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Surface chemical immobilization of parylene C with thermosensitiveblock copolymer brushes based on N-isopropylacrylamide a...
inertness, solvent resistance, good mechanical strength, and     was significantly reduced with higher pNIPAM amount onlow ...
ORIGINAL RESEARCH REPORTFIGURE 1. Schematic diagram illustrating the process of Friedel-Crafts acylation reaction on PC su...
FIGURE 2. Schematic description of surface ATRP reaction of pNIPAM, pNTBAM and the procedure to graft their block copolyme...
ORIGINAL RESEARCH REPORTgrowing the homo- and co-polymer brushes. ATRP reactions                groups onto the aromatic r...
FIGURE 4. Comparative ATR-FTIR spectra of PC, PC-Cl, PC-NI, P-NT,                                                         ...
ORIGINAL RESEARCH REPORTcmÀ3) and pNTBAM (1.04 g cmÀ3). The higher grafted                      in pNTBAM brushes that les...
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 ...
ORIGINAL RESEARCH REPORTFIGURE 6. (A) Water contact angle of PC, PC-Cl, PC-NI, PC-NT, and PC-NT-NI as a function of temper...
(Figure 8). After 24-h culture, a low cell density was found                                                              ...
ORIGINAL RESEARCH REPORTFIGURE 8. Phase contrast microscopic images of the cell attachment on polymer films after 24 h incu...
exhibit LCST at 22 C, and grafted pNIPAM on PC-NI exhib-                 12. Chang TY, Yadav VG, De Leo S, Mohedas A, Raja...
ORIGINAL RESEARCH REPORT      poly[(N-isopropylacrylamide)-r-((3-(methacryloylamino) propyl)-di-    51. Kubota K, Fujishig...
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Surface chemical immobilization of parylene c with thermosensitive block copolymer brushes based on n isopropylacrylamide and n-tert-butylacrylamide synthesis characterization and cell adhesion detachment

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Surface chemical immobilization of parylene c with thermosensitive block copolymer brushes based on n isopropylacrylamide and n-tert-butylacrylamide synthesis characterization and cell adhesion detachment

  1. 1. Surface chemical immobilization of parylene C with thermosensitiveblock copolymer brushes based on N-isopropylacrylamide and N-tert-butylacrylamide: Synthesis, characterization, and cell adhesion/detachmentChanghong Zhang,1,2 P. Thomas Vernier,3 Yu-Hsuan Wu,4 Wangrong Yang31 Department of Chemistry, University of Southern California, Los Angeles, California 900892 Kansas Polymer Research Center, Pittsburg State University, Pittsburg, Kansas 667623 Department of Electrical Engineering, University of Southern California, Los Angeles, California 900894 Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089Received 9 January 2011; revised 8 July 2011; accepted 20 July 2011Published online 9 November 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31941Abstract: Poly(N-isopropylacrylamide) (pNIPAM), poly(N-tert- and 35% of the cells were found detached respectively, andbutylacrylamide) (pNTBAM), and their copolymer brushes the unattached cells aggregated on the substrate. In compari-were covalently immobilized onto parylene C (PC) surfaces son, cells cultured on the tissue culture petri dish (TCP)via surface initiated atom transfer radical polymerization exhibited no quantity and morphology changes at the culture(ATRP). Contact angle measurement between 13 and 40 C temperatures of 37, 24, and 6 C. This study showed that: (1)showed that the hydrophobicity of the modified PC surfaces immobilization of PC with nonthermal sensitive pNTBAMwas thermally sensitive. Among these samples, PC grafted could provide PC surface thermal sensitive hydrophilicity; (2)with pNIPAM (PC-NI), PC grafted with pNTBAM (PC-NT) and the chlorines on the polymer brushes of PC-NT could bePC grafted with copolymer brushes containing pNTBAM and used to further initiate the ATRP pNIPAM and form block co-pNIPAM (PC-NT-NI) exhibited the lower critical solution tem- polymer brushes; (3) the incorporation of pNTBAM into pNI-perature (LCST) at 29, 22, and 24 C, respectively. Cytocom- PAM on PC-NT-NI could change the surface thermalpatibility study for the modified surfaces was performed by 5 hydrophilicity property, and be further applied to decreasedays human skin fibroblast culture at 37 C. Data showed that the LCST of the modified PC surface; (4) grafted pNIPAMonly a very small amount of cells adhered on the PC and PC- brushes on PC-NI by ATRP showed very low cell adhesionNI surfaces, while a significantly higher amount of cell adhe- and proliferation in 5 days fibroblast culture at 37 C, and cellsion and growth was observed on PC-NT and PC-NT-NI surfa- detached at 24 C; (5) the incorporation of pNTBAM into pNI-ces. Furthermore, cell detachment at the temperatures of 24 PAM on PC-NT-NI decreased the thermal sensitivity of celland 6 C were studied after the substrates were cultured with adhesion/detachment, cell detached at 6 C, but the cell adhe-cells at 37 C for 24 h. The results showed that the cells on sion and proliferation were significantly improved at a widePC-NI formed the aggregations and loosely attached on the temperature range. V 2011 Wiley Periodicals, Inc. J Biomed Mater Csubstrate after 30-min culture at 24 C, while no significant Res Part B: Appl Biomater 100B: 217–229, 2012.cell detachment was observed for PC-NT and PC-NT-NI sam-ples at this temperature. By continuing the cell culture for Key Words: cell adhesion, block copolymer, surface modifica-additional 100 min at 6 C for PC-NT and PC-NT-NI, about 10 tion, polymerization, cell–material interactionsHow 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 methylsiloxane) (PDMS), have exhibited their advantages inImplantable medical devices exhibit wide applications, such biocompatibility, chemical versatility, biological functionality,as health monitoring sensors, wireless medical signal devi- and mechanical strength.3,4 These polymers have been usedces, and electrode arrays for nerve function restoration.1,2 for decades as the coating materials to make the implantedThe materials for implantable medical devices must be bio- medical devices suitable to human tissue environments.5–8compatible, and survive long-term exposure to the compli- Among them, poly(2-chloro xylylene), also named as pary-cated and harsh human body environment. Polymers, such lene-C (PC), has several outstanding properties such as bio-as polyurethane, parylene, polymethyacrylate, and poly(di- compatibility, biostability, low water permeability, chemicalCorrespondence to: C. Zhang; e-mail: changhong.zh@gmail.comV 2011 WILEY PERIODICALS, INC.C 217
