Immobilization of poly(acrylamide) brushes onto poly(caprolactone) surface by combining ATRP and “click” chemistry: Synthesis, characterization and evaluation of protein adhesion

Applied Surface Science 329 (2015) 223–233

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Immobilization of poly(acrylamide) brushes onto poly(caprolactone) surface by combining ATRP and “click” chemistry: Synthesis, characterization and evaluation of protein adhesion Yuhao Ma a , Xinxiu Bian a , Liu He a , Mengtan Cai a , Xiaoxiong Xie a , Xianglin Luo a,b,∗ a b

College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, PR China State Key Laboratory of Polymer Material and Engineering, Sichuan University, Chengdu 610065, PR China

a r t i c l e

i n f o

Article history: Received 28 September 2014 Received in revised form 19 December 2014 Accepted 22 December 2014 Available online 26 December 2014 Keywords: ATRP “Click” chemistry Poly(acrylamide) Fouling-resistance Blends

a b s t r a c t Developments of poly(caprolactone) in blood-contacting applications are often restricted due to its intrinsic hydrophobicity. One common way to improve its hemocompatibility is to attach hydrophilic polymers. Here we developed a non-destructive method to graft hydrophilic poly(acrylamide) (PAAm) onto poly(caprolactone) (PCL) surface. In this strategy, azido-ended PCL with low molecular weights was synthesized and blended with PCL to create a surface with “clickable” property. Alkyne-ended poly(acrylamide)s with controlled chain lengths were then synthesized by atom transfer radical polymerization (ATRP), and finally were immobilized onto PCL surface by “click” reaction. The occurrence of immobilization was verified qualitatively by water contact angle measurement and quantitatively by X-ray photoelectron spectroscopy (XPS). The PAAm grafted surface exhibited fouling resistant properties, as demonstrated by reduced bovine serum albumin (BSA) and fibrinogen (Fg) adhesion. © 2014 Elsevier B.V. All rights reserved.

1. Introduction One challenge in implanted devices is to prevent nonspecific adsorption of microorganism or biomolecules such as protein, which may lead to poor healing effect and device failure [1]. Even a small amount of non-specific protein adsorption on device surfaces can bring about unwanted bio-fouling. For example, for implanted devices in blood contacting environments, fibrinogen adsorption, even at low levels, is able to mediate platelet adhesion, potentially leading to thrombosis [2]. Other applications like biosensor or marine coating also require surface resistance to non-specific protein adsorption. The trend to address the issues of non-specific protein adsorption is to construct hydrophilic surfaces against fouling. PEG based polymers are suitable as nonfouling materials and most extensively studied [3,4]. Nevertheless, they are still far from ideal materials. They are susceptible to oxidative degradation. It is reported that the surfaces modified with or without PEG polymers exhibit same

∗ Corresponding author at: College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, PR China. Tel.: +86 0288 5466166; fax: +86 02885405402. E-mail address: [email protected] (X. Luo). http://dx.doi.org/10.1016/j.apsusc.2014.12.149 0169-4332/© 2014 Elsevier B.V. All rights reserved.

degree of fouling in vivo [5]. There have been significant efforts to search for alternatives of PEG [6,7,8]. Zwitterionic polymers, as one class of nonfouling materials newly developed, have attracted extensive attention in these years due to their biomimic nature and charge neutrality [9]. However, the difficulties in preparing monomers and achieving high molecular weight limit their commercial potentials. For these reasons, alternative nonfouling materials with higher stability and accessibility are developed. Poly(acrylamide) may be another good anti-fouling candidate, since it meets several principles for surfaces resisting protein adsorption – (i) hydrophilic, (ii) electrically neutral, (iii) “hydrogen bond forming” [3]. As a hydrophilic and electrically neutral material, poly(acrylamide) has the capability to form a tightly bonded water layer when grafted onto surface. Several studies have demonstrated the ultralow fouling properties of poly(acrylamide) against proteins [10,11] and microorganisms [12]. The poly(acrylamide)-grafted surfaces remained highly resistant to protein adsorptions, even in a wide range of tested pH values (5.2–8.4) and ionic strengths (10–150 mM) [13]. As a traditional material, poly(acrylamide) has been widely used in cosmetic formulations, subdermal fillers for aesthetic facial surgery, drug delivery carriers, and tissue implants [14,15]. Poly(acrylamide) is found to be atoxic and noncarcinogenic, as demonstrated in the acute and chronic toxicity studies in animals [19].

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By “graft-from” or “graft-to” method, the materials, which are generally hydrophobic such as PCL, are grafted using nonfouling materials to obtain surfaces resisting protein adsorption. PCL, a biodegradable semi-crystal polyester, has been suggested for a wide field of applications such as drug delivery systems [16,17], vascular tissue engineering [18,19], tissue-engineered skin (plain film), and scaffolds for supporting fibroblast and osteoblast growth [20,21]. To facilitate its application in vascular grafts, further modification to introduce fouling-resistant or bioadhesive motif is often required [22,23]. Successful attempts have been made in terms of hydrolysis [24] and aminolysis based grafting (electrostatic selfassembly [23], surface initiated polymerization [25] and chemical conjugation [26,27]). For example, alkyl bromide was covalently bond to surface NH2 introduced after aminolysis of PCL substrate and served as ATRP initiators, then were zwitterionic polymers brushes grown on surface [25]. However, aminolysis and hydrolysis on PCL surface, due to their destructive nature, will cause degradation of polyester backbone and removal of the top modified layer, exposing the underneath unmodified surface [28,29]. We here developed a non-destructive method to modify the PCL surface by tethering poly(acrylamide) based on terminal substitution of poly(caprolactone) [Scheme 1]. Before blending and casting, the hydroxyl groups of PCL with low molecular weights were converted into bromides and then into azido moieties, which introduced the clickable groups to finally obtained surface. Alkyne-poly(acrylamide) was used as the nonfouling material and was then “clicked” to surface through copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction. The surface composition and topography were determined by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Unlike surface initiated polymerization (SIP), atom transfer radical polymerization (ATRP) in solution allows for control over molecular weight and polydispersity of poly(acrylamide) [30]. We use water contact angle (WCA) to evaluate the hydrophilicity before and after modification. We here obtained alkyne ended poly(acrylamide) with different molecular weight and immobilized them onto PCL surface. With determined molecular weight of poly(acrylamide), we are able to investigate the protein resist ability in terms of chain length through its impact on grafting reaction and modified surface.

