Biological and Structural Characterization of a Naturally InspiredMaterial Engineered from Elastin as a Candidate for TissueEngineering ApplicationsMassimo 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, Universita 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, Universita della Basilicata, Via Ateneo Lucano 10, Potenza, Italy¶Department of Occupational Medicine, Ergonomics and Disability, Salvatore Maugeri Foundation (FSM), IRCCS, Laboratory ofNanotechnology, Pavia, Italy

ABSTRACT: The adoption of a biomimetic approach in thedesign and fabrication of innovative materials for biomedicalapplications is encountering a growing interest. In particular,new molecules are being engineered on the basis of proteinspresent in the extracellular matrix, such as fibronectin,collagen, or elastin. Following this approach scientists expectto be able not only to obtain materials with tailoredmechanical properties but also to elicit specific biologicalresponses inherited by the mimicked tissue. In the presentwork, a novel peptide, engineered starting from the sequence encoded by exon 28 of human tropoelastin, was characterized froma 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 seemsto be a good candidate to play a relevant role in future biomedical applications with special reference to vascular surgery.

■ INTRODUCTION

The development of innovative biomimetic materials is gaininggreat interest in tissue engineering and regenerative medicine.Naturally inspired molecules are often the key component ofnew multifunctional materials tailored to serve as scaffold forcell culture or innovative bioactive coatings. Among all, aspecial interest was cast on materials able to emulate the nativeextracellular matrix properties and, therefore, to elicit specificcellular responses and to guide new tissue formation orregeneration. In particular, biomaterials, inspired to naturallyoccurring systems, such as collagen and elastin, are emerging aspivotal components in functional tissue engineering. Theircapability to intrinsically incorporate biological activity and toregulate growth factor signaling, as well as to promote cellproliferation, migration, and differentiation, make theminnovative and popular materials. They have been employedin various forms as coatings and films, as hydrogels and aspolymers. Especially, elastin and its derived molecules,exhibiting remarkable properties, such as elasticity, self-aggregation, and biological activity, have proved to be attractivefor a wide variety of biomedical applications, including skinsubstitutes,1 vascular grafts,2,3 heart valves,4 and elasticcartilage.5 They have been employed as insoluble fibers,6,7

hydrolyzed soluble form,8 as recombinant tropoelastin,9,10 asrepeats of synthetic sequences,11,12 and also in combinationwith other biopolymers.13−15

From a biological point of view, mimetic materials inspiredby extracellular matrix components are expected to be able toguide the migration, growth, and organization of cells duringregeneration processes. Indeed, it has been shown that elastinor its derivatives interact and favor the growth of endothelialcells,16 inhibit smooth muscle cell proliferation,17 and result tobe antitrombogenic.18,19

The remarkable mechanical properties provided by elastininto connective tissue (such as large arteries, elastic ligaments,lungs, and skin) is mainly due to the abundance of hydrophobicdomains, rich of apolar amminoacids such as Pro, Val, Ala, andLeu. Moreover, the peculiar resistance to the rupture of elastinis mainly associated to the presence of hydrophilic domains,lysine-rich, and of a cross-link network. In particular, this highlycross-linked nature is mainly responsible for the insolubility ofelastin that renders the protein difficult to study in solution by

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

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spectroscopy and to manage for laboratory applications.However, nowadays recombinant DNA technologies allow forthe synthesis of tailored polypeptides, derived from elastin orhydrophobic domains of tropoelastin, taking the advantage of amonodisperse polypeptide product. A large number of tailoredpolypeptides have been synthesized and their biophysicalproperties were studied. Large variants of polypeptides havingthe pentapeptide motif VPGXG (where X is any amino aciddifferent from proline) n-fold repeated have been produced,because they are highly soluble in aqueous solution.Furthermore, they possess the property of self-aggregating ata critical temperature forming cross-linked gel-like networksorganized in fibrillar and fibrous supramolecular structures andgiving rise to a polymeric material with mechanical andsupramolecular properties very similar to those of the nativeelastin. As a relevant example, a recombinant elastin-derivedpolypeptide consisting of sequences coded by exon 20, 21, and24 of the human tropoelastin gene was synthesized andinvestigated as nontrombogenic coatings for small diametervascular grafts. This polypeptide showed to be highly stable,fiber forming, low immunogenic, and it reduced the capabilityto stimulate both platelets and smooth muscle cells, high-lighting a strong potential as a component of a potentialengineered vascular conduit.12

