Surface chemical immobilization of parylene c with thermosensitive block copolymer brushes based on...

Surface chemical immobilization of parylene C with thermosensitiveblock copolymer brushes based on N-isopropylacrylamide and N-tert-butylacrylamide: Synthesis, characterization, and cell adhesion/detachment

Changhong Zhang,1,2 P. Thomas Vernier,3 Yu-Hsuan Wu,4 Wangrong Yang3

1Department of Chemistry, University of Southern California, Los Angeles, California 900892Kansas Polymer Research Center, Pittsburg State University, Pittsburg, Kansas 667623Department of Electrical Engineering, University of Southern California, Los Angeles, California 900894Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089

Received 9 January 2011; revised 8 July 2011; accepted 20 July 2011

Published online 9 November 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31941

Abstract: Poly(N-isopropylacrylamide) (pNIPAM), poly(N-tert-

butylacrylamide) (pNTBAM), and their copolymer brushes

were covalently immobilized onto parylene C (PC) surfaces

via surface initiated atom transfer radical polymerization

(ATRP). Contact angle measurement between 13 and 40�Cshowed that the hydrophobicity of the modified PC surfaces

was thermally sensitive. Among these samples, PC grafted

with pNIPAM (PC-NI), PC grafted with pNTBAM (PC-NT) and

PC grafted with copolymer brushes containing pNTBAM and

pNIPAM (PC-NT-NI) exhibited the lower critical solution tem-

perature (LCST) at 29, 22, and 24�C, respectively. Cytocom-

patibility study for the modified surfaces was performed by 5

days human skin fibroblast culture at 37�C. Data showed that

only a very small amount of cells adhered on the PC and PC-

NI surfaces, while a significantly higher amount of cell adhe-

sion and growth was observed on PC-NT and PC-NT-NI surfa-

ces. Furthermore, cell detachment at the temperatures of 24

and 6�C were studied after the substrates were cultured with

cells at 37�C for 24 h. The results showed that the cells on

PC-NI formed the aggregations and loosely attached on the

substrate after 30-min culture at 24�C, while no significant

cell detachment was observed for PC-NT and PC-NT-NI sam-

ples at this temperature. By continuing the cell culture for

additional 100 min at 6�C for PC-NT and PC-NT-NI, about 10

and 35% of the cells were found detached respectively, and

the unattached cells aggregated on the substrate. In compari-

son, cells cultured on the tissue culture petri dish (TCP)

exhibited no quantity and morphology changes at the culture

temperatures of 37, 24, and 6�C. This study showed that: (1)

immobilization of PC with nonthermal sensitive pNTBAM

could provide PC surface thermal sensitive hydrophilicity; (2)

the chlorines on the polymer brushes of PC-NT could be

used to further initiate the ATRP pNIPAM and form block co-

polymer brushes; (3) the incorporation of pNTBAM into pNI-

PAM on PC-NT-NI could change the surface thermal

hydrophilicity property, and be further applied to decrease

the LCST of the modified PC surface; (4) grafted pNIPAM

brushes on PC-NI by ATRP showed very low cell adhesion

and proliferation in 5 days fibroblast culture at 37�C, and cell

detached at 24�C; (5) the incorporation of pNTBAM into pNI-

PAM on PC-NT-NI decreased the thermal sensitivity of cell

adhesion/detachment, cell detached at 6�C, but the cell adhe-

sion and proliferation were significantly improved at a wide

temperature range. VC 2011 Wiley Periodicals, Inc. J Biomed Mater

Res Part B: Appl Biomater 100B: 217–229, 2012.

Key Words: cell adhesion, block copolymer, surface modifica-

tion, polymerization, cell–material interactions

How to cite this article: Zhang C, Thomas Vernier P., Wu Y-H, Yang W. 2012. Surface chemical immobilization of parylene C withthermosensitive block copolymer brushes based on N-isopropylacrylamide and N-tert-butylacrylamide. J Biomed Mater ResPart B 2012:100B:217–229.

INTRODUCTION

Implantable medical devices exhibit wide applications, suchas health monitoring sensors, wireless medical signal devi-ces, and electrode arrays for nerve function restoration.1,2

The materials for implantable medical devices must be bio-compatible, and survive long-term exposure to the compli-cated and harsh human body environment. Polymers, suchas polyurethane, parylene, polymethyacrylate, and poly(di-

methylsiloxane) (PDMS), have exhibited their advantages inbiocompatibility, chemical versatility, biological functionality,and mechanical strength.3,4 These polymers have been usedfor decades as the coating materials to make the implantedmedical devices suitable to human tissue environments.5–8

Among them, poly(2-chloro xylylene), also named as pary-lene-C (PC), has several outstanding properties such as bio-compatibility, biostability, low water permeability, chemical

Correspondence to: C. Zhang; e-mail: [email protected]

VC 2011 WILEY PERIODICALS, INC. 217

inertness, solvent resistance, good mechanical strength, andlow dielectric constant9; it has been approved by US Foodand Drug Administration (FDA) as class VI biocompatiblematerial and widely used as coating material for the medi-cal implants. PC can be conveniently coated onto the com-plex substrate by a nonsolvent involved chemical-vapor-dep-osition (CVD) technique, forming a thin, strong, and pinhole-free membrane layer at room temperature, making it a goodcandidate as a coating material for many long-term implant-able devices.10,11 However, the high hydrophobicity and lowpolarity of PC prevent it from adhering to the cells and tis-sues, which makes it an ideal packaging material, but limitits use on the implanted medical devices that need to beanchored to tissue.12 Very limited methods, such as surfacesculpturing, plasma treatment, activated water vapor treat-ment, photo-oxidation and surface chemical modification,have been employed to overcome this shortfall.13–17 How-ever, most of them were focused on the generation of ionicgroups on PC, not on forming a molecular or polymeric ad-hesive coating that is important for some medical implants,such as electrodes of the biosensor in nerve system, to formlong-term adhesion to the cells and surrounding tissues.