2.2. Preparation of PCL–N3 PCL with low molecular weights was synthesized through typical ring-opening polymerization of ␧-caprolactone in bulk. Briefly, ␧-CL, dodecanol (M:I = 20, mol/mol) was mixed with Sn(Oct)2 and degassed after purged with nitrogen three times. The mixture was maintained at 120 ◦ C for 24 h. The product was then dissolved in dichloromethane and precipitated in cold methanol twice for purification. The product (PCL20 ) collected was dried under vacuum (yield = 94.7%). The obtained PCL (4.0 g, 1.62 mmol) was dissolved in 15 ml toluene containing 0.677 ml of triethylamine. After cooling to 0 ◦ C 2-bromoisobutyryl bromide (1.117 g, 4.86 mmol, 0.6 ml) in 5 ml toluene was added dropwise to the mixture in 30 min. The mixture was kept in ice–water bath for 1 h, and then stirred at room temperature for 24 h. The product was filtered to remove undissolved salt and then concentrated and precipitated into cold methanol. The product (PCL20 –Br) was then vacuumed overnight to constant weight (yield = 80.2%). PCL–N3 was obtained via an azide nucleophilic exchange reaction. PCL20 –Br (2.85 g, 1.06 mmol) and NaN3 (0.3 g, 4.62 mmol) were co-dissolved in 20 ml DMF. Then the reaction mixture was kept at 50 ◦ C and stirred for 24 h under darkness. The mixture was filtered to remove undissolved salts and then precipitated into large excess of ethanol. The product was then dried under vacuum (yield = 68.4%). 2.3. PCL–N3 /PCL blended film PCL–N3 /PCL blended film was prepared by dissolving 1.1 g of PCL pellet and 0.5 g of PCL–N3 in 8.1 ml distilled 1,4-dioxane. The mixture was then spread onto a 10 cm × 10 cm glass plate. The film deposition was achieved by solvent evaporation under room temperature for 24 h and further melting at 70 ◦ C for 12 h. After cooling to room temperature, a half-translucent PCL film with smooth surface was obtained. The film was then cut into pieces of 5 mm × 5 mm and washed by ethanol/H2 O (1/1, v/v) mixture for 2–3 h and dried under vacuum. 2.4. Fluorescence labeling of surface azide groups

2. Experimental 2.1. Materials ␧-Caprolactone (␧-CL) (Sigma–Aldrich, New Jersey, USA) was pretreated with calcium hydride and distilled under reduced pressure. Cuprous chloride (Chengdu Kelong Chemicals Ltd., China) was treated by hydrochloric acid and precipitated in water before use. Toluene and dioxane were purified by refluxing over sodium before use. Triethylamine (TEA), dichloromethane (CH2 Cl2 ) and N, N-dimethyl formamide (DMF) were purified by distillation under vacuum over CaH2 before utilization. Poly(caprolactone) (Mw ∼14,000, average Mn ∼10,000 by GPC) was purchased from Aldrich sigma. Stannous 2-ethyl hexanoate (Sn(Oct)2 , 95%), dodecanol, 2-bromoisobutyryl bromide (BIBB, 97%), fluorescein isothiocyanate (FITC), 3-butyn-1-ol and sodium azide (NaN3 ) were purchased from Sigma (USA). Sodium D-isoascorbate was provided by Aladdin. Acrylamide was provided by Biosharp-Sigma A-885e and used as received. Tris(2-aminoethyl) amine was purchased from Alfa Aesar (Tianjin, China) and 2-chloropropionyl chloride was provided by TCI (Shanghai, China). BCA Protein Assay Kit was purchased from Thermo Fisher Scientific Inc. Bovine serum albumin (BSA) and fibrinogen (Fg) were purchased from Biosharp Co. All other solvents and chemicals were purchased from Chengdu Kelong Chemicals Ltd.

