Biological and Structural Characterization of a Naturally Inspired

Dec 5, 2013 - Istituto di Fisica, Universitá Cattolica del Sacro Cuore, Roma, Italy. ∥. Department of Molecular Medicine, Center for Tissue Enginee...
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Biological and Structural Characterization of a Naturally Inspired Material Engineered from Elastin as a Candidate for Tissue Engineering Applications Massimo Vassalli,*,† Francesca Sbrana,† Alessandro Laurita,‡ Massimiliano Papi,§ Nora Bloise,∥,¶ Livia Visai,∥,¶ and Brigida Bochicchio⊥ †

Institute of Biophysics, National Research Council, Genova, Italy CIGAS, University of Basilicata, Potenza, Italy § Istituto di Fisica, Universitá Cattolica del Sacro Cuore, Roma, Italy ∥ Department of Molecular Medicine, Center for Tissue Engineering (CIT), INSTM UdR of Pavia, University of Pavia, Pavia, Italy ⊥ Department of Science, Università della Basilicata, Via Ateneo Lucano 10, Potenza, Italy ¶ Department of Occupational Medicine, Ergonomics and Disability, Salvatore Maugeri Foundation (FSM), IRCCS, Laboratory of Nanotechnology, Pavia, Italy ‡

ABSTRACT: The adoption of a biomimetic approach in the design and fabrication of innovative materials for biomedical applications is encountering a growing interest. In particular, new molecules are being engineered on the basis of proteins present in the extracellular matrix, such as fibronectin, collagen, or elastin. Following this approach scientists expect to be able not only to obtain materials with tailored mechanical properties but also to elicit specific biological responses inherited by the mimicked tissue. In the present work, a novel peptide, engineered starting from the sequence encoded by exon 28 of human tropoelastin, was characterized from a chemical, physical, and biological point of view. The obtained molecule was observed to aggregate at high temperatures, forming a material able to induce a biological effect similar to what elastin does in the physiological context. This material seems to be a good candidate to play a relevant role in future biomedical applications with special reference to vascular surgery.

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hydrolyzed soluble form,8 as recombinant tropoelastin,9,10 as repeats of synthetic sequences,11,12 and also in combination with other biopolymers.13−15 From a biological point of view, mimetic materials inspired by extracellular matrix components are expected to be able to guide the migration, growth, and organization of cells during regeneration processes. Indeed, it has been shown that elastin or its derivatives interact and favor the growth of endothelial cells,16 inhibit smooth muscle cell proliferation,17 and result to be antitrombogenic.18,19 The remarkable mechanical properties provided by elastin into connective tissue (such as large arteries, elastic ligaments, lungs, and skin) is mainly due to the abundance of hydrophobic domains, rich of apolar amminoacids such as Pro, Val, Ala, and Leu. Moreover, the peculiar resistance to the rupture of elastin is mainly associated to the presence of hydrophilic domains, lysine-rich, and of a cross-link network. In particular, this highly cross-linked nature is mainly responsible for the insolubility of elastin that renders the protein difficult to study in solution by

INTRODUCTION The development of innovative biomimetic materials is gaining great interest in tissue engineering and regenerative medicine. Naturally inspired molecules are often the key component of new multifunctional materials tailored to serve as scaffold for cell culture or innovative bioactive coatings. Among all, a special interest was cast on materials able to emulate the native extracellular matrix properties and, therefore, to elicit specific cellular responses and to guide new tissue formation or regeneration. In particular, biomaterials, inspired to naturally occurring systems, such as collagen and elastin, are emerging as pivotal components in functional tissue engineering. Their capability to intrinsically incorporate biological activity and to regulate growth factor signaling, as well as to promote cell proliferation, migration, and differentiation, make them innovative and popular materials. They have been employed in various forms as coatings and films, as hydrogels and as polymers. Especially, elastin and its derived molecules, exhibiting remarkable properties, such as elasticity, selfaggregation, and biological activity, have proved to be attractive for a wide variety of biomedical applications, including skin substitutes,1 vascular grafts,2,3 heart valves,4 and elastic cartilage.5 They have been employed as insoluble fibers,6,7 © 2013 American Chemical Society

