Protein immobilization on the surface of poly-L-lactic acid films for ...

European Polymer Journal 38 (2002) 2279–2284www.elsevier.com/locate/europolj<strong>Protein</strong> <strong>immobilization</strong> on the surface of poly-L-lacticacid films for improvement of cellular interactionsZuwei Ma, Changyou Gao * , Jian Ji, Jiacong ShenDepartment of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, ChinaReceived 13 August 2001; received in revised form 8 April 2002; accepted 16 April 2002AbstractTo covalently immobilize gelatin or collagen type I on poly-L-lactic acid (PLLA) film surfaces poly(hydroxyethylmethacrylate) (PHEMA) or poly(methacrylic acid) (PMAA) was grafted via photooxidization and subsequent UVinducedpolymerization [Makromol. Chem. 186 (1985) 1533.1]. For films grafted with PHEMA, methyl sulfonylchloride was used to activate the hydroxyl groups and for films grafted with PMAA 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to activate the carboxyl groups. Gelatin and collagen were finally reacted with the activatedhydroxyl or carboxyl groups to obtain covalently immobilized protein layers. Grafting of PHEMA, PMAA and proteinon the surfaces was confirmed using ATR-IR and XPS. Surface wettability of the modified films was improved. Theprotein immobilized PLLA may be widely used as a biocompatible material.Ó 2002 Elsevier Science Ltd. All rights reserved.Keywords: Poly-L-lactic acid; Surface modification; <strong>Protein</strong> <strong>immobilization</strong>; Biocompatibility1. IntroductionIn recent years, many biodegradable polymers havebeen used in tissue engineering to build three-dimensionalporous materials to provide scaffolds for cellstowards regeneration of tissue engineered organs such asliver [2], articulate cartilage [3] and artificial skin [4].Among these biodegradable polymers, poly-L-lactic acid(PLLA) is widely used due to its biodegradability, goodmechanical properties and proper degradation ratewhich is often comparable with the healing time of thedamaged human tissues. Transplantation of isolatedcells seeded in biodegradable PLLA scaffold has beeninvestigated as a means of producing biologic substitutesto regenerate or replace the damaged tissue such as articulatecartilage [5].* Corresponding author. Tel.: +86-571-87951108; fax: +86-571-87951948.E-mail address: cygao@mail.hz.zj.cn (C. Gao).However, one great limitation of PLLA is the lack ofcompatibility for cells. It is difficult to transplant isolatedcells in PLLA scaffold because cell attachment onPLLA is rather low due to its hydrophobicity [6]. Oneapproach to solve this problem is to immobilize a biocompatiblelayer on the surface of the polymer to improvecell-material interactions. In recent years proteinor oligopeptide <strong>immobilization</strong> on polymer surfaces toimprove biocompatibility is of much interest. Immobilizationof some special biologically active molecules onsynthetic materials is of critical importance since theycan in principle elicit some specific, predictable andcontrolled responses from the cells seeded on the materials.To covalently immobilize protein molecules on achemically inert polymer surface such as PLLA, it isnecessary to introduce some reactive groups such ashydroxyl (–OH), carboxyl (–COOH) or amino groupson the polymer surface. Many methods like plasmatreatment in ammonia [7], plasma induced graftingpolymerization [8], photo-induced grafting polymerization[9] and ozone oxidization [10] have been used to0014-3057/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.PII: S0014-3057(02)00119-2

2280 Z. Ma et al. / European Polymer Journal 38 (2002) 2279–2284Fig. 1. Schematic representation of the reaction protocol for <strong>immobilization</strong> of protein on PLLA film surfaces, where H 2 N–Prepresents a protein molecule.introduce the above reactive groups on polymer surfaces.