Previous studies on elastin were mainly confined to solublederivatives such as alpha- and kappa- elastin or to smallpeptides, the sequences of which were found as a repeatedmotif into the elastin primary structure, obtained by chemicalsynthesis. It is interesting to observe that even smallpentapeptides showed the propensity to self-aggregate intotwisted-rope structures similar to those exhibited by the entireprotein, highlighting an intrinsic redundance and self-similarityof the protein biological role. This functional finding is reflectedby the structure of the protein that is made by repetitivesequences found at different scales along the entire sequence ofthe molecule. Taking this finding into account, it can beexpected that polypeptide sequences encoded by single exonsof the human tropoelastin gene (HTE) could adoptautonomous and independent functions related to the specificconformations. This picture was validated by a reductionistapproach consisting in the study of each polypeptide sequenceencoded by each exon of HTE and considered useful forobtaining insights into the structural properties of the protein.20

These studies were realized by accomplishing the exon-by-exonfull chemical synthesis of human tropoelastin and carrying out acomplete conformational study on the synthetic polypeptides.In particular, this procedure led to the discovery that some ofthe polypeptide sequences encoded by the proline-richdomains, such as exon 18, 20, and 24 were able to coacervateand give rise to filaments with a supramolecular organizationsimilar to that exhibited by the entire protein.20 So far, only acharacterization at the molecular level was performed on thesepromising polypeptides, while biological compatibility tests arestill lacking.21,22

In this study, the structure and biological activity of anengineered peptide inspired by the sequence encoded by exon28 of the human tropoelastin gene was investigated. Inparticular, a conformational and morphological study wascarried out of the networked fibrillar material obtained whenheating the peptide. This material was also characterized from abiological point of view in order to assess its effectiveness fortissue engineering applications.

■ MATERIALS AND METHODSPeptide Synthesis and Purification. The Ex28K-coded N-

acetylated peptide (EX28K-LIN) was synthesized by solid phasemethodology using an automatic synthesizer (Applied Biosystems 431A). The peptide sequence is Ac-GKAAVPGVLGGLGALGGV-GIPGGVKV, 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) ofWang resin (Novabiochem, Laufelfingen, Switzerland). The Fmoc-amino acids were purchased from Nova Biochem (Laufelfingen,Switzerland) and from Inbios (Pozzuoli, Italy). The cleavage of thepeptide from the resin was achieved by using an aqueous mixture of95% trifluoroacetic acid (TFA). The peptide was lyophilized andpurified by reversed-phase HPLC (high-performance liquid chroma-tography). 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 ofthe 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 oftemperature on a Cary UV50 spectrophotometer equipped with aPeltier temperature controller using 1 cm path length quartz cells andreported as turbidimetry on apparent absorbance (TAA) versustemperature. The solution temperature was increased from 20 to 70°C with 2 °C steps, while monitoring the absorbance at 440 nm after 5min equilibration at each temperature point. Then the solutiontemperature was decreased back from 70 to 20 °C with 5 °C steps,monitoring the absorbance at 440 nm after 5 min. The materialobtained after the thermal cycle will be hereafter indicated as EX26K-F1B.

Circular Dichroism (CD). CD spectra for the peptide wereobtained using a Jasco J-600 Spectropolarimeter at various temper-atures and at concentrations of 0.1 mg/mL in water by using cells of0.1 cm. Spectra were acquired in the range 190 −250 nm by takingpoints every 0.1 nm, with 20 nm min−1 scan rate, integration time of 2s, and 1 nm bandwidth. The data are expressed in terms of [Θ], themolar ellipticity in units of deg cm2 dmol−1.

Fourier Transform Infrared Spectroscopy (FTIR). EX28K-LINpeptide was examined by FTIR as a lyophilized powder and itsaggregated form, EX28K-FIB, in the solid state in KBr pellets (1 mg/100 mg). The spectra were recorded on a Jasco FTIR-460 PLUS usinga resolution of 4 cm−1 and then smoothed by using the Savitzky-Golayalgorithm.23 The decomposition of FTIR spectra was obtained usingthe peak fitting module implemented in the Origin Software(Microcalc Inc.) using the second derivative method. In the curvefitting procedure, the Voigt peak shape has been used for all peaks.The Voigt shape is a combination of the Gaussian and Lorentzian peakshapes and accounts for the broadening present in the FTIR spectrum.

Environmental Scanning Electron Microscopy (ESEM) ofEX28K-FIB. EX28K-FIB after the thermal cycle was observed byESEM using a Philips XL ESEM microscope under controlled pressureranging between 2.0 Torr and 5.2 Torr. For environmental conditions,the sample was filtered under vacuum (0.45 μm) before ESEMmeasurements; for standard SEM measurements, the lyophilized pelletcollected after centrifugation was observed.