Bulk pNIPAM and substrates coated with pNIPAM thinfilms have been intensively studied in a range of biomedicalapplications, due to its beneficial temperature-dependentadhesive properties.18–21 Below the lower critical solutiontemperature (LCST) of 32�C, pNIPAM exhibits extended con-formation and high solubility in water; above 32�C, pNIPAMbecomes aggregated and hydrophobic.22 Homo pNIPAM hasbeen produced into different bulk copolymers for specificbiomedical applications, and these copolymers also exhib-ited different LCSTs than that of homo pNIPAM. For exam-ple, homopolymer of pNIPAM copolymerized with hydropho-bic poly(N-tert-butylacrylamide) (pNTBAM) exhibitedimproved cell attachment and proliferation with LCST below32�C23; pNIPAM copolymerized with hydrophilic polyethyl-ene glycol (PEG) showed more rapid cell detachment upontemperature decrease than pNIPAM, and the LCST is below32�C.24,25 The methods to covalently anchor the homo pNI-PAM on the substrates have also been developed recently tofunctionalize the surface with thermal sensitive properties,these methods included electron beam initiation, plasma-deposition, UV irradiation and surface atom transfer radicalpolymerization (ATRP). These modification methods havebeen suggested to have their roles in medical applications,such as anti-biofouling, temperature responsive biosensors,controlled drug release, thermal responsive chromatography,and reversible cell adhesion/detachment.26–35 Among thisapplications, homo pNIPAM grafted substrates has beenintensely applied for mammalian cell adhesion/detachmentstudy.34,36,37 Below LCST, it has been found that the cells donot adhere to the pNIPAM-coated substrate. However, thereports for the cell adhesion above LCST were contradic-tory: some papers reported that large amount cells adheredand proliferated on the substrates, some papers reportedthat cell adhesion was low; and some researchers foundthat cell coverage was high on the substrate with sparsepNIPAM amount (7.9 lg cm�2 or less), while cell adhesion

was significantly reduced with higher pNIPAM amount onsome substrates, such as polystyrene and silicon wafer.30,34

Recently, copolymer brushes containing pNIPAM weregrafted on the substrates to approach the rapid cell detach-ment, including poly(N-isopropylacrylamide-co-ethylene gly-col) on silicon wafer, and poly(N-isopropylacrylamide-co-eth-ylene glycol monoacrylate) on polystyrene.33,38 It has beenfound that the incorporation of polyethylene glycol unitsinto the pNIPAM chains on substrate resulted in more rapidfibroblast detachment during the temperature transition,but the polymers containing polyethylene glycol (PEG) havelong been demonstrated with low protein and cell bindingability at 37�C in biological environment,39 thus PEG seg-ments in the coating may facilitate the cell detachmentaround LCST, but limit substrates from forming tight andlong-term cell adhesion above LCST. Moreover, as manyresearches have focused on the short-term rapid cell detach-ment on pNIPAM-based substrate, no study has been per-formed for the improvement of long term cell adhesion andgrowth for pNIPAM contained substrates.

Some researchers has found that the substrates graftedwith pNIPAM brushes could effectively adhere to the porcineretina tissue at 37�C and lost adhesion at room temperature of24�C without damage to the surrounding tissues, this phenom-enon is believed to be related to the cell, protein and biomate-rial interaction.40 However, the LCST of the modified PC surfacehas been expected to go further lower to maintain the deviceadhesion below 22�C, so does the tissue detachment below10�C (or lower); this property could be essential for some med-ical devices, such as subcutaneous electrodes and drug deliverydevices in conjunctiva area that may frequently be exposed to alow temperature environment, and still need to be tissue adhe-sive. Being an important coating material for medical devices inrecent years, so far there is no study for generation of pNIPAMcopolymer brushes on the PC-based substrates for adjustmentof LCSTs, as well as the study for the cell proliferation and ther-mal induced cell adhesion/detachment.

This article extends the work to graft pNIPAM-based copol-ymer brushes on PC, and the LCSTs were further adjusted byformation of the copolymer brushes. We proposed to graft PCsurfaces with pNIPAM, pNTBAM, and their block copolymerbrushes by surface-initiated ATRP reaction. The surfacechemical composition, topography, and thermal sensitivecontact angle were characterized. Fibroblasts proliferation andadhesion/detachment were observed for each substrate, andthe condition was compared at incubation temperatures of 37,24, and 6�C to investigate the cell adhesion/detachment upontemperature change. We expect that this study will furtherprovide an effective method to immobilize the PC surface witha thermal responsive tissue adhesive layer.

MATERIALS AND METHODS

MaterialsN-isopropylacrylamide (NIPAM) and N-tert-butylacrylamide(NTBAM) from Sigma-Aldrich were recrystallized twice fromhexane:toluene (6:1, v/v). Azobisisbutyronitrile (AIBN) waspurchased from Sigma-Aldrich and recrystallized from meth-anol before use. The 2-chloropropionyl chloride (CPC) from

218 ZHANG ET AL. SURFACE CHEMICAL IMMOBILIZATION OF PARYLENE C

Sigma-Aldrich was distilled to remove impurities. Dichloro-methane and DMF were distilled with calcium hydrogen(CaH2) to remove the impurities. Ethanol was distilled bycalcium oxide (CaO) before use. Anhydrous aluminum tri-chloride (AlCl3, Fluka), copper (I) chloride (CuCl, Sigma-Aldrich), 1,1,4,7,10,10-hexamethyltriethylenetetramine(HMTETA, Sigma-Aldrich), were used as received withoutfurther purification.

PC films in round shape (10 lm in thickness and 22mm in diameter) were prepared by deposition of di(chloro-p-xylylene) onto micro cover slips (VWR) using PDS2010Labcoater (Specialty Coating System Company, Indianapolis,IN). The films were peeled from slips, sonicated in dimethyl-formamide (DMF) and acetone for 30 min, respectively, andthen vacuum dried at room temperature prior to surfacemodification.

Synthesis of poly(N-isopropylacrylamide) and poly(N-tert-butylacrylamide)Bulk pNIPAM and pNTBAM were synthesized separately byfree radicals polymerization. NIPAM or NTBAM was mixedwith AIBN at the molar ratio of 100:1 in ethanol to form a20 wt % solution; the solutions were then heated to 60–70�C for 12–16 h reaction under nitrogen protection. Theresulting solutions were concentrated, redissolved in smallamount of tetrahydrofuran (THF) and precipitated in etheror hexane. The collected polymers were vacuum dried at40�C, and used as the standard control to measure the ap-proximate amount of pNIPAM or pNTBAM brushes graftedon PC films by spectrometry method.