Received: August 28, 2013 Revised: December 5, 2013 Published: December 5, 2013 15898

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spectroscopy and to manage for laboratory applications. However, nowadays recombinant DNA technologies allow for the synthesis of tailored polypeptides, derived from elastin or hydrophobic domains of tropoelastin, taking the advantage of a monodisperse polypeptide product. A large number of tailored polypeptides have been synthesized and their biophysical properties were studied. Large variants of polypeptides having the pentapeptide motif VPGXG (where X is any amino acid different from proline) n-fold repeated have been produced, because they are highly soluble in aqueous solution. Furthermore, they possess the property of self-aggregating at a critical temperature forming cross-linked gel-like networks organized in fibrillar and fibrous supramolecular structures and giving rise to a polymeric material with mechanical and supramolecular properties very similar to those of the native elastin. As a relevant example, a recombinant elastin-derived polypeptide consisting of sequences coded by exon 20, 21, and 24 of the human tropoelastin gene was synthesized and investigated as nontrombogenic coatings for small diameter vascular grafts. This polypeptide showed to be highly stable, fiber forming, low immunogenic, and it reduced the capability to stimulate both platelets and smooth muscle cells, highlighting a strong potential as a component of a potential engineered vascular conduit.12 Previous studies on elastin were mainly confined to soluble derivatives such as alpha- and kappa- elastin or to small peptides, the sequences of which were found as a repeated motif into the elastin primary structure, obtained by chemical synthesis. It is interesting to observe that even small pentapeptides showed the propensity to self-aggregate into twisted-rope structures similar to those exhibited by the entire protein, highlighting an intrinsic redundance and self-similarity of the protein biological role. This functional finding is reflected by the structure of the protein that is made by repetitive sequences found at different scales along the entire sequence of the molecule. Taking this finding into account, it can be expected that polypeptide sequences encoded by single exons of the human tropoelastin gene (HTE) could adopt autonomous and independent functions related to the specific conformations. This picture was validated by a reductionist approach consisting in the study of each polypeptide sequence encoded by each exon of HTE and considered useful for obtaining insights into the structural properties of the protein.20 These studies were realized by accomplishing the exon-by-exon full chemical synthesis of human tropoelastin and carrying out a complete conformational study on the synthetic polypeptides. In particular, this procedure led to the discovery that some of the polypeptide sequences encoded by the proline-rich domains, such as exon 18, 20, and 24 were able to coacervate and give rise to filaments with a supramolecular organization similar to that exhibited by the entire protein.20 So far, only a characterization at the molecular level was performed on these promising polypeptides, while biological compatibility tests are still lacking.21,22 In this study, the structure and biological activity of an engineered peptide inspired by the sequence encoded by exon 28 of the human tropoelastin gene was investigated. In particular, a conformational and morphological study was carried out of the networked fibrillar material obtained when heating the peptide. This material was also characterized from a biological point of view in order to assess its effectiveness for tissue engineering applications.