Atomic Force Microscopy (AFM) Imaging. Small 5 μL drops ofEX28K-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 (ParkSystems Inc., CA, U.S.A.) in intermittent contact mode in air underambient condition. Rectangular silicon cantilevers (PPP-NCHR,Nanosensors, Neuchatel, Switzerland) with a nominal tip radius of10 nm and 330 kHz resonance frequency were used. Topographyimages were acquired at a resolution of 512 pixels per line using a scanrate of 0.4 Hz. AFM scanner performance and calibration wasroutinely checked by using a reference grid model STR3-180P (VLSIStandards, CA, U.S.A.) with a lateral pitch of 3 μm and step height of18 nm. AFM images were preprocessed for tilt correction and scarsremoval with Gwyddion software.24 With respect to SEM images, to

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appreciate the morphology of isolated filaments on EX28K-FIBsamples 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 onEX28K-FIB samples (see previous section) were processed to calculatefibrillar features. Using a semiautomated procedure implemented in adedicated freeware software,25 the mathematical path r(s) describingthe contour of single fibers was extracted and relevant geometricalparameters were calculated. In particular, if the EX28K-FIB polymer issupposed to behaves as a thermally driven semiflexible polymer, itsconformation is statistically described by the wormlike chain (WLC)model.26 If the tangent versor t(s) is obtained from the parametrizedcurve

= ∂∂

t ss

sr( ) ( )

its correlation function is expected to have an exponential decay

σ⟨ + ⟩ =σ−t s t s e( ) ( ) s P/

The decay length P is the so-called persistence length P that carriesinformation about the bending elasticity of the polymer (the longerthe P, the harder the rod). In the analysis described in Results, onlyfibers for which L/P > 4 were considered to remain in a reasonablerange of validity of the WLC model.27

Cell Cultures. The murine fibroblast cells line L929 (ATCC: CRL-2148) and the human osteosarcoma cell line SAOS-2 (ATCC:HTB85) 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% fetalbovine serum, 1% L-glutamine, 1% sodium pyruvate, and 1%antibiotics (Sigma-Aldrich, St.Louis, MO, U.S.A.). SAOS-2 cells werecultured in McCoy’s 5A modified medium with 1% L-glutamine and 25mM HEPES (Cambrex Bio Science, Baltimore, MD), supplementedwith 15% fetal bovine serum, 1% sodium pyruvate, and 1% antibiotics.Both types of cells were cultured at 37 with 5% CO2, routinelytrypsinized after confluency, counted, and seeded on 96 multiwellculture plates at 1 × 104 cells/well. After 4 h incubation, differentconcentrations (25, 50, 100, and 250 μg/ml) of EX28K-LIN orEX28K-FIB, previously UV sterilized for 1 h, were added to the seededcells and further incubated for 24 h and 7 days. No medium changewas performed until the end of the culture incubation. The positivecontrol was represented by both cell types without EX28K-LIN orEX28K-FIB and incubated for the same period of time.3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium Bro-

mide (MTT) Assay. To evaluate the mitochondrial activity ofuntreated (control) and treated SAOS-2 and L929 cells a colorimetricassay with MTT (Sigma-Aldrich) was performed after 24 h and 7 days(end of the culture period).28 At both time points, the medium wasremoved and replaced by 100 μL of fresh culture medium withoutserum. To each well of cultured cells containing the fresh medium, 10μL of MTT (5 mg/mL) in phosphate-buffered saline (PBS) (137 mMNaCl, 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 humidified5% CO2 incubator at 37°C for 4 h. Viable cells are able to reduce MTTinto formazan crystals. At the end of incubation, the MTT solutionwas removed and 100 μL of acidic isopropanol (0.1 M HCl in absoluteisopropanol) were added to solubilize the fromazan products for 30min at 37 °C in dark. Aliquots of 200 μL were sampled, and theabsorbance values were measured at 595 nm by Biorad iMarkmicroplate reader (BioRad Laboratories, Hercules, CA). All measure-ments regarding cell viability were tested in triplicate. Standard curvecell viability for each type of cell was used and the results expressed aspercentage 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 seededon sterile Thermanox plastic coverslips (polyolefin) (Nalge NuncInternational, Rochester, NY) and then incubated as indicated above.At the end of each incubation time (24 h and 7 days), cells treatedwith EX28K-LIN and EX28K-FIB, such as control untreated cells,

were fixed with 2.5% (v/v) glutaraldehyde solution (Sigma-Aldrich) ina 0.1 M Na-cacodylate buffer (pH 7.2) for 1 h at 4 °C, washed withNa-cacodylate buffer, and then dehydrated at room temperature in agradient EtOH series up to 100%.29 The samples were kept in 100%EtOH for 15 min and then critical-point-dried with CO2. Thespecimens were sputter-coated with gold and observed at 250× and1500× magnification using a Leica Cambridge Stereoscan 440microscope (Leica Microsystems, Benshein, Germany) at 8 kV.