In order to label azide groups, azide groups must be converted into amine groups. PCL–N3 /PCL films were immersed in 10 g/L PPh3 solution (EtOH 10 ml, H2 O 0.1 ml) for 12 h in order to reduce the azides into amines. Pristine PCL and reduced PCL–N3 /PCL films were transferred to a FITC (10 mg) solution of 10 ml ethanol in dark at room temperature for 24 h and then were washed with copious amount of ethanol and dried under vacuum. Fluorescence observation was carried out by an Olympus IX51 microscope. FITC-labeled films were visualized using the characteristic wavelength of fluorescein (ex = 496 nm; em = 518 nm), and the image was obtained at the fixed exposing time. 2.5. Preparation of alkyne-ended PAAm 2.5.1. Synthesis of Me6 TREN Me6 TREN was prepared as reported [31]. Briefly, to prepare the salt (ClNH3 CH2 CH2 )3 NHCl, 15 ml of 3.0 M HCl in methanol was added dropwise to 2 ml of tris(2-aminoethyl) amine in 25 ml of methanol. After stirring for 1 h, the precipitate was washed three times with methanol and dried under vacuum. Then (ClNH3 CH2 CH2 )3 NHCl, 5 ml H2 O, 25 ml formic acid and 23 ml formaldehyde were mixed and refluxed at 120 ◦ C for 6 h. The mixture was spin-dried to remove volatile component. To the solid residue was then added 50 ml of 10 wt.% NaOH aqueous solution. The resulting aqueous phase was extracted with 50 ml diethyl ether

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Scheme 1. Illustration of “Click” chemistry on PCL20 –N3 /PCL blended film for anti-fouling performance.

for four times. The organic layer were passed through anhydrous NaOH and concentrated by rotary evaporation. After vacuum distillation at 62 ◦ C, 0.8 g of the product was obtained as colorless oil at a yield of 40%. 1 H NMR (CDCl3 , 400 MHz) [Fig. S1 in supporting information]: ı 2.20 (s, 18H, CH3 ); ı 2.36 (m, 6H, CH2 ); ı 2.58 (m, 6H, CH2 ).

2.5.2. Synthesis of 3-butynyl 2-chloropropanoate 3-Butynyl 2-chloroisobutyrate was prepared by esterification of 3-butyn-1-ol with 2-chloropropionyl chloride. Briefly, 1 ml of 3-butyn-1-ol accompanied with 1.8 ml of triethylamine was dissolved in 10 ml dichloromethane. After the mixture was cooled to 0 ◦ C, 1.8 ml of 2-chloropropionyl chloride in 5 ml dichloromethane was added dropwise over 2 h. Then the mixture was stirred overnight at room temperature. The mixture was washed and extracted with H2 O for five times. The organic phase was collected and passed over silicon gel before concentrated by rotary evaporation. The obtained residue was distilled under reduced pressure, yielding colorless oil at a yield of 89%. 1 H NMR (CDCl3 , 400 MHz) [Fig. S2 in supporting information]: ı 1.70 (d, 3H, CH3 ); ı 2.02 (t, 1H, CH); ı 2.58 (m, 2H, CH2 ); ı 4.29 (t, 2H, CH2 ); ı 4.43 (m, 1H, CH).

2.5.3. Synthesis of alkyne-ended PAAm To synthesize alkyne-ended poly(acrylamide), acrylamide (1.2 g, 17.40 mmol) and Me6 TREN (96 ␮L,0.35 mmol) were dissolved in a mixture of deionized water (9.2 ml) and ethanol (3.5 ml) and the solution was degassed by nitrogen bubbling for 30 min. The initiator (52 ␮L, 0.35 mmol) in 0.5 ml of degassed ethanol was then added via syringe 1 min after CuCl (35 mg, 0.35 mmol) addition. The mixture was allowed to stir at room temperature for 1 h. The reaction was terminated by exposing to air and finally precipitating into acetone (10 folds). The acetone solution containing precipitate was centrifuged and the concentrated solid was redissolved in water before 3 days of dialysis. The product was obtained by freezedrying and yielded as a white solid. 1 H NMR (D2 O, 400 MHz) [Fig. 1]: ı 2.45–2.00 (d, 1H, CO CH ), ı 1.95–1.35 (d, 2H, CH2 ).

2.6. Grafting of the alkynyl-PAAm onto PCL–N3 /PCL films via click reaction For a typical click reaction of alkynyl-PAAm onto PCL–N3 /PCL film surface, PCL–N3 /PCL films were immersed in a mixture of 4 ml DMSO, 4 ml H2 O and 0.5 ml Ethanol, then CuSO4 ·5H2 O (1 mg, 6 ␮mol) and alkynyl-PAAm (40 mg) were added. After bubbling the solution with N2 for 30 min, sodium d-isoascorbate (3 mg, 15 ␮mol) was added into the solution. The reactor was sealed and kept in a water bath at 37 ◦ C for 24 h. After the reaction, the obtained films were rinsed with large amount of ultrapure water and dried in a vacuum oven.