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MATERIALS AND METHODS

Peptide Synthesis and Purification. The Ex28K-coded Nacetylated peptide (EX28K-LIN) was synthesized by solid phase methodology using an automatic synthesizer (Applied Biosystems 431 A). The peptide sequence is Ac-GKAAVPGVLGGLGALGGVGIPGGVKV, where the reactive lysine residues, highlighted in bold, are inserted in the proximity of the two peptide extremities. Fmoc/ DCC/HOBT chemistry was used, starting from 222 mg (0.25 mM) of Wang resin (Novabiochem, Laufelfingen, Switzerland). The Fmocamino acids were purchased from Nova Biochem (Laufelfingen, Switzerland) and from Inbios (Pozzuoli, Italy). The cleavage of the peptide from the resin was achieved by using an aqueous mixture of 95% trifluoroacetic acid (TFA). The peptide was lyophilized and purified by reversed-phase HPLC (high-performance liquid chromatography). Binary gradient was used and the solvent were H2O (0.1% TFA) and CH3CN (0.1% TFA). Turbidimetry Assay. Turbidimetry of 1.5 ml of 5 mM solution of the EX28K-LIN peptide in TBS solution (Tris 50 mM, NaCl 1.5 M, and CaCl2 1.0 mM, pH 7.0) was measured at 440 nm as a function of temperature on a Cary UV50 spectrophotometer equipped with a Peltier temperature controller using 1 cm path length quartz cells and reported as turbidimetry on apparent absorbance (TAA) versus temperature. The solution temperature was increased from 20 to 70 °C with 2 °C steps, while monitoring the absorbance at 440 nm after 5 min equilibration at each temperature point. Then the solution temperature was decreased back from 70 to 20 °C with 5 °C steps, monitoring the absorbance at 440 nm after 5 min. The material obtained after the thermal cycle will be hereafter indicated as EX26KF1B. Circular Dichroism (CD). CD spectra for the peptide were obtained using a Jasco J-600 Spectropolarimeter at various temperatures and at concentrations of 0.1 mg/mL in water by using cells of 0.1 cm. Spectra were acquired in the range 190 −250 nm by taking points every 0.1 nm, with 20 nm min−1 scan rate, integration time of 2 s, and 1 nm bandwidth. The data are expressed in terms of [Θ], the molar ellipticity in units of deg cm2 dmol−1. Fourier Transform Infrared Spectroscopy (FTIR). EX28K-LIN peptide was examined by FTIR as a lyophilized powder and its aggregated form, EX28K-FIB, in the solid state in KBr pellets (1 mg/ 100 mg). The spectra were recorded on a Jasco FTIR-460 PLUS using a resolution of 4 cm−1 and then smoothed by using the Savitzky-Golay algorithm.23 The decomposition of FTIR spectra was obtained using the peak fitting module implemented in the Origin Software (Microcalc Inc.) using the second derivative method. In the curve fitting procedure, the Voigt peak shape has been used for all peaks. The Voigt shape is a combination of the Gaussian and Lorentzian peak shapes and accounts for the broadening present in the FTIR spectrum. Environmental Scanning Electron Microscopy (ESEM) of EX28K-FIB. EX28K-FIB after the thermal cycle was observed by ESEM using a Philips XL ESEM microscope under controlled pressure ranging between 2.0 Torr and 5.2 Torr. For environmental conditions, the sample was filtered under vacuum (0.45 μm) before ESEM measurements; for standard SEM measurements, the lyophilized pellet collected after centrifugation was observed. Atomic Force Microscopy (AFM) Imaging. Small 5 μL drops of EX28K-FIB were deposited on clean Si 100 substrates for 5 min, rinsed with filtered Milli-Q water, and then air-dried for AFM imaging. Data acquisition was carried out by using a XE-120 microscope (Park Systems Inc., CA, U.S.A.) in intermittent contact mode in air under ambient condition. Rectangular silicon cantilevers (PPP-NCHR, Nanosensors, Neuchatel, Switzerland) with a nominal tip radius of 10 nm and 330 kHz resonance frequency were used. Topography images were acquired at a resolution of 512 pixels per line using a scan rate of 0.4 Hz. AFM scanner performance and calibration was routinely checked by using a reference grid model STR3-180P (VLSI Standards, CA, U.S.A.) with a lateral pitch of 3 μm and step height of 18 nm. AFM images were preprocessed for tilt correction and scars removal with Gwyddion software.24 With respect to SEM images, to 15899