■ RESULTSThermal Aggregation of EX28K-LIN. The thermal

evolution of the peptide was evaluated by turbidimetrymeasurements. EX28K-LIN was warmed and cooled asdescribed in Materials and Methods in the 20−70 °Ctemperature range, and the experimental results are reportedin figure 1. While warming, the absorbance has a clear sigmoidal

transition just over 70 °C, indicating that the peptide is startingto aggregate toward structures of more than few 100 nm (thusscattering more light at 440 nm, enhancing the absorbance).Interestingly, this transition seems to be irreversible and thepeptide remains in an aggregated state even after the coolingprocess. The term EX28K-FIB will be used throughout thepaper to indicate this aggregated state, obtained from EX28K-LIN after thermal cycle.

Structural Change of EX28K-LIN upon Heating. Tohave some insights into the aggregation transition highlightedby the abrupt change in absorbance at high temperatures(figure 1), finer conformational details were investigated bymeans of CD spectroscopy. Figure 2 shows the CD spectra ofEX28K-LIN in different buffers (H2O and TFE) as a functionof temperature.In aqueous solution at 0 °C, the spectrum is characterized by

a shoulder at about 216 nm and by a strong negative band at197 nm. While increasing the temperature to 25and 60 °C, theshoulder at 216 nm progressively disappears and the negativeband is reduced. Overall, CD spectra exhibit features quitecompatible with the presence of Polyproline-II (PPII)structure,30 together with unordered conformation. Howeverthe lack of a well-defined isoelliptic point suggests thecontribution of at least one other conformation. In fact, thedifference between the CD spectra at 60 and 0 °C for H2O

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

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buffer, reported in the inset in Figure 2, clearly shows theappearance of the signature of antiparallel beta-structures with anegative band at around 217 nm and a positive one at about195 nm, that are thus starting to participate to the conformerspopulation.The microenvironment that determines the conformation of

an amino acid sequence is not known a priori and can bedifferent from the bulk macroscopic solution conditions (i.e.,physiological conditions). Predicting the functional solventenvironment for insoluble elastin is particularly difficult and, inaddition, the protein hydrophobicity and highly cross-linkednature suggest a less polar internal environment than thesurrounding solvent. For this reason, the experiments in thisstudy 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-inducingsolvent because it favors intramolecular hydrogen bonding, thuspromoting folded conformations such as helices and beta-turns.While the real local solution conditions for insoluble elastin areunknown, it is likely to be intermediate between the twosolvent extremes of water and TFE.20 CD spectra of EX28K-LIN in TFE indicates the presence of α-helical structures. In

particular at 0 °C, the spectrum shows a negative band at 220nm due to n−π* electronic transition, a negative one at about208 nm and a positive band at 180 nm due to parallel andperpendicular 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] valueof the positive band. The increase of the temperature to 25 °Cdoes not affect the π−π* electronic transitions thus leavingsubstantially unchanged the bands at 208 and 190 nm. On thecontrary, the band at 222 nm slightly decreases. The furtherincrease to 60 °C induces the reduction of the positive bandand the increase of both negative bands thus suggesting thedestabilization of the α-helical conformation.

EX28K-FIB Structural Components. While CD spectros-copy provides information on the structural changes induced inEX28K-LIN upon heating, it cannot be used after the transitionto EX28K-FIB that induces aggregation and the solutionbecomes dense and opaque. To gain some insight on theconformation acquired by EX28K-FIB, solid-state FTIRspectroscopy was applied. This technique allows the mappingof full vibrational spectra in the infrared region and in particularthe amide I region is mainly due to the stretching vibrations ofthe CO group of the polypeptide backbone and is the mostinformative on protein secondary conformation. Furthermore,it is stronger than the amide II component, which originatesfrom the combination of the N−H bending and COstretching modes. The analysis of FTIR spectra wasconcentrated on these two bands, carrying the most relevantinformation on the polypeptide secondary structure.31

The decomposed FTIR spectra of the amide I and II regionsof EX28K-LIN and EX28K-FIB are shown in Figure 3. Thedecomposed FTIR spectrum of the amide I region shows forEX28K-LIN a component at 1640 cm−1, attributed to theabsorption of water, usually found between 1640−1650 cm−1.The component at 1661 cm−1 is assigned to unorderedconformations. The component at 1680 cm−1 is assigned to β-turns.32 The remaining component at 1698 cm−1 could be alsoassigned 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 1680cm−1, indicative of β-pleated sheet conformations. Thepresence of both components allows to exclude thecontribution of hydrogen bonded β-turns, also showing aband in the 1625 and 1634 cm−1 range, in favor of the presenceof antiparallel β-sheet conformation.33 Finally, the band at 1654

Figure 2. (a) CD spectra of EX28K-LIN peptide at growingtemperatures in H2O (full squares 0 °C ; full circles 25 °C ; fulltriangles 60 °C) and in TFE (empty squares 0 °C ; empty circles 25°C ; empty triangles 60 °C). (b) Inset: Difference of CD spectra inH2O between 60 and 0 °C in water.

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. Dashedline, experimental spectrum; solid line, calculated spectrum.

