DOI: 10.1021/la100207q 9875Langmuir 2010, 26(12), 9875–9884 Published on Web 03/29/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Surface Investigation onBiomimeticMaterials toControl Cell Adhesion: The

Case of RGD Conjugation on PCL

Filippo Causa,* Edmondo Battista, RaffaellaDellaMoglie, Daniela Guarnieri,Maria Iannone, andPaolo A. Netti

Interdisciplinary Research Centre on Biomaterials (CRIB) University Federico II, Piazzale Tecchio 80, 80125,Naples, Italy, and Italian Institute of Technology (IIT) Via Morego, 30 Genoa, Italy

Received January 15, 2010. Revised Manuscript Received March 15, 2010

The cell recognition of bioactive ligands immobilized onpolymeric surfaces is strongly dependent on ligandpresentationat the cell/material interface.While small peptide sequences such asArg-Gly-Asp (RGD) are beingwidely used to obtainbiomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptorsdeserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(ε-caprolactone) (PCL), alargely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface character-istics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact anglemeasurements, surface free energy determination) and spectrophotometric assays specially adapted for the analyticalquantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of anumber, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymericsubstrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymercrystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesionstudies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such workproposes a morphological and analytical approach in surface characterization to study the surface treatment and thedistribution of ligands immobilized on polymeric substrates.

Introduction

In biological tissues, cells are immersed in the extracellularmatrix (ECM) that is a coacervate of glycosaminoglycans andproteins with various mechanical and signaling functions. Inparticular, fibroblast and osteoblast cells are known to expressvarious integrins, each component having a large extracellulardomain responsible for ligandbinding, a transmembrane domain,and a short cytoplasmic domain responsible for interacting withthe actin cytoskeleton.1 Integrin heterodimers bind to specificamino acid sequences, such as the arginine-glycine-aspartic acid(Arg-Gly-Asp or RGD) recognition motif that is largely pre-sent in many ECM proteins, including fibronectin, vitronectin,bone sialoprotein, and osteopontin.2 Small synthetic peptides(a few hundred daltons) that contain amino acid sequence RGDcan thus mediate cell attachment as well as the large parentalmolecule (a hundred thousand dalton). On the basis of this,biomimetic approaches have been developed to immobilize shortpeptides, such as RGD, onto synthetic or natural surfaces, toproduce biofunctional materials able to promote and enhance cellattachment.1,3 In particular, it has been found that a minimumRGDdensity of 1.0� 10-15 mol/cm2, corresponding to a spacingof about 140 nm between peptide ligands, is sufficient to promotecell spreading, while a density of 1.0 � 10-14 mol/cm2 is neededto promote the formation of focal contacts.4 However, such

parameters strongly depend on peptide presentation and, in turn,from chemical and physical characteristics of the substrate.

Moreover, spatial distribution and the aggregation of RGDpeptides at the micro- and nanoscale significantly affect cellresponses. For example, nanoscale clustering of RGD peptidescan induce integrins to cluster, thus triggering complete cellsignaling.5,6

Poly(ε-caprolactone) (PCL), a biodegradable aliphatic poly-ester,7,8 has been suggested for a wide field of applications such asdrug delivery systems,9,10 tissue-engineered skin (plain film), andscaffolds for supporting fibroblast and osteoblast growth.11,12

However, as any other synthetic polymer it does not presentmolecular motifs for cell biological recognition, and therefore it,lacks a friendly interface with living cells.13 A way to obtain PCLbiomimetic surfaces in promoting cell adhesion was themodifica-tion of polymeric backbone to introduce functional groups for thefollowing RGD conjugation.14,15 Marletta et al. demonstratedthat, when adsorbed onto PCL surface, RGD seems to have only

*To whom correspondence should be addressed. InterdisciplinaryResearch Centre on Biomaterials (CRIB), University Federico II, PiazzaleTecchio 80, 80125, Naples, Italy. Telephone number: þ39-081-7682100, faxnumber: þ39-081-7682404; e-mail address: causa@unina.it.(1) Hersel, U.; Dahmen, C.; Kessler, H. Biomaterials 2003, 24, 4385–4415.(2) Garcia, A. J.; Reyes, C. D. J. Dent. Res. 2005, 84, 407–413.(3) El-Amin, S. F.; Kofron, M. D.; Attawia, M. A.; Lu, H. H.; Tuan, R. S.;

Laurencin, C. T. Clin. Orthop. Rel. Res. 2004, 427, 220–225.(4) Massia, S. P.; Hubbell, J. A. J.Cell. Biol. 1991, 114, 1089–1100.

(5) Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G.J. Cell. Sci. 2000, 113, 1677–1686.

(6) Yang, H.; Kao, W. J. Int. J. Nanomed. 2007, 2, 89–99.(7) Eldsater, C.; Erlandsson, B.; Renstad, R. A.; Albertsson, C.; Karlsson, S.

Polymer 2000, 41, 1297–1304.(8) Choi, E. J.; Kim, C. H.; Park, J. K. Macromolecules 1999, 32, 7402–7408.(9) Zhong, Z. K.; Sun, X. Z. S. Polymer 2001, 42, 6961–6969.(10) Allen, C.; Han, J.; Yu, Y.; Maysinger, D.; Eisenberg, A. J. Controlled

Release 2000, 63, 275–286.(11) Ng, K. W.; Hutmacher, D. W.; Schantz, J. T.; Ng, C. S.; Too, H. P.; Lim,

T. C.; Phan, T. T.; Teoh, S. H. Tissue Eng. 2001, 7, 441–455.(12) Hutmacher, D.W.; Schantz, T.; Zein, I.; Ng, K.W.; Teoh, S. H.; Tan, K. C.

J. Biomed. Mater. Res. 2001, 55, 203–216.(13) Croll, T. I.; O’Connor, A. J.; Stevens, G. W.; Cooper-White, J. J.

Biomacromolecules 2004, 5, 463–473.(14) Healy, K. E.; Tsai, D.; Kim, J. E. Mater. Res. Soc. Symp. Proc. 1992, 252,

109–114.(15) McConachie, A.; Newman, D.; Tucci, M.; Puckett, A.; Tsao, A.; Hughes,

J.; Benghuzzi, H. Biomed. Sci. Instrum. 1999, 35, 45–50.