  2. 2. inertness, solvent resistance, good mechanical strength, and was significantly reduced with higher pNIPAM amount onlow dielectric constant9; it has been approved by US Food some substrates, such as polystyrene and silicon wafer.30,34and Drug Administration (FDA) as class VI biocompatible Recently, copolymer brushes containing pNIPAM werematerial and widely used as coating material for the medi- grafted on the substrates to approach the rapid cell detach-cal implants. PC can be conveniently coated onto the com- ment, including poly(N-isopropylacrylamide-co-ethylene gly-plex substrate by a nonsolvent involved chemical-vapor-dep- col) on silicon wafer, and poly(N-isopropylacrylamide-co-eth-osition (CVD) technique, forming a thin, strong, and pinhole- ylene glycol monoacrylate) on polystyrene.33,38 It has beenfree membrane layer at room temperature, making it a good found that the incorporation of polyethylene glycol unitscandidate as a coating material for many long-term implant- into the pNIPAM chains on substrate resulted in more rapidable devices.10,11 However, the high hydrophobicity and low fibroblast detachment during the temperature transition,polarity of PC prevent it from adhering to the cells and tis- but the polymers containing polyethylene glycol (PEG) havesues, which makes it an ideal packaging material, but limit long been demonstrated with low protein and cell bindingits use on the implanted medical devices that need to be ability at 37 C in biological environment,39 thus PEG seg-anchored to tissue.12 Very limited methods, such as surface ments in the coating may facilitate the cell detachmentsculpturing, plasma treatment, activated water vapor treat- around LCST, but limit substrates from forming tight andment, photo-oxidation and surface chemical modification, long-term cell adhesion above LCST. Moreover, as manyhave been employed to overcome this shortfall.13–17 How- researches have focused on the short-term rapid cell detach-ever, most of them were focused on the generation of ionic ment on pNIPAM-based substrate, no study has been per-groups on PC, not on forming a molecular or polymeric ad- formed for the improvement of long term cell adhesion andhesive coating that is important for some medical implants, growth for pNIPAM contained substrates.such as electrodes of the biosensor in nerve system, to form Some researchers has found that the substrates graftedlong-term adhesion to the cells and surrounding tissues. with pNIPAM brushes could effectively adhere to the porcine Bulk pNIPAM and substrates coated with pNIPAM thin retina tissue at 37 C and lost adhesion at room temperature offilms have been intensively studied in a range of biomedical 24 C without damage to the surrounding tissues, this phenom-applications, due to its beneficial temperature-dependent enon is believed to be related to the cell, protein and biomate-adhesive properties.18–21 Below the lower critical solution rial interaction.40 However, the LCST of the modified PC surfacetemperature (LCST) of 32 C, pNIPAM exhibits extended con- has been expected to go further lower to maintain the deviceformation and high solubility in water; above 32 C, pNIPAM adhesion below 22 C, so does the tissue detachment belowbecomes aggregated and hydrophobic.22 Homo pNIPAM has 10 C (or lower); this property could be essential for some med-been produced into different bulk copolymers for specific ical devices, such as subcutaneous electrodes and drug deliverybiomedical applications, and these copolymers also exhib- devices in conjunctiva area that may frequently be exposed to aited different LCSTs than that of homo pNIPAM. For exam- low temperature environment, and still need to be tissue adhe-ple, homopolymer of pNIPAM copolymerized with hydropho- sive. Being an important coating material for medical devices inbic poly(N-tert-butylacrylamide) (pNTBAM) exhibited recent years, so far there is no study for generation of pNIPAMimproved cell attachment and proliferation with LCST below copolymer brushes on the PC-based substrates for adjustment32 C23; pNIPAM copolymerized with hydrophilic polyethyl- of LCSTs, as well as the study for the cell proliferation and ther-ene glycol (PEG) showed more rapid cell detachment upon mal induced cell adhesion/detachment.temperature decrease than pNIPAM, and the LCST is below This article extends the work to graft pNIPAM-based copol-32 C.24,25 The methods to covalently anchor the homo pNI- ymer brushes on PC, and the LCSTs were further adjusted byPAM on the substrates have also been developed recently to formation of the copolymer brushes. We proposed to graft PCfunctionalize the surface with thermal sensitive properties, surfaces with pNIPAM, pNTBAM, and their block copolymerthese methods included electron beam initiation, plasma- brushes by surface-initiated ATRP reaction. The surfacedeposition, UV irradiation and surface atom transfer radical chemical composition, topography, and thermal sensitivepolymerization (ATRP). These modification methods have contact angle were characterized. Fibroblasts proliferation andbeen suggested to have their roles in medical applications, adhesion/detachment were observed for each substrate, andsuch as anti-biofouling, temperature responsive biosensors, the condition was compared at incubation temperatures of 37,controlled drug release, thermal responsive chromatography, 24, and 6 C to investigate the cell adhesion/detachment uponand reversible cell adhesion/detachment.26–35 Among this temperature change. We expect that this study will furtherapplications, homo pNIPAM grafted substrates has been provide an effective method to immobilize the PC surface withintensely applied for mammalian cell adhesion/detachment a thermal responsive tissue adhesive layer.study.34,36,37 Below LCST, it has been found that the cells donot adhere to the pNIPAM-coated substrate. However, the MATERIALS AND METHODSreports for the cell adhesion above LCST were contradic- Materialstory: some papers reported that large amount cells adhered N-isopropylacrylamide (NIPAM) and N-tert-butylacrylamideand proliferated on the substrates, some papers reported (NTBAM) from Sigma-Aldrich were recrystallized twice fromthat cell adhesion was low; and some researchers found hexane:toluene (6:1, v/v). Azobisisbutyronitrile (AIBN) wasthat cell coverage was high on the substrate with sparse purchased from Sigma-Aldrich and recrystallized from meth-pNIPAM amount (7.9 lg cmÀ2 or less), while cell adhesion anol before use. The 2-chloropropionyl chloride (CPC) from218 ZHANG ET AL. SURFACE CHEMICAL IMMOBILIZATION OF PARYLENE C