Surface modification of PC filmsImmobilization of the chloropropionyl groups on PC sur-face via Friedel-Crafts acylation reaction. PC films wereimmersed into 75 mL dichloromethane solution containing2.5 g (18.8 mmol) AlCl3 and 1.79 ml (18.8 mmol) CPC withnitrogen protection. This reaction was performed at 0�C forfirst 6 h and followed by another 10 h at room temperaturewith mild agitation as illustrated in Figure 1. After that, thefilms were thoroughly washed with DMF for 2 h and acetonefor another 2 h, and then vacuum dried at room temperaturefor 4 h prior to surface polymerization. The aromatic ringsin PC films were substituted with certain amount of chloro-propionyl groups containing labile chlorine atoms, and thesemodified PC films were designated as PC-Cl.

Surface initiated atom transfer radical polymerization(ATRP) of NIPAM, NTBAM, and their block copolymerbrushes on PC films. To graft pNIPAM brushes on PC surfa-ces, PC-Cl films were added into 20 mL DMF/water (3:1 v/v)cosolvent containing NIPAM (3 g, 23.6 mmol) and HMTETA(149 lL, 0.531 mmol). This mixture was mildly agitatedunder nitrogen flow for at least 15 min to remove the oxy-gen, and CuCl (53 mg, 0.53 mmol) was then added. Followingthat, the temperature was increased to 50–55�C and main-tained for another 22 h to polymerize NIPAM on PC surfaces.These PC films grafted with pNIPAM brushes were desig-nated as PC-NI.

To graft pNTBAM brushes on PC surfaces, a proceduresimilar to that of PC-NI was applied. NTBAM (3 g, 26.5mmol), HMTETA (132 lL, 0.472 mmol), PC-Cl films andCuCl (47 mg, 0.47 mmol) were mixed with 20 mL DMF, thesurface ATRP reaction was performed at 45–50�C for 22 hunder nitrogen protection. Final PC films grafted withpNTBAM were designated as PC-NT.

To prepare the PC surfaces with block copolymerbrushes containing pNIPAM and pNTBAM segments, PC-NTfilms were used as initiator to polymerize NIPAM undersame ATRP reaction condition as described above for thepreparation of PC-NI, but the reaction time was set at 55–60�C to maintain the initiation reactivity; the resulting PCfilms grafted with copolymer brushes were designated asPC-NT-NI. Similarly, PC-NI films were also used as initiatorto polymerize NTBAM at 50–55�C; the resulting films weredesignated as PC-NI-NT. These films were rinsed by acetoneand dried in vacuum oven before characterization. TheATRP polymerization reactions on PC surfaces are illus-trated in Figure 2.

Surface characterization methodsX-ray photoelectron spectroscopy (XPS, Surface ScienceInstrument, M-probe Surface Spectrometer) was used to fordetailed information about surface chemical composition. Allmeasurements were taken on the center of the sample atroom temperature. Monochromatic X-rays were incident at35� to the sample surface, and the emitted electrons werecollected at a takeoff angle of 35� from the plane of thesample surface. ESCA-2000 software was used to collectand analyze the data. To get an overview of the speciespresent in the sample, survey scans were run from 0 to1000 binding eV.

FIGURE 1. Schematic diagram illustrating the process of Friedel-Crafts acylation reaction on PC surfaces. Aromatic rings of PC were substituted

with chloropropionyl groups, which were able to initiate surface ATRP reaction.

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JAN 2012 VOL 100B, ISSUE 1 219

Attenuated total reflection Fourier transform infraredspectroscopy (ATR-FTIR, Perkin Elmer, Model Spectrum 200with germanium crystal at 45� angle) was used for surfacecomposition characterization and polymer brushes quantifica-tion, the penetration depths for this technique are between0.17 and 0.99 lm.10 The amount of polymer brushes graftedon the substrate was measured by comparing the peak ratiosto the PC films with the solvent-cast polymer layer.

Surface topographic properties of the modified PC filmswere studied by atomic force microscopy (AFM, Digital Instru-ment, Dimension 3100, Santa Barbara, CA) at 24�C in the air;one side of the sample films was fixed on a flat metal plate bya double-sided adhesive tape, and a smooth area of the otherside were chosen for AFM characterization. The square area of1 lm � 1 lm on the films was scanned in the tapping mode.Prior to the AFM test, sample surfaces were cleaned by ace-tone and water, and followed by vacuum dry at 24�C over-night. The calculated arithmetic mean of the surface roughness(Ra) was derived from the roughness profile from AFM image.

Static contact angle were measured by contact angle goni-ometer (Tantec, IL) for the polymeric films. The sample holderwas modified into a flat metal plate embedded with cooling–heating coils, which were connected to a temperature-adjusta-ble water bath. A membrane thermocouple connected with adigital reader (Omega Inc, CN76000) was glued to the sampleholder to detect surface temperature. The temperature of sam-ple surface was slowly adjusted from 13 to 42�C, at each tem-perature point the contact angle was recorded.

Cell culture on the surface modified PC filmsRound-shaped polymer films (diameter in 22 mm) werethoroughly rinsed by acetone for 1 h and water for another

1 h, followed by vacuum dry at room temperature for 2days to remove all the volatile small molecules from thesurface. The sample films were sterilized by 75% (V/V)ethanol for 20 min, then by UV radiation for another 15min, finally rinsed with sterile PBS solution for three timesbefore cell culture. The sterilized films were put into 12-well tissue culture plates (BD Science), human skin fibro-blast cells were seeded at concentration of 6 � 104 cells/well on the polymer surfaces with 200 lL medium [Dulbec-co’s modified Eagle medium (DMEM) supplemented with10% fetal bovine serum (GIBCO), 1.8 mM L-glutamine(GiBCO), 45 U mL�1 penicillin and 45 lg mL�1 streptomycin(GIBCO)]. For cell attachment and proliferation study, thefilms seeded with cells were incubated at 37�C for 5 daysand polystyrene tissue culture petri dishes (TCPs) wereused as controls.

The study for cell detachment at low temperature of 24and 6�C was performed after 1-day cell culture at 37�C. Thesamples were first moved to a 24�C environment for 30-min incubation, and then were moved to a 6�C environmentfor additional 100-min incubation, cell number and mor-phology were studied at different temperatures. The cellnumber on the polymer films was quantitated by countingfour different areas on the film observed in the microscopefield; for each sample, three films were used and the cellnumber were averaged. TCPs and pristine PC films wereused as controls in this study. The cell morphology wasobserved by phase contrast microscopy.