9876 DOI: 10.1021/la100207q Langmuir 2010, 26(12), 9875–9884

Article Causa et al.

a minor effect on the cell response.16 However, both ion irradia-tion and RGD adsorption on PCL surfaces modulated theexpression of integrins involved in human osteoblast (hOB)growth and function.17 However, the majority of these modifica-tion methods have several drawbacks including low level offunctional groups, lack of control of peptide presentation onPCL surface, and the possibility of unknown and uncontrolleddegradation products. Further, such methods leave the bioactivegroups merely adsorbed onto the surface (i.e., they are notcovalently attached), meaning that there is a danger of beingexchanged or removed upon introduction into existing in vitroculture or in vivo implantation. Conversely, chemical methodswere demonstrated to be successful at bioactivating polymersurface. The introduction of functional groups on biodegradablepolyester surfaces and in particular on PCL has also previouslybeen achieved by hydrolysis,13,18 aminolysis,13 plasma treat-ment,19,20 or copolymerization.21 Amine groups were graftedonto film surfaces through treatment with a diamine, beforeattaching peptide sequences such as RGD by using either carbo-diimide, glutaraldehyde, or epoxy-amine chemistry.22 Subsequentcell adhesion studies have demonstrated an increase in adhesionand spreading of cells on these modified surfaces,23 in particular,on PCL containing laminin-derived peptides sequences IKVAV,RGD, or YIGSR covalently attached to the surface of thepolymer. Peptides were attached to the surface of the polymerusing a two-step procedure that employs a treatment with1,6-hexanediamine followed by the use of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide.23 Recent work has also modifiedPCLwith poly(ethylene oxide) grafts before coupling with RGD-containing peptides, again resulting in enhanced cellular re-sponses.24 PCL was also functionalized with RGD after amino-lysis for three-dimensional PCL scaffold.25,26 However, in allabove-mentioned works, there is a lack of control of effectivepresentation of immobilized peptide toward cell, and surfacecharacteristics after peptide conjugation are not adequatelyinvestigated. Thus, a systematic study on peptide ligand organiza-tion and spatial distribution on polymer surfaces could representa step forward in engineering bioactive interfaces and, in parti-cular, to evaluate the effective peptide distribution and presenta-tion able to trigger specific cell function such as adhesion ordifferentiation.

Here, we performed the grafting ofGRGDYpeptide onto solidPCL with a wet chemistry consisting of a two-step procedure:polymer aminolysis to graft functional groups (primary amines) onthe film surface and a following conjugation of the RGD motif.Even though the procedure is similar to others already reported inthe literature,22,23 each step was controlled through functional

group determination as well as chemical and physical parameterevaluation. The aminolysis reaction was carried out for PCLsurfaces and monitored. Morphological and topological charac-teristics of RGD presenting surfaces were deeply studied in termsof peptide surface density and distribution onto PCL surface aswell as penetration depth in the polymer substrate. Finally, inorder to investigate cell recognition of bioactive peptide, adhesionof NIH3T3 fibroblasts to the proposed PCL substrates wasperformed in serum-free media to exclude any role played byserum proteins in cell recognition.

Materials and Methods

The poly(ε-caprolactone) (PCL) pellets used in this study,(Mw = 65000 g mol-1, 181 609) are a product of Sigma-Aldrich,St. Louis, MO. The following reagents were purchased fromSigma-Aldrich, St. Louis,MO: 1,6-hexanediamine (DEA), hexyl-amine, glutaraldehyde solution grade I 25% (GA), glycerol,tritolyl phosphate (mixture of isomers, 90%, 268 917), sodiumcyanoborohydride (NaBH3CN, Fluka, 71435), ethanolamine,Tween 20, QuantiPro BCA assay kit (kit component: QuantiProBuffer QA 250 mL, QuantiPro BCA QB 250 mL, 4% copper(II)sulfate pentahydrate solution 12 mL), Kaiser test kit (Fluka,60017, the test kit contains 50mL each of the following solutions:phenol∼80% in ethanol, KCN inH2O/pyridin, ninhydrin 6% inethanol), fluoresceinamine isomer I (Fluka, 07980) (FLUO),Rhodamine B isothiocyanate mixed isomers (Sigma, R1755)(RBITC), phosphate buffered saline (PBS), dichloromethane(DCM), water (CHROMASOLV Plus, for HPLC, 34877), etha-nol, 2-propanol (IPA). The peptidesGRGDYandGYDGRwerepurchased from INBIOS S.r.l., Naples, Italy.

Aminolysis of Poly(ε-caprolactone) Plates. Poly(ε-caprol-actone) (PCL) pellets were processed to form thin sheets by hotcompression above polymer melting temperature (70 �C) for 2 hand equilibrated at room temperature.As prepared, the sheetswerethoroughly washed with water and isopropanol (IPA) and flushed(or dried) with nitrogen.

A procedure already described by Zhu et al.27 was followed inorder to incorporate amino groups onto the surface of PCL using1,6-hexanediamine (DEA) (Scheme 1). Briefly, aminolysis wasconducted by immersing the sheets in a 10% (w/w) 1,6-hexane-diamine/isopropanol (DEA/IPA) solution in a custom-madereactor thermostatted in a water bath at various temperature withadequate magnetic stirring for suitable period time. After amino-lysis treatment, the samples were rinsed extensively with a 0.3%Tween 20 solution and deionized water until neutral reaction.Subsequently, the sheets were dried in a vacuum desiccator atroom temperature for 24 h.

In order to evaluate kinetic parameters of reaction, PCLsamples were aminolyzed in 10% DEA/IPA at 25, 37, and40 �C for 30 min. Activation energy (Ea) of aminolysis reactionwas calculated according to the Arrhenius equation by fitting theplot of grafting rate against 1/T. The rate of grafting wascalculated in the first 30 min from the slope of the curve at eachtemperature (see Supporting Information for more details). Theaminolyzed PCL (PCL-NH2) plates were subsequently weighedto record mass loss and processed for physicochemical character-ization.

Spectroscopy after Aminolysis. ATR/FTIR measurementswere performed by using a Thermo Nicolet 6700 spectrometerequipped with Smart Perfomer accessory by using a Ge crystalwith incident angle of 45� and a sampling area of 2mm.Thin filmswere pressed onto the Ge crystal at 12 psi (82.5 kPa) and spectrawere collected in the range 4000-650 cm-1 at 8 cm-1 resolutionwith 128 scans. To detect at the molecular level the presence ofprimary amines on PCL surface, ATR/FTIR analysis was per-formeddirectly on several points of the PCL-NH2 sheets dried in a

(16) Marletta, G.; Ciapetti, G.; Satriano, C.; Pagani, S.; Baldini, N.Biomaterials2005, 26, 4793–4803.(17) Amato, I.; Ciapetti, G.; Pagani, S.; Marletta, G.; Satriano, C.; Baldini, N.;

Granchi, D. Biomaterials 2007, 28, 3668–3678.(18) Cai, K.; Yao, K.; Cui, Y.; Yang, Z.; Li, X.; Xie, H.; Qing, T.; Gao, L.

Biomaterials 2002, 23, 1603–1611.(19) Yang, J.; Bei, J.; Wang, S. Biomaterials 2002, 23, 2607–2614.(20) Chu, P. K.; Chen, J. Y.; Wang, L. P.; Huang, N. Mater. Sci. Eng., R: Rep

2002, R36, 143-206.(21) Zhu, Y.; Chian, K. S.; Chan-Park, M. B.; Mhaisalkara, P. S.; Ratner, B. D.