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appreciate the morphology of isolated filaments on EX28K-FIB samples AFM imaging was performed on strongly diluted samples. AFM Image Processing for Features Extraction. Squared AFM images of a defined lateral dimension (10 μm × 10 μm) taken on EX28K-FIB samples (see previous section) were processed to calculate fibrillar features. Using a semiautomated procedure implemented in a dedicated freeware software,25 the mathematical path r(s) describing the contour of single fibers was extracted and relevant geometrical parameters were calculated. In particular, if the EX28K-FIB polymer is supposed to behaves as a thermally driven semiflexible polymer, its conformation is statistically described by the wormlike chain (WLC) model.26 If the tangent versor t(̂ s) is obtained from the parametrized curve t (̂ s) =

were fixed with 2.5% (v/v) glutaraldehyde solution (Sigma-Aldrich) in a 0.1 M Na-cacodylate buffer (pH 7.2) for 1 h at 4 °C, washed with Na-cacodylate buffer, and then dehydrated at room temperature in a gradient EtOH series up to 100%.29 The samples were kept in 100% EtOH for 15 min and then critical-point-dried with CO2. The specimens were sputter-coated with gold and observed at 250× and 1500× magnification using a Leica Cambridge Stereoscan 440 microscope (Leica Microsystems, Benshein, Germany) at 8 kV.

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RESULTS Thermal Aggregation of EX28K-LIN. The thermal evolution of the peptide was evaluated by turbidimetry measurements. EX28K-LIN was warmed and cooled as described in Materials and Methods in the 20−70 °C temperature range, and the experimental results are reported in figure 1. While warming, the absorbance has a clear sigmoidal

∂ r(s) ∂s

its correlation function is expected to have an exponential decay ⟨t (̂ s + σ )t (̂ s)⟩σ = e−s / P The decay length P is the so-called persistence length P that carries information about the bending elasticity of the polymer (the longer the P, the harder the rod). In the analysis described in Results, only fibers for which L/P > 4 were considered to remain in a reasonable range of validity of the WLC model.27 Cell Cultures. The murine fibroblast cells line L929 (ATCC: CRL2148) and the human osteosarcoma cell line SAOS-2 (ATCC:HTB 85) were obtained from the American Type Culture Collection (Rockville, MD). L929 cells were cultured in RPMI-1640 medium (Cambrex Bio Science, Baltimore, MD) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, and 1% antibiotics (Sigma-Aldrich, St.Louis, MO, U.S.A.). SAOS-2 cells were cultured in McCoy’s 5A modified medium with 1% L-glutamine and 25 mM HEPES (Cambrex Bio Science, Baltimore, MD), supplemented with 15% fetal bovine serum, 1% sodium pyruvate, and 1% antibiotics. Both types of cells were cultured at 37 with 5% CO2, routinely trypsinized after confluency, counted, and seeded on 96 multiwell culture plates at 1 × 104 cells/well. After 4 h incubation, different concentrations (25, 50, 100, and 250 μg/ml) of EX28K-LIN or EX28K-FIB, previously UV sterilized for 1 h, were added to the seeded cells and further incubated for 24 h and 7 days. No medium change was performed until the end of the culture incubation. The positive control was represented by both cell types without EX28K-LIN or EX28K-FIB and incubated for the same period of time. 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. To evaluate the mitochondrial activity of untreated (control) and treated SAOS-2 and L929 cells a colorimetric assay with MTT (Sigma-Aldrich) was performed after 24 h and 7 days (end of the culture period).28 At both time points, the medium was removed and replaced by 100 μL of fresh culture medium without serum. To each well of cultured cells containing the fresh medium, 10 μL of MTT (5 mg/mL) in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCL, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) was added and the cell cultures were incubated in dark in a humidified 5% CO2 incubator at 37°C for 4 h. Viable cells are able to reduce MTT into formazan crystals. At the end of incubation, the MTT solution was removed and 100 μL of acidic isopropanol (0.1 M HCl in absolute isopropanol) were added to solubilize the fromazan products for 30 min at 37 °C in dark. Aliquots of 200 μL were sampled, and the absorbance values were measured at 595 nm by Biorad iMark microplate reader (BioRad Laboratories, Hercules, CA). All measurements regarding cell viability were tested in triplicate. Standard curve cell viability for each type of cell was used and the results expressed as percentage to untreated cells that were set to 100%. Scanning Electron Microscopy (SEM) Analysis of Cell Cultures. For SEM analysis, SAOS-2 and L929 cells were seeded on sterile Thermanox plastic coverslips (polyolefin) (Nalge Nunc International, Rochester, NY) and then incubated as indicated above. At the end of each incubation time (24 h and 7 days), cells treated with EX28K-LIN and EX28K-FIB, such as control untreated cells,