RESULTS AND DISCUSSION

The approach to covalently anchor the polyacrylamides tothe PC surface involves the use of ATRP methods for

FIGURE 2. Schematic description of surface ATRP reaction of pNIPAM, pNTBAM and the procedure to graft their block copolymer brushes on

PC-Cl surfaces. PC-NT and PC-NI were used as macroinitiator to initiate NIPAM and NTBAM respectively. * PC-NI-NT could not be formed.


growing the homo- and co-polymer brushes. ATRP reactionsare typically initiated by the reaction of a copper complexwith a halide initiator. Although a large number of chlorineatoms exist on pristine PC, they lack sufficient reactivity toinitiate the ATRP reaction. By covalently binding chloropro-pionyl groups to the PC aryl groups via Friedel-Crafts reac-tion (Figure 1), the labile chlorines exhibited higher reactiv-ity and thus effectively initiated ATRP reaction, givingacrylamide-based polymer brushes on PC (Figure 2). In thisstudy, the control of Friedel-Crafts reaction condition is notonly important to obtain a high yield of labile chlorineatoms, but also to maintain the bulk mechanical property ofPC. It has been found that low concentration of CPC-AlCl3ligand, low reaction temperature (0�C to room temperature)and moderate reaction time (14–16 h) efficiently promotesthe generation of labile chlorines, and the resulting PC-Clfilms exhibited slightly yellow color, insignificant loss of themechanical strength, and ability to surface initiate ATRP atroom temperature; while the PC films that were treatedhigh concentration of CPC-AlCl3 ligand, high temperature orextended time became dark brown and very brittle.

The reaction of PC-Cl with NIPAM and NTBAM mono-mers under ATRP conditions led to the formation of pNI-PAM and pNTBAM brushes on the PC surface. The tempera-ture of ATRP reactions was maintained between 40 and45�C for PC-NT and 50 and 55�C for PC-NI to reach themaximum grafting amount. It has been observed that lowertemperature did not well initiate the ATRP, resulting in lowsurface coverage, while the high temperature above 65�Ccauses the decomposition of copper–ligand complex duringATRP reaction.41 Based on the report for bulk polymeriza-tion of pNIPAM, cosolvent of DMF/water was used for ATRPof PC-NI to obtain high molecular weight of grafted pNIPAMbrushes.42 The terminal chlorines at the polymer brushes ofPC-NI and PC-NT were further applied as macro-initiator toinitiate ATRP of the second monomer of NTBAM and NIPAMrespectively, to form copolymer brushes. Cosolvent of DMF/water was used for ATRP of PC-NT-NI and dry DMF as sol-vent for PC-NI-NT. As will be discussed below by XPS andFT-IR analysis, PC-NT-NI was formed but PC-NI-NT was notafter second step of ATRP.

XPS and FT-IR characterization of polymer brushesgrafted on PCThe resultant polyacrylamide thin films were characterizedby XPS and FT-IR methods. The XPS survey scan spectra ofPC, PC-Cl, PC-NI, PC-NT, and PC-NT-NI are shown in Figure3(A), XPS analysis were performed at several areas onmodified PC surface, and showed similar data, indicating ahomogenous grafting of the polymer brushes on the surface.The characteristic peaks of carbon and chlorine wereobserved at 281.8 eV (C 1s), 268.4 eV (Cl 2s), and 197.8 eV(Cl 2p) for PC, and the XPS surface elemental analysisshowed the ratio of C:Cl at 89.4%:10.6%, close to the theo-retical atomic ratio of pure PC for C:Cl at 8:1 derived fromits molecular formula of (C8H7Cl)n. After Friedel-Crafts reac-tion, an additional peak of O 1s could be observed for PC-Clat 529.5 eV due to the incorporation of propionyl chloride

groups onto the aromatic rings of PC, and the surface ele-mental ratio of C:O:Cl was 76.9%:11.0%:12.1%. Assumingthat only single chloropropionyl group is bound per aro-matic ring of PC, the substitution ratio of aromatic rings onPC-Cl is about 30% according to the elemental ratio changefrom PC to PC-Cl. The binding energies of chlorine on PC-Clalso appeared to decrease slightly to 267.8 eV (Cl 2s) and197.7 eV (Cl 2p), indicating the introduction of labile chlo-rine atoms onto PC surfaces. After ATRP reactions of eitherNIPAM or NTBAM on PC-Cl, additional N 1s peaks could beclearly observed at about 396.0 eV for PC-NI and PC-NT,suggesting the successful immobilization of the homo pNI-PAM or pNTBAM brushes onto PC via ATRP. The surface ele-mental ratio of C:O:N of PC-NI was measured at about75.6%:12.0%:12.4%, close to the theoretical atomic value ofC:O:N for pure pNIPAM at 6:1:1 derived from its molecularformula of (C6H9ON)n; and C:O:N:Cl of PC-NT was about76.7%:10.7%:9.9%:2.7%, the ratio of C:O:N was close to thetheoretical atomic value of C:O:N for pure pNTBAM at 7:1:1derived from its molecular formula of (C7H11ON)n, indicat-ing a full coverage of the polymer brushes on these modi-fied substrates.

A chlorine signal from PC-NI could not be observed inXPS spectra. The loss of the chlorine signal from the aro-matic rings of the substrates can be attributed to the forma-tion of flexible, hydrophilic and thick pNIPAM coatings,which is beyond XPS sampling depth (regular 7.5 nm in anorganic matrix),43 preventing the chlorine atoms on the sub-strate from detection. The loss of the labile chlorine signalsfrom the ending groups of the hydrophilic pNIPAM brushescan be attributed to the accelerated hydrolysis in the cosol-vent of ATRP reaction as reported previously.44,45

Additional information for the immobilized polymerbrushes on the substrate was obtained from XPS high reso-lution scan of C 1s. As shown in Figure 3(B), the strongestpeak at 281.0 eV in PC spectra was attributed to methyleneand aryl carbons. The C 1s peak of PC-Cl was increased dueto the incorporation of amide (C¼¼O) groups. Peak-fittingwas not performed for surface modified PC due to the exis-tence of various carbons bonds and interaction in the poly-mer brushes and between the substrates, but the shake-upsatellite signals (p–p* transition) for PC-NI, PC-NT and PC-NT-NI could be clearly observed, indicating the incorpora-tion of the pNIPAM and pNTBAM and their copolymerbrushes onto PC surface.