Biomaterials 2006, 27, 68–78.(22) Gabriel, M.; Van Nieuw Amerongen, G. P.; Van Hinsbergh, V. W. M.;

VanNieuwAmerongen, A. V.; Zentner, A. J. Biomater. Sci. Polym. 2006, 17, 567–577.(23) Santiago, L. Y.; Nowak, R. W.; Rubin, J. P.; Marra, K. G. Biomaterials

2006, 27, 2962–2969.(24) Taniguchi, I.; Kuhlman, W. A.; Mayes, A. M.; Griffith, L. G. Polym. Int.

2006, 55, 1385–1397.(25) Dalton, P. D.; Woodfield, T.; Hutmacher, D. W. Biomaterials 2009, 30,

701–702.(26) Lam, C. X.; Savalani, M. M.; Hutmacher, D. W. Biomed. Mater. 2008, 3,

034108. (27) Zhu, Y.; Gao, C.; Liu, X.; Shen, J. Biomacromolecules 2002, 3, 1312–1319.

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Causa et al. Article

vacuum desiccator without any additional treatment (n = 4 foreach measurements). The penetration depth of an evanescentwave in the sample near the regionmore diagnostic (λ 1650 cm-1)was calculated by applying following equation:28

dp ¼ λ

2πn1

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffisin2 θ- ðn2=n1Þ2

qwhere n2 (∼1.5) is the refractive index generally assumed forpolymers, while n1 (4.0) is the refractive index of Ge crystal.28 Inparticular, single internal reflection element (IRE) of Ge with anincident angle (θ) of 45� allows us to reach qualitative informationabout samples in thin layer as high as 0.4 μm.

Determination ofEngraftedAmines afterAminolysis.Theoverall amino groups grafted onto PCL-NH2 plates were quanti-fied by using theKaiser test kit.29 In this assay, ninhydrin reactingwith free amino groups emerging from the polymer surfaceproduces a purple pigment (Ruhemann’s purple) detected usinga spectrophotometer (Lambda 25, Perkin-Elmer) in the range550-570 nm. The assay was conducted in homogeneous phase bydissolving the polymer in the organic component of the kit, and atthe final stage of reaction, as reported by others,23,27,30 developingthe pigment in anorganic solvent (see Supporting Information fordetails). The amino density per sample area was calculated bycomparing the absorbance at 551 nm with a previously obtainedcalibration curve (Supporting Information Figure S1).

Weight Loss, Mechanical and Thermal Properties after

Aminolysis. To evaluate the effects of the aminolysis on the bulkproperties of the polymer, PCL substrates of about 60mgwith anaverage areaof 8 cm2 and rectangular shapewere treated in a 10%DEA/IPA solution for a predetermined period of time rangingfrom 15 to 24 hs. Residual mass after aminolysis treatment wasevaluated by using the gravimetric method on an electronicbalance with a resolution of 0.1 mg (Gibertini, E50S). Driedplateswereweighed at each timepoint of treatment, anddatawerereported as mass loss related to the projection of the treatedsurface area as shown in the following equation:

%RM ¼ 1-ðMi -MfÞ=MiÞ

A

� �� 100

where Mi is the initial mass, Mf is the final mass, and A is thesample area.

Mechanical tests were performed to assess tensile properties ofdried thin sheets in accordance with the ASTM D1708-06astandard at room temperature. A set of samples with an averagearea of 20 cm2 and a cross section of 0.18μmwere aminolyzed in a10% DEA/IPA solution at 37 �C and cut in dog-bone shapes asprescribed by ASTM standards. All measurements were carriedout by Instron 5566 dynamometer with a 1 kN load-cell and across-head speed of 100 mm/min. The elastic modulus wasobtained from stress-strain (σ-ε) curves as the slope of initiallinear portion corrected by toe compensation. Five samples weretested for each measurement.

Measurements of differential scanning calorimetry (DSC) wereperformed with a Perkin-Elmer Jade DSC covering the tempera-ture range 25-100 �C.The sampleswere heated at 10 �C/min, anddata collected were processed by Perkin-Elmer Pyris 6 software.From the melting peak of the polymer, the crystallinity degreeXc% of the polymer was calculated according to the equation:31

Xc% ¼ ΔHm

ΔH�m

!� 100

where ΔHm is the enthalpy of melting measured in the first runand ΔH�m the enthalpy of melting of a totally crystalline PCL(ΔH�m = 139 J/g).32

Peptide Conjugation. PCL-NH2 sheetswere bioactivated in atwo-step method by using glutaraldehyde (GA) as cross-linkingagent to immobilize GRGDY and GYDGR peptides. In the firststep, the sheets were treated with a 2% glutaraldehyde (GA) in10 mM phosphate buffer (PBS) (pH 7.4) at room temperature inan orbital shaker for 3 h. The reaction was stopped by rinsing thesamples extensively with deionized water. Afterward, the peptideamino-terminus was covalently linked by reductive aminationonto PCL aldehyde activated surfaces. The coupling solutionswere made by 50 mM carbonate (pH 8.5) buffer with 0.2 mg/mLof peptides and 5 mM of NaBH3CN. The coupling step wasallowed to react for 4 h with gentle shaking and then stopped,rinsing surfaces with copious amount of deionized water. Un-reacted aldehyde groups were blocked by treating the polymersurface with 0.2M ethanolamine in carbonate buffer (50mM, pH8.5) for 30 min at room temperature. Finally, the polymer-coupled peptide surfaces (PCL-GA-GRGDY, PCL-GA-GYDGR) were rinsed with water containing 0.3% Tween 20and with deionized water; then, samples were stored dry untilfurther use. Afterward, the disks were washed in PBS buffer andsterilized for cell attachment studies or thoroughly washed with

Scheme 1. Synthetic Scheme Showing the Two Steps Procedure Used to Immobilize Peptide Sequence on PCL Surfacea

a (1) Aminolysis of PCL substrate by diamine solutions, (2) primary amine presenting PCL substrate, (3) tether (GA) insertion and following peptide(GRGDY or GYDGR) conjugation.

(28) Urban, M. W. Attenuated Total Reflectance Spectroscopy of PolymersTheory and Practice; American Chemical Society: Washington, DC, 1996.(29) Sarin, V. K.; Kent, S. B.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981,

117, 147–157.(30) Zhang, H.; Lin, C.-Y; Hollister, S. J. Biomaterials 2009, 30, 4063–4069.

(31) Taddei, P.; Simoni, R.; Fini, G. J. Mol. Struct. 2001, 317, 565–566.(32) Crescenzi, V.; Manzini, G.; Calzolari, G.; Borri, C. Eur. Polym. J. 1972, 8,

449–463.

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ultrapure water and dried under vacuum for surface characteriza-tion.

Analysis of Surface Topology. The surface topography aswell as the roughness of all the prepared surfaces was measuredwith a diCaliber atomic force microscope (Veeco Instruments) intapping mode in air with a standard silicon tip. During themeasurements, the relative room humidity was about 30%, andthe room temperature was 25 �C. Images were recorded usingheight and phase-shift channels with 256 � 256 measurementpoints (pixels). Measurements were made several times on differ-ent zones of each sample on a scanning area of 90 μm � 90 μm,and roughness parameter (arithmetic averageRa, and root-mean-square, rms) calculation and image processing were performedusing the SPMLabAnalysis software (Veeco Instruments).

