Conjugation of Monocarboxybetaine Molecules on Amino-Poly‑p‑xylylene Films to Reduce Protein Adsorption and CellAdhesionHsiu-Wen Chien,† Ming-Chun Keng,† Meng-Jiy Wang,‡ Hsien-Yeh Chen,† Sheng-Tung Huang,*,§

and Wei-Bor Tsai*,†

†Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4,Roosevelt, Rd., Taipei 106, Taiwan‡Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan§Institute of Biotechnology, National Taipei University of Technology, Taipei 10617, Taiwan

*S Supporting Information

ABSTRACT: A surface that resists protein adsorption and cell adhesion ishighly desirable for many biomedical applications such as blood-contact devicesand biosensors. In this study, we fabricated a carboxybetaine-containing surfaceand evaluated its antifouling efficacy. First, an amine-containing substrate wascreated by chemical vapor deposition of 4-aminomethyl-p-xylylene-co-p-xylylene(Amino-PPX). Aldehyde-ended carboxybetaine molecules were synthesized andconjugated onto Amino-PPX. The carboxybetaine-PPX surface greatly reducedprotein adsorption and cell adhesion. The attachment of L929 cells on thecarboxybetaine-PPX surface was reduced by 87% compared to the cell adhesionon Amino-PPX. Furthermore, RGD peptides could be conjugated oncarboxybetaine-PPX to mediate specific cell adhesion. In conclusion, wedemonstrate that a surface decoration with monocarboxybetaine molecules isuseful for antifouling applications.

1. INTRODUCTION

Nonfouling biomaterials surfaces that resist protein adsorptionand cell adhesion are highly desirable for many biomedicalapplications such as blood-contacting devices and biosensors. Acommon feature for nonfouling materials is the ability to bindwater molecules tightly. Therefore, many hydrophilic materialssuch as poly(ethylene glycol) (PEG),1 polyHEMA,2 poly-acrylamide,3 dextran,4 and zwitterionic polymers5 have beenused for antifouling to prevent accumulation of proteins andcells. In comparison with PEG and dextran that achievehydration via hydrogen bonding, zwitterionic materials, such asphosphobetaine, sulfobetaine, and carboxybetaine, bind watermolecules more strongly via electrostatically induced hydra-tion.5 Decoration of surfaces with zwitterionic polymers greatlyreduces nonspecific protein adsorption.5,6 For example, apoly(sulfobetaine)-graft surface exhibited undetectable non-specific protein adsorption (

(20−100 nm) coatings on a wide variety of materials.20 PPXcoatings have been successfully applied to biomedical devicessuch as stents and cardiac pacemakers20 due to the advantagesof this techniques such as the solvent-free fabricationenvironment, the absence of initiators and plasticizers, andthe ability to render custom-tailored surface modifications. Aremarkable feature of PPX coatings is that versatile functionalPPX could be formed, which could anchor biomolecules forbiomedical applications.21−23

In this study, an amino-PPX coating was used for conjugationof monocarboxybetaine molecules. Surface amino groups couldbe used for the conjugation of molecules containing carboxylicacids, alkyl halides, or aldehydes by the formation of amide orimine bonds.24−27 We synthesized an aldehyde-ended carbox-ybetaine for immobilization on the amino-PPX surface. Thecarboxyl functionality of carboxybetaine could be used for laterconjugation of biomolecules via carbodiimide reaction in orderto mediate specific cell responses.28,29 The antifouling ability ofthe carboxybetaine-immobilized amino-PPX coating was firstevaluated. RGD peptides were later conjugated on thecarboxybetaine surfaces to demonstrate that biomoleculescould be conjugated on the substrate in order to specificallycontrol cell behavior.