2.7. Characterization ATR-FTIR spectra of pristine PCL and PCL–N3 /PCL films and FTIR spectra of PCL/PCL–Br/PCL–N3 and alkynl-poly(acrylamide) were obtained from a Fourier transform infrared spectrometer (FTIR, Thermo Nicolet 6700) with a resolution of 4 cm−1 in absorbance mode. X-ray Photoelectron Spectroscopy (XPS) was performed in an AXIS ULTRA spectrometer (Shimadzu Co., Japan) equipped with a monochromated Al·K␣ X-ray source at a power of 144 W (12 mA, 12 kV). Survey spectra were acquired at a pass energy of 320 eV with a scan area of 300 ␮m × 700 ␮m. Sessile drop method was taken to evaluate the hydrophilicity of the surface. Drop image was analyzed by drop shape analysis (DSA) software and the contact angle was determined by tangent method. Surface topography and roughness were studied by Cypher S Atom Force Microscope (AFM) in tapping mode and scan area of all samples was set to be 50 ␮m × 50 ␮m. The data was processed by Nanoscope Analysis v 1.20. The chemical structure of the synthesized material was characterized by 400 MHz H NMR (AVANCETM III HD 400, Bruker). Molecular weight distribution of the poly(acrylamide) was obtained using Agilent LC1100 system equipped with PL AQUAGELOH MIXED-M (7.5 × 300 mm, 8 ␮m) column. Narrowly distributed PEG standards were used for calibration. 2.8. Protein adhesion study 2.8.1. Bicinchoninic acid (BCA) protein assay The PCL–N3 /PCL films and poly(acrylamide)-grafted PCL–N3 /PCL films were placed in individual wells of a 24 well tissue culture plate. After equilibration by PBS for 1 h, the films were exposed to single protein solutions of BSA and Fg (20 mg/ml in PBS) for 3 h in a shaker at 37 ◦ C, respectively. After rinsing the films with fresh PBS several times to remove detached protein, the films were transferred to new wells and washed with an aqueous solution of 0.4 ml 0.1 wt% SDS at 37 ◦ C for 30 min to extract adsorbed proteins from surfaces. Based on the principle of the bicinchoninic acid (BCA) protein assay kit method, the protein concentration in the SDS solution was determined by a Microplate Reader, and the optical absorbance was recorded at a wavelength of 578 nm. The reported data were calculated from values of four parallel samples for each film. 2.8.2. FITC labeled protein adsorption To label BSA and Fg with FITC, each protein was dissolved in Na2 CO3 buffer solution (pH = 9.0). FITC (10 folds) in DMSO was then added dropwise into protein solution. The mixture was then kept in 4 ◦ C for 24 h and then dialysis against Na2 CO3 buffer/Ethanol (2:1, v/v). The protein solution was lyophilized after absorbance at 495 nm for dialysate was undetectable. The obtained protein was then dissolved in PBS buffer (pH = 7.2, 20 mg/ml for BSA and 10 mg/ml for Fg). For protein adsorption, films were exposed to single protein solutions of BSA and Fg for 3 h at 37 ◦ C respectively

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Fig. 1. Spectroscopy characterization of PCL20 , PCL20 –Br and PCL–N3 by (a) 1 H NMR (400 MHz, CDCl3 ) and (b) FTIR.

following equilibration by PBS for 1 h. Then the films were rinsed with copious PBS and ethanol to remove unattached protein and unlinked FITC. Fluorescence observation was taken on an Olympus IX51 microscope, and the image was obtained at the fixed exposing time by excitation of the fluorophore with a 496 nm laser. The fluorescence intensity was acquired using image-pro plus. 3. Results and discussion To develop a non-destructive method to modify PCL surface, PCL–N3 with low molecular weights was firstly mixed with standard commercial PCL to obtain films with a “clickable” surface. A smooth surface with constant root mean square roughness (rms) values of 20.8 nm and no evident pore could be obtained, which has almost no difference with that of generally obtained PCL casting film [25,32]. Alkyne-poly(acrylamide) was then “clicked” to surface through copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. To accomplish this, PCL–N3 with low molecular weight and alkyne-ended poly(acrylamide) with well-defined chain length and molecular weight distribution were synthesized.

of TEA, PCL–Br presented anchoring of the bromoisobutyryl moiety by the downfield shift of the hydroxymethylene of PCL–OH ( CH2 CH2 OH) from 3.62 ppm to 4.18 ppm ( CH2 OCOC(CH3 )2 Br) and by the presence of a sharp singlet ( C(CH3 )2 Br, peak g) at 1.92 ppm, corresponding to the two methyl groups adjacent to the bromine atom. In addition, the complete disappearance of the characteristic peaks at 3.62 ppm (peak e) and the appearance of the new peak at 4.18 ppm (peak e’) confirmed that PCL–OH has been quantitatively end-functionalized by the bromocompound. Finally, PCL–N3 was obtained via a replacing reaction of bromine in PCL–Br by NaN3 in DMF. The substitution of the bromide group into the azide functions was represented by the shift of terminal methyl signal from 1.93 ppm (peak f’) to 1.44 ppm (peak f”). In addition, appearance of characteristic peaks at 645 cm−1 (␯ C–Br) in FTIR spectra of PCL–Br also confirmed the success of the esterification reaction. Conversion of ended bromide into azide groups was also verified by characteristic azide peak at 2100 cm−1 (␯ N N N) in PCL–N3 spectrum in Fig. 1b.

3.2. Synthesis of alkyne-PAAm by ATRP 3.1. Synthesis of PCL20 –N3 The synthesis of PCL–N3 with low molecular weights was composed of three steps: (1) the preparation of PCL–OH by ringopening polymerization of ␧-caprolactone using dodecanol as an initiator; (2) the esterification reaction between PCL–OH and 2bromoisobutyryl bromide to prepare PCL–Br; (3) the substitution reaction with NaN3 . Fig. 1a demonstrated the 1 H NMR spectra of the synthesized polymers. The structure of PCL–OH displayed the characteristic peaks at 1.38 ppm ( OCOCH2 CH2 CH2 , peak c), 1.65 ppm ( OCOCH2 CH2 CH2 CH2 , peaks b and d) and 2.31 ppm ( OCOCH2 , peak a). The peak at 1.25 ppm was attributed to the residual of initiator dodecanol, CH3 (CH2 )11 . The average degree of polymerization (DP) of PCL was determined by comparing the integrated area of peak a ( OCOCH2 , methylene protons) to peak e ( CH2 CH2 OH, terminal methylene protons). The DP was 20, which corresponded to the theoretical value. After esterification reaction between PCL–OH and 2-bromoisobutyryl bromide in the presence