Contact Angle Measurements and Surface Free Energy

(SFE) Evaluation. The static contact angle of ultrapure waterover the surface of PCL, PCL-NH2, and PCL-GA-GRGDYwasmeasured with automatic video-based measurements of contactangle performed at 25 �C and 65% relative humidity by using anCAM 200 instrument (KSV, Finland). For this measurement,2 μL of ultrapure water was initially placed over the surface of thepolymer.

The pictures were processed by KSV-CAM software to calcu-late the contact angle with the surface polymer by applying theYoung/Laplace method. Eight independent measurements wereperformed per treatment. The statistical significance of the con-tact angle value was assessed by performing one-way ANOVAwith Tukey’s post hoc comparison (INERST v1.3 script in Excel)by comparing bothRGD-conjugated and aminatedPCL surfaceswith respect to the plain polymer.A significance level of 99%(p=0.001) was chosen for all the tests.

Measurements of SFE were performed by evaluating staticcontact angles of three different liquids (Milliporewater, glycerol,and tritolyl phosphate (Aldrich)) onto the different surfaces. Atleast five measurements were made for each sample and thenaveraged.TheSFEvalueswere evaluatedbyusing theChaudhury-Good-van Oss model,33 where the total SFE (γTOT) is given bythe sum of apolar Lifshitz-van der Waals (γLW) and polar Lewisacid-base (γAB) components:

γTOT ¼ γABþ γLW

The contribution of individual acid (γA) and basic (γB) to totalpolar Lewis acid-base (γAB) were calculated according to thefollowing equation:

γAB ¼ 2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiγA 3γB

qDetermination of Conjugated Peptide. The immobilized

RGD-peptide was determined directly on solid support by usingMicroBCA assay (Sigma-Aldrich) as described from Tyllianakiset al.34 In particular, the number of peptide bonds and thepresence of four particular amino acids (cysteine, cystine, trypto-phan, and tyrosine) are able to reduce one ion Cu2þ to Cu1þ,which forms a chelate complex with two molecules of bicincho-ninic acid (BCA) absorbing at 562 nm.35 This moderate purple-colored complex allows a spectrophotometric determination ofnanomolar quantities of functional groups in aqueous solution.This evaluation is finally carried out through standard curves ofappropriate substance. The amount of reduction is proportionalto peptide bonds present.

Inour case, the amount of immobilized short peptides, contain-ing the enhancing tyrosine residue (GRGDY andGYDGR), was

performed by adding the MicroBCA working solution directlyonto the samples in a reduced volumetric form of the assay (seemore in Supporting Information). The determination of thenanomolar peptide concentration was carried out by calibrationcurve, previously obtained at the same conditions (Figure S2). Anominal density was calculated by taking into account the area ofeach treated sample and referred to as RGD nmol/cm2. Such anominal density has to be considered as the overall number ofpeptide moles immobilized on a polymer chain rather than aneffective RGD surface density. In addition, in order to finelyinvestigate the effect of any contaminant on the assay colorformation, aminolyzed substrates and pure polymer were alsotested.

Spatial Distribution of Surface Treatment. Confocal laserscanning microscopy (CLSM) was used to investigate the pene-tration depth of treatment by LSM 510 Zeiss confocal invertedmicroscope equipped with a Zeiss 20�/3 NA objective and anargon laser (excitation = 541 nm; emission = 572 nm). In orderto follow the advance of bioactivation in the two stages ofprocesses (aminolysis and peptide bound polymer), sample sur-faces were covalently coupled with two different fluorescentlabels. First, PCL-NH2 sheets were treated with 0.1 mg/mL ofRhodamine B isothiocyanate (RBITC) in IPA overnight at 4 �C.Afterward, plates were rinsed thoroughly with IPA and then with0.3% Tween 20 and water for 24 h to remove any noncovalentlybound dye molecule. Conversely, fluoresceinamine (FLUO) waslinked to the aldehyde activated polymer surfaces in the sameconditions used for peptide conjugation (0.2 mg/mL of dye and5mMofNaBH3CN in 50mMcarbonate buffer, pH 8.5). Finally,sheets were removed from reaction wells and copiously rinsedwith 0.3% Tween 20 and d.i. water for 24 h to remove anynoncovalently linked molecules. The films were then left to dryovernight in a vacuum desiccator before analysis. Nonfunctiona-lized PCL surfaces were equally processed as control. Samplesafter Rhodamine B isothiocyanate or fluoresceinamine conjuga-tion were respectively visualized using the characteristic wave-length ofRhodamine (λex=543nm; λem=572nm) or fluorescein(λex = 496 nm; λem = 518 nm).

To compare the results, CLSM settings, in particular laserpower, pinhole aperture, detector gain, and amplifier offset, werekept constant for both kinds of observations.Z-stack acquisitionswere preformed through the samples by starting from the outerpart with optical slices of 1.46μm. Intensity profiles of fluorescentdyes as a function of penetration depth were obtained along theline drawn in direction perpendicular to the surface.

Cell Adhesion Study. Mouse embryo fibroblasts NIH3T3were maintained at 37 �C and 5% CO2 in Dulbecco’s modifiedEagle medium (DMEM) supplemented with 10% fetal bovineserum (FBS, BioWhittaker, Walkersville, MD), 2 mM L-gluta-mine (Sigma, St. Louis, MO), 1000 U/L penicillin (Sigma,St. Louis, MO), and 100 mg/L streptomycin (Sigma, St. Louis,MO). For the experiments, 70-80% confluent cells were used.

For cell adhesion experiments, PCL, PCL-NH2, PCL-GA-GYDGR, and PCL-GA-GRGDY substrates were sterilized withantibiotics and preincubated in serum-free medium for 16-18 h.After incubation, 5 � 104 cells were seeded on the samples andgrown in DMEM-w/o FBS to avoid unspecific cell adhesiondepending on serum protein adsorption.

In order to evaluate cell adhesion and shape, scanning electronmicroscopy (SEM) analysis was performed by Leica 420 by usinga beam energy of 5 KeV. After 24 h from cell seeding, the sheetswere rinsed with PBS and fixed with 2.5% glutaraldehyde (pH7.4) (Sigma-Aldrich) for 2 h at room temperature. The cell-plateconstructs were dehydrated in graded ethanol concentrations(from 50% to 100% v/v in ethanol), air-dried, gold sputtered,and analyzed by SEM. For CLSM analysis, samples were fixed,after 24 h from cell seeding, with 4% para-formaldehyde for20 min at RT, rinsed twice with PBS buffer, and incubated withPBS-BSA0.5%toblock unspecific binding.Actinmicrofilamentswere stainedwithphalloidin tetramethylrhodamineB isothiocyanate

(33) van Oss, C. J. Colloids Surf., B: Biointerf. 1995, 5, 91–110.(34) Tyllianakis, E. P.; Kakabakos, S. E.; Evangelatos, G. P.; Ithakissios, D. S.