To further characterize the surface chemical composi-tion, ATR-FTIR was also performed to analyze the infraredabsorption of the functional groups on the substrates.Because of the higher penetration depth of the IR radiationbeam in ATR-FTIR technique (1–5 lm) than the samplingdepth of XPS (<7.5 nm),46 the appearance of carbonylbands for PC-Cl in FTIR (Figure 4) suggested that the Frie-del-Crafts reaction was not only confined to the near surfaceregion, the CPC-AlCl3 ligands also penetrated into a depth ofPC surface structure, reacting with the aromatic rings. Itwas found that the PC films became dark brown and brittleafter an extended Friedel-Crafts reaction time (>24 h) or ata high temperature (> 45�C), this may be attributed to the



decrement of the polymer chain alignment by the over-reac-tion of polymer with polar CPC-AlCl3 ligands. Comparison ofthe surface FTIR spectra gave more information about Frie-del-Crafts reaction and ATRP reaction. Formation of the acylgroups on PC-Cl is included in a broad band between 1693and 1710 cm�1 (C¼¼O stretch), this broad C¼¼O absorptionband can be attributed to the coexistence of alkylation andacylation reaction by the active chlorine groups of CPC dur-ing Friedel-Crafts reaction. Both chlorine atoms on CPC par-ticipated in the electrophilic substitution of the hydrogenatoms on the aromatic rings of PC, resulting in the mono-substituted aromatic rings with propionyl chloride groups(i.e., ACH2CH2C(O)Cl) or with 2-chloropropionyl groups (i.e.,AC(O)CH2CH2Cl), the chlorine atoms on these short ali-phatic chains could further react with other aromatic ringsto form a-acetonic group (i.e., AC(O)CH2CH2A) betweenmultiple aromatic rings; besides, the propionyl chloridegroups could further be hydrolyzed into propionic acid by

moisture in the air, which can be indicated by the broad ab-sorbance peak between 1693 and 1710 cm�1, and theappearance of an absorbance peak at about 2940 cm�1 inFTIR spectra. Among them, only the chlorine atoms in chlor-opropionyl groups exhibited high reactivity to initiate ATRP,and the following ATRP can occur at room temperature orhigher. Interestingly, maintenance of the Freidel-Crafts reac-tion of PC at 22–24�C could lead to the formation of astrong absorption at 1694 cm�1 with broad band (spectranot shown), but the resulting PC-Cl exhibited low reactivityand could only initiate the ATRP at the temperature above50�C, this may be due to the higher amount propionic acidon PC-Cl by alkylation reaction.

In this study, cosolvent of DMF/water was used in ATRPof NIPAM to obtain the hydrophilic polymer brushes withmaximum length according to the study of bulk pNIPAM po-lymerization,47 and dry DMF was used as solvent for theATRP of NTBAM to form the hydrophobic polymer brushes.The peaks of amide bond (amide I band, secondary amideC¼¼O stretch) in the polymer brushes were observed in PC-NI spectra at 1646 cm�1, while in PC-NT spectra at lowervalue of 1654 cm�1 due to the lower chains flexibility, po-larity and existence of weaker intermolecular hydrogenbond interaction of the pNTBAM brushes.

To roughly quantify the pNIPAM and pNTBAM on thesubstrate, the strength of the absorption peaks from amidebonds of polymer brushes on PC-NI or PC-NT was used, andthe absorption peak at 1607 cm�1 arising from aromaticrings of substrate was used as the reference. The character-istic peak strength ratio of pNIPAM/substrate (I1646/I1607)for PC-NI or pNTBAM/substrate (I1654/I1607) of PC-NT wascompared to the solvent-cast polymer layer with knownamount of the polyacrylamide. Calculated results showed �7.2 lg cm�2 pNTBAM on PC-NT and 9.7 lg cm�2 pNIPAMon PC-NI; the approximate thickness of grafted pNTBAMand pNIPAM on the substrates were calculated at about 70and 88 nm by using the density of bulk pNIPAM (1.10 g

FIGURE 3. (A) XPS survey scan analysis of PC, PC-Cl, PC-NT, PC-NI,

PC-NT-NI, and PC-NI-NT. No chlorine peaks (Cl 2s and Cl 2p) were

observed for PC-NI, indicating PC-NI was unable to further initiate the

ATRP of NTBAM to form the block copolymer brushes. * PC-NI-NT

could not be formed and no chlorine signals were shown in its XPS

spectra. (B) High-resolution XPS spectra of the carbon C 1s signal for

each substrate. The shake-up satellite signal (p–p* transition) pointed

by arrow heads can be clearly observed for PC-NI, PC-NT, and PC-NT-

NI due to the grafted polymer brushes.

FIGURE 4. Comparative ATR-FTIR spectra of PC, PC-Cl, PC-NI, P-NT,

and PC-NT-NI. Acyl groups on PC-Cl can be observed between 1693

and 1710 cm�1 (C¼¼O stretch), amide group on PC-NI and PC-NT can

be found at 1646 and 1654 cm�1 (C¼¼O stretch), respectively, PC-NT-NI

shows an amide bond absorption peak between 1646 and 1654 cm�1.


cm�3) and pNTBAM (1.04 g cm�3). The higher graftedamount of the pNIPAM brushes on PC-NI could be attrib-uted to the lower steric hinderance of the propyl groups inNIPAM, which could assist the formation of the longer andmore flexible chains than that of NTBAM with butyl groups.

A key characteristic of the ATRP reaction is the preser-vation of the active labile halogen atoms throughout the po-lymerization. These halogens are effective initiators for thegrowth of a second polymer chain, from a different mono-mer, forming a block copolymer structure (Figure 2).48 Onlyone of the two polymer brushes discussed above retainstheir Cl initiators. XPS data for PC-NT show the presence ofCl, while it is absent in PC-NI. These chloride initiators atthe termini of the pNTBAM brushes of PC-NT allowed theuse of ATRP with NIPAM to add pNIPAM chains to the PC-NT materials. Unfortunately, the lack of chloride initiatorson PC-NI made it impossible for us to prepare PC-NI-NT.

