11718
`
`Langmuir 2007, 23, 11718-11725
`
`Cell and Protein Compatibility of Parylene-C Surfaces
`Tracy Y. Chang,†,# Vikramaditya G. Yadav,‡,¶,# Sarah De Leo,§ Agustin Mohedas,^
`Bimal Rajalingam,£ Chia-Ling Chen,| Selvapraba Selvarasah,| Mehmet R. Dokmeci,| and
`Ali Khademhosseini*,£,¶
`
`Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,
`Department of Chemical Engineering, UniVersity of Waterloo, Waterloo, Canada N2L 3G1, Department of
`Biological Engineering, Louisiana State UniVersity, Baton Rouge, Louisiana 70808, Department of
`Biomedical Engineering, Texas A&M UniVersity, College Station, Texas 77843, Center for Biomedical
`Engineering, Department of Medicine, Brigham and Women’s Hospital, HarVard Medical School, Boston,
`Massachusetts 02139, Department of Electrical and Computer Engineering, Northeastern UniVersity,
`Boston, Massachusetts 02115, and HarVard-MIT DiVision of Health Sciences and Technology,
`Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
`ReceiVed June 11, 2007. In Final Form: August 9, 2007
`
`Parylene-C, which is traditionally used to coat implantable devices, has emerged as a promising material to generate
`miniaturized devices due to its unique mechanical properties and inertness. In this paper we compared the surface
`properties and cell and protein compatibility of parylene-C relative to other commonly used BioMEMS materials.
`We evaluated the surface hydrophobicity and roughness of parylene-C and compared these results to those of tissue
`culture-treated polystyrene, poly(dimethylsiloxane) (PDMS), and glass. We also treated parylene-C and PDMS with
`air plasma, and coated the surfaces with fibronectin to demonstrate that biochemical treatments modify the surface
`properties of parylene-C. Although plasma treatment caused both parylene-C and PDMS to become hydrophilic, only
`parylene-C substrates retained their hydrophilic properties over time. Furthermore, parylene-C substrates display a
`higher degree of nanoscale surface roughness (>20 nm) than the other substrates. We also examined the level of BSA
`and IgG protein adsorption on various surfaces and found that surface plasma treatment decreased the degree of protein
`adsorption on both PDMS and parylene-C substrates. After testing the degree of cell adhesion and spreading of two
`mammalian cell types, NIH-3T3 fibroblasts and AML-12 hepatocytes, we found that the adhesion of both cell types
`to surface-treated parylene-C variants were comparable to standard tissue culture substrates, such as polystyrene.
`Overall, these results indicate that parylene-C, along with its surface-treated variants, could potentially be a useful
`material for fabricating cell-based microdevices.
`
`1. Introduction
`Polymeric biomaterials are widely used in therapeutics1,2 and
`diagnostics3,4 as micro- and nanobiosensors for cell-based assays,
`drug delivery, and tissue-engineering applications.5 Polymeric
`microdevices are capable for analyzing cells and proteins,6-8
`
`* Corresponding author. E-mail: alik@mit.edu.
`† Department of Biology, Massachusetts Institute of Technology.
`‡ University of Waterloo.
`§ Louisiana State University.
`^ Texas A&M University.
`£ Harvard Medical School.
`| Northeastern University.
`¶ Harvard-MIT Division of Health Sciences and Technology, Mas-
`sachusetts Institute of Technology.
`# Denotes equal contributions.
`(1) Langer, R. Drug delivery. Drugs on target. Science 2001, 293 (5527),
`58-59.
`(2) Langer, R.; Vacanti, J. P. Tissue engineering. Science 1993, 260 (5110),
`920-926.
`(3) Bashir, R. BioMEMS: state-of-the-art in detection, opportunities and
`prospects. AdV. Drug DeliVery ReV. 2004, 56 (11), 1565-1586.
`(4) Byrne, M. E.; Park, K.; Peppas, N. A. Molecular imprinting within hydrogels.
`AdV. Drug DeliVery ReV. 2002, 54 (1), 149-161.
`(5) Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Microscale
`technologies for tissue engineering and biology. Proc. Natl. Acad. Sci. U.S.A.
`2006, 103 (8), 2480-2487.
`(6) Folch, A.; Ayon, A.; Hurtado, O.; Schmidt, M. A.; Toner, M. Molding of
`deep polydimethylsiloxane microstructures for microfluidics and biological
`applications. J. Biomech. Eng. 1999, 121 (1), 28-34.
`(7) Khademhosseini, A.; Suh, K. Y.; Jon, S.; Eng, G.; Yeh, J.; Chen, G. J.;
`Langer, R. A soft lithographic approach to fabricate patterned microfluidic channels.
`Anal. Chem. 2004, 76 (13), 3675-3681.
`(8) Sia, S. K.; Whitesides, G. M. Microfluidic devices fabricated in poly-
`(dimethylsiloxane) for biological studies. Electrophoresis 2003, 24 (21), 3563-
`3576.
`
`generating tissue-engineering scaffolds,9-11 and miniaturizing
`bioassays for high-throughput experimentation.12 With the recent
`emergence of soft
`lithography, elastomers, such as poly-
`(dimethylsiloxane) (PDMS), have become enabling materials
`for the widespread fabrication and the use of microfabricated
`systems. PDMS offers numerous advantages over traditional
`biomaterials. It is relatively inexpensive, inert, nontoxic, and
`can be easily molded to form microstructures.13 Despite these
`desirable characteristics, PDMS has a number of shortcomings.