Anal. Biochem. 1994, 219, 335–340.(35) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner,

F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D.C. Anal. Biochem. 1985, 150, 76–85.

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(Sigma-Aldrich). Phalloidin was diluted in PBS-BSA 0.5% andincubated for 30 min at RT. Images were acquired by using aHe-Ne excitation laser at the wavelength of 543 nmby using a 20�objective. Moreover, cell viability and proliferation on proposedsubstrates were evaluated by usingAlamarBlue assay (AbDSerotecLtd., UK) and compared to plain PCL surfaces (see SupportingInformation).

Results

PCL surfaces were bioactivated by covalently linking RGDmotifs directly on surfaces enriched in amino groups afteraminolysis (Scheme 1). The aminolysis represents an easy-to-perform chemical technique to engraft amino groups alongpolyester chains, and its limited influence on bulk materialproperties is often used in surface functionalization of scaffoldsfor tissue engineering.13,21-23,27,30 In order to study the aminolysiseffects onto PCL, a very smooth and nonporous surface wasfabricated with a controlled crystallinity degree and surfacemorphology. PCL pellets were shaped in slight sheets with anaverage thickness about 200 μm by hot compression moldingabove the polymer fusion temperature. Such treatment allowsreaching Ra and rms average values, respectively, of 0.029 and0.036 μm without any apparent pore present on the surface. Thefusion enthalpy of pure PCL sheets evaluated byDSCwas 65.2(5.4 J/g, and the average calculated degree of crystallization was46.9 ( 3.9%.

Chemical modification of PCL sheets was carried out byreaction with a bifunctional amine (DEA) in mild conditionsabove the amine melting temperature (28 �C). The reactionmechanism was investigated through analysis of surface beforeand after aminolysis in the proposed reaction conditions.Mechanisms of Aminolysis Reaction and Amine Determi-

nation. ATR spectrum of neat PCL showed characteristic peaksof carbonyl and aliphatic groups, respectively, at 1725 cm-1

(ν CO) and 2944, 2865 cm-1 (νs, νas C-H), whereas spectra ofaminolized surfaces highlighted more diagnostic peaks related tothe presence of amide groups (Figure 1). Peaks more significantare centered at 3338 cm-1 (ν NH), 1641 cm-1 (ν CO), and 1541cm-1 (δ NH) ascribable to amide I and amide II bands.36

Aminolysis reaction, indeed, takes place by nucleophilic attackat the carbonyl of PCL by diamine. Under alkaline conditions, inan aprotic, polar solvent, the tetrahedral intermediate is deproto-nated leading to the formation of an amide and an alcohol.37

To quantify the amine density onto polymer surfaces, a slightmodification procedure based on Kaiser test was performed. It isa fast and convenient methodology routinely used to evaluate theamount of peptide growing directly onto resins in solid-phasepeptide synthesis.29,38 Differently from others,23,27,30 here theaminolized polymer sheets were brought in solution in order tomeasure the overall amount of amino groups engrafted onto thechains. By adding first potassiumcyanide in pyridine solution andthen the other kit components directly onto PCL samples, thereaction between ninhydrin and amino groups takes place in thehomogeneous phase. At the final stage of the assay, the purplepolymer mixture is suspended in a DCM/ethanol (1:1) mixture,being careful to add first the organic solvent and then ethanoldropwise to avoid polymer precipitation. Dichloromethaneresulted in a good solvent for PCL at room temperatureand does not affect UV absorption of Ruhemann’s purple

pigment. Evaluation of amino functional groups was carriedout through the calibration curve obtained as described in detailin Supporting Information (Figure S1). The nominal densityvalue was obtained by dividing the number of moles obtainedfor each sample by its surface area (expressed in cm2). It is worthnoticing that in the case of a polymeric substrate such as PCL thisvalue has to be referred to as overall amino groups engrafted ontochains rather than areal density.

A plot of amine nominal density against time of treatment wasobtained at different temperatures (data not shown). The rates ofgrafting at 24, 37, and 40 �C, respectively, of 1.01, 3.27, and 3.74�10-9 mol/cm2 min were calculated from the initial slopes of thecurves between 0 and 30 min. On this basis (see SupportingInformation, Figure S3), the activation energy (Ea) of the amino-lysis reaction of 69.5 ( 4.6 kJ/mol was evaluated by calculatingthe linear fitting of log-log (basis e) curves of rates as a functionof the temperature.39 This positive value of Ea suggests a strongdependence of the amination as a function of temperature.

As already reported in the literature by Croll et al.13 for PLGAand by Bech et al.39 for PET, the Ea is affected by the size ofdiamine, reported as mobility of molecule, by the solvent, andeventually by the substituent groups flanking the ester. For alinear polyester polymer such as PCL, the chosen aminolysisconditions, such as the 1,6-hexandiamine/isopropanol solutionand 37 �C, are a good compromise between the achievement of afast treatment (rate of grafting of 3.27� 10-9 mol/cm2min) and atemperature low enough to avoid the softening of the samples.

Amaximumof nominal amino density is obtained after 30minof treatment (Figure 2A), reaching a value around 100 nmol/cm2.With increasing aminolysis time, the density drops down at anominal concentration value around 66 nmol/cm2. At the sametime, AFM measurements evidenced an increase in roughnessparameters in the case of long surface treatment time (longer that30 min). In particular, Ra and rms were almost constant byincreasing the treatment time with a stepwise gain in surfaceroughness parameters represented by the time of 30 min at 37 �C(Figure 2A). According to the results already reported in theliterature, aminolysis has to be referred to as a degradationreaction occurring upon contact with diamine solution. In thefirst stage, indeed, the reaction starts preferentially at the amor-phous regions of the polymer.40 At longer aminolysis time, thedecrease of bound NH2 may be caused by chain scission, forma-tion of oligomers and other lowmass fragments that are removed

Figure 1. ATR-FTIR of plain and aminolyzed PCL substrates.PCL substrate show characteristic extra peaks relative to thepresence of amide and amine covalently linked to the polymersurface.

(36) Coates, J. InEncyclopedia of Analytical Chemistry; Meyers, R. A.; Wiley J. &Sons Ltd: Chichester, 2000; pp 10815-10837.(37) Bunnett, J. F.; Davis J. Am. Chem. Soc. 1960, 82, 665–674.(38) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.

1970, 34, 595–598.

(39) Bech, L.; Leipottvin, B.; Roger, P. J. Polym. Sci., Part A 2007, 45, 2172–2183.

(40) von Burkersrod, F.; Schedl, L.; G€opferich, A. Biomaterials 2002, 23, 4221–4231.

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from the surface during reaction, and the rinsing process.39 Theobtained result could thus be due to the scission and detachmentof polymer chains present on the surface and the presentation offresh polymer located beneath. The proposed mechanism couldalso lead to a plateau value in amine density occurring at longertime of treatment.Influence of Aminolysis Treatment on Bulk Properties.

