2. Bochicchio B, Pepe A, Tamburro AM. Investigating by CD the molecular mechanism of elasticity of elastomeric proteins. Chirality 2008; 20: 985–94. 3. Chow D, Nunalee ML, Lim DW, Simnick AJ, Chilkoti A. Peptide-based biopolymers in biomedicine and biotechnology. Mater Sci Eng R Rep 2008; 62: 125–55. 4. Asai D, Xu D, Liu W, Garcia Quiroz F, Callahan DJ, Zalutsky MR, Craig SL, Chilkoti A. Protein polymer hydrogels by in situ, rapid and reversible self-gelation. Biomaterials 2012; 33: 5451–8. 5. Keeley FW, Bellingham CM, Woodhouse KA. Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin. Philos Trans R Soc Lond B Biol Sci 2002; 357: 185–9. 6. Samouillan V, Lamure A, Maurel E, Dandurand J, Lacabanne C, Ballarin F, Spina M. Characterisation of elastin and collagen in aortic bioprostheses. Med Biol Eng Comput 2000; 38: 226–31. 7. Lin Y, Xia X, Wang M, Wang Q, An B, Tao H, Xu Q, Omenetto F, Kaplan DL. Genetically programmable thermoresponsive plasmonic gold/silkelastin protein core/shell nanoparticles. Langmuir 2014; 30: 4406–14. Chirality DOI 10.1002/chir
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