The block copolymer brushes were also examined onPC-NT-NI by XPS and FT-IR. Interestingly, although the tend-ency of hydrolysis of chlorine atoms on the grafted pNIPAMsegments of PC-NT-NI, the chlorine signals were still clearlyobserved, even though PC-NI showed no chlorine signal.XPS elemental analysis showed the ratio of C:O:N:Cl on PC-NT-NI at 76.2%:11.2%:10.9%:1.7%, a lower chlorine contentthan that on PC-NT (2.7%). The chlorine signals on PC-NT-NI could be attributed to the unreacted chlorines sur-rounded by the hydrophobic pNTBAM brushes and werenot hydrolyzed.

Figure 4 shows that PC-NT-NI has an amide bondabsorption at 1649 cm�1, it is located between the absorp-tion peak of 1646 cm�1 in PC-NI and 1654 cm�1 in PC-NT,and shows greater intensity than either of them, indicatingan overlap of amide bonds signal from pNTBAM and pNI-PAM segments in PC-NT-NI. The approximate molar ratio ofthe pNTBAM and pNIPAM at 58:42 could be obtained fromthe area ratio of fitted peak at 1646 and 1654 cm�1 in PC-NT-NI spectra; the amount of pNIPAM segment on PC-NT-NIcould be further calculated at �5.9 lg cm�2, thus the totalthickness of the grafted copolymer brushes on PC-NT-NI isat �123 nm.

Surface morphology characterization by AFMThe sample film surfaces were thoroughly washed by ace-tone and dried at vacuum at room temperature prior toAFM measurement. As shown in Figure 5, PC films preparedby CVD process were uniform and smooth, with averageroughness value Ra of 1.8 nm. An increase of surface rough-ness (Ra ¼ 2.3 nm) was found for PC-Cl films with the for-mation of an irregular granule structure. This increase canbe explained by the breakdown of the polymer chains in adepth of the substrate surface by the CPC-AlCl3 ligand asdiscussed in FTIR analysis. The Ra values of PC-NI, PC-NT,and PC-NT-NI were about 2.8, 3.8, and 3.4 nm, respectively.Among PC-NI, PC-NT, and PC-NT-NI, PC-NI showed the low-est roughness, which can be attributed to the lowest sterichinderance of isopropyl groups and the highest chain flexi-bility of pNIPAM, while PC-NT showed a highest roughnessdue to the highest rigidity of the tert-butylacrylate groups

in pNTBAM brushes that less regularly accumulated on thesubstrate. The medium roughness of PC-NT-NI between thatof PC-NT and PC-NI could be attributed to the moderateconformational rigidity of the pNTBAM-co-pNIPAM brushesof on substrates.

Surface contact angle measurement in response to envi-ronmental temperature changeThe water droplet contact angle values measured for PC,PC-Cl, PC-NI, PC-NT, and PC-NT-NI were plotted as a func-tion of temperatures from 13 to 40�C in Figure 6. Asexpected, the contact angles of PC and PC-Cl remained con-stant at roughly 90 and 80�, respectively, with no changeover the temperature range used here. The PC-Cl films givea lower surface contact angle than that of PC films due tothe hydrogen bonding (albeit weak) observed from the car-bonyl oxygen.

PC-NI shows a temperature dependent hydrophilicity,with the contact dropping from 71 to 52� with a tempera-ture decrease from 35 to 22�C. The LCST for PC-NI is esti-mated to be 29�C, which was lower than the LCST value(32�C) of bulk pNIPAM. The lower LCST of PC-NI could beattributed to the enhanced hydrophobic interaction betweenthe grafted pNIPAM brush and the hydrophobic PC sub-strate. The thermal induced phase transition of the homopNIPAM results from the change of the inter- and intrachaininteractions. The pNIPAM chains of the brush are partiallyimmobilized onto the hydrophobic PC surface, and thisincreases the hydrophobic interactions between the sub-strate and propyl groups in pNIPAM, which in turn limitsthe motility and hydrodynamic radius of the polymer brush.The net effect is an easy collapse of the polymer brush intothe hydrophobic, dense phase at a lower temperature thanobserved for bulk pNIPAM.49–51

Bulk pNTBAM is hydrophobic with low swelling ratio(<0.05 wt %) and shows no thermal shifts of its surfaceproperties. The grafted pNTBAM brushes on PC-NT surface,however, exhibit a rapid decrease in contact angle, from 77to 60�, when the temperature is decreased from 30 to 20�C.The LCST of PC-NT was found to be 24�C. Similar tempera-ture-related phase transition was also found for otherpoly(N-substituted acrylamide) brush–water systems,52 andthe temperature dependence of these systems could beattributed to the changes of the molecular interactionsamong polymer chains and water. The thermal independenthydrophobicity of bulk pNTBAM is attributed to the highintramolecular interaction of the polymer chains, whichovercomes the extramolecular interaction between polymerchains and water. However, by grafting of the pNTBAMbrushes onto the substrate, the quantity and the interactionof the immobilized pNTBAM chains were significantlyreduced, it resulted in the enhanced extramolecular interac-tion between polymer chains and surrounding water, thusthe grafted pNTBAM exhibited water solubility at low tem-perature. The change of the surface energy was further con-firmed by comparing the surface contact angles among thebulk pNTBAM (85�), PC-NT (77�), and PC (91�) at 24�C.The lowest contact angle value of PC-NT indicated the



highest surface energy, as well as the stronger hydrogenbonding interaction between the grafted pNTBAM brushesand water.

The PC-NT-NI that was grafted with copolymer brushesof pNIPAM and pNTBAM also showed thermal sensitivehydrophilicity. The contact angle of PC-NT-NI decreasedfrom 74 to 54� with the temperature decrease from 34 to15�C. The LCST of PC-NT-NI was found to be 22�C, which islower than that of either PC-NI or PC-NT. The lower LCST insuch copolymers has attributed to the limited motility ofpNIPAM chains at low temperature and enhanced hydropho-bic interaction between pNIPAM and water, which is causedby the reduced amount of structured water in the pNIPAMwhen it is copolymerized with other hydrophobic poly-mers.22,53 As illustrated in Figure 6(B), the grafted copoly-mer brushes on PC-NT-NI exhibited an extended ‘coil’ struc-ture below LCST and aggregated ‘‘globule’’ structure aboveLCST. The contact angle for PC-NT-NI also showed a slowdecrease from 32 to 25�C, and a rapid decrease from 25 to15�C; the slow decrease can be attributed to the interferedphase transition of PNIPAM by hydrophobic PNTBAM seg-

ments, and the rapid decrease can be explained by the syn-ergic hydrophilic effects of the PNTBAM and PNIPAMsegments.