The influence on bulk properties of aminolysis of PCL sheets wasinvestigated through an analysis of degradation kinetics in termofpolymer weight loss as well as an evaluation of mechanical andthermal properties during the treatment. A slight decrease (lowerthan 0.3%) inmass is recorded up to 24 h (Figure 2B left). The rateof degradation is more pronounced in the first 3 h of aminolysis,reaching a mass loss value of about 0.2% after 1 h. A longer timeof treatment does not considerably affect the mass. The samedegradation kinetics was evaluated by mechanical tests in tensilecondition. In particular, elastic modulus variation with treatmenttime ranging from 15 min to 24 h is reported (inset of Figure 2Aright). As result, elastic modulus drops at the early stage of treat-ment around 3 h; in particular, in the first 2 h the elastic modulusis roughly constant around a value of 300MPa as reported in the

literature in the case of untreated PCL.41 Evaluation of fusionenthalpy during the the aminolysis process did not show anysignificant change (p=0.48, n=8, unpaired t test), highlightingno appreciable modification in the degree of crystallinity. Allthe experimental results, thus, demonstrate that the proposedaminolysis treatment does not affect the bulk properties of thenative PCL.Evaluation of Surface FreeEnergy andHydrophilic Char-

acteristics of Treated Surfaces. The change of the polarcharacteristics of the PCL surface after aminolysis reaction andgrafting with RGD-based peptide was investigated in terms ofcontact angle measurements and of the SFE (with dispersive andpolar components) evaluation. The results obtained from contactanglemeasurements reveal the increase of PCL surfacewettabilityafter aminolysis reaction with respect to the untreated PCLsurface (Figure 3A) (statistical relevance p < 0.01). The treatedsurfaces (37 �C, 30 min, DEA 10% w/w) showed an increase inhydrophilic nature of the PCL substrate with a mean contactangle decreasing from about 78� to 60� confirming the incorpora-tion of highly hydrophilic amine groups on the polymer surface.Instead, the subsequent link of the RGD peptide through GAinsertion determines only a slight increase of the mean value ofthe contact angle without any statistically relevant difference withrespect to aminated surfaces. The difference between plain and

Figure 2. (a) Plot of nominal amine density and surface roughness parameters (Ra, rms) for aminolyzed PCL substrate for different reactionlengths. Data are reported in terms of mean, and bars represent standard deviation. (b) Residual weight and elastic modulus kinetics duringaminolysis treatment (left). A focus on the first 3 h of treatment (right). Data are reported in terms of mean, and bars represent standarddeviation.

(41) Rosa, D. S.; Guedes, C. G. F.; Bardi, M. A. G. Polym. Test. 2007, 26, 209–215.

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peptide-conjugated substrates can be ascribable to the presence ofthe RGD peptide partially neutralizing the charges on the PCL-GA-GRGDY sample surfaces.

Moreover, while the total SFE (γtot) remains about the sameafter the aminolysis and RGD peptide treatment the relativeweight of the Lifshitz-van der Waals (γLW) and Lewis acid-base(γAB) component changes (Figure 3A). Similar data were re-ported in the case of adsorption of RGD on PCL surface afterplasma treatment.16 In particular, the presence of amine groupsand peptide sequences influence acid-base contributions to SFE.This last effect is due to the combined decrease of the acid term γA

and the small, but critical, increase of the γB component in termsof the well-know Chaudhury-Good-van Oss equation. RGD-functionalized surfaces thus exhibited a slightly higher polarcharacter (a higher acid/base contribution) when compared tothe untreated surface. This may be attributed to the presence ofpolar amino acids such as arginine (guanidine group) and asparticacid (carboxyl group).Evaluation of Grafted Peptide and Morphological Map-

ping. One-pot colorimetric assay based on the BCA-Cuþ purplecolor complex was used to determine directly on solid PCLsupport the amount of immobilized peptides. This assay is widelyused to measure proteins both in solution and on adsorbing solidsubstrates with high sensitivity (picomolar scale) and in a veryreproducible way. Furthermore, this method can measure shortpeptides incorporating cysteine, cystine, tryptophan, and tyrosinethat are recognized as cuprous reduction enhancer residues. Yuet al.42 used the BCA-assay to quantify peptides covalentlyimmobilized on hydrogel scaffolds indirectly subtracting theassayed amount of unbound peptide from the known amountof starting (or prebound) peptide. Tyllianakis et al.34 describedone incubation step method based on BCA assay as a useful tool

for the determination of solid supports (agarose beads andsepharose gel) functionalized with cysteine and tyrosine. Thedetection limit of the assay (0.7 nmol/tube for solid tyrosineligands functional support) was shown as well as the stability ofbicinchonic-Cuþ complex and its absorbance in the operativecondition and with a series of contaminants (i.e., Tween, SDS,etc.). The quantitation of the different groups was finally carriedout through standard curves of an appropriate substance.

Here, a MicroBCA assay was used to directly quantify thesurface concentration of adhesion peptide covalently bound onPCL substrates by comparison with the calibration curve ob-tained using the corresponding peptide as standard. The immo-bilized signal (GRGDY) is an RGD-like sequence containing atthe carboxyl end the bulky and lipophilic tyrosine residue thatmediates adhesion with high affinity via Rvβ3 and RIIbβ3 integrinreceptors1 and increase the sensitivity of the assay.34 The con-centration of peptide covalently coupled to the PCL was calcu-lated by taking into account a correction factor obtained bytopological analysis. The sample surface area resulted to be about1.1% higher than the projected area, and then the final resultattained a nominal density of 2.81 ( 0.35 nmol/cm2.

Surface spatial distribution as well as the penetration depth oftreatment was studied by probing the presence of fluorescentmoieties. To this purpose, two fluorescent molecules were used asa model to investigate the spatial distribution and penetrationdepth during each step of the proposed treatment. RBITC wasused to label primary amines presented on the PCL substrate,while FLUO was used as a tracer of aldehyde functional groupson which peptide sequences can attach in the last step ofconjugation after tether insertion.

Confocal microscopy images showed an uneven spatial dis-tribution of FLUO onto PCL surfaces that seems to mimic theamorphous portion of semicrystalline polymer surface (Figure 3Bright) reproducing the topology of spherulities at the surfaceafter aminolysis. Such a result suggests a clustering of peptidesequences immobilized on PCL substrate.