  3. 3. ORIGINAL RESEARCH REPORTFIGURE 1. Schematic diagram illustrating the process of Friedel-Crafts acylation reaction on PC surfaces. Aromatic rings of PC were substitutedwith chloropropionyl groups, which were able to initiate surface ATRP reaction.Sigma-Aldrich was distilled to remove impurities. Dichloro- Surface initiated atom transfer radical polymerizationmethane and DMF were distilled with calcium hydrogen (ATRP) of NIPAM, NTBAM, and their block copolymer(CaH2) to remove the impurities. Ethanol was distilled by brushes on PC films. To graft pNIPAM brushes on PC surfa-calcium oxide (CaO) before use. Anhydrous aluminum tri- ces, PC-Cl films were added into 20 mL DMF/water (3:1 v/v)chloride (AlCl3, Fluka), copper (I) chloride (CuCl, Sigma- cosolvent containing NIPAM (3 g, 23.6 mmol) and HMTETAAldrich), 1,1,4,7,10,10-hexamethyltriethylenetetramine (149 lL, 0.531 mmol). This mixture was mildly agitated(HMTETA, Sigma-Aldrich), were used as received without under nitrogen flow for at least 15 min to remove the oxy-further purification. gen, and CuCl (53 mg, 0.53 mmol) was then added. Following PC films in round shape (10 lm in thickness and 22 that, the temperature was increased to 50–55 C and main-mm in diameter) were prepared by deposition of di(chloro- tained for another 22 h to polymerize NIPAM on PC surfaces.p-xylylene) onto micro cover slips (VWR) using PDS2010 These PC films grafted with pNIPAM brushes were desig-Labcoater (Specialty Coating System Company, Indianapolis, nated as PC-NI.IN). The films were peeled from slips, sonicated in dimethyl- To graft pNTBAM brushes on PC surfaces, a procedureformamide (DMF) and acetone for 30 min, respectively, and similar to that of PC-NI was applied. NTBAM (3 g, 26.5then vacuum dried at room temperature prior to surface mmol), HMTETA (132 lL, 0.472 mmol), PC-Cl films andmodification. CuCl (47 mg, 0.47 mmol) were mixed with 20 mL DMF, the surface ATRP reaction was performed at 45–50 C for 22 h under nitrogen protection. Final PC films grafted withSynthesis of poly(N-isopropylacrylamide) and poly(N- pNTBAM were designated as PC-NT.tert-butylacrylamide) To prepare the PC surfaces with block copolymerBulk pNIPAM and pNTBAM were synthesized separately by brushes containing pNIPAM and pNTBAM segments, PC-NTfree radicals polymerization. NIPAM or NTBAM was mixed films were used as initiator to polymerize NIPAM underwith AIBN at the molar ratio of 100:1 in ethanol to form a same ATRP reaction condition as described above for the20 wt % solution; the solutions were then heated to 60– preparation of PC-NI, but the reaction time was set at 55–70 C for 12–16 h reaction under nitrogen protection. The 60 C to maintain the initiation reactivity; the resulting PCresulting solutions were concentrated, redissolved in small films grafted with copolymer brushes were designated asamount of tetrahydrofuran (THF) and precipitated in ether PC-NT-NI. Similarly, PC-NI films were also used as initiatoror hexane. The collected polymers were vacuum dried at to polymerize NTBAM at 50–55 C; the resulting films were40 C, and used as the standard control to measure the ap- designated as PC-NI-NT. These films were rinsed by acetoneproximate amount of pNIPAM or pNTBAM brushes grafted and dried in vacuum oven before characterization. Theon PC films by spectrometry method. ATRP polymerization reactions on PC surfaces are illus- trated in Figure 2.Surface modification of PC filmsImmobilization of the chloropropionyl groups on PC sur-face via Friedel-Crafts acylation reaction. PC films were Surface characterization methodsimmersed into 75 mL dichloromethane solution containing X-ray photoelectron spectroscopy (XPS, Surface Science2.5 g (18.8 mmol) AlCl3 and 1.79 ml (18.8 mmol) CPC with Instrument, M-probe Surface Spectrometer) was used to fornitrogen protection. This reaction was performed at 0 C for detailed information about surface chemical composition. Allfirst 6 h and followed by another 10 h at room temperature measurements were taken on the center of the sample atwith mild agitation as illustrated in Figure 1. After that, the room temperature. Monochromatic X-rays were incident atfilms were thoroughly washed with DMF for 2 h and acetone 35 to the sample surface, and the emitted electrons werefor another 2 h, and then vacuum dried at room temperature collected at a takeoff angle of 35 from the plane of thefor 4 h prior to surface polymerization. The aromatic rings sample surface. ESCA-2000 software was used to collectin PC films were substituted with certain amount of chloro- and analyze the data. To get an overview of the speciespropionyl groups containing labile chlorine atoms, and these present in the sample, survey scans were run from 0 tomodified PC films were designated as PC-Cl. 1000 binding eV.JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JAN 2012 VOL 100B, ISSUE 1 219
  4. 4. FIGURE 2. Schematic description of surface ATRP reaction of pNIPAM, pNTBAM and the procedure to graft their block copolymer brushes onPC-Cl surfaces. PC-NT and PC-NI were used as macroinitiator to initiate NIPAM and NTBAM respectively. * PC-NI-NT could not be formed. Attenuated total reflection Fourier transform infrared 1 h, followed by vacuum dry at room temperature for 2spectroscopy (ATR-FTIR, Perkin Elmer, Model Spectrum 200 days to remove all the volatile small molecules from thewith germanium crystal at 45 angle) was used for surface surface. The sample films were sterilized by 75% (V/V)composition characterization and polymer brushes quantifica- ethanol for 20 min, then by UV radiation for another 15tion, the penetration depths for this technique are between min, finally rinsed with sterile PBS solution for three times0.17 and 0.99 lm.10 The amount of polymer brushes grafted before cell culture. The sterilized films were put into 12-on the substrate was measured by comparing the peak ratios well tissue culture plates (BD Science), human skin fibro-to the PC films with the solvent-cast polymer layer. blast cells were seeded at concentration of 6  104 cells/ Surface topographic properties of the modified PC films well on the polymer surfaces with 200 lL medium [Dulbec-were studied by atomic force microscopy (AFM, Digital Instru- co’s modified Eagle medium (DMEM) supplemented withment, Dimension 3100, Santa Barbara, CA) at 24 C in the air; 10% fetal bovine serum (GIBCO), 1.8 mM L-glutamineone side of the sample films was fixed on a flat metal plate by (GiBCO), 45 U mLÀ1 penicillin and 45 lg mLÀ1 streptomycina double-sided adhesive tape, and a smooth area of the other (GIBCO)]. For cell attachment and proliferation study, theside were chosen for AFM characterization. The square area of films seeded with cells were incubated at 37 C for 5 days1 lm  1 