<strong>Protein</strong> molecules can be subsequently immobilizedusing chemical methods such as carbodiimidechemistry [11] or sulfonyl chloride chemistry [12].Collagen is one of the most important biologicalmacromolecules of the extracellular matrix in tissues, andhas been used successfully to produce commercializedbiomaterials for a wide range of applications includingburn dressings, hemostats and soft tissue augmentation[13]. Haw Suh has grafted type I atelocollagen on ozoneoxidized PLLA films and the compatibility of the modifiedfilms for osteoblasts was improved significantly [10].Gelatin, which is the product of hydrolysis of collagen,has also been used in modifying synthetic biomaterials.Yamaoka has immobilized gelatin on the surface ofPLLA by reacting the material directly with an alkalinesolution of gelatin. The modified PLLA has better cellattachment property for 3T3 fibroblasts [14]. The successof gelatin and collagen may be attributed to their naturalorigin and low immunogenicity [15].In this study, PLLA films grafted with poly(hydroxyethylmethacrylate) (PHEMA) or poly(methacrylicacid) (PMAA) were prepared via the photooxidizationand subsequent UV-induced polymerization [1]. Gelatinor collagen was then covalently immobilized on the filmsurfaces via the reacting groups (OH or COOH), asshown in Fig. 1.2. Experiment2.1. MaterialsPLLA was synthesized using the method described in[16]. PLLA films were prepared by casting a 1,4-dioxanesolution containing 6 wt.% of PLLA (M n ¼ 200,000,M w ¼ 400,000) onto a stainless steel plate. Films weredried to constant weight and had a thickness of 0.5 mm.Hydroxyethyl methacrylate (HEMA, Sigma) and methacrylicacid (MAA, Sigma) were purified by distillationunder vacuum. Water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) andmethyl sulfonyl chloride were purchased from Aldrich.Gelatin and collagen type I were purchased from Sigma.2.2. Photooxidization and graft polymerizationThe PLLA film was placed in 40 ml hydrogen peroxidesolution (30%) and irradiated with UV light generatedfrom a high-pressure mercury lamp (250 W) for40 min at 50 °C. The photooxidized film was rinsed withdeionized water and dried at room temperature in vacuumfor 4 h to remove excess hydrogen peroxide. Thefilm was then immersed into an aqueous monomer solutionwith a given concentration in a Pyrex glass tubepurged with nitrogen. Graft polymerization was carriedout under UV irradiation at a distance of 12.5 cm for 60min at 50 °C. The grafted film was rinsed with deionizedwater at 70 °C for 24 h to remove the homopolymers[17].2.3. Immobilization of protein2.3.1. Method IThe PLLA-g-PHEMA film was introduced into aglass tube containing 2 ml methyl sulfonyl chloride and20 ml diethylether. After incubation at 20 °C for 2 hgelatin or collagen solution with given concentrationwas reacted with the activated film for 24 h at 30 °C.

Z. Ma et al. / European Polymer Journal 38 (2002) 2279–2284 22812.3.2. Method IIThe COOH residues on the film surface were activatedat 0 °C for 4 h in EDAC phosphate buffer solution (10mg/ml, pH 7.4). Then the gelatin or collagen in phosphatebuffer solution (pH 4.5) with given concentrationwas reacted with the activated film for 24 h at 0 °C.The protein immobilized PLLA films were rinsed withdeionized water at 37 °C for 24 h. The film was slightlybrushed with a cotton tampon to aid the removal ofnone-grafted (adsorbed) protein. To confirm that theadsorbed protein could be removed completely, PLLA,PLLA-g-PHEMA and PLLA-g-PMAA films were directlyimmersed in gelatin or collagen solution andrinsed as described above. In the XPS spectra of thesefilms there were no nitrogen peaks. However, peaks ofnitrogen were detected in XPS spectra of the proteingrafted PLLA films.2.4. CharacterizationThe content of hydroperoxide groups on the filmsurface was determined by the iodometry method [18].ATR-IR spectra were obtained on a Nicolet Magna-IR560 machine. XPS spectra were recorded with aESCA LAB Mark II spectrometer employing AlK a excitationradiation. The charging shift was referred to theC 1s line emitted from the saturated hydrocarbon at 285.0eV. The take off angle of the XPS was 30°. To calculatethe atomic ratio of N to C on the outer-most layer of theprotein immobilized PLLA films, the collecting factor of1.77:1 (N:C) were used. The surface density of thegrafted protein on the PLLA films was determined bythe ninhydrin method [19]. Each value was averagedfrom five times measurements.Static water contact angles were obtained on aKRUSS DSA10-MK machine. The sessile contact anglewas determined by placing a drop of water (0.8 ll) onthe surface and recording the angle between the horizontalplane and the tangent to the drop