2. MATERIALS AND METHODS2.1. Materials. Most of reagents were received from Sigma−

Aldrich (St. Louis, MO) unless specified otherwise. β-Propiolactonewas received from Alfa Chemistry (Stony Brook, NY). 4-Amino-methyl-[2.2]paracyclophane was synthesized from [2.2]-paracyclophane (Sigma-Aldrich) as described elsewhere.30 RGD-containing peptides (GYGRGDSP) were purchased from KelownaInc. (Taipei, Taiwan). L929 cells were received from Food IndustryResearch and Development Institute (Hsinchu, Taiwan).Phosphate-buffered saline (PBS) was prepared with 137 mM NaCl,

2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.4). 2-(N-morpholino)ethanesulfonic acid (MES) buffer contained 100 mMNaCl and 10 mM MES (pH 5.5). N-Cyclohexyl-2-aminoethanesul-fonic acid (CHES) buffer was composed of 10 mM NaCl and 50 mMCHES (pH 9.0).2.2. Synthesis of 3-((3-(4-Formylphenoxy)propyl)-

dimethylammonio)propanoate. 4-(3-(dimethylamino)propxy)-benzaldehyde (DMPBA) and 3-((3-(4-formylphenoxy)propyl)-dimethylammonio)propanoate (CBBA) were synthesized as describedin Scheme 1. In the synthesis of DMPBA, 4-hydroxybenzaldehyde(8.55 g, 70 mmol) and potassium carbonate (14.4 g, 101 mmol) were

dissolved in 20 mL of dimethylformamide at 120 °C for 30 min. 3-Dimethylamino-1-propyl chloride hydrochloride (5.53 g, 35 mmol)was then added in the solution for 5 h reaction. The mixture wasplaced in an ice bath to terminate the reaction. The mixture wasdiluted with 200 mL of deionized water, and DMPBA was thenextracted by ethyl acetate. The organic phase was collected, andresidual water was removed by solid MgSO4. The product was verifiedby 1H NMR (D-methanol 500 MHz), as shown in SupportingInformation Figure S1; δ (ppm): 9.81 (s, 1H, CH), 7.85 (d, 2H, CH),7.07 (d, 2H, CH), 4.11 (t, 2H, CH2), 2.51 (t, 2H, CH2), 2.27(s, 6H,CH3), 1.99 (p, 2H, CH2).

In the synthesis of CBBA, DMPBA (2.41 g, 1.16 mmol) and β-propiolactone (0.99 g, 1.37 mmol) were reacted in 15 mL ofanhydrous tetrahydrofuran at 0 °C in N2 atmosphere for 8 h. Theproduct was precipitated as white crystals and purified by filtration.The product was identified by 1H NMR (D-methanol, 500 MHz) andby its mass spectrum, as shown in Supporting Information Figures S1and S2; δ (ppm): 9.84 (s, 1H, CH), 7.88 (d, 2H, CH), 7.13 (d, 2H,CH), 4.21 (t, 2H, CH2), 3.63 (t, 2H, CH2), 3.52 (t, 2H, CH2), 3.19 (s,6H, CH3), 2.66 (t, 2H, CH2), 2.30 (p, 2H, CH2). MS-ESI (m/z) for[C15H21NO4]

+: 279.15 and 280.2.2.3. Fabrication of Carboxybetaine-Containing Surfaces.

The process of surface immobilization of carboxybetaine moleculesis illustrated in Figure 1. An Amino-PPX coating was prepared ontissue culture polystyrene (TCPS), glass, or gold using CVDpolymerization of 4-aminomethyl-[2,2]paracyclophane.27 Briefly, 4-aminomethyl-[2,2]paracyclophane was sublimed at 100−150 °C and areduced pressure of 0.5 mbar. The reactants were then transportedinto a pyrolysis chamber (720 °C) and then into a deposition chamber(15 °C) in which the substrates were placed. A thin 4-aminomethyl-p-xylylene-co-p-xylylene (Amino-PPX) layer was formed on thesubstrates.

CBBA was conjugated on Amino-PPX via imine formation betweenCBBA’s aldehyde and surface amines. CBBA was dissolved indeionized water in the presence of excess NaBH3CN. The CBBAsolution was added to Amino-PPX for 8 h. The substrates were thenrinsed with deionized water to remove unreacted CBAA. The CBAA-conjugated substrates from a CBAA solution with 10, 50, or 100 mg/mL were referred to as CBBA-10, CBBA-50, and CBBA-100,respectively.