lm on the films was scanned in the tapping mode. and polystyrene tissue culture petri dishes (TCPs) werePrior to the AFM test, sample surfaces were cleaned by ace- used as controls.tone and water, and followed by vacuum dry at 24 C over- The study for cell detachment at low temperature of 24night. The calculated arithmetic mean of the surface roughness and 6 C was performed after 1-day cell culture at 37 C. The(Ra) was derived from the roughness profile from AFM image. samples were first moved to a 24 C environment for 30- Static contact angle were measured by contact angle goni- min incubation, and then were moved to a 6 C environmentometer (Tantec, IL) for the polymeric films. The sample holder for additional 100-min incubation, cell number and mor-was modified into a flat metal plate embedded with cooling– phology were studied at different temperatures. The cellheating coils, which were connected to a temperature-adjusta- number on the polymer films was quantitated by countingble water bath. A membrane thermocouple connected with a four different areas on the film observed in the microscopedigital reader (Omega Inc, CN76000) was glued to the sample field; for each sample, three films were used and the cellholder to detect surface temperature. The temperature of sam- number were averaged. TCPs and pristine PC films wereple surface was slowly adjusted from 13 to 42 C, at each tem- used as controls in this study. The cell morphology wasperature point the contact angle was recorded. observed by phase contrast microscopy.Cell culture on the surface modified PC films RESULTS AND DISCUSSIONRound-shaped polymer films (diameter in 22 mm) were The approach to covalently anchor the polyacrylamides tothoroughly rinsed by acetone for 1 h and water for another the PC surface involves the use of ATRP methods for220 ZHANG ET AL. SURFACE CHEMICAL IMMOBILIZATION OF PARYLENE C
  5. 5. ORIGINAL RESEARCH REPORTgrowing the homo- and co-polymer brushes. ATRP reactions groups onto the aromatic rings of PC, and the surface ele-are typically initiated by the reaction of a copper complex mental ratio of C:O:Cl was 76.9%:11.0%:12.1%. Assumingwith a halide initiator. Although a large number of chlorine that only single chloropropionyl group is bound per aro-atoms exist on pristine PC, they lack sufficient reactivity to matic ring of PC, the substitution ratio of aromatic rings oninitiate the ATRP reaction. By covalently binding chloropro- PC-Cl is about 30% according to the elemental ratio changepionyl groups to the PC aryl groups via Friedel-Crafts reac- from PC to PC-Cl. The binding energies of chlorine on PC-Cltion (Figure 1), the labile chlorines exhibited higher reactiv- also appeared to decrease slightly to 267.8 eV (Cl 2s) andity and thus effectively initiated ATRP reaction, giving 197.7 eV (Cl 2p), indicating the introduction of labile chlo-acrylamide-based polymer brushes on PC (Figure 2). In this rine atoms onto PC surfaces. After ATRP reactions of eitherstudy, the control of Friedel-Crafts reaction condition is not NIPAM or NTBAM on PC-Cl, additional N 1s peaks could beonly important to obtain a high yield of labile chlorine clearly observed at about 396.0 eV for PC-NI and PC-NT,atoms, but also to maintain the bulk mechanical property of suggesting the successful immobilization of the homo pNI-PC. It has been found that low concentration of CPC-AlCl3 PAM or pNTBAM brushes onto PC via ATRP. The surface ele-ligand, low reaction temperature (0 C to room temperature) mental ratio of C:O:N of PC-NI was measured at aboutand moderate reaction time (14–16 h) efficiently promotes 75.6%:12.0%:12.4%, close to the theoretical atomic value ofthe generation of labile chlorines, and the resulting PC-Cl C:O:N for pure pNIPAM at 6:1:1 derived from its molecularfilms exhibited slightly yellow color, insignificant loss of the formula of (C6H9ON)n; and C:O:N:Cl of PC-NT was aboutmechanical strength, and ability to surface initiate ATRP at 76.7%:10.7%:9.9%:2.7%, the ratio of C:O:N was close to theroom temperature; while the PC films that were treated theoretical atomic value of C:O:N for pure pNTBAM at 7:1:1high concentration of CPC-AlCl3 ligand, high temperature or derived from its molecular formula of (C7H11ON)n, indicat-extended time became dark brown and very brittle. ing a full coverage of the polymer brushes on these modi- The reaction of PC-Cl with NIPAM and NTBAM mono- fied substrates.mers under ATRP conditions led to the formation of pNI- A chlorine signal from PC-NI could not be observed inPAM and pNTBAM brushes on the PC surface. The tempera- XPS spectra. The loss of the chlorine signal from the aro-ture of ATRP reactions was maintained between 40 and matic rings of the substrates can be attributed to the forma-45 C for PC-NT and 50 and 55 C for PC-NI to reach the tion of flexible, hydrophilic and thick pNIPAM coatings,maximum grafting amount. It has been observed that lower which is beyond XPS sampling depth (regular 7.5 nm in antemperature did not well initiate the ATRP, resulting in low organic matrix),43 preventing the chlorine atoms on the sub-surface coverage, while the high temperature above 65 C strate from detection. The loss of the labile chlorine signalscauses the decomposition of copper–ligand complex during from the ending groups of the hydrophilic pNIPAM brushesATRP reaction.41 Based on the report for bulk polymeriza- can be attributed to the accelerated hydrolysis in the cosol-tion of pNIPAM, cosolvent of DMF/water was used for ATRP vent of ATRP reaction as reported previously.44,45of PC-NI to obtain high molecular weight of grafted pNIPAM Additional information for the immobilized polymerbrushes.42 The terminal chlorines at the polymer brushes of brushes on the substrate was obtained from XPS high reso-PC-NI and PC-NT were further applied as macro-initiator to lution scan of C 1s. As shown in Figure 3(B), the strongestinitiate ATRP of the second monomer of NTBAM and NIPAM peak at 281.0 eV in PC spectra was attributed to methylenerespectively, to form copolymer brushes. Cosolvent of DMF/ and aryl carbons. The C 1s peak of PC-Cl was increased duewater was used for ATRP of PC-NT-NI and dry DMF as sol- to the incorporation of amide (C¼ ¼O) groups. Peak-fittingvent for PC-NI-NT. As will be discussed below by XPS and was not performed for surface modified PC due to the exis-FT-IR analysis, PC-NT-NI was formed but PC-NI-NT was not tence of various carbons bonds and interaction in the poly-after second step of ATRP. mer brushes and between the substrates, but the shake-up satellite signals (p–p* transition) for PC-NI, PC-NT and PC-XPS and FT-IR characterization of polymer brushes NT-NI could be