at the point ofcontact with the substrate. Captive bubble contact angles(CBCA) were measured by observing the air bubblein water at room temperature within 30 s after it contactedthe polymer surface. For both methods, eachvalue was averaged from 15 measurements.3. Results and discussion3.1. Grafting of PHEMA and PMAA onto the surfaces ofPLLA filmsAs the first step of the <strong>immobilization</strong> of protein,hydroperoxide groups were introduced on the PLLAfilm surfaces via photooxidization in hydrogen peroxidesolution under UV light. The surface density of the hydroperoxidegroups was 1:5 0:1 10 6 mol/cm 2 asdetermined with the iodometry method [18], whichwould represent about 90 hydroperoxide groups persquare A. Giving such a high degree of PLLA oxidization,it also seemed unreasonable that there was nodifference in water contact angle between control PLLAfilm and the oxidized one (Table 2). The reasonableexplanation could be that even though PLLA is hydrophobic,the hydrogen peroxide molecules could stillpermeate into the bulk PLLA at 50 °C so that thephotooxidization of PLLA films occurred not only onthe surface but also in the bulk material. The hydroperoxidegroups in the inner layer could also be decomposedand induce the graft polymerization.Under UV light the hydroperoxide groups on the filmsurface decomposed into macromolecular radicals andfree hydroxyl radicals. The macromolecular radicals caninitiate grafting while the hydroxyl radicals initiatehomopolymerization. The ATR spectra of the PLLAfilms grafted with PHEMA or PMAA are shown in Fig.2. The broad absorption between 3000 and 3700 cm 1was assigned to the stretching vibration of O–H in hydroxylgroups of PHEMA or carboxyl groups ofPMAA. On the PMAA grafted film some carboxylFig. 2. ATR-IR spectra of the control and modified PLLA films: (a) method I and (b) method II.

2282 Z. Ma et al. / European Polymer Journal 38 (2002) 2279–2284Fig. 3. XPS spectra of the control and modified PLLA films: (a) C 1s core level scan spectra of the control and modified PLLA films and(b) survey scan spectrum of the protein grafted PLLA film.groups were transferred to carboxylate groups (–COO )and part of the carbonyl absorption was moved from1750 to 1700 cm 1 (Fig. 2). The alteration of the surfacechemical composition was further investigated byXPS (Fig. 3). The C 1s spectra of the control PLLA,PLLA-g-PHEMA and PLLA-g-PMAA films all gavethree main peaks with binding energies at 285.0,286.6 and 289.0 eV, corresponding to carbon atomsof saturated hydrocarbons, carbon atoms with a singlebond to oxygen (O–C–C@O or–C–O–C@O) and carbonatoms in carbonyl groups (–C@O) [20,21]. In theC 1s spectra of PLLA-g-PHEMA and PLLA-g-PMAAfilms, the peaks at 285.0 eV were larger than that of thecontrol film because both PHEMA and PMAA havehigher content of saturated hydrocarbons (50% and75%, respectively) than PLLA (33%). In the C 1s spectrumof the film grafted with PMAA, the peak at 286.6eV was much smaller because PMAA does not havecarbon atoms with a single bond to oxygen. These dataconfirm the occurrence of grafting of PHEMA orPMAA on the PLLA film surfaces.3.2. Immobilization of proteins on the PLLA filmsGelatin and collagen were covalently immobilized onthe PLLA surfaces using two methods. In method I thehydroxyl groups on the PLLA-g-PHEMA surfaces werereacted with methyl sulfonyl chloride to form sulfonategroups. Then, amino groups of the protein moleculeswere reacted with the sulfonate groups to produce acovalent combination. In method II, EDAC was used toactivate the carboxyl groups on the PLLA-g-PMAA filmsurfaces. The activated carboxyl groups were then reactedwith amino groups of protein molecules.ATR spectra of the protein immobilized PLLA filmsare shown in Fig. 2. Absorption at 1600 cm 1 confirmedthe presence of amide bonds on the protein immobilizedfilm surfaces. In the XPS spectra of the protein immobilizedfilms there appeared N 1s peaks (Fig. 3), which directlyconfirmed the grafting of protein. The atomicratios of N to C on the surface of the gelatin-grafted filmsare shown in Fig. 4. The surface density of the graftedproteins on the PLLA films is listed in Table 1.Fig. 4. Atomic ratios of N to C on the outer-most layer of the gelatin immobilized PLLA films: (a) method I and (b) method II.