2.4. Surface Characterization. Surface wettability was evaluatedby static water contact angle (WCA) measurement (FTA-125, FirstTen Angstroms, Portsmouth, VA) using deionized water (5 μL each).The samples for WCA measurement were prepared on glass. At leastsix locations were measured for each sample. The conjugation ofCBBA on the amino-PPX substrates was also analyzed with anattenuated total reflection-Fourier transform infrared spectroscopy(ATR-FTIR, Nicolet NEXUS 470, Thermo, Waltham, MA). Thesamples for ATR-FTIR measurement were prepared on gold.

The surface chemical composition of each sample was characterizedby electron spectroscopy for chemical analysis (ESCA). The samplesfor ESCA were prepared on tissue culture polystyrene plates. ESCAspectra were recorded on a VG ESCA Scientific Theta Probe (U.K.)with an Al Kα X-ray source radiation (1486 eV) at a takeoff angle of53°. The atomic compositions of the surfaces were calculated from thehigh-resolution spectrum of each element.

2.5. Adhesion of L929 Cells to PPX Surfaces. The substrates forcell culture were prepared in 96-well TCPS. The substrates weresterilized in 70% ethanol prior to cell experiments. L929 cells wereseeded on the samples at a density of 104 cells/cm2 for 6 h. L929 cellculture medium contained alpha minimum essential medium (αMEM;HyClone, Logan, UT) supplemented with 10% fetal bovine serum(Biological Industry, Israel), 2 mg/mL NaHCO3, 0.5% fungizone(GIBCO), 0.25% gentamycin (GIBCO), and 0.679% β-mercaptoe-thanol. The nuclei of the attached cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed under afluorescence microscope. Six images were taken from each sample(100× objective magnification). The density of adherent cells wasdetermined from the microscopic images by using ImageJ software(NIH).

Scheme 1. Synthesis Process of 3-((3-(4-Formylphenoxy)propyldimethylammonio)propanoate)(CBBA)

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2.6. Determination of Protein Adsorption Using QuartzCrystal Microbalance (QCM). QCM (ANTQ300, ANT Inc., Taipei,Taiwan) was used to determine protein adsorption. Amino-PPX wasdeposited on gold substrates of QCM quartz crystal chips (ca. 9 MHzresonance frequency), followed by the conjugation of CBBA. Proteinadsorption was recorded and calculated based on a total sensing areaof 0.1 cm2. PBS was first continuously delivered into the flow channelat a flow rate of 50 μL/min until the resonance frequency reached astate of equilibrium. Subsequently, the cell culture medium containing10% fetal bovine serum flew into the QCM chamber at a flow rate of50 μL/min and the time-dependent change of frequency resonancefrequency was real-time monitored. After the resonance frequencybecame stable, PBS was injected into the chamber at a flow rate of 50μL/min for rinsing the surface until the resonance frequency reachedequilibrium again. The adsorbed mass of serum proteins was calculatedfrom the frequency shift according to the Sauerbrey equation.31

2.7. Peptide Conjugation to CBBA Substrates. RGDconjugation to CBBA substrates was performed via carbodiimidereaction. The carboxyl groups of CBBA substrates were activated byincubation in 100 μL of MES buffer containing 50 mM NHS and 100mM EDC for 1 h at room temperature, and then incubated with 100μL of 1 mM RGD solution in CHES buffer for 6 h. The RGD-conjugated surfaces were sterilized with 70% ethanol for 30 min andrinsed with sterilized PBS prior to cell seeding.2.8. Statistical Analysis. The data was reported as means ±

standard deviation (SD). The statistical analyses between differentgroups were determined using Student’s t test. Probabilities of p ≤0.05 were considered a significant difference. All statistical analyses

were performed using GraphPad Instat 3.0 program (GraphPadSoftware, La Jolla, CA).