Figure 1. Turbidimetry assay of 5 mM EX28K-LIN peptide in TBS solution (pH 7.0); absorbance is reported as a function of temperature for warming (triangles) and cooling (rhombus) processes.

transition just over 70 °C, indicating that the peptide is starting to aggregate toward structures of more than few 100 nm (thus scattering more light at 440 nm, enhancing the absorbance). Interestingly, this transition seems to be irreversible and the peptide remains in an aggregated state even after the cooling process. The term EX28K-FIB will be used throughout the paper to indicate this aggregated state, obtained from EX28KLIN after thermal cycle. Structural Change of EX28K-LIN upon Heating. To have some insights into the aggregation transition highlighted by the abrupt change in absorbance at high temperatures (figure 1), finer conformational details were investigated by means of CD spectroscopy. Figure 2 shows the CD spectra of EX28K-LIN in different buffers (H2O and TFE) as a function of temperature. In aqueous solution at 0 °C, the spectrum is characterized by a shoulder at about 216 nm and by a strong negative band at 197 nm. While increasing the temperature to 25and 60 °C, the shoulder at 216 nm progressively disappears and the negative band is reduced. Overall, CD spectra exhibit features quite compatible with the presence of Polyproline-II (PPII) structure,30 together with unordered conformation. However the lack of a well-defined isoelliptic point suggests the contribution of at least one other conformation. In fact, the difference between the CD spectra at 60 and 0 °C for H2O 15900

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particular at 0 °C, the spectrum shows a negative band at 220 nm due to n−π* electronic transition, a negative one at about 208 nm and a positive band at 180 nm due to parallel and perpendicular components of π−π* electronic transition, respectively. The whole spectral features are typical of type I/ III β-turn conformation as also suggested by the high [Θ] value of the positive band. The increase of the temperature to 25 °C does not affect the π−π* electronic transitions thus leaving substantially unchanged the bands at 208 and 190 nm. On the contrary, the band at 222 nm slightly decreases. The further increase to 60 °C induces the reduction of the positive band and the increase of both negative bands thus suggesting the destabilization of the α-helical conformation. EX28K-FIB Structural Components. While CD spectroscopy provides information on the structural changes induced in EX28K-LIN upon heating, it cannot be used after the transition to EX28K-FIB that induces aggregation and the solution becomes dense and opaque. To gain some insight on the conformation acquired by EX28K-FIB, solid-state FTIR spectroscopy was applied. This technique allows the mapping of full vibrational spectra in the infrared region and in particular the amide I region is mainly due to the stretching vibrations of the CO group of the polypeptide backbone and is the most informative on protein secondary conformation. Furthermore, it is stronger than the amide II component, which originates from the combination of the N−H bending and CO stretching modes. The analysis of FTIR spectra was concentrated on these two bands, carrying the most relevant information on the polypeptide secondary structure.31 The decomposed FTIR spectra of the amide I and II regions of EX28K-LIN and EX28K-FIB are shown in Figure 3. The decomposed FTIR spectrum of the amide I region shows for EX28K-LIN a component at 1640 cm−1, attributed to the absorption of water, usually found between 1640−1650 cm−1. The component at 1661 cm−1 is assigned to unordered conformations. The component at 1680 cm−1 is assigned to βturns.32 The remaining component at 1698 cm−1 could be also assigned to β-turns and unordered conformations, respectively. The FTIR decomposed spectrum of EX28K-FIB shows a strong band at 1630 cm−1, together with a minor one at 1680 cm−1, indicative of β-pleated sheet conformations. The presence of both components allows to exclude the contribution of hydrogen bonded β-turns, also showing a band in the 1625 and 1634 cm−1 range, in favor of the presence of antiparallel β-sheet conformation.33 Finally, the band at 1654