It is well known that atom transfer radical polymerization (ATRP) technique can be easily adopted to prepare polymers with precisely controlled chain length and molecular weight distribution. A large number of monomers with only a few exceptions have been successfully polymerized by this process. Acrylamides and methacrylamides are the notable monomers not satisfactorily amenable to ATRP. However, when more active catalyst ligand Me6 TREN than ligands commonly used in ATRP such as bipyridine was used, Teodorescu and Matyjaszewski [33] obtained PAAm with linear increase of molecular weights along with conversion. In our work, we synthesized Me6 TREN used as a catalyst ligand [Fig. S1 in supporting information], which was synthesized by methylation of tris(2-aminoethyl) amine. In order to synthesize alkyne-PAAm by ATRP, an initiator,3butynyl 2-chloroisobutyrate, was prepared by esterification of 3-butyn-1-ol with 2-chloropropionyl chloride. The structure of the initiator was verified by 1 H NMR [Fig. S2 in supporting information].

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Table 1 Polymerization data of poly(acrylamide) synthesized by ATRP. Sample

M/I

DPa

Mwb

PAAm-58 PAAm-168 PAAm-329

50 250 500

58 168 329

3499 8110 12,858

a b

Mnb 2305 5864 10,165

PDIb 1.52 1.38 1.26

Obtained from 1 H NMR. Obtained from GPC.

ATRP synthesis of alkyne-PAAm was carried out by using 3-butynyl 2-chloroisobutyrate as an initiator, acrylamide as monomers and CuCl/Me6 TREN as catalyst system in a mixture of deionized water and ethanol. The structure of alkyne-PAAm was confirmed by 1 H NMR shown in Fig. 2. The split peaks e at 1.4 ∼ 1.9 ppm and f at 2.1 ∼ 2.5 ppm were assigned to methlyene and methane protons of poly(acrylamide) respectively, and the split of the peaks was caused by tacticity [34]. Peak a, b, c, d and e were assigned to the residual of the initiator, among which the methane and alkyne signals largely overlap with signals of poly(acrylamide) protons. Besides, the signal of alkyne could be mitigated by the substitution nature of active alkyne proton in deuterated solvent. With above considerations, Peaks b and e were used for DP determination by their integration ratio for the obtained polymers. In our study, poly(acrylamide)s with different [M0 ]/[I0 ] were synthesized. The calculated DP of each sample was listed in Table 1.

Fig. 2. Characterization of alkyne-poly(acrylamide) by 1 H NMR (400 MHz, D2 O).

The GPC data were obtained using PEG as calibration standards. However, since the hydrodynamic volume of PEG might differ from that of PAAm, the molecular weights determined by GPC were not accurate, only PDI data was used [35]. The GPC traces of synthesized

Fig. 3. (a) ATR-FTIR spectra of pristine PCL and PCL–N3 /PCL and (b) fluorescent image of FITC labeled PCL–N3 /PCL and pristine PCL.

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Table 2 Element content and static water contact angle of all samples. Sample

Atomic content (%)

PCL–N3 /PCL PCL–PAAm58 PCL–PAAm168 PCL–PAAm329

[N]/[C]

C

O

N

Exp.

theory

73.89 73.94 71.66 72.09

25.65 22.33 22.39 21.32

0.46 3.73 5.95 6.60

5.4 × 10−3 0.050 0.083 0.092

6.7 × 10−3 – – –



*

Conformation

WCA

– 0.28 0.18 0.10

– 0.36 0.10 0.04

– Mushroom Brush Brush

90.1 38.3 34.1 20.2

± ± ± ±

1.3 4.6 1.6 3.7

poly(acrylamide)s were all single sharp peaks (not shown here), indicating controlled chain length and narrow molecular weight distribution of the poly(acrylamide)s. Table 1 shows that PDI of the synthesized alkyne-PAAm decreased with the ratio increase of [M0 ]/[I0 ]. PDI was up to 1.52 when [M0 ]/[I0 ] was 50, while PDI was 1.38 and 1.26 when [M0 ]/[I0 ] was 200 and 500. Meanwhile, DP was also relative to the ratio of [M0 ]/[I0 ]. In the first case, the amount of the initiator was large so that not all initiator molecules and initiated chains could prolongate, leading to DP be higher than theoretic value and to PDI be wider comparing with the another two [M0 ]/[I0 ] ratio. Nevertheless, reasonably controlled ATRP of acrylamide with end group alkyne was achieved in a mixed solvent of deionized water and ethanol.