Z. Ma et al. / European Polymer Journal 38 (2002) 2279–2284 2283Table 1Surface density of the grafted proteins on PLLA filmsSampleThe atomic ratios of N to C on the outer-most layer ofthe protein immobilized PLLA films determined by XPScan be used to estimate the surface density of the graftedprotein because there are no nitrogen atoms in PLLA.Films with higher atomic ratio of N to C should havehigher surface density of protein. However, the correlationbetween these two parameters is not simply linear.Fig. 4 showed that the atomic ratio of N to C of thegelatin grafted films increased with monomer concentrationand gelatin concentration. Previous study hasdemonstrated that the grafting degree of the UV-inducedgraft polymerization increased with the monomer concentration[22]. With higher monomer concentrationmore functional groups (hydroxyl or carboxyl) were introducedon the film surface and were reacted with moreprotein molecules. <strong>Protein</strong> concentration also had aneffect on the surface density of the grafted protein. Withhigher protein concentration more protein moleculescould be grafted onto the film surfaces.3.3. WettabilityGraftingmethodDensity of graftedprotein (lg/cm 2 )PLLA-g-gelatin I 5:0 0:6PLLA-g-gelatin II 3:6 0:4PLLA-g-collagen I 2:6 0:8PLLA-g-collagen II 2:0 0:6The concentration of the proteins (gelatin or collagen) and themonomers (HEMA or MMA) is 4 mg/ml and 5 vol.%,respectively.The wettability of the control and modified films wasstudied using static water contact angles (Table 2).Surface wettability of the modified PLLA films wasobviously enhanced compared with the control film. Theunmodified PLLA surface showed a relatively highcontact angle. Such a hydrophobic polymer has beenknown to be unfavorable for cell attachment [23]. Theadhesion of human endothelial cells on unmodifiedPLLA is only 8% after 30 min and 10% after 1 h, comparedto a corresponding 43% and 59% on tissue culturepolystyrene with a CBCA of 35° [24]. Many works reportedthat cells attached and spread more easily andeffectively on surfaces with proper hydrophilicity thanon hydrophobic surfaces [24–26].The protein immobilized films had a higher sessiledrop contact angle than the PLLA-g-PHEMA andPLLA-g-PMAA films because PHEMA and PMAA aremore hydrophilic than gelatin and collagen. However,no big difference exists between the CBCA of the PLLAg-PHEMA,PLLA-g-PMAA and the protein graftedfilms, probably because all the grafted hydrophilicmacromolecules on PLLA surface could rearrange theirconformation in water to let the hydrophilic groups facetowards the water to form a hydrated layer persisting inthe interface between the material and water [27].4. ConclusionReactive groups (hydroxyl or carboxyl) were introducedat the chemically inert PLLA surface throughphoto-induced grafting of PHEMA or PMAA. The reactivegroups were subsequently used to graft gelatin orcollagen type I through sulfonyl chloride chemistry orcarbodiimide chemistry. ATR-IR and XPS measurementsconfirmed the occurrence of the grafting. Thewettability of the protein immobilized films was improvedobviously. This provides an opportunity toadapt the grafting strategy to a chosen biomacromoleculetherefore to potentially improve the cellular interactionon synthetic biomaterials.AcknowledgementsThis work is financially supported by The Major StateBasic Research Program of China (G1999054305).Table 2Water contact angles of the control and modified PLLA filmsSampleGraftingmethodSessile dropcontact angleCBCAControl PLLA 81:5 2:0 71:0 1:6Photooxidized81:5 2:8 71:9 3:1PLLAPLLA-g-PHEMA 51:1 5:5 40:1 3:6PLLA-g-PMMA 51:0 3:1 39:8 4:1PLLA-g-gelatin I 61:4 2:7 40:2 5:7PLLA-g-gelatin II 59:3 3:3 44:5 0:5PLLA-g-collagen I 69:0 2:9 35:0 3:4PLLA-g-collagen II 66:1 5:8 41:3 4:2References[1] Feng XD, Sun YH, Qiu KY. Makromol Chem 1985;186:1533.1.[2] Johnson LB, Aiken J, Mooney D, Schloo B, Griffith-CimaL, Langer R, et al. The mesentery as a laminated vascularbed for hepatocyte transplantation. Cell Transplant 1993;3:273–81.[3] Freed LE, Marquis JC, Nohria A, Emmanuel J, MikosAG, Langer R. Neocartilage formation in vitro and in vivousing cells cultured on synthetic biodegradable polymers.J Biomed Mater Res 1993;27:11–23.[4] Matsuda K, Suzuki S, Isshiki N, Yoshioka K, Okada T,Ikada Y. Influence of glycosaminoglycans on the collagen

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