`For example, although PDMS has been shown to be compatible
`for short-term culturing of cells,14 little is known of its long-term
`stability in tissue-engineering applications and in vivo diagnostics.
`Therefore, it may be important to explore alternative biomaterials
`that can be used to fabricate biomedical microdevices. Poly-
`
`(9) Bianchi, F.; Vassalle, C.; Simonetti, M.; Vozzi, G.; Domenici, C.; Ahluwalia,
`A. Endothelial cell function on 2D and 3D micro-fabricated polymer scaffolds:
`applications in cardiovascular tissue engineering. J. Biomater. Sci., Polym. Ed.
`2006, 17 (1-2), 37-51.
`(10) Fidkowski, C.; Kaazempur-Mofrad, M. R.; Borenstein, J.; Vacanti, J. P.;
`Langer, R.; Wang, Y. Endothelialized microvasculature based on a biodegradable
`elastomer. Tissue Eng. 2005, 11 (1-2), 302-309.
`(11) Kaihara, S.; Borenstein, J.; Koka, R.; Lalan, S.; Ochoa, E. R.; Ravens,
`M.; Pien, H.; Cunningham, B.; Vacanti, J. P. Silicon micromachining to tissue
`engineer branched vascular channels for liver fabrication. Tissue Eng. 2000, 6
`(2), 105-117.
`(12) Thorsen, T.; Maerkl, S. J.; Quake, S. R. Microfluidic large-scale integration.
`Science 2002, 298 (5593), 580-584.
`(13) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D. E. Soft
`lithography in biology and biochemistry. Annu. ReV. Biomed. Eng. 2001, 3, 335-
`373.
`(14) Lee, J. N.; Jiang, X.; Ryan, D.; Whitesides, G. M. Compatibility of
`mammalian cells on surfaces of poly(dimethylsiloxane). Langmuir 2004, 20 (26),
`11684-11691.
`
`10.1021/la7017049 CCC: $37.00 © 2007 American Chemical Society
`Published on Web 10/04/2007
`
`Downloaded via COPYRIGHT CLEARANCE CTR DCMT DLVRY on September 16, 2020 at 15:25:26 (UTC).
`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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`

`Biocompatibility of Parylene-C
`
`Langmuir, Vol. 23, No. 23, 2007 11719
`
`(chloro-p-xylylene), also referred to as parylene-C, is one such
`potential candidate for fabricating biomedical devices.
`Parylene-C is a thermoplastic, crystalline, and transparent
`polymer that is extensively used as a coating for insulating
`implantable biomedical devices.15 In addition, parylene-C is
`chemically inert and nonbiodegradable. Parylene-C is synthesized
`from a low-molecular weight (MW) dimer, dichloro-di(p-
`xylylene), using a process that involves the decomposition of
`p-xylylene to yield chloro-p-xylylene, followed by the polym-
`erization of chloro-p-xylylene to parylene-C.16 Parylene-C can
`be vapor-deposited onto substrates to generate uniform, pinhole-
`free membranes that can be subsequently dry-etched using oxygen
`plasma to yield microscale features and patterns that are ideal
`for culturing cells.17 The all-carbon structural backbone, high-
`MW, and nonpolar entities make parylene-C highly resistant to
`most chemicals, as well as to fungal and bacterial growth. In
`addition to having conducive biochemical properties, parylene-C
`has a Young’s modulus of (cid:24)4 GPa18 (compared to 0.75 MPa for
`PDMS14)smaking it mechanically robust and highly suitable
`for fabricating stable and reusable microfluidic devices or
`stencils.17-22 Recent studies have shown parylene-C to be more
`hemocompatible and less thrombogenic than silicon.23 Parylene-C
`has also demonstrated high stability in vivo for a variety of
`applications, such as cardiovascular implants.24,25 Furthermore,
`parylene-C is a potentially useful material for in vitro cell culture
`studies. For example, we have developed the use of parylene-C
`stencils for patterning cells and proteins and for generation of
`cocultures with control over the degree of homotypic and
`heterotypic cell-cell interactions.26,27 Another recent study
`provides the methodology for making nanoscale sculptured thin
`films (STFs) out of parylene-C.28 Due to the high surface area
`to volume ratio of the STF, the parylene-C STF supports high
`level of cell adhesion.28 However, despite the apparent bio-
`compatibility of parylene-C, there has been no direct comparison
`of parylene-C to PDMS and other materials commonly used in
`BioMEMS.
`In this study, we compared the biocompatibility of parylene-C
`membranes with PDMS, glass, and optically clear virgin
`polystyrene by analyzing protein adsorption, cell adhesion, and
`cell morphology characteristics on each of these surfaces. In
`addition, we treated parylene-C and PDMS with air plasma and
`coated the surfaces of these substrates with fibronectin to study
`the effects of surface treatments on protein adsorption, cell
`
`(15) Loeb, G. E.; Walker, A. E.; Uematsu, S.; Konigsmark, B. W. Histological
`reaction to various conductive and dielectric films chronically implanted in the
`subdural space. J. Biomed. Mater. Res. 1977, 11 (2), 195-210.
`(16) Hahn, A. W.; Yasuda, H. K.; James, W. J.; Nichols, M. F.; Sadhir, R.
`K.; Sharma, A. K.; Pringle, O. A.; York, D. H.; Charlson, E. J. Glow discharge
`polymers as coatings for implanted devices. Biomed. Sci. Instrum. 1981, 17,