clearly observed, indicating the incorpora-grafted on PC tion of the pNIPAM and pNTBAM and their copolymerThe resultant polyacrylamide thin films were characterized brushes onto PC surface.by XPS and FT-IR methods. The XPS survey scan spectra of To further characterize the surface chemical composi-PC, PC-Cl, PC-NI, PC-NT, and PC-NT-NI are shown in Figure tion, ATR-FTIR was also performed to analyze the infrared3(A), XPS analysis were performed at several areas on absorption of the functional groups on the substrates.modified PC surface, and showed similar data, indicating a Because of the higher penetration depth of the IR radiationhomogenous grafting of the polymer brushes on the surface. beam in ATR-FTIR technique (1–5 lm) than the samplingThe characteristic peaks of carbon and chlorine were depth of XPS (7.5 nm),46 the appearance of carbonylobserved at 281.8 eV (C 1s), 268.4 eV (Cl 2s), and 197.8 eV bands for PC-Cl in FTIR (Figure 4) suggested that the Frie-(Cl 2p) for PC, and the XPS surface elemental analysis del-Crafts reaction was not only confined to the near surfaceshowed the ratio of C:Cl at 89.4%:10.6%, close to the theo- region, the CPC-AlCl3 ligands also penetrated into a depth ofretical atomic ratio of pure PC for C:Cl at 8:1 derived from PC surface structure, reacting with the aromatic rings. Itits molecular formula of (C8H7Cl)n. After Friedel-Crafts reac- was found that the PC films became dark brown and brittletion, an additional peak of O 1s could be observed for PC-Cl after an extended Friedel-Crafts reaction time (24 h) or atat 529.5 eV due to the incorporation of propionyl chloride a high temperature ( 45 C), this may be attributed to theJOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JAN 2012 VOL 100B, ISSUE 1 221
  6. 6. 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 cmÀ1 (C¼O stretch), amide group on PC-NI and PC-NT can ¼ be found at 1646 and 1654 cmÀ1 (C¼O stretch), respectively, PC-NT-NI ¼ shows an amide bond absorption peak between 1646 and 1654 cmÀ1. moisture in the air, which can be indicated by the broad ab- sorbance peak between 1693 and 1710 cmÀ1, and the appearance of an absorbance peak at about 2940 cmÀ1 in FTIR 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 or higher. Interestingly, maintenance of the Freidel-Crafts reac- tion of PC at 22–24 C could lead to the formation of a strong absorption at 1694 cmÀ1 with broad band (spectra not shown), but the resulting PC-Cl exhibited low reactivity and could only initiate the ATRP at the temperature aboveFIGURE 3. (A) XPS survey scan analysis of PC, PC-Cl, PC-NT, PC-NI, 50 C, this may be due to the higher amount propionic acidPC-NT-NI, and PC-NI-NT. No chlorine peaks (Cl 2s and Cl 2p) wereobserved for PC-NI, indicating PC-NI was unable to further initiate the on PC-Cl by alkylation reaction.ATRP of NTBAM to form the block copolymer brushes. * PC-NI-NT In this study, cosolvent of DMF/water was used in ATRPcould not be formed and no chlorine signals were shown in its XPS of NIPAM to obtain the hydrophilic polymer brushes withspectra. (B) High-resolution XPS spectra of the carbon C 1s signal foreach substrate. The shake-up satellite signal (p–p* transition) pointed maximum length according to the study of bulk pNIPAM po-by arrow heads can be clearly observed for PC-NI, PC-NT, and PC-NT- lymerization,47 and dry DMF was used as solvent for theNI due to the grafted polymer brushes. ATRP of NTBAM to form the hydrophobic polymer brushes. The peaks of amide bond (amide I band, secondary amidedecrement of the polymer chain alignment by the over-reac- ¼O C¼ stretch) in the polymer brushes were observed in PC-tion of polymer with polar CPC-AlCl3 ligands. Comparison of NI spectra at 1646 cmÀ1, while in PC-NT spectra at lowerthe surface FTIR spectra gave more information about Frie- value of 1654 cmÀ1 due to the lower chains flexibility, po-del-Crafts reaction and ATRP reaction. Formation of the acyl larity and existence of weaker intermolecular hydrogengroups on PC-Cl is included in a broad band between 1693 bond interaction of the pNTBAM brushes.and 1710 cmÀ1 (C¼ stretch), this broad C¼ absorption ¼O ¼O To roughly quantify the pNIPAM and pNTBAM on theband can be attributed to the coexistence of alkylation and substrate, the strength of the absorption peaks from amideacylation reaction by the active chlorine groups of CPC dur- bonds of polymer brushes on PC-NI or PC-NT was used, anding Friedel-Crafts reaction. Both chlorine atoms on CPC par- the absorption peak at 1607 cmÀ1 arising from aromaticticipated in the electrophilic substitution of the hydrogen rings of substrate was used as the reference. The character-atoms on the aromatic rings of PC, resulting in the mono- istic peak strength ratio of pNIPAM/substrate (I1646/I1607)substituted aromatic rings with propionyl chloride groups for PC-NI or pNTBAM/substrate (I1654/I1607) of PC-NT was(i.e., ACH2CH2C(O)Cl) or with 2-chloropropionyl groups (i.e., compared to the solvent-cast polymer layer with knownAC(O)CH2CH2Cl), the chlorine atoms on these short ali- amount of the polyacrylamide. Calculated results showed $phatic chains could further react with other aromatic rings 7.2 lg cmÀ2 pNTBAM on PC-NT and 9.7 lg cmÀ2 pNIPAMto form a-acetonic group (i.e., AC(O)CH2CH2A) between on PC-NI; the approximate thickness of grafted pNTBAMmultiple aromatic rings; besides, the propionyl chloride and pNIPAM on the substrates were calculated at about 70groups could further be hydrolyzed into propionic acid by and 88 nm by using the density of bulk pNIPAM (1.10 g222 ZHANG ET AL. SURFACE CHEMICAL IMMOBILIZATION OF PARYLENE C
  7. 7. ORIGINAL RESEARCH REPORTcmÀ3) and pNTBAM (1.04 g cmÀ3). The higher grafted in pNTBAM brushes that less regularly accumulated on theamount of the pNIPAM brushes on PC-NI could be attrib- substrate. The medium roughness of PC-NT-NI between thatuted to the lower steric hinderance of the propyl groups in of PC-NT and PC-NI could be attributed to the moderateNIPAM, which could assist the formation of the longer and conformational rigidity of the pNTBAM-co-pNIPAM brushesmore flexible chains than that of NTBAM with butyl groups. of on substrates. A key characteristic of the ATRP reaction is the preser-vation of the active labile halogen atoms throughout the po- Surface contact angle measurement in response to envi-lymerization. These halogens are effective initiators for the ronmental temperature changegrowth of a second polymer chain, from a different mono- The water droplet contact angle values measured for PC,mer, forming a block copolymer structure (Figure 2).48 Only PC-Cl, PC-NI, PC-NT, and PC-NT-NI were plotted as a func-one of the two