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cm−1 is assigned to unordered conformation. The amide IIcomponents at 1527 and 1551 cm−1 could be attributed toantiparallel β-sheets and unordered conformations, respectively.Morphology of EX28K-FIB Material. SEM and AFM

analysis were used to characterize the morphology of EX28K-FIB samples. As shown in Figure 4a, ESEM visualizationrevealed an interconnected network of bundle rope denselypacked into a monolithic structure. The ultrastructure of theaggregate can be observed after dehydration of the sample andimaging in SEM (Figure 4b), showing the presence of acomplex network of thin interconnected filaments. Toappreciate the morphology of isolated filaments, AFM imagingwas 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 therange of 300 nm to 1 μm. In addition, it is also possible toobserve 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 withboth fibrils and fibers (as in Figure 5a) were acquired.

Mechanical Properties of EX28K-FIB Fibrils. To have aninsight into the mechanical properties of the fibrils composingthe EX28K-FIB material, a statistical approach was applied. Ifthe fibrils are thought to behave as elastic filaments in solution,the shape they assume upon deposition on a surface relates tothe energy of the bath (the temperature T) and to theirflexibility: if the fibrils are very rigid, they are expected todeposit as straight rods, while in the opposite case of extremelyflexible filaments they are expected to appear rolled up with acurved shape. In the framework of the WLC model (introducedin Materials and Methods), the elasticity of the fibrils (theirYoung’s modulus) can be measured starting from a knowledgeof the persistence length P.14 In particular, it can be shownthat34

=YI k TPB

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

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 selectedregions of the image in (a), represented with a different look-up table, to highlight specific features. (d) Distribution of the Young’s modulusestimated for a set of 84 fibers. (e) Distribution of the height over lateral dimension ratio α for selected fibers (see text for details).

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of inertia of the fibril, and P is its persistence length. Supposingto describe the fibril as an elastic cylinder with an elliptic crosssection, this equation leads to

π=Y

k Tab

P4 B

3

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

with all values ranging between 0.4 and 3.5 MPa and a peak at0.7 MPa.To evaluate the stability of the measurements, an

independent evaluation of b was obtained. This parameter isparticularly critical because it appears at the power of 3 in theequation and it is evaluated from a profile that suffers of AFMtip convolution problems. A subset of fibers for which the valueof b was highly stable among the 10 measured profile waschosen and the ratio α = a/b was calculated, obtaining a peakeddistribution (see histogram in Figure 5e) around α = 0.17. Thisvalue was then used to calculate the moment of inertia I for allfibrils

π πα

= =I ab a4 4

33

4

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 andd) cells treated with increasing concentrations of peptide (panels a and c) and EX28K-FIB (panels b and d). The values represented are the means ofthe 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 azoom 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 afterincubation, 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).

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and thus to obtain a determination of the Young’s modulusindependent from each measure of b

απ

=Yk Ta

P4 B

3

4

The histogram for Y calculated from this procedure was fullycompatible with the previous determination (data not shown),giving rise to a peak value of 0.9 MPa in the same range ofvalues.Biocompatibility of EX28K-LIN and Biological Effect of

EX28K-FIB. The biocompatibility of EX28K-LIN was tested byMTT assay and morphological inspection on L929 (fibroblasts)and SAOS-2 (osteoblast) cell lines. A dose−response curve wasevaluated at 24 h and 7 days by MTT assay (see Figure 6a,c).For both cell lines, the viability was substantially dose-independent and for longer times (7 days) the enzymaticactivity in the presence of the EX28K-LIN peptide was a bithigher than for control cells (zero dose), showing a tendency ofthe cellular systems to be stimulated by the presence of inEX28K-LIN solution. Similarly, incubation of L929 and SAOS-2 cells with 25 or 250 μg of EX28K-LIN did not reveal anyspecific morphological changes with respect to control after 24h (data not shown) such as after 7 days (Figure 7 panels c ande, with respect to controls in panel a). This result was similarlyconfirmed at higher magnification (inset in panels c and e ofFigure 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 dose-dependence for both cell lines (Figure 6 panels b and d) withclearly stronger effect on SAOS-2 cells. In any case, for thehighest dose the viability of cells under EX28K-FIB treatmentwas never higher than 60% of the control. Moreover, markedmorphological changes were observed when both cell typeswere treated with EX28K-FIB: the cells were lower in numberfor both adherent fibroblast and osteoblast cells, and theyshowed 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 withEX28K-FIB did not show the typical cytoplasmic elongationsand were somewhat detached.