Figure 2. (a) CD spectra of EX28K-LIN peptide at growing temperatures in H2O (full squares 0 °C ; full circles 25 °C ; full triangles 60 °C) and in TFE (empty squares 0 °C ; empty circles 25 °C ; empty triangles 60 °C). (b) Inset: Difference of CD spectra in H2O between 60 and 0 °C in water.

buffer, reported in the inset in Figure 2, clearly shows the appearance of the signature of antiparallel beta-structures with a negative band at around 217 nm and a positive one at about 195 nm, that are thus starting to participate to the conformers population. 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 and, in addition, the protein hydrophobicity and highly cross-linked nature suggest a less polar internal environment than the surrounding solvent. For this reason, the experiments in this study were performed in water but also in trifluoroethanol (TFE) that is a significantly less polar solvent than water. Moreover, TFE is usually considered a structure-inducing solvent because it favors intramolecular hydrogen bonding, thus promoting folded conformations such as helices and beta-turns. 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.20 CD spectra of EX28KLIN in TFE indicates the presence of α-helical structures. In

Figure 3. FTIR spectra of EX28K-LIN (a) and EX28K-FIB (b) in KBr pellet. The band fitting results of amide I and II regions are shown. Dashed line, experimental spectrum; solid line, calculated spectrum. 15901

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Figure 4. Representative ESEM (a) and SEM (b) image of EX28K-FIB. Acquisition details and scale bars are reported in the legend of the pictures.

Figure 5. (a) Representative AFM image of EX28K-FIB at low concentration (vertical range = 40 nm). (b,c) Successive AFM acquisitions of selected regions of the image in (a), represented with a different look-up table, to highlight specific features. (d) Distribution of the Young’s modulus estimated for a set of 84 fibers. (e) Distribution of the height over lateral dimension ratio α for selected fibers (see text for details).

cm−1 is assigned to unordered conformation. The amide II components at 1527 and 1551 cm−1 could be attributed to antiparallel β-sheets and unordered conformations, respectively. Morphology of EX28K-FIB Material. SEM and AFM analysis were used to characterize the morphology of EX28KFIB samples. As shown in Figure 4a, ESEM visualization revealed an interconnected network of bundle rope densely packed into a monolithic structure. The ultrastructure of the aggregate can be observed after dehydration of the sample and imaging in SEM (Figure 4b), showing the presence of a complex network of thin interconnected filaments. To appreciate the morphology of isolated filaments, AFM imaging was performed on EX28K-FIB after dilution (Figure 5a−c). Everywhere in the samples, short cylindrical filaments, putatively fibrils, are clearly visible (see region c in Figure 5) with a diameter of about 20−30 nm and a disperse length in the range of 300 nm to 1 μm. In addition, it is also possible to observe larger fibers that seem to be made by twisted fibrils (see detail Figure 5c) with a pitch of 110−160 nm. While fibrils

are common, fibers are more rare and only few images with both fibrils and fibers (as in Figure 5a) were acquired. Mechanical Properties of EX28K-FIB Fibrils. To have an insight into the mechanical properties of the fibrils composing the EX28K-FIB material, a statistical approach was applied. If the fibrils are thought to behave as elastic filaments in solution, the shape they assume upon deposition on a surface relates to the energy of the bath (the temperature T) and to their flexibility: if the fibrils are very rigid, they are expected to deposit as straight rods, while in the opposite case of extremely flexible filaments they are expected to appear rolled up with a curved shape. In the framework of the WLC model (introduced in Materials and Methods), the elasticity of the fibrils (their Young’s modulus) can be measured starting from a knowledge of the persistence length P.14 In particular, it can be shown that34 YI = kBTP

where Y is the Young’s modulus, kB is the Boltzmann constant, T is the absolute temperature, I is the cross sectional moment 15902

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Figure 6. Cell viability results at two incubation times, as determined by MTT test performed on L929 (panels a and b) and SAOS-2 (panels c and d) cells treated with increasing concentrations of peptide (panels a and c) and EX28K-FIB (panels b and d). The values represented are the means of the results of each sample performed in duplicate and repeated in three separated experiments. Error bars indicate standard errors of the means.