polymer brushes discussed above retains tion of temperatures from 13 to 40 C in Figure 6. Astheir Cl initiators. XPS data for PC-NT show the presence of expected, the contact angles of PC and PC-Cl remained con-Cl, while it is absent in PC-NI. These chloride initiators at stant at roughly 90 and 80 , respectively, with no changethe termini of the pNTBAM brushes of PC-NT allowed the over the temperature range used here. The PC-Cl films giveuse of ATRP with NIPAM to add pNIPAM chains to the PC- a lower surface contact angle than that of PC films due toNT materials. Unfortunately, the lack of chloride initiators the hydrogen bonding (albeit weak) observed from the car-on PC-NI made it impossible for us to prepare PC-NI-NT. bonyl oxygen. The block copolymer brushes were also examined on PC-NI shows a temperature dependent hydrophilicity,PC-NT-NI by XPS and FT-IR. Interestingly, although the tend- with the contact dropping from 71 to 52 with a tempera-ency of hydrolysis of chlorine atoms on the grafted pNIPAM ture decrease from 35 to 22 C. The LCST for PC-NI is esti-segments of PC-NT-NI, the chlorine signals were still clearly mated to be 29 C, which was lower than the LCST valueobserved, even though PC-NI showed no chlorine signal. (32 C) of bulk pNIPAM. The lower LCST of PC-NI could beXPS elemental analysis showed the ratio of C:O:N:Cl on PC- attributed to the enhanced hydrophobic interaction betweenNT-NI at 76.2%:11.2%:10.9%:1.7%, a lower chlorine content the grafted pNIPAM brush and the hydrophobic PC sub-than that on PC-NT (2.7%). The chlorine signals on PC-NT- strate. The thermal induced phase transition of the homoNI could be attributed to the unreacted chlorines sur- pNIPAM results from the change of the inter- and intrachainrounded by the hydrophobic pNTBAM brushes and were interactions. The pNIPAM chains of the brush are partiallynot hydrolyzed. immobilized onto the hydrophobic PC surface, and this Figure 4 shows that PC-NT-NI has an amide bond increases the hydrophobic interactions between the sub-absorption at 1649 cmÀ1, it is located between the absorp- strate and propyl groups in pNIPAM, which in turn limitstion peak of 1646 cmÀ1 in PC-NI and 1654 cmÀ1 in PC-NT, the motility and hydrodynamic radius of the polymer brush.and shows greater intensity than either of them, indicating The net effect is an easy collapse of the polymer brush intoan overlap of amide bonds signal from pNTBAM and pNI- the hydrophobic, dense phase at a lower temperature thanPAM segments in PC-NT-NI. The approximate molar ratio of observed for bulk pNIPAM.49–51the pNTBAM and pNIPAM at 58:42 could be obtained from Bulk pNTBAM is hydrophobic with low swelling ratiothe area ratio of fitted peak at 1646 and 1654 cmÀ1 in PC- (0.05 wt %) and shows no thermal shifts of its surfaceNT-NI spectra; the amount of pNIPAM segment on PC-NT-NI properties. The grafted pNTBAM brushes on PC-NT surface,could be further calculated at $5.9 lg cmÀ2, thus the total however, exhibit a rapid decrease in contact angle, from 77thickness of the grafted copolymer brushes on PC-NT-NI is to 60 , when the temperature is decreased from 30 to 20 C.at $123 nm. The LCST of PC-NT was found to be 24 C. Similar tempera- ture-related phase transition was also found for otherSurface morphology characterization by AFM poly(N-substituted acrylamide) brush–water systems,52 andThe sample film surfaces were thoroughly washed by ace- the temperature dependence of these systems could betone and dried at vacuum at room temperature prior to attributed to the changes of the molecular interactionsAFM measurement. As shown in Figure 5, PC films prepared among polymer chains and water. The thermal independentby CVD process were uniform and smooth, with average hydrophobicity of bulk pNTBAM is attributed to the highroughness value Ra of 1.8 nm. An increase of surface rough- intramolecular interaction of the polymer chains, whichness (Ra ¼ 2.3 nm) was found for PC-Cl films with the for- overcomes the extramolecular interaction between polymermation of an irregular granule structure. This increase can chains and water. However, by grafting of the pNTBAMbe explained by the breakdown of the polymer chains in a brushes onto the substrate, the quantity and the interactiondepth of the substrate surface by the CPC-AlCl3 ligand as of the immobilized pNTBAM chains were significantlydiscussed in FTIR analysis. The Ra values of PC-NI, PC-NT, reduced, it resulted in the enhanced extramolecular interac-and PC-NT-NI were about 2.8, 3.8, and 3.4 nm, respectively. tion between polymer chains and surrounding water, thusAmong PC-NI, PC-NT, and PC-NT-NI, PC-NI showed the low- the grafted pNTBAM exhibited water solubility at low tem-est roughness, which can be attributed to the lowest steric perature. The change of the surface energy was further con-hinderance of isopropyl groups and the highest chain flexi- firmed by comparing the surface contact angles among thebility of pNIPAM, while PC-NT showed a highest roughness bulk pNTBAM (85 ), PC-NT (77 ), and PC (91 ) at 24 C.due to the highest rigidity of the tert-butylacrylate groups The lowest contact angle value of PC-NT indicated theJOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JAN 2012 VOL 100B, ISSUE 1 223
  8. 8. 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 brusheson PC, an increase of the surface roughness can be obviously observed. Ra: average roughness.highest surface energy, as well as the stronger hydrogen ments, and the rapid decrease can be explained by the syn-bonding interaction between the grafted pNTBAM brushes ergic hydrophilic effects of the PNTBAM and PNIPAMand water. segments. The PC-NT-NI that was grafted with copolymer brushesof pNIPAM and pNTBAM also showed thermal sensitive Cytocompatibility and cell adhesion/detachment studyhydrophilicity. The contact angle of PC-NT-NI decreased In this study, we further studied the cell adhesion/detach-from 74 to 54 with the temperature decrease from 34 to ment on parylene substrates coated with pNIPAM and pNIT-15 C. The LCST of PC-NT-NI was found to be 22 C, which is BAM brushes (PC-NI, PC-NT and PC-NT-NI) in response to alower than that of either PC-NI or PC-NT. The lower LCST in change of environmental temperature. To evaluate the cyto-such copolymers has attributed to the limited motility of compatibility of the surface modified films, human skinpNIPAM chains at low temperature and enhanced hydropho- fibroblasts were seeded onto the membrane samples of PC,bic interaction between pNIPAM and water, which is caused PC-Cl, PC-NI, PC-NT, and PC-NT-NI and incubated at 37 Cby the reduced amount of structured water in the pNIPAM for 5 days in a combination