Cytocompatibility and cell adhesion/detachment studyIn this study, we further studied the cell adhesion/detach-ment on parylene substrates coated with pNIPAM and pNIT-BAM brushes (PC-NI, PC-NT and PC-NT-NI) in response to achange of environmental temperature. To evaluate the cyto-compatibility of the surface modified films, human skinfibroblasts were seeded onto the membrane samples of PC,PC-Cl, PC-NI, PC-NT, and PC-NT-NI and incubated at 37�Cfor 5 days in a combination of cell culture medium and fetalbovine serum. Tissue culture petridishes (TCPs) were usedas controls. During cell proliferation, fibroblasts were innormal flattened appearance but grew on all the samples atvarying densities, dependent on the substrates, indicatingthe existence of the different cell-material interactions forthese substrates. The quantitative data in Figure 7 showedthat proliferation rate of fibroblasts on PC-NT, PC-NT-NI,and TCP control are significantly higher (>10 times) than

FIGURE 5. Three dimensional AFM images of: (A) PC, (B) PC-Cl, (C) PC-NI, (D) PC-NT, and (E) PC-NT-NI. After surface graft with polymer brushes

on PC, an increase of the surface roughness can be obviously observed. Ra: average roughness.


that on PC and PC-NI; and cell proliferation rate on PC-Clwas lower than that on PC-NT but still five times higherthan that on PC or PC-NI; no statistical difference of cellnumbers was found on PC and PC-NI films during culture.The cells on TCP reached confluence at about 7 days, andon PC-NT and PC-NT-NI surface reached confluence after 9and 10 days culture in the incubator.

The difference in cell density on each substrate can beattributed to the various factors of the cell-material inter-face, such as surface hydrophobicity, energy, topography,surface charge, and chemical composition.54–57 For thesesubstrates, surface hydrophobicity and surface chemicalcomposition are expected to be the main factors. At 37�C,PC film was very hydrophobic (contact angle of 91�) withlow surface polarity, leading to a low cell attachment andproliferation rate (about 7% of that of TCP at day 5). ThePC-Cl surface containing polar carbonyl groups exhibited a

decreased hydrophobicity (contact angle: 81�) and increasedcell attachment amount (about 35% of that of TCP atday 5). By grafting polymer brushes onto substrates, PC-NI,PC-NT and PC-NT-NI showed a further reduction of hydro-phobicity, which was reflected by the decrease of contactangles to 71� , 77�, and 74�, respectively, at 37�C. Although anumber of cells adhered to PC-NI in first 4 h after seeding, itwas also found that PC-NI did not support the sustained celladhesion after day 1. A significantly lower cell number wasobserved on PC-NI than that of PC-NT, PC-NT-NI, and TCP.This result is consistent to the previous research reported byOkano et al., for polystyrene grafted with thick pNIPAM layer,where those researchers observed very little cell adhesion forfilms above 7.9 lg cm�2.30 The PC-NI films studied here hadgrafted pNIPAM amount at about 9.7 lg cm�2. In contrast,cell adhesion and proliferation thus showed a significantincrease on PC-NT and PC-NT-NI, reaching 50–70% of the

FIGURE 6. (A) Water contact angle of PC, PC-Cl, PC-NI, PC-NT, and PC-NT-NI as a function of temperature. (B) Schematic illustration of polymer

brushes changes on PC-NT-NI below and above LCST in aqueous solution. The polymer brushes became extended below LCST and collapsed

above LCST.



cell density observed for TCP at day 5. The pNIPAM layer inPC-NT-NI was 5.9 lg cm�2, below the maximum level deter-mined by Okano for promoting cell adhesion. Considering thefact that the hydrophobicity of the three polymer brush sys-tems are comparable, the cell-substrate interaction on thesesubstrates is clearly also dependent on surface chemical com-position.58 A recent study by Lynch et al., examined cell adhe-sion on bulk samples of pNIPAM, pNTBAM and their copoly-mers, and found very different properties for the differentpolymers.59 They found that the cell adhesion and prolifera-tion on pNIPAM and pNTBAM were indistinguishable in theabsence of serum, but showed marked differences when se-rum was added to the growth medium (as was the case withour experiments). The principal source of the difference inthe behavior of the different materials is in the ability ofthese polymers to bind fibronectin and albumin. Lynch et al.showed that high pNIPAM content leads to high binding af-finity for albumin and low affinity for fibronectin, while highpNTBAM content leads to low binding affinity for albuminand high affinity for fibronecton. It is known that the fibro-nectin supports the cell proliferation, while albumin reducesit.60 Lynch et al., proposed that the tert-butyl groups ofpNTBAM sterically block access to the amide NAH groups,preventing the majority of the serum proteins from binding,with the exception of fibronectin, which binds most effec-tively to hydrophobic surfaces.59,61 Both pNIPAM andpNTBAM based materials give initial cell adhesion, asobserved here, but pNTBAM gives markedly more efficientcell proliferation. The thin pNIPAM film of PC-NT-NI partiallyblocks the underlying pNTBAM film, limiting serum exposureto the underlying pNTBAM, thus showing a moderate cellattachment and proliferation, markedly better than PC-NI, butnot as good as PC-NT-NI.

Cell detachment study at the reduced temperatures (24and 6�C) was performed after 24-h cell culture on each sub-strate at 37�C. PC, PC-Cl, and TCP were used as controls