Figure 7. Representative SEM images of L929 (left) and SAOS-2 (right) cells cultured for 7 days. Panels a and b show the control culture (a) and a zoom on it (b). Cells treated with low (25 μg) and high (250 μg) dose of are shown in panels c and e, while panels d and f report the results after incubation, imaged at 250×. Scale bars represent 10 μm (panels a, c, d, e and f) and 3 μm (panel b and the insets in panels c, d, e, and f).

of inertia of the fibril, and P is its persistence length. Supposing to describe the fibril as an elastic cylinder with an elliptic cross section, this equation leads to Y=

4kBT πab3

with all values ranging between 0.4 and 3.5 MPa and a peak at 0.7 MPa. To evaluate the stability of the measurements, an independent evaluation of b was obtained. This parameter is particularly critical because it appears at the power of 3 in the equation and it is evaluated from a profile that suffers of AFM tip convolution problems. A subset of fibers for which the value of b was highly stable among the 10 measured profile was chosen and the ratio α = a/b was calculated, obtaining a peaked distribution (see histogram in Figure 5e) around α = 0.17. This value was then used to calculate the moment of inertia I for all fibrils

P

in which a and b are the radii of the elliptical section with a being in the vertical direction and b in the lateral one. To obtain a statistically relevant evaluation of the Young’s modulus of EX28K-FIB fibrils, more than 80 isolated fibrils were analyzed by extracting P from a semiautomated software procedure (see Materials and Methods) and measuring a and b from 10 profiles. The results are reported in Figure 5d in which a skewed distribution o the Young’s modulus can be observed,

I= 15903

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presence of the acetyl group (see Materials and Methods) does not alter this main feature of the peptide, that is mechanically outperforming as an ideal spring.35 Interestingly, imposing a thermal cycle to EX28K-LIN and taking the peptide over about 60 °C induces an irreversible transition, as monitored by turbidimetry. This process converts the isolated peptide in a strongly aggregated form, named EX28K-FIB. The first steps of this pathway were monitored by CD, showing that it undergoes a conformational change from the mainly unordered structure to a β-rich state that is aggregation-prone, probably following a hierarchical mechanism previously observed in elastin-like peptides.21,36 This finding was also monitored by FTIR spectroscopy that confirmed the presence of structuring β-sheets in the EX28KFIB material. The use of high-resolution microscopy (SEM and AFM) allowed to characterize the morphology of showing the presence of elongated fibrils inside the material that can twist and further aggregate to even form big supramolecular aggregates. Even though no independent spectroscopic measurements were performed, it is not unlikely that the aggregation pathway followed by EX28K-LIN toward EX28KFIB is amyloidogenic in its nature.21 In addition, AFM imaging was also exploited to obtain a quantification of the elastic properties of EX28K-LIN fibrils in EX28K-FIB, measuring a Young’s modulus of few MPa, completely in agreement with similar studies performed in literature on elastin-like or elastininspired materials14,15,37 showing that EX28K-FIB is mimicking the properties of the inspiring molecule, elastin, also from a macroscopic mechanical point of view. Furthermore, EX28K-LIN and EX28K-FIB were also characterized from a biological point of view by means of enzymatic MTT test and direct morphological inspection. The biomimetic approach that was adopted in the synthesis of the peptide resulted, as expected, in a molecule that is highly biocompatible and, besides a very small activation of the cellular populations for higher times, it is almost biologically inactive. On the contrary, EX28K-FIB is inducing a relevant biological response, reducing cell viability of fibroblasts and, even more markedly, of osteoblast cells. This behavior mimics one of the features for which elastin has raised interest in regenerative medicine and, in particular, in vascular applications. In fact, elastin, which constitutes the main component of the tunica intima, the innermost layer of blood vessels, has the ability to strongly reduce the proliferation of unwanted cells (in particular smooth muscles17 and fibroblasts), potentially acting as an antitrombogenic molecule.18,19

and thus to obtain a determination of the Young’s modulus independent from each measure of b Y=