of cell culture medium and fetalwhen it is copolymerized with other hydrophobic poly- bovine serum. Tissue culture petridishes (TCPs) were usedmers.22,53 As illustrated in Figure 6(B), the grafted copoly- as controls. During cell proliferation, fibroblasts were inmer brushes on PC-NT-NI exhibited an extended ‘coil’ struc- normal flattened appearance but grew on all the samples atture below LCST and aggregated ‘‘globule’’ structure above varying densities, dependent on the substrates, indicatingLCST. The contact angle for PC-NT-NI also showed a slow the existence of the different cell-material interactions fordecrease from 32 to 25 C, and a rapid decrease from 25 to these substrates. The quantitative data in Figure 7 showed15 C; the slow decrease can be attributed to the interfered that proliferation rate of fibroblasts on PC-NT, PC-NT-NI,phase transition of PNIPAM by hydrophobic PNTBAM seg- and TCP control are significantly higher (10 times) than224 ZHANG ET AL. SURFACE CHEMICAL IMMOBILIZATION OF PARYLENE C
  9. 9. ORIGINAL RESEARCH REPORTFIGURE 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 polymerbrushes changes on PC-NT-NI below and above LCST in aqueous solution. The polymer brushes became extended below LCST and collapsedabove LCST.that on PC and PC-NI; and cell proliferation rate on PC-Cl decreased hydrophobicity (contact angle: 81 ) and increasedwas lower than that on PC-NT but still five times higher cell attachment amount (about 35% of that of TCP atthan that on PC or PC-NI; no statistical difference of cell day 5). By grafting polymer brushes onto substrates, PC-NI,numbers was found on PC and PC-NI films during culture. PC-NT and PC-NT-NI showed a further reduction of hydro-The cells on TCP reached confluence at about 7 days, and phobicity, which was reflected by the decrease of contacton PC-NT and PC-NT-NI surface reached confluence after 9 angles to 71 , 77 , and 74 , respectively, at 37 C. Although aand 10 days culture in the incubator. number of cells adhered to PC-NI in first 4 h after seeding, it The difference in cell density on each substrate can be was also found that PC-NI did not support the sustained cellattributed to the various factors of the cell-material inter- adhesion after day 1. A significantly lower cell number wasface, such as surface hydrophobicity, energy, topography, observed on PC-NI than that of PC-NT, PC-NT-NI, and TCP.surface charge, and chemical composition.54–57 For these This result is consistent to the previous research reported bysubstrates, surface hydrophobicity and surface chemical Okano et al., for polystyrene grafted with thick pNIPAM layer,composition are expected to be the main factors. At 37 C, where those researchers observed very little cell adhesion forPC film was very hydrophobic (contact angle of 91 ) with films above 7.9 lg cmÀ2.30 The PC-NI films studied here hadlow surface polarity, leading to a low cell attachment and grafted pNIPAM amount at about 9.7 lg cmÀ2. In contrast,proliferation rate (about 7% of that of TCP at day 5). The cell adhesion and proliferation thus showed a significantPC-Cl surface containing polar carbonyl groups exhibited a increase on PC-NT and PC-NT-NI, reaching 50–70% of theJOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JAN 2012 VOL 100B, ISSUE 1 225
  10. 10. (Figure 8). After 24-h culture, a low cell density was found on PC-NI, similar to the 5-day cell culture. The cells attached 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 chemical modification with mechanical agitation. As the temperature was decreased to 24 C, the attached cells completely detached from the PC-NI substrate and floated above the substrate after incubating for 40 min at (Figure 8D,D’), while the cells on PC-Cl, PC-NT, PC-NT-NI, and TCP control exhibited no significant change (photos were not shown). As the incubation temperature was reduced to 6 C and main- tained for another 100 min, the cell coverage on PC-NT was reduced. The remained cell number on the surface wasFIGURE 7. Quantitative assays of cell proliferation on PC, PC-Cl, PC- counted and the results showed that roughly 10% 6 5% ofNI, PC-NT, PC-NT-NI films, and TCP control. Data represent the meanof three samples (p 0.05). Cells were seeded at 6 Â 104 cells/well the cells on PC-NT detached and floating cells could beand cultured at 37 C in the incubator for 1, 3, and 5 days. Cell showed observed in the medium (Figure 8E,E’); similarly, $35% 6significantly higher attachment and proliferation on PC-NT, PC-NT-NI, 5% of the cells on PC-NT-NI detached, with some of theand TCP control in comparison to PC, PC-Cl, and PC-NI films. Very detached cells forming clumps above the substrate (Figuresmall 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 8F,F’). The cells on TCP control, PC and PC-Cl remained nor-and PC-NI. mal spread shape without significant detachment (Figure 8A,A’,B,B’,C,C’) after 100 min at 6 C. It has been suggestedcell density observed for TCP at day 5. The pNIPAM layer in previously that the exposure of NAH groups of the poly-PC-NT-NI was 5.9 lg cmÀ2, below the maximum level deter- meric brushes to the serum containing medium could causemined by Okano for promoting cell adhesion. Considering the a reduction in cell attachment.60 At a temperature belowfact that the hydrophobicity of the three polymer brush sys- LCST, the hydrophilicity of PC-NI, PC-NT, and PC-NT-NI weretems are comparable, the cell-substrate interaction on these significantly increased, resulting in a higher percentage ofsubstrates is clearly also dependent on surface chemical com- their NAH groups being exposed to the media. The cells onposition.58 A recent study by Lynch et al., examined cell adhe- PC-NT and PC-NT-NI also showed slower detachment ratession on bulk samples of pNIPAM, pNTBAM and their copoly- and higher cell adhesion density than that on PC-NI, andmers, and found very different properties for the different these could be attributed to the slower change of polymerpolymers.59 They found that the cell adhesion and prolifera- chains and relatively higher amount of the unexposed NAHtion on pNIPAM and pNTBAM were indistinguishable in the groups that was caused by the high steric hindrance ofabsence of serum, but showed marked differences when se- butyl groups on pNTBAM segments of PC-NT and PC-NT-NI.rum was added to the growth medium (as was the case with Interestingly, the detached cells returned to grow on theour experiments). The principal source of the difference in polymer brush coated substrates or TCP after 2-day incuba-the behavior of the different materials is in the ability of tion at 37 C, and the cell adhesion/detachment study couldthese