3. RESULTS AND DISCUSSION

3.1. Surface Characterization of CBBA ConjugatedAmino-PPX. We designed a route to synthesize an aldehyde-containing carboxybetaine molecule (Scheme 1) for immobi-lization on Amino-PPX. The NMR and mass spectra indicatethat CBBA was successfully synthesized (Supporting Informa-tion Figures S1 and S2).Conjugation of CBBA molecules on Amino-PPX was first

verified by surface wettability using static WCA measurement.The deposition of Amino-PPX to TCPS decreased the WCAfrom 68° to 54.4° (p < 0.01, Table 1). After the conjugation ofCBBA with 10, 50, and 100 mg/mL, the WCA was increasedfrom 53.0°, 62.4°, to 68°, respectively. The observation wasunexpected because carboxybetaine is a highly hydrophilicmolecule and thus surface conjugation of CBBA is expected toincrease surface hydrophilicity. We later realized that theincrease in surface hydrophobility after the conjugation of high-density CBBA might come from the aromatic moiety in CBBA.In the design of the reaction route to synthesize CBBA, weused 4-hydroxybenzaldehyde as a starting material due to itsavailability and reactivity. We suggest that the hydrophobicaromatic moiety counteracts the hydrophilicity of carboxybe-

Figure 1. Illustration of the conjugation of aldehyde-ended carboxybetaine to Amino-PPX via amine-aldehyde condensation. (a) Chemical vapordeposition (CVD) polymerization of 4-aminomethyl-[2.2]paracyclophane to form 4-aminomethyl-p-xylylene-co-p-xylylene (Amino-PPX). (b)Conjugation of 3-((3-(4-Formylphenoxy)propyl)dimethylammonio)propanoate (CBBA) to Amino-PPX via imine formation.

Table 1. Surface Characterization of CBBA-Modified Amino-PPX and the Amount of the Adsorption of Serum Protein on theSubstrates

atom %a

C O N WCA (deg)b ΔHz protein adsorption (ng/cm2)c

Amino-PPX 84.3 11.3 4.4 54.4 ± 3.4 −103.2 563.7CBBA-10 82.4 13.2 4.5 53.0 ± 3.4 −77.8 425.0CBBA-50 79.3 16.1 4.6 62.4 ± 4.9* −61.8 337.6CBBA-100 78.6 17.1 4.3 68.0 ± 2.3** −52.3 285.7

aThe atomic percentage was characterized by electron spectroscopy for chemical analysis (ESCA). bWater contact angles (WCA). n = 5, value =mean ± standard deviation. *p < 0.05. **p < 0.01 vs Amino-PPX. cProtein adsorption was determined from the frequency shift (ΔHz) of QCM.

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taine, so the surface hydrophilicity is decreased with increasingCBAA densities.Surface modification by conjugation of CBBA was further

analyzed by ATR-FTIR (Figure 2). The stretching of the N−H

bonds at 3400 cm−1 in the spectrum for Amino-PPX indicatesthe presence of amines on Amino-PPX. After CBBAconjugating, two peaks appeared. One is for CO stretchband at 1680 cm−1, and the other is for carboxylic acid O−Hstretch at 3350 cm−1, which are consistent with the character-istic absorption bands of CBBA. The intensities of these peakswere increased with increasing CBBA concentrations.ESCA analysis was used to analyze the elemental

composition of the substrates after Amino-PPX depositionand CBBA conjugation (Table 1). Oxygen was unexpectedfound on Amino-PPX. Since 4-aminomethyl-[2.2]-paracyclophane is only composed of carbon and nitrogen, theAmino-PPX film should not contain the oxygen element. Wesuspect that the oxygen comes from TCPS.32 On the otherhand, CBBA is composed of carbon, nitrogen, and oxygen, sothe conjugation of CBBA to Amino-PPX should increase thesurface oxygen content. After the conjugation of 10, 50, or 100mg/mL of CBBA, the oxygen content was increased to 13.2%,16.1%, or 17.1%, respectively. The increase in the oxygencontents confirmed that aldehyde-ended carboxybetaine isindeed conjugated on Amino-PPX, and the amount ofcarboxybetaine is increased with increasing CBAA concen-trations.3.2. Antifouling Ability of the CBBA-Modified PPX.