3.3. PCL–N3 /PCL blended film Because of the compatibility of PCL–N3 with PCL, the blended film of PCL–N3 /PCL with a smooth surface and without evident pore could be obtained. The pristine PCL film was prepared in the same condition as a control. The chemical compositions of pristine PCL and PCL–N3 /PCL were characterized by ATR-FTIR. The spectrum of pristine PCL showed characteristic peaks of carbonyl and aliphatic groups, respectively, at 1725 cm−1 (␯ CO) and 2944, 2865 cm−1 (␯s , ␯as C H), whereas spectra of PCL–N3 /PCL surfaces gives an extra peak at 2100 cm−1 which is ascribed to azide stretching vibration [36,37], as shown in Fig. 3a. In order to further verify the presence of the surface azide groups on PCL–N3 /PCL, azido moieties were reduced by PPh3 into amines, which could be visualized after FITC labeling. As seen in Fig. 3b, PCL20 –N3 /PCL film exhibited significant fluorescence, while fluorescence on pristine PCL was almost invisible, indicating absence of amines or other groups to be labeled. Signals of C, O, and N were appeared on the XPS spectra of PCL20 –N3 /PCL in Fig. 4. In the spectrum, the absence of sodium signal proved that N 1s peaks came from PCL20 –N3 instead of adsorption of unreacted NaN3 . The absence of bromide signal at 69.0 eV was seen as another indication of complete transformation of bromide during final azido functionalization reaction. The N 1s spectrum Fig. 4b revealed two distinct peaks centered at 399.8 eV and 404.2 eV with roughly area ratio of 2:1. The higher energy peak was assigned to the central, electron-deficient nitrogen of the azide group [38]. Table 2 presented the surface chemical composition of PCL20 –N3 /PCL membranes. Nitrogen element was only from azide groups in PCL20 –N3 /PCL, and N/C atomic ratio was stable about 0.47% ± 0.02% in different scan areas for all determined samples, which indicates the azido moieties were distributed equally on surface. Therefore, the surface azide density might be calculated from N/C atomic ratio by the following equation (take 7.5 nm as the X-ray penetration depth): Aazido =

 V ∗  PCL 114 g/mol

∗6∗

N 1 ∗ C 3

(1)

Fig. 4. XPS spectra of PCL–N3 /PCL (a) wide scan (b) N 1s detail scan.

where Aazido represented the surface area density of azido moieties on surface, V was defined as an volume of 7.5 nm*1 cm2 , PCL represented the bulk density of PCL (1.145 g/cm3 ), N/C represented the N/C ratio in Table 2 (determined from peak area of N 1s and C 1s core-level scan corrected by sensitivity factor of corresponding elements). The surface area density of azide groups from calculation was 0.5/nm2 . Despite this, the azido moieties were distributed among the whole bulk, not just the surface. So the bulk density of azido moieties should be calculated according to the ratio of PCL20 –N3 in the blended film. The bulk density of azido moieties was therefore given by the following equation:  azido =

mb ∗ r 2662 g/mol ∗ Vb

(2)

where ’azido represented the bulk density of azido moieties, mb and Vb the mass and volume of each film. The ratio of mb and Vb equaled the density of PCL, namely PCL . r represented mass percentage of PCL20 –N3 , and 2662 g/mol was the molar mass of PCL20 –N3 . The

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229

Fig. 5. XPS wide scan of (a) PCL–PAAm58 (b) PCL–PAAm168 (c) PCL–PAAm329 and C 1s core-level scan of (a’) PCL–PAAm58 (b’) PCL–PAAm168 (c’) PCL–PAAm329.

obtained ’azido was 0.084/nm2 , compared with bulk density of azido moieties (azido ) estimated on the surface from the following equation: azido ∗ 7.5 nm = Aazido

(3) 0.068/nm3 ,

which is close The calculated azido was found to be to ’azido . Therefore the density of azido moieties, whether in bulk or near surface, has a relative constant value. Neither surface aggregation [39] nor spatial unevenness was observed in this case. Unlike the surface aminolysis process [27], functional groups for coupling could be introduced uniformly with the depth by a non-destructive method.

To obtain grafting density of poly(acrylamide), we have attempted to measure the weight difference before and after PAAm modification. However, no readable mass change could be detected. This might be due to steric hindrance of grafted coils that results in a low grafting density of PAAm [41]. Nevertheless, the grafting density of polyacrylamide could be estimated by XPS data. Here, we suppose the grafted layer is thin enough to not affect X-ray penetration, calculating the grafting density of poly(acrylamide) was therefore possible by the following equation: =

 V ∗  PCL 114 g/mol

∗R

(4)

3.4. Click reaction of alkyne-PAAm onto PCL surface The click reaction of alkynyl-PAAm onto PCL–N3 /PCL film surface carried out in a mixture of DMSO/H2 O/Ethanol. Use of DMSO in the mixed solvent was to decrease the viscosity of aqueous PAAm solution, while adding ethanol was to achieve better approaching of reacting molecules to PCL films. The success of immobilization of poly(acrylamide) on PCL–N3 /PCL film surface was confirmed by XPS. In Fig. 5, N signals of all of three samples in wide scan appeared and were stronger than that of PCL–N3 /PCL. The C 1s core-level scan can be curve-fitted into four peak components with binding energies (B.E.) at about 284.6, 285.9, 287.9 and 288.6 eV, attributed to the C H, C O, O C N and O C O species. The O C N species were associated with amide groups in grafted poly(acrylamide). The single peak at 399.4 eV in N 1s detail scan [Fig. S3 in supporting information] was attributed to the NHx structure of the grafted PAAm [40]. The amount of nitrogen increased with increasing PAAm DP in Table 1, indicating the increase of the grafted acrylamide unit numbers.

Fig. 6. Static water contact angle of pristine PCL, PCL–N3 /PCL, PCL–PAAm58, PCL–PAAm168 and PCL–PAAm329.