■ DISCUSSION

Tropoelastin is a modular protein organized in a cassette-likestructure in which each domain exploits specific functions thatare redundant along the whole molecule. In general, two mainclasses of domains can be identified, based on their function:mechanical domains, that confer to elastin its uniquemechanical properties, and cross-linking domains, thatparticipate in the formation of the supramolecular networkedstructure. Exon 28 from which EX28K-LIN was mainlyinspired, is a mechanical domain whose ability to extendupon stretch and recover the initial length when the stimulus isreleased is based on its random coil structure. In fact, anunstructured polypeptide in solution acts as a reservoir ofentropic energy, that is, the main reversible source of energy(elastic energy) in biopolymers, while enthalpy is associated totransitions in the internal degrees of freedom that are normallyirreversible. The structural analysis performed by CD and FTIRconfirm the biomimetic nature of the EX28K-LIN that shows amain presence of unstructured regions and a strong similaritywith peptides coded by other elastin mechanical exons.20 Theaddition of the lysines in the engineered sequence and the

presence of the acetyl group (see Materials and Methods) doesnot alter this main feature of the peptide, that is mechanicallyoutperforming as an ideal spring.35

Interestingly, imposing a thermal cycle to EX28K-LIN andtaking the peptide over about 60 °C induces an irreversibletransition, as monitored by turbidimetry. This process convertsthe isolated peptide in a strongly aggregated form, namedEX28K-FIB. The first steps of this pathway were monitored byCD, showing that it undergoes a conformational change fromthe mainly unordered structure to a β-rich state that isaggregation-prone, probably following a hierarchical mecha-nism previously observed in elastin-like peptides.21,36 Thisfinding was also monitored by FTIR spectroscopy thatconfirmed the presence of structuring β-sheets in the EX28K-FIB material. The use of high-resolution microscopy (SEM andAFM) allowed to characterize the morphology of showing thepresence of elongated fibrils inside the material that can twistand further aggregate to even form big supramolecularaggregates. Even though no independent spectroscopicmeasurements were performed, it is not unlikely that theaggregation pathway followed by EX28K-LIN toward EX28K-FIB is amyloidogenic in its nature.21 In addition, AFM imagingwas also exploited to obtain a quantification of the elasticproperties of EX28K-LIN fibrils in EX28K-FIB, measuring aYoung’s modulus of few MPa, completely in agreement withsimilar studies performed in literature on elastin-like or elastin-inspired materials14,15,37 showing that EX28K-FIB is mimickingthe properties of the inspiring molecule, elastin, also from amacroscopic mechanical point of view.Furthermore, EX28K-LIN and EX28K-FIB were also

characterized from a biological point of view by means ofenzymatic MTT test and direct morphological inspection. Thebiomimetic approach that was adopted in the synthesis of thepeptide resulted, as expected, in a molecule that is highlybiocompatible and, besides a very small activation of the cellularpopulations for higher times, it is almost biologically inactive.On the contrary, EX28K-FIB is inducing a relevant biologicalresponse, reducing cell viability of fibroblasts and, even moremarkedly, of osteoblast cells. This behavior mimics one of thefeatures for which elastin has raised interest in regenerativemedicine and, in particular, in vascular applications. In fact,elastin, which constitutes the main component of the tunicaintima, the innermost layer of blood vessels, has the ability tostrongly reduce the proliferation of unwanted cells (inparticular smooth muscles17 and fibroblasts), potentially actingas an antitrombogenic molecule.18,19

■ CONCLUSIONSA new elastin-inspired peptide, EX28K-LIN, was synthesizedand characterized from a chemical, physical, and biologicalpoint of view. This molecule has a relevant tendency to self-aggregate at high temperatures and to form an irreversiblenetwork of fibrils and fibres, EX28K-FIB. This material was alsocharacterized from a biological point of view, showing a clearantiproliferative activity that can be seen as an inheritance ofthe bare elastin on which EX28K-LIN was inspired. Moreover,lysine residues were added in the sequence of the peptide toprovide a sort of molecular hook, ready for exploiting thechemistry of the amino side chain to modulate cross-linkingparameters38 or to obtain covalent linkages to technologicalsurfaces. In perspective, this material will constitute aninteresting platform in vascular applications, keeping many ofthe beneficial properties of elastin and providing a technological

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

■ AUTHOR INFORMATIONCorresponding Author*E-mail: massimo.vassalli@cnr.it.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thanks Dr. Neluta Ibris for collaboration on AFMimages (CIGAS, University of Basilicata) and to Dr. MarinaLorusso for her technical assistance. The financial support fromMIUR (PRIN 2010-Project 2010L (SH3K)) is gratefullyacknowledged. We are grateful to Dr. Picenoni (Politecnicodi Milano, Milano, Italy) for technical assistance in SEMstudies.