The resistance of the CBBA-modified PPX to cell adhesion andprotein adsorption was evaluated. Figure 3A shows that theattachment of L929 cells to Amino-PPX was apparentlydecreased after CBBA conjugation (Figure 3A). Thequantitative data indicate that the cell adhesion to Amino-PPX (1.19 × 104 cells/cm2) was significantly lower than the cellnumbers on TCPS (2.39 × 104 cells/cm2) (Figure 3B).Conjugation of CBBA at a concentration of 10 mg/mL did notsignificantly decrease cell adhesion on the PPX surface (1.03 ×104 cells/cm2). The cell resistance was increased when the

conjugation of CBBA was increased. The cell adhesion wassignificantly decreased to 69% and 87% on CBBA-50 andCBBA-100, respectively, compared with Amino-PPX (p <0.001, Figure 3B). The results demonstrate that conjugation ofCBBA molecules on Amino-PPX increases surface cell-resistance.Cell adhesion to an artificial surface is usually mediated by

surface-adsorbed cell-adhesion proteins from serum proteins, sowe determined the adsorption of serum proteins from cellculture medium to the CBBA-modified surfaces by QCMmeasurement. The frequency shifts were −103.2, −77.8, −61.8,and −52.3 Hz on Amino-PPX, CBBA-10, CBBA-50, andCBBA-100, respectively (Table 1). According to the Sauerbreyequation, the amounts of adsorbed proteins were 563.7, 425.0,337.6, and 285.7 ng/cm2 for Amino-PPX, CBBA-10, CBBA-50,and CBBA-100, respectively. The amount of the surface-boundserum proteins was decreased with increasing concentrations ofCBBA, consistent with the trend of cell adhesion.

3.3. Cell Adhesion on RGD-Functionalized Surfaces.An advantage of carboxybetaine surfaces is that the carboxylgroups can be used for conjugation of bioactive molecules. Inthis study, RGD peptides were conjugated to CBBA-100 toevaluate whether cell adhesion could be restored. RGD is themost commonly applied cell-binding peptide, which is found infibronectin and vitronectin, to mediate cell adhesion via bindingto cell membrane integrins.33 We found that after RGDconjugation on CBBA-100, cell adhesion was increased from0.073 × 104 to 1.10 × 104 cells/cm2 (Figure 4). On the otherhand, when CBBA-100 was incubated with RGD in the absenceof EDS/NHS, cell adhesion was not restored (data not shown).The carboxylates on CBBA could be functionalized viacarbodiimide reaction, and then mediate specific cell response.

Figure 2. FTIR spectra of Amino-PPX, CBBA-10, CBBA-50, andCBBA-100 substrates.

Figure 3. Adhesion, 6 h, of L929 cells on Amino-PPX that wasconjugated with 10 (CBBA-10), 50 (CBBA-50) or 100 (CBBA-100)mg/mL of CBBA. (A) Fluorescent images of DAPI-stained nuclei.Scale bar = 100 μm. (B) Cell numbers were determined by countingthe numbers of nuclei from the fluorescent images. n = 5, value = mean± standard deviation. ***p < 0.001 vs Amino-PPX.