Moreover, a high depth penetration of treatments was verified.The profile density reported (Figure 4A,B) showed a remarkablepenetration that could be ascribable to molecule (FLUO andRBITC) permeability within the polymer. In particular, a crosssection of functionalized samples showed a fluorescence emissiondown to about 60 μm from the upper surface in the case ofaminolysis for RBITC conjugation, while the penetration wasreduced to 50μmin the case of FLUOconjugationwith a significantreduction of intensity at about 30 μm. Such results emphasize theoverestimation of evaluated densities for both amine and peptide.Bioactivation Assessment Through NIH3T3 Fibroblast

Adhesion Tests. In order to study the bioactivation of PCLsubstrates at the cell-material interface, their interaction withfibroblast cells was studied. SEM images revealed that, after 24 hfrom seeding, NIH3T3 cells adhere on all the substrates. How-ever, cell morphology drastically changes on different samples.In particular, on nontreated PCL, PCL-NH2, and PCL-GA-GYDGR surfaces, cells showed a round shape, indicating a scarceadhesion to the substrate (respectively Figure 5 A-E, B-F, andC-G). Conversely, on PCL bioactivated withRGDpeptide, cellswere correctly adhered and well-spread on the surface, showing agood interaction with the material (Figure 5D,H). Moreover, athigher magnification, the formation of filopodia was observed(Figure 5I). The effect of PCL functionalization in enhancing celladhesion was further confirmed by actin cytoskeleton staining.This qualitative analysis indicated that cells were better adheredon RGD bioactivated substrates (Figure 5M), as demonstratedby the presence of stress fibers, compared to cells seeded on

Figure 3. (a) WCA and SFE evaluation for each step of theprocedure reported as mean and standard deviation. (b) Topolo-gical mapping of treated surface and spatial distribution of con-jugated molecules. Topology (left) observed by AFM in tappingmode of PCL-NH2 surface after 30 min of aminolysis in 10%DEA/IPA solution. Fluorescentmicroscopy images (right) of PCLafter FLUO conjugation.

(42) Yu, T. T.; Shoichet, M. S. Biomaterials 2005, 26, 1507–1514.

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nontreated PCL, PCL-NH2, and PCL-GA-GYDGR surfaces

(Figure 5J,K,L), where there is no evidence of cytoskeleton

organization. Moreover, Alamar blue assay (see Figure 4 in

Supporting Information) confirmed a significant improvement in

cell proliferation of RGD-conjugated substrate after two days of

culture when compared to plain PCL.

Discussion

The binding of amino acid sequences encoded along extra-cellular protein backbones (such as fibronectin, collagen, vitro-nectin, and laminin) to cell membrane integrins is one of the keymechanisms by which these cells recognize the material.1 Themost studied peptide-functional materials are those that mimic

Figure 4. Intensity profiles of fluorescent molecules in the depth during the two-step conjugation procedure: (a) CLSM intensity profile as afunction of depth in the z-direction of PCL-NH-RBITC. (b) CLSM intensity profile as a function of depth in the z-direction of PCL-GA-FLUO samples.

Figure 5. Scanning electronmicroscopemicrographs (AandEPCL;BandFPCL-NH2;CandGPCL-GA-GYDGR;D,H,and IPCL-GA-GRGDY). Confocal laser scanningmicroscope images of phalloidin staining ofmicrofilaments (J PCL;KPCL-NH2; L PCL-GA-GYDGR;and M PCL-GA-GRGDY). Bar 50 μm.

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the integrin binding RGD sequences. More importantly, thediscussion about the importance of spatial distribution of thepresenting peptide signal, its density, and tether choice in influen-cing cell response is still open as well as chemistry with respect toroughness contribution to cell adhesion.43

Chemical methods and, in particular, surface chemical mod-ification rather than chemi- or physisorption represents a robust,repeatable, and easy to perform procedure to bind peptidesequence on polymeric surfaces. However, to realize bioactiveinterfaces it is mandatory to investigate the surface characteristicsas well as the presentation of immobilized signals with respect tothe polymeric substrate.

Here, we monitored the evolution of chemical and physicalproperties of polymeric surface during each step of the bioconju-gation. In particular, the synthesis consisted of the aminolysis ofthe polyester substrate to obtain functional groups on the surface,on which peptide sequences were coupled by a homobifunctionalcross-linker in a second step.

Aminolysis is already reported as a procedure to introduceprimary amines on polyesters such as PLGA,13 PET, or PCL.27

Data already reported in the literature fromGabriel et al.22 aswellas Zhu et al.27 showed, in different treatment conditions, lower oralmost equal amine density on PCL: respectively, 7.9 � 10-9 and2 � 10-7 mol/cm2, while an increase of roughness after amino-lysis was also evidenced on PCL23,27 and on PET fibers by Rogeret al.39 In particular, as already described in the literature,27 themaximum NH2 density yielded at 30 min is about 10-7 mol/cm2,that in the case of flat surfaces and all amino groups laid as singlelayer should correspond to an average area per amino-terminatedchain of about 0.1 A2. Such a result represents an impossibly highconcentration. Indeed, even though surface roughness is nottaken into account, data presented in this work attaining around60 nmol/cm2 should be referred to as a surface layer of about60 μm (see Figure 4A) and, thus, corrected on the basis of densityprofile along the depth of the sample. Taking into accountpenetration of aminolysis treatment and, in particular, supposinga constant level of amine in the first 60 μm of PCL sheets ispossible to estimate the extent of primarymodification equivalentto about 0.16% by considering polymer density of 1.12 g/cm3. Asreported by Croll et al.13 for PLGA substrates, a level of primarymodification below 1% is theoretically required to allow theattachment of a complete monolayer of proteins via covalentbinding. In our case, a coverage of about 10% should be achievedon the PCL surface. Differently, our estimation should be morerealistic by considering the amount of amine distributed in amicrometric thickness rather than distributed merely on thesurface. It was also reported that the aminolysis resulted in theformation of an amide bond and an alcoholic group on the PCLbackbone with the following lysis of chain segments undergoingfunctionalization.27 Such a phenomenon could bring about aplateau value after a peak in the number of amines present onPCL due to the detachment of the interested portion of the PCLchain at a longer time of treatment. Crack formation as well as amassive loss of mechanical properties after partial degradationduring aminolysis is already reported, mainly in the case ofPET.44,45 The influence on bulk properties and the control overthe surface chemistry and evolution during the treatment of itsphysicochemical properties were studied as key aspects. Inparticular, it was demonstrated that the bulk mechanical proper-ties are not significantly affected by aminolysis in the proposed

reaction conditions. A negligible decrease of elastic modulus(∼11%) and a very slight reduction of mass (lower than 0.3%)were obtained after 3 h of treatment. However, it worth noticingthat aminolysis of PCL also presents some limitations concerningthe range of engrafted amine groups, falling in the range from afew tens to one hundred nmol/cm2, as well as the lack of control oftheir spatial distribution, impairing a uniform distribution ofsignals on the polymeric surface because of its semicrystallinenature.

As for the bioactivation after RGD conjugation, there is ageneral consensus for solid and rigid materials, such as glass,assuming that the amount of peptide is evenly distributed over thesurface of the substrates. It is generally considered that only amodification of surface is achieved and that the reagent penetra-tion depth is the same as the integrin accessible depth of 10 nm,treating results in a two-dimensional manner.46 In the case ofpolymers, instead, the depth of the surface modification aftertreatment will depend upon polymer crystallinity, size of pore,and swelling capability of polymer at surface.43 Here, we found ahigh penetration depth that could determine a large overestima-tion of peptide density that can contribute to cell adhesion.A corrected density can be obtained by calculating the penetra-tion depth of conjugated fluorescent dye (assumed as peptidemolecule) into the polymer bulk immobilized at the same condi-tions used for the GRGDY conjugation. In detail, the densityof bioactive GRGDY peptide covalently immobilized through atether represented by a short aldehyde were first obtainedby the MicroBCA method; it was assumed that the onlyGRGDYmolecules available for the cytoskeletal-associated trans-membrane receptors, represented by integrins, were located in anouter layer of 10 nm of polymer.46 Such a distance can beconsidered a threshold distance for the integrin engagement;afterward, the amounts of molar of GRGDY obtained byMicroBCA were corrected on the basis of the profile densityobtained along the depth (fluorescent intensity profile was ob-tained by FLUO as a model molecule). A corrected surfacedensity of about 1 pmol/cm2 was estimated as the number ofmolecules per area available for the integrin engagement.