■ REFERENCES(1) Lamme, E. N.; van Leeuwen, R. T.; Jonker, A.; van Marle, J.;Middelkoop, E. Living skin substitutes: survival and function offibroblasts seeded in a dermal substitute in experimental wounds. J.Invest. Dermatol. 1998, 111, 989−995.(2) Boland, E. D.; Matthews, J. A.; Pawlowski, K. J.; Simpson, D. G.;Wnek, G. E.; Bowlin, G. L. Electrospinning collagen and elastin:preliminary vascular tissue engineering. Front. Biosci. 2004, 9, 1422−1432.(3) Bashur, C. A.; Venkataraman, L.; Ramamurthi, A. Tissueengineering and regenerative strategies to replicate biocomplexity ofvascular elastic matrix assembly. Tissue Eng., Part B 2012, 18, 203−217.(4) Neuenschwander, S.; P Hoerstrup, S. Heart valve tissueengineering. Transplant Immunol. 2004, 12, 359−365.(5) Xu, J.-W.; Johnson, T. S.; Motarjem, P. M.; Peretti, G. M.;Randolph, M. A.; Yarem-chuk, M. J. Tissue-engineered exible ear-shaped cartilage. Plast. Reconstr. Surg. 2005, 115, 1633−1641.(6) Scott, M.; Vesely, I. Aortic valve cusp microstructure: the role ofelastin. Ann. Thorac. Surg. 1995, 60, S391−S394.(7) Kielty, C. M.; Stephan, S.; Sherratt, M. J.; Williamson, M.;Shuttleworth, C. A. Applying elastic fibre biology in vascular tissueengineering. Philos. Trans. R. Soc. London, Ser. B 2007, 362, 1293−1312.(8) Leach, J. B.; Wolinsky, J. B.; Stone, P. J.; Wong, J. Y. Crosslinkedα-elastin biomaterials: towards a processable elastin mimetic scaffold.Acta Biomater. 2005, 1, 155−164.(9) Ciofani, G.; Genchi, G. G.; Liakos, I.; Athanassiou, A.; Mattoli, V.;Bandiera, A. Human recombinant elastin-like protein coatings formuscle cell proliferation and differentiation. Acta Biomater. 2013, 9(2), 5111−5121.(10) Bandiera, A.; Sist, P.; Urbani, R. Comparison of ThermalBehavior of Two Recombinantly Expressed Human Elastin-LikePolypeptides for Cell Culture Applications. Biomacromolecules 2010,11, 3256−3265.(11) Nettles, D. L.; Chilkoti, A.; Setton, L. A. Applications of elastin-like polypeptides in tissue engineering. Adv. Drug Delivery Rev. 2010,62, 1479−1485.(12) Woodhouse, K. A.; Klement, P.; Chen, V.; Gorbet, M. B.;Keeley, F. W.; Stahl, R.; Fromstein, J. D.; Bellingham, C. M.Investigation of recombinant human elastin polypeptides as non-thrombogenic coatings. Biomaterials 2004, 25, 4543−4553.(13) Bax, D. V.; Rodgers, U. R.; Bilek, M. M.; Weiss, A. S. Celladhesion to tropoelastin is mediated via the C-terminal GRKRK motifand integrin αVβ3. J. Biol. Chem. 2009, 284, 28616−28623.(14) Bracalello, A.; Santopietro, V.; Vassalli, M.; Marletta, G.; DelGaudio, R.; Bochicchio, B.; Pepe, A. Design and Production of aChimeric Resilin-, Elastin-, and Collagen-Like Engineered Polypeptide.Biomacromolecules 2011, 12, 2957−2965.