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Fig. 7. Surface topography of (a) PCL–N3 /PCL, (b) PCL–PAAm58, (c) PCL–PAAm168 and (d) PCL–PAAm329 on a 50 ␮m * 50 ␮m scan area.

where  represented the surface density of poly(acrylamide), R represented the surface density ratio between poly(acrylamide) and poly(caprolactone), determined from N/C ratio and unit number of poly(acrylamide). The results were listed in Table 2. To determine the critical density (*), which appears at the limit between the

mushroom and brush regime, we used one recognized method for grafted PAAm chains [12]:

 ∗ ≡ 46.43 ∗ N −6/5

(5)

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where N is the unit number of grafted chains, obtained from 1 H NMR data. The calculated * were listed in Table 2. A grafting layer with  above the threshold * has a brush chain package, hence, the grafted polyacrylamide presents brush conformation on PCL–PAAm168, PCL–PAAm329 and mushroom conformation on PCL–PAAm58. The hydrophilicity before and after the click reaction of alkynylPAAm onto PCL–N3 /PCL film surface was determined by water contact angle measurement (Fig. 6). The surface of PCL20 –N3 /PCL was more hydrophobic (90.1◦ ) than that of pristine PCL (70.2◦ ), which was possibly due to the hydrophobicity of surface azide. After the click reaction, the contact angle dramatically decreased to be lower than 50◦ , indicating surface covered with poly(acrylamide). The difference of contact angle values of three PAAm grafted PCL films might be caused by different acrylamide amounts. The surface topography before and after the click reaction was characterized by AFM. As shown in Fig. 7, change was very slight after modification by PAAm58 and PAAm168 in comparison with one before the “click” reaction, while a significant morphology change is usually inevitable in related studies [32,42]. The rms increased mildly from 20.8 nm for unmodified surface to 25.8 nm and to 29.7 nm for PCL–PAAm58, and PCL–PAAm168. However, the immobilization of PAAm329 (Fig. 7d) causes significant change of the surface topography and rms (42.8 nm for PCL–PAAm329), leaving large amount of scattered aggregates as high as 150∼400 nm. We speculated that PAAm with long chain may self aggregate on the surface, since that long chain PAAm may facilitate interchain/intrachain hydrogen bond formation.

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Fig. 8. Protein adsorption of BSA and Fg on PCL–N3 /PCL, PCL–PAAm58, PCL–PAAm168 and PCL–PAAm329 by BCA assay. Error bars represent means ± standard deviation (n = 3) (*p < 0.05, compared with PCL–N3 /PCL).

3.5. Protein adhesion The fouling-resistant property after click reaction was verified through the amounts of adsorbed proteins on films. Bovine serum albumin (BSA) and fibrinogen (Fg) solution were used

Fig. 9. Fluorescent image of adsorbed FITC-BSA on (a) PCL–N3 /PCL, (b) PCL–PAAm58 and FITC-Fg on (c) PCL–N3 /PCL, (d) PCL–PAAm58.

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separately in the test. We first evaluated the amounts of absorbed proteins on films through typical BCA method. The results in Fig. 8 indicated that lower absorbed amounts for both proteins were achieved by PAAm grafting on PCL–N3 /PCL film surfaces. One interesting phenomenon we observed is that in our FITC-protein adsorption test, we used ethanol to remove free FITC. As we see in Fig. 9a and c, ethanol is erosive to PCL surface and caused many cracks, while in poly(acrylamide) grafted surface Fig. 9b and d no crack was found. Here the grafted layer may also serve as a protective layer against erosion to PCL substrate. Unlike antifouling surface prepared by surface initiated polymerization, we did not obtain an almost non-fouling surface as Liu did on gold [43]. There are not too many works for polymer surface modification by poly(acrylamide). Fujimoto [10] used plasma induced graft polymerization to modify polyurethane surface. A minimum adsorption of bovine serum albumin (∼200 ng/cm2 ) on grafted polyurethane surface was obtained, and in our study we obtained a similar value for PCL–PAAm58 through BCA assay (Fig. 8). However, the ability to resist protein adsorption did not increase as longer molecular chains of poly(acrylamide) were grafted. Larger amount of both BSA and Fg proteins were adsorbed to PCL–PAAm168 and PCL–PAAm329 surface. That trend was also observed in FITC labeled protein adsorption [Fig. S4 in supporting information]. Therefore, for all samples, PCL–PAAm58 immobilized by short chain poly(acrylamide) exhibits the lowest protein foulings. The merits of short chain grafting to resist fouling have been proved by other studies [44,45]. It has been reported that low molecular weight PEG (∼2000 D) grafted on PU substrate reached the low limit of protein adsorption, further increase of chain length did not bring about greater reduction of adsorbed proteins. For weaker protein repelling performance of longer chain polyacrlamide, we provided several possible explanations as the reason: (1) hydration layer might not well cover the surface in this case. One study pointed out that the interchain hydrogen bond formation might be favored for grafted long chains, which would weaken polymer interactions with water and result in a less effective hydration layer for nonfouling properties [46]. Unfortunately, it did not provide any further evidence to verify the weaker interaction with water, whereas our water contact angle results indicates stronger polymer–water interaction when long chains were grafted, which means that hydrogen bond formation with water was not affected too much by interchain interactions. The weaker protein repellence could be caused by incomplete surface coverage of hydration layer with the influence of interchain interactions. The lower grafting density of longer poly(acrylamide) on PCL (Table 2) also suggests this possibility. (2) The immobilized long chain poly(acrylamide) increased the surface roughness, which may allow protein entrapment in its groove regions. In this study, undesirable nonspecific-protein adsorption on PCL substrate was significantly reduced by immobilization of short chain poly(acrylamide) without affecting surface morphology. Such surface could be employed as a precursor for many biomedical applications. To construct a hemocompatible surface, rapid endothelialisation process is often required. Our poly(acrylamide) immobilized surface contains a lot of end-chloride groups, and they could easily be further substituted by azido moieties and serve as active “clickable” sites to conjugate bio-functional molecules to induce rapid endothelialisation or for other applications.