(Figure 8). After 24-h culture, a low cell density was foundon PC-NI, similar to the 5-day cell culture. The cellsattached on PC-NT and PC-NT-NI showed slight heterogene-ity due to the low amount of the cultured cells and uneven-ness of the surface caused by multiple steps of chemicalmodification with mechanical agitation. As the temperaturewas decreased to 24�C, the attached cells completelydetached from the PC-NI substrate and floated above thesubstrate after incubating for 40 min at (Figure 8D,D’),while the cells on PC-Cl, PC-NT, PC-NT-NI, and TCP controlexhibited no significant change (photos were not shown). Asthe incubation temperature was reduced to 6�C and main-tained for another 100 min, the cell coverage on PC-NT wasreduced. The remained cell number on the surface wascounted and the results showed that roughly 10% 6 5% ofthe cells on PC-NT detached and floating cells could beobserved in the medium (Figure 8E,E’); similarly, �35% 65% of the cells on PC-NT-NI detached, with some of thedetached cells forming clumps above the substrate (Figure8F,F’). The cells on TCP control, PC and PC-Cl remained nor-mal spread shape without significant detachment (Figure8A,A’,B,B’,C,C’) after 100 min at 6�C. It has been suggestedpreviously that the exposure of NAH groups of the poly-meric brushes to the serum containing medium could causea reduction in cell attachment.60 At a temperature belowLCST, the hydrophilicity of PC-NI, PC-NT, and PC-NT-NI weresignificantly increased, resulting in a higher percentage oftheir NAH groups being exposed to the media. The cells onPC-NT and PC-NT-NI also showed slower detachment ratesand higher cell adhesion density than that on PC-NI, andthese could be attributed to the slower change of polymerchains and relatively higher amount of the unexposed NAHgroups that was caused by the high steric hindrance ofbutyl groups on pNTBAM segments of PC-NT and PC-NT-NI.Interestingly, the detached cells returned to grow on thepolymer brush coated substrates or TCP after 2-day incuba-tion at 37�C, and the cell adhesion/detachment study couldbe repeated with similar results, these results are consistentwith the research reported by Dr. Okano et al. in their celladhesion/detachment study for pNIPAM grafted polystyrenesurface, indicating the maintenance of the cell metabolismand viability after cell adhesion/detachment test.62,63

All these results from in-vitro cell study will be helpfulin use of the modified PC as a coating material for theimplantable medical devices for tissue adhesion. The coateddevice is cytocompatible above LCST and can be controlledto attach or detach the cells and organ with small or no tis-sue damage by simply adjusting the environmental tempera-ture. Incorporation of pNTBAM into pNIPAM on the PCresulted in a lower LCST and significantly improved cell ad-hesion and growth, it will be a benefit for the long-term fix-ation of the medical devices; furthermore, this tissue–mate-rial adhesion can also be reduced or released at a reducedtemperature around the tissue during surgery. By decreaseof the LCST of the grafted polymer brushes, PC can beapplied for more implants that are frequently exposed to alow temperature but with no unexpected detachment. Bycombining other methods, for example, of increasing or

FIGURE 7. Quantitative assays of cell proliferation on PC, PC-Cl, PC-

NI, PC-NT, PC-NT-NI films, and TCP control. Data represent the mean

of three samples (p < 0.05). Cells were seeded at 6 � 104 cells/well

and cultured at 37�C in the incubator for 1, 3, and 5 days. Cell showed

significantly higher attachment and proliferation on PC-NT, PC-NT-NI,

and TCP control in comparison to PC, PC-Cl, and PC-NI films. Very

small amount of cells proliferated on PC-NI and PC for 5-day culture;

while cell number on PC-Cl showed a higher cell quantity than PC

and PC-NI.


decreasing the divalent cations, such as Ca2þ, Mg2þ and/ortheir chelating agents in the media, cell adhesion to the sub-strate could be affected,64,65 these factors may provide addi-tional ‘‘tuning’’ for the temperature sensitive binding to thedifferent substrates. Moreover, the incorporation of pNTBAMexhibited an improvement of cell attachment and prolifera-tion on pNIPAM contained layer, this will not only assist thetissue repair after implantation surgery, but also improvethe long-term implant–tissue adhesion. The related studyfor short-term and long-term tissue adhesion/detachment in

response to the environmental temperature is in perform-ance and will be reported later.

SUMMARY

Our purpose is to chemically modify the PC surfaces withtemperature controlled cell adhesion/detachment, andimproved cell adhesion and proliferation above LCST. Theseproperties have been obtained by covalently grafting pNI-PAM, pNTBAM, and their copolymer brushes onto PC viachemical reaction. Hydrophobic pNTBAM brushes on PC-NT

FIGURE 8. Phase contrast microscopic images of the cell attachment on polymer films after 24 h incubation at 37�C and followed by 40-min incu-

bation at 24�C and 100-min incubation at 6�C, cell were initially seeded at 6 � 104 cells/well. (A) and (A’) TCP control at 37�C and 6�C. (B) and (B’)

PC films at 37 and 6�C. (C) and (C’) PC-Cl films at 37 and 6�C. (D) and (D’) PC-NI films at 37 and 24�C. (E) and (E’) PC-NT films at 37 and 6�C. (F)and (F’) PC-NT-NI films at 37 and 6�C. Cells amount on PC-NI was observed low at 37�C, and the cells were completely detached after 40-min cul-

ture at 24�C; cells on PC-NT and PC-NT-NI showed a high attachment at 37�C, and partially detached from PC-NT-NI and PC-NT after 100-min cul-

ture at 6�C, some cells were observed clumped and dangling on PC-NT-NI. No obvious cell detachment was observed for PC-Cl, PC-NT, and TCP

control after 100-min culture at 6�C. Arrows show the attached cells on PC, PC-Cl, and PC-NI, and the dangling cells clumps on PC-NT-NI.



exhibit LCST at 22�C, and grafted pNIPAM on PC-NI exhib-ited the LCST at 29�C. The copolymer brush containingpNTBAM and pNIPAM on PC-NT-NI, obtained by two-stepATRP, showed the LCST at 24�C.

In cell culture study, PC-NI showed very low cell adhe-sion and proliferation; while the copolymerized layer on PC-NT-NI exhibited a significantly improved cell adhesion andproliferation. All the substrates grafted with polymerbrushes showed thermal sensitive cell adhesion/detachmentbut varied according to the substrates; PC-NI showed apoor cell adhesion at 37�C but complete detachment at24�C; cells on PC-NT showed a high amount of attachmentand proliferation at 37�C, and cell detachment increased astemperature was reduced to 6�C; while PC-NT-NI exhibiteda strong cells attachment at 37�C, as well as up to 35% celldetachment at 6�C. As PC is in a rapid growth as an effec-tive coating material of many medical devices, this studyexplored a way to enhance the PC coated implants with ad-justable LCST, improved cytocompatibility, as well as ther-mal controlled tissue adhesion.

ACKNOWLEDGMENTS

The authors appreciate the help of Mr. Christian Gutierrez, Bio-engineering Department at University of Southern California,for providing parylene C films; Dr. Mark Thompson, ChemistryDepartment at University of Southern California, for correctingthe grammar and providing the lab tools.

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