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3.4. Amino-PPX Platform for Conjugation of Antifoul-ing Carboxybetaine. Functionalized PPX constitutes aversatile class of reactive polymers that can be used for thedecoration of bioactive molecules on biomaterials.21−23 Thistechnology could be applied to a wide variety of materials suchas polymers, metals, titanium alloy, silicon, glass, and ceramics.Several functionalities of PPX including amine, thiol, alkyene,and benzoyl have been successfully established, providing anexcellent platform for conjugation of different biomolecules.In this work, we demonstrate that a surface decoration with

monocarboxybetaine molecules is useful for antifoulingapplications. Aldehyde-ended carboxybetaine molecules werecoupled to Amino-PPX, and reduced cell adhesion and proteinadsorption. However, the antifouling ability of the CBBA-conjugated PPX is still inferior to the zwitterionic surfaces thatare created using surface-initiatd atom transfer radical polymer-ization18,34,35 or self-assembled monolayer.9,10 We conjecturethat the antifouling capability of our carboxybetaine-immobi-lized surface might be improved by increasing the density andhydrophilicity of the aldehyde-ended carbetatine molecules.The conjugation density of carboxybetaine molecules on

Amino-PPX might not be high enough to completely inhibitprotein adsorption and cell adhesion. The conjugation ofCBBA is limited by the amount of surface amines of Amino-PPX. We tried to determine the amount of amines on Amino-PPX, but failed to find a suitable method. In fact, we hardly finda literature providign accurate quantification of functionalgroups of PPX. Recently, Chang et al. determined the tatalweight of PPX coating by using QCM, and then estimated theamount of functional groups.36 The surface density offunctional groups was estimated as 5.30 ± 0.11 nmol/cm2.However, the actual density of functional groups on theoutmost layer of PPX films should be much lower since PPXcoating is not a monolayer. We may use other types of vapor-deposited functionalized coatings, for example, plasmapolymerization, to obtain a surface with high-density amines.For example, plasma polymerization of allylamine creates aminesubstrates.37 It is previously shown that conjugation of PEG to

poly(allylamine) inhibits protein adsorption and cell adhe-sion.38

Another strategy to enhance the antifouling ability of ourcarboxybetaine-conjugated surfaces is to incease hydrophilicityof the aldehyde-ended carbetatine molecules. As shownpreviously, the conjugation of CBBA increases the hydro-phobicity of Amino-PPX. CBBA contains a hydrophobicaromatic moiety, which may be responsible for the decreasein surface hydrophilicity. An aldehyde-ended carboxybetainemolecule without an aromatic ring might enhance thehydrophilicity of carboxybetaine-conjugated PPX. Furthermore,oligo-carboxybetaine molecules might be more resistant toprotein adsorption than monocarboxybetaine molecules. Rede-sign the aldehyde-ended carboxybetaine should enhance theantifouling abilit.

4. CONCLUSIONAldehyde-ended carboxybetaine molecules were synthesizedand conjugated onto an amine-containing substrate that werefabricated via CVD polymerization of 4-aminomethyl-[2.2]-paracyclophane. Cell adhesion and protein adsorption weregreatly reduced on the carboxybetaine-conjugated PPX. Wedemonstrate that surface decoration with monocarboxybetainemolecules is useful for antifouling applications.

■ ASSOCIATED CONTENT*S Supporting Information1H NMR spectra of DMPBA and CBBA; mass spectrum ofCBBA. This material is available free of charge via the Internetat http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*(W.-B.T.) Tel: +886-2-3366-3996. Fax: +886- 2-2362-3040.E-mail: weibortsai@ntu.edu.tw.*(S.-T.H.) Tel: +886-2-2771-2171-2525. Fax: +886- 2-2731-7117. E-mail: ws75624@ntut.edu.tw.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank the Ministry of Science and Technology,Taiwan for financial support (Grant Number: 100-2221-E-002-114-MY2).

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Figure 4. Amino-PPX was conjugated with 100 mg/mL CBBA, andthen RGD peptides were immobilized. (A) After 6 h of culture, themorphology of L929 cells on Amino-PPX, CBBA, or RGD-CBBA wasobserved under a phase-contrast microscope. Scale bar = 50 μm. (B)Cell numbers were counted from the microscopic images. ***p <0.001 vs Amino-PPX and RGD-CBBA substrate. n = 5, value = mean± standard deviation.

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dx.doi.org/10.1021/la502813n | Langmuir XXXX, XXX, XXX−XXXF

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