4. Conclusions In conclusion, a method for preparing poly(caprolactone) with “clickable” surface was created. It is applicable for coupling various materials to construct functional surfaces. Poly(acrylamide) of different chain lengths were synthesized, and were then immobilized

onto such surface, as verified by XPS and WCA results. The PAAm coupled surface showed protein resistant properties when using BSA and Fg as model proteins. The density of adsorbed protein was found to be correlated with chain length of anchored PAAm and was lowest when grafting short chain PAAm. This research provides a novel insight into anti-nonspecific protein adsorption features of a traditional hydrophilic polymer, and may direct a new way for surface modification of PCL substrate. Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 51473099 and 51273125). We would also like to appreciate our laboratory members for the generous help, and gratefully acknowledge the Analytical and Testing Center at Sichuan University for test. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsusc. 2014.12.149. References [1] S. Jiang, Z. Cao, Ultralow-fouling, functionalizable, and hydrolyzable zwitterionic materials and their derivatives for biological applications, Adv. Mater. 22 (2010) 920–932. [2] W.B. Tsai, J.M. Grunkemeier, T.A. Horbett, Human plasma fibrinogen adsorption and platelet adhesion to polystyrene, J. Biomed. Mater. Res. 44 (1999) 130–139. [3] S. Chen, L. Li, C. Zhao, J. Zheng, Surface hydration: principles and applications toward low-fouling/nonfouling biomaterials, Polymer 51 (2010) 5283–5293. [4] Q. Yu, Y. Zhang, H. Wang, J. Brash, H. Chen, Anti-fouling bioactive surfaces, Acta Biomater. 7 (2011) 1550–1557. [5] M. Shen, L. Martinson, M.S. Wagner, D.G. Castner, B.D. Ratner, T.A. Horbett, PEO-like plasma polymerized tetraglyme surface interactions with leukocytes and proteins: in vitro and in vivo studies, J. Biomater. Sci. Polym. Ed. 13 (2002) 367–390. [6] S. Martwiset, A.E. Koh, W. Chen, Nonfouling characteristics of dextrancontaining surfaces, Langmuir 22 (2006) 8192–8196. [7] S.L. McArthur, K.M. McLean, P. Kingshott, H.A.W. St John, R.C. Chatelier, H.J. Griesser, Effect of polysaccharide structure on protein adsorption, Colloids Surf. B: Biointerfaces 17 (2000) 37–48. [8] B. Mrabet, M.N. Nguyen, A. Majbri, S. Mahouche, M. Turmine, A. Bakhrouf, M.M. Chehimi, Anti-fouling poly(2-hydoxyethyl methacrylate) surface coatings with specific bacteria recognition capabilities, Surf. Sci. 603 (2009) 2422–2429. [9] L. Mi, M.T. Bernards, G. Cheng, Q. Yu, S. Jiang, pH responsive properties of non-fouling mixed-charge polymer brushes based on quaternary amine and carboxylic acid monomers, Biomaterials 31 (2010) 2919–2925. [10] K. Fujimoto, H. Tadokoro, Y. Ueda, Y. Ikada, Polyurethane surface modification by graft polymerization of acrylamide for reduced protein adsorption and platelet adhesion, Biomaterials 14 (1993) 442–448. [11] K.M. DeFife, M.S. Shive, K.M. Hagen, D.L. Clapper, J.M. Anderson, Effects of photochemically immobilized polymer coatings on protein adsorption, cell adhesion, and the foreign body reaction to silicone rubber, J. Biomed. Mater. Res. 44 (1999) 298–307. [12] I. Cringus-Fundeanu, J. Luijten, H.C. van der Mei, H.J. Busscher, A.J. Schouten, Synthesis and characterization of surface-grafted poly(acrylamide) brushes and their inhibition of microbial adhesion, Langmuir 23 (2007) 5120–5126. [13] L. Lingyun, L. Qingsheng, S. Anuradha, Poly(acrylamide): evaluation of ultralow fouling properties of a traditional material, in: Proteins at Interfaces III State of the Art, American Chemical Society, 2012, pp. 661–676. [14] W.F. Bergfeld, D.V. Belsito, J.G. Marks Jr., F.A. Andersen, Safety of ingredients used in cosmetics, J. Am. Acad. Dermatol. 52 (2005) 125–132. [15] T.-H. Yang, Recent applications of poly(acrylamide) as biomaterials, Recent Pat. Mater. Sci. 1 (2008) 29–40. [16] C. Eldsäter, B. Erlandsson, R. Renstad, A.C. Albertsson, S. Karlsson, The biodegradation of amorphous and crystalline regions in film-blown poly (∈caprolactone), Polymer 41 (2000) 1297–1304. [17] E.-J. Choi, C.-H. Kim, J.-K. Park, Synthesis and characterization of starch-gpoly(caprolactone) copolymer, Macromolecules 32 (1999) 7402–7408. [18] S. Sarkar, G.Y. Lee, J.Y. Wong, T.A. Desai, Development and characterization of a porous micro-patterned scaffold for vascular tissue engineering applications, Biomaterials 27 (2006) 4775–4782. [19] M.C. Serrano, M.T. Portolés, M. Vallet-Regí, I. Izquierdo, L. Galletti, J.V. Comas, R. Pagani, Vascular endothelial and smooth muscle cell culture on NaOH-treated poly(␧-caprolactone) films: a preliminary study for vascular graft development, Macromol. Biosci. 5 (2005) 415–423.

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