4α 3kBT πa 4

P

The histogram for Y calculated from this procedure was fully compatible with the previous determination (data not shown), giving rise to a peak value of 0.9 MPa in the same range of values. Biocompatibility of EX28K-LIN and Biological Effect of EX28K-FIB. The biocompatibility of EX28K-LIN was tested by MTT assay and morphological inspection on L929 (fibroblasts) and SAOS-2 (osteoblast) cell lines. A dose−response curve was evaluated at 24 h and 7 days by MTT assay (see Figure 6a,c). For both cell lines, the viability was substantially doseindependent and for longer times (7 days) the enzymatic activity in the presence of the EX28K-LIN peptide was a bit higher than for control cells (zero dose), showing a tendency of the cellular systems to be stimulated by the presence of in EX28K-LIN solution. Similarly, incubation of L929 and SAOS2 cells with 25 or 250 μg of EX28K-LIN did not reveal any specific morphological changes with respect to control after 24 h (data not shown) such as after 7 days (Figure 7 panels c and e, with respect to controls in panel a). This result was similarly confirmed at higher magnification (inset in panels c and e of Figure 7 with respect to control in panel b). The same analysis was repeated after treating the cells with EX28K-FIB. In this condition, MTT assay showed a dosedependence for both cell lines (Figure 6 panels b and d) with clearly stronger effect on SAOS-2 cells. In any case, for the highest dose the viability of cells under EX28K-FIB treatment was never higher than 60% of the control. Moreover, marked morphological changes were observed when both cell types were treated with EX28K-FIB: the cells were lower in number for both adherent fibroblast and osteoblast cells, and they showed an altered shape, as reported in Figure 7 panels d and f (zooms are shown in the insets) with respect to their control (panels a and b). In particular, SAOS-2 cells treated with EX28K-FIB did not show the typical cytoplasmic elongations and were somewhat detached.

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DISCUSSION Tropoelastin is a modular protein organized in a cassette-like structure in which each domain exploits specific functions that are redundant along the whole molecule. In general, two main classes of domains can be identified, based on their function: mechanical domains, that confer to elastin its unique mechanical properties, and cross-linking domains, that participate in the formation of the supramolecular networked structure. Exon 28 from which EX28K-LIN was mainly inspired, is a mechanical domain whose ability to extend upon stretch and recover the initial length when the stimulus is released is based on its random coil structure. In fact, an unstructured polypeptide in solution acts as a reservoir of entropic energy, that is, the main reversible source of energy (elastic energy) in biopolymers, while enthalpy is associated to transitions in the internal degrees of freedom that are normally irreversible. The structural analysis performed by CD and FTIR confirm the biomimetic nature of the EX28K-LIN that shows a main presence of unstructured regions and a strong similarity with peptides coded by other elastin mechanical exons.20 The addition of the lysines in the engineered sequence and the

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CONCLUSIONS A new elastin-inspired peptide, EX28K-LIN, was synthesized and characterized from a chemical, physical, and biological point of view. This molecule has a relevant tendency to selfaggregate at high temperatures and to form an irreversible network of fibrils and fibres, EX28K-FIB. This material was also characterized from a biological point of view, showing a clear antiproliferative activity that can be seen as an inheritance of the bare elastin on which EX28K-LIN was inspired. Moreover, lysine residues were added in the sequence of the peptide to provide a sort of molecular hook, ready for exploiting the chemistry of the amino side chain to modulate cross-linking parameters38 or to obtain covalent linkages to technological surfaces. In perspective, this material will constitute an interesting platform in vascular applications, keeping many of the beneficial properties of elastin and providing a technological 15904

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advancement in terms of potential high throughput production. In particular, specific applications as coating material for indwelling vascular devices, such as stents, can be foreseen.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thanks Dr. Neluta Ibris for collaboration on AFM images (CIGAS, University of Basilicata) and to Dr. Marina Lorusso for her technical assistance. The financial support from MIUR (PRIN 2010-Project 2010L (SH3K)) is gratefully acknowledged. We are grateful to Dr. Picenoni (Politecnico di Milano, Milano, Italy) for technical assistance in SEM studies.

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