The relevance of the quantity of peptide per area canbe comparedwith literature reported showing that aminimumofRGDdensity of1.0� 10-15mol/cm2 is sufficient in the case of fibroblast to promotecell spreading, while only a density of 1.0 � 10-14 mol/cm2 issufficient to promote the formation of focal contact in the case ofglass substrates.4Equally,more recentpublication reportedadensityof 10-12 mol/cm2 of conjugated RGD peptide on PET surfaces assufficient for an improvement in cell adhesion.47However, it isworthemphasizing that such results are related to the particular surfacecharacteristics and chemistry; that is, in our case, a higher roughnessand different presentation of RGD-based peptide to cell integrinscould determine different thresholds levels.

Moreover, it is widely reported in the literature that, beyondligand density, the morphology and spatial organization of thepresenting solid signals at the surface and, in particular, of thebioactive peptide strongly affect cell behavior and fate. Recently,it was also demonstrated that integrin clustering and cell adhesioninduced by RGD ligands is dependent on ligand arrangementand, in particular, that the cell adhesion is inhibited for a RGDnanopattern order while activated by a nanopattern disorderif ligand spacing is higher than 70 nm.48 On this basis, the

(43) Perlin, L.; MacNeil, S.; Rimmer, S. Soft Matter 2008, 4, 2331–2349.(44) Haghighat Kish, M.; Borhani, S. J. Appl. Polym. Sci. 2000, 78, 1923–1931.(45) Holmes, S. A. J. Appl. Polym. Sci. 1996, 61, 255–260.

(46) Brandley, B. K.; Schnaar, R. L. Anal. Biochem. 1988, 172, 270–278.(47) Chollet, C.; Chanseau, C.; Remy, M.; Guignandon, A.; Bareille, R.;

Labrug�ere, C.; Bordenave, L.; Durrieu, M. C. Biomaterials 2009, 30, 711–720.(48) Huang, J.; Gr€ater, S. V.; Corbellini, F.; Rinck, S.; Bock, E.; Kemkemer, R.;

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organization of the bioactive ligands immobilized on the polymersurface was studied through topological investigation and fluore-scence mapping. A fluorescent molecule (FLUO) was selected tomimic the peptide in studying its distribution on the PCL surface.The presence of uneven distribution of fluorescent signal ontreated surfaces demonstrated a non-uniformmolecular conjuga-tion at the micrometric level (Figure 3B right), while a disorderedtopography can be recognized at the nanometric level (Figure 3Bleft). The lack of spatial uniformity in peptide conjugation can beascribable to the intermediate step of aminolysis, resulting inenhancement of the amorphous portion of the polymer surface.49

Thismotivates a similar appearance at the surface of the topologyof aminated PCL (Figure 3B left) with the fluorescence images ofFLUO-conjugated PCL (Figure 3B right).

Furthermore, the overall gain in hydrophilic characteristicswas already demonstrated to improve cell adhesion on polymericsurface.50,51 In our case, the same presence of a peptide sequencecan alter the physical and chemical properties of the back-ground materials and affect the biological properties of thematerials through nonspecific means. The control, representedby scrambled peptide sequence, confirms the specific binding ofimmobilized peptides. Moreover, adhesion tests were carried outin serum-free conditions. The effect of serum absorption at thecell-material interface can, indeed, potentially interfere with thepeptide engagement by integrin receptors. Even though far fromin vivo conditions, the serum free conditionwas chosen to rule outany effect of serum proteins in the adhesion mechanisms, beingmore appropriatewhen investigating the presentation of bioactivemolecule to cell integrins. The enhancement in cell adhesion isthus fully ascribable to the effective engagement of RGDsequence by integrin receptors (presumably Rvβ3 and RIIbβ3

1)present on NIH3T3 cell membrane. Moreover, viability testsconfirmed that the conjugated peptides significantly improvethe NIH3T3 cell proliferation on PCL substrate, indicating thatthe proposed conjugation of GRGDY peptide can play a crucialrole in building up biomimetic materials, especially for tissueengineering applications.

Conclusions

It is becoming increasingly apparent that the future of syntheticbiomaterials will depend on the ability to develop materials totrigger a specific function in cell behavior at the cell-materialinterface. To do this, a fundamental step is represented by theinvestigation of effective presentation of ligands toward cellcomponent and the control of surface characteristics after bio-material treatment.

In cell-material interaction, cell-adhesion represents a funda-mental cell function to be regulated. To this regard, we investi-gated the immobilization of RGD peptide on PCL surfaces bymonitoring the surface characteristics after each step of treatmentand evaluating ligand distribution on the surface and penetrationin the polymer substrate.

Here, we demonstrated that aminolysis represents an easy route tointroduceprimary amineswithhigh yield that canbe easily optimizedwith respect toprocessing conditions.More importantly, conjugationof amine-terminated peptides by means of reductive amination aftertether insertion showed a specific recognition of the solid signal toNIH3T3 integrin cell receptors highlighting a correct presentation ofthepeptide sequences. Inparticular,GRGDYconjugationontoPCLrevealed a typical spatial distribution that traces the boundary ofpolymer crystallite at the surface. Moreover, the investigation ontreatment penetration revealed that the thickness of immobilizedligand spans tens of micrometers reducing the “effective” signalimmobilized on the surface but not affecting bulk mechanicalproperties of the polymer. A determination of amines and peptideseffectively engrafted on polymer was carried out and discussed.

These approaches provide a useful procedure to investigate poly-meric surfaces aftermodificationwith specificmolecules andhighlightthe potential for realizing and characterizing surfaces capable ofimprovingcell attachmentor, in thecaseofdifferentpeptide sequencesor structure, promote specific cell-material interactions.

Acknowledgment. The authors thank Dr. Antonio Gloria formechanical tests, Dr. Gobind Das for helping us in spectroscopicmeasurements, and Dr. Maurizio Ventre for statistical analysis.

Supporting Information Available: Additional informationand figures as described in the text. This material is availablefree of charge via the Internet at http://pubs.acs.org.

(49) Zeronian, S. H.; Collins, M. J. Text Prog. 1989, 20, 1.(50) Lim, J. Y.; Shaughnessy, M. C.; Zhou, Z.; Noh, H.; Vogler, E. A.;

Donahue, H. J. Biomaterials 2008, 29, 1776–1784.(51) Arima, Y.; Iwata, H. Biomaterials 2007, 28, 3074–3082.