(15) Sbrana, F.; Fotia, C.; Bracalello, A.; Baldini, N.; Marletta, G.;Ciapetti, G.; Bochicchio, B.; Vassalli, M. Multiscale characterization ofa chimeric biomimetic polypeptide for stem cell culture. BioinspirationBiomimetics 2012, 7, 046007.(16) Ito, S.; Ishimaru, S.; Wilson, S. E. Effect of coacervated α-elastinon proliferation of vascular smooth muscle and endothelial cells.Angiology 1998, 49, 289−297.(17) Karnik, S. K.; Brooke, B. S.; Bayes-Genis, A.; Sorensen, L.;Wythe, J. D.; Schwartz, R. S.; Keating, M. T.; Li, D. Y. A critical rolefor elastin signaling in vascular morphogenesis and disease. Develop-ment 2003, 130, 411−423.(18) Ito, S.; Ishimaru, S.; Wilson, S. E. Application of Coacervated[alpha]-Elastin to Arterial Prostheses for Inhibition of AnastomoticIntimal Hyperplasia. ASAIO J. 1998, 44, M501−M505.(19) Jordan, S. W.; Chaikof, E. L. Novel thromboresistant materials.J. Vasc. Surg. 2007, 45, A104−A115.(20) Tamburro, A. M.; Bochicchio, B.; Pepe, A. Dissection of HumanTropoelastin: Exon-By-Exon Chemical Synthesis and RelatedConformational Studies. Biochemistry 2003, 42, 13347−13362.(21) Bochicchio, B.; Pepe, A.; Flamia, R.; Lorusso, M.; Tamburro, A.M. Investigating the Amyloidogenic Nanostructured Sequences ofElastin: Sequence Encoded by Exon 28 of Human Tropoelastin Gene.Biomacromolecules 2007, 8, 3478−3486.(22) Tamburro, A. M.; Pepe, A.; Bochicchio, B.; Quaglino, D.;Ronchetti, I. P. Supramolecular amyloid-like assembly of thepolypeptide sequence coded by exon 30 of human tropoelastin. J.Biol. Chem. 2005, 280, 2682−2690.(23) Savitzky, A.; Golay, M. J. Smoothing and differentiation of databy simplified least squares procedures. Anal. Chem. 1964, 36, 1627−1639.(24) Necas, D.; Klapetek, P. Gwyddion: an open-source software forSPM data analysis. Cent. Eur. J. Phys. 2012, 10, 181−188.(25) Roiter, Y.; Minko, S. AFM Single Molecule Experiments at theSolid-Liquid Interface: In Situ Conformation of Adsorbed FlexiblePolyelectrolyte Chains. J. Am. Chem. Soc. 2005, 127, 15688−15689.(26) (a) Feldman, D. The Theory of Polymer Dynamics; Doi, M.; andEdwards, S. F., Eds.; Clarendon Press, Oxford University Press: NewYork, 1986; p 391. (b) J. Polym. Sci., Part C: Polym. Lett. 1989, 27,239−240.(27) Kulic, I. M.; Mohrbach, H.; Lobaskin, V.; Thaokar, R.; Schiessel,H. Apparent persistence length renormalization of bent DNA. Phys.Rev. E 2005, 72, 041905.(28) Saino, E.; Grandi, S.; Quartarone, E.; Maliardi, V.; Galli, D.;Bloise, N.; Fassina, L.; De Angelis, M. G. C.; Mustarelli, P.; Imbriani,M.; Visai, L. In vitro calcified matrix deposition by human osteoblastsonto a zinc-containing bioactive glass. Eur. Cell Mater. 2011, 21, 59−72 discussion 72..(29) Saino, E.; Maliardi, V.; Quartarone, E.; Fassina, L.; Benedetti, L.;De Angelis, M. G. C.; Mustarelli, P.; Facchini, A.; Visai, L. In VitroEnhancement of SAOS-2 Cell Calcified Matrix Deposition onto RadioFrequency Magnetron Sputtered Bioglass-Coated Titanium Scaffolds.Tissue Eng., Part A 2010, 16, 995−1008.(30) Adzhubei, A. A.; Sternberg, M. J.; Makarov, A. A. Polyproline-IIHelix in Proteins: Structure and Function. J. Mol. Biol. 2013, 425,2100−2132.(31) Cai, S.; Singh, B. R. A Distinct Utility of the Amide III InfraredBand for Secondary Structure Estimation of Aqueous ProteinSolutions Using Partial Least Squares Methods. Biochemistry 2004,43, 2541−2549.(32) Jackson, M.; Mantsch, H. H. The use and misuse of FTIRspectroscopy in the deter-mination of protein structure. Crit. Rev.Biochem. Mol. Biol. 1995, 30, 95−120.(33) Miyazawa, T.; Blout, E. The Infrared Spectra of Polypeptides inVarious Conformations: Amide I and Ii Bands. J. Am. Chem. Soc. 1961,83, 712−719.(34) (a) Lumry, R.; Rosenberg, A. Macromolecules in solution;Hertbert Morawetz, Interscience: New York, 1965; p495. + xvi. (b)Biopolymers 1966, 4, 1053−1053.

Langmuir Article

dx.doi.org/10.1021/la403311x | Langmuir 2013, 29, 15898−1590615905

(35) Sbrana, F.; Lorusso, M.; Canale, C.; Bochicchio, B.; Vassalli, M.Effect of chemical cross-linking on the mechanical properties ofelastomeric peptides studied by single molecule force spectroscopy. J.Biomech. 2011, 44, 2118−2122.(36) del Mercato, L. L.; Maruccio, G.; Pompa, P. P.; Bochicchio, B.;Tamburro, A. M.; Cin-golani, R.; Rinaldi, R. Amyloid-like Fibrils inElastin-Related Polypeptides: Structural Characterization and ElasticProperties. Biomacromolecules 2008, 9, 796−803.(37) Gosline, J.; Lillie, M.; Carrington, E.; Guerette, P.; Ortlepp, C.;Savage, K. Philos. Trans. R. Soc. London, Ser. B 2002, 357, 121−132.(38) Betre, H.; Ong, S. R.; Guilak, F.; Chilkoti, A.; Fermor, B.;Setton, L. A. Chondrocytic differentiation of human adipose-derivedadult stem cells in elastin-like polypeptide. Biomaterials 2006, 27, 91−99.

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dx.doi.org/10.1021/la403311x | Langmuir 2013, 29, 15898−1590615906