polymers to bind fibronectin and albumin. Lynch et al. be repeated with similar results, these results are consistentshowed that high pNIPAM content leads to high binding af- with the research reported by Dr. Okano et al. in their cellfinity for albumin and low affinity for fibronectin, while high adhesion/detachment study for pNIPAM grafted polystyrenepNTBAM content leads to low binding affinity for albumin surface, indicating the maintenance of the cell metabolismand high affinity for fibronecton. It is known that the fibro- and viability after cell adhesion/detachment test.62,63nectin supports the cell proliferation, while albumin reduces All these results from in-vitro cell study will be helpfulit.60 Lynch et al., proposed that the tert-butyl groups of in use of the modified PC as a coating material for thepNTBAM sterically block access to the amide NAH groups, implantable medical devices for tissue adhesion. The coatedpreventing the majority of the serum proteins from binding, device is cytocompatible above LCST and can be controlledwith the exception of fibronectin, which binds most effec- to attach or detach the cells and organ with small or no tis-tively to hydrophobic surfaces.59,61 Both pNIPAM and sue damage by simply adjusting the environmental tempera-pNTBAM based materials give initial cell adhesion, as ture. Incorporation of pNTBAM into pNIPAM on the PCobserved here, but pNTBAM gives markedly more efficient resulted in a lower LCST and significantly improved cell ad-cell proliferation. The thin pNIPAM film of PC-NT-NI partially hesion and growth, it will be a benefit for the long-term fix-blocks the underlying pNTBAM film, limiting serum exposure ation of the medical devices; furthermore, this tissue–mate-to the underlying pNTBAM, thus showing a moderate cell rial adhesion can also be reduced or released at a reducedattachment and proliferation, markedly better than PC-NI, but temperature around the tissue during surgery. By decreasenot as good as PC-NT-NI. of the LCST of the grafted polymer brushes, PC can be Cell detachment study at the reduced temperatures (24 applied for more implants that are frequently exposed to aand 6 C) was performed after 24-h cell culture on each sub- low temperature but with no unexpected detachment. Bystrate at 37 C. PC, PC-Cl, and TCP were used as controls combining other methods, for example, of increasing or226 ZHANG ET AL. SURFACE CHEMICAL IMMOBILIZATION OF PARYLENE C
  11. 11. ORIGINAL RESEARCH REPORTFIGURE 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 Â 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 TCPcontrol 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.decreasing the divalent cations, such as Ca2þ, Mg2þ and/or response to the environmental temperature is in perform-their chelating agents in the media, cell adhesion to the sub- ance and will be reported later.strate could be affected,64,65 these factors may provide addi-tional ‘‘tuning’’ for the temperature sensitive binding to the SUMMARYdifferent substrates. Moreover, the incorporation of pNTBAM Our purpose is to chemically modify the PC surfaces withexhibited an improvement of cell attachment and prolifera- temperature controlled cell adhesion/detachment, andtion on pNIPAM contained layer, this will not only assist the improved cell adhesion and proliferation above LCST. Thesetissue repair after implantation surgery, but also improve properties have been obtained by covalently grafting pNI-the long-term implant–tissue adhesion. The related study PAM, pNTBAM, and their copolymer brushes onto PC viafor short-term and long-term tissue adhesion/detachment in chemical reaction. Hydrophobic pNTBAM brushes on PC-NTJOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JAN 2012 VOL 100B, ISSUE 1 227
  12. 12. exhibit LCST at 22 C, and grafted pNIPAM on PC-NI exhib- 12. Chang TY, Yadav VG, De Leo S, Mohedas A, Rajalingam B, Chenited the LCST at 29 C. The copolymer brush containing CL, Selvarasah S, Dokmeci MR, Khademhosseini A. Cell and pro- tein compatibility of parylene-C surfaces. Langmuir 2007;23:pNTBAM and pNIPAM on PC-NT-NI, obtained by two-step 11718–11725.ATRP, showed the LCST at 24 C. 13. Chang TY, Yadav VG, De Leo S, Mohedas A, Rajalingam B, Chen In cell culture study, PC-NI showed very low cell adhe- CL, Selvarasah S, Dokmeci MR, Khademhosseini A. Cell and pro- tein compatibility of parylene-C surfaces. Langmuir 2007;23:sion and proliferation; while the copolymerized layer on PC- 11718–11725.NT-NI exhibited a significantly improved cell adhesion and 14. Pruden KG, Sinclair K, Beaudoin S. Characterization of parylene-Nproliferation. All the substrates grafted with polymer and parylene-C photooxidation. J Polym Sci A Polym Chem 2003;brushes showed thermal sensitive cell adhesion/detachment 41:1486–1496. 15. Lange K, Grimm S, Rapp M. Chemical modification of parylene Cbut varied according to the substrates; PC-NI showed a coatings for SAW biosensors. Sens Actuat B Chem 2007;125:poor cell adhesion at 37 C but complete detachment at 441–446.24 C; cells on PC-NT showed a high amount of attachment 16. Demirel MC, So E, Ritty TM, Naidu SH, Lakhtakia A. Fibroblastand proliferation at 37 C, and cell detachment increased as cell attachment and growth on nanoengineered sculptured thin films. J Biomed Mater Res B Appl Biomater 2007;81B:219–223.temperature was reduced to 6 C; while PC-NT-NI exhibited 17. Cetinkaya M, Boduroglu S, Demirel MC. Growth of nanostruc-a strong cells attachment at 37 C, as well as up to 35% cell tured thin films of poly (p-xytylene) derivatives by vapor deposi-detachment at 6 C. As PC is in a rapid growth as an effec- tion. Polymer 2007;48:4130–4134.tive coating material of many medical devices, this study 18. Cheng X, Wang Y, Hanein Y, Bohringer KF, Ratner BD. Novel cell patterning using microheater-controlled thermoresponsiveexplored a way to enhance the PC coated implants with ad- plasma films. J Biomed Mater Res A 2004;70:159–168.justable LCST, improved cytocompatibility, as well as ther- 19. Ohya S, Nakayama Y, Matsuda T. Thermoresponsive artificialmal controlled tissue adhesion. extracellular matrix for tissue engineering: Hyaluronic acid bio- conjugated with poly(N-isopropylacrylamide) grafts. Biomacromo- lecules 2001;2:856–863.ACKNOWLEDGMENTS 20. Okajima S, Yamaguchi T, Sakai Y, Nakao S. Regulation of cell ad-The authors appreciate the help of Mr. Christian Gutierrez, Bio- hesion using a signal-responsive membrane substrate. Biotechnolengineering Department at University of Southern California, Bioeng 2005;91:237–243.for providing parylene C films; Dr. Mark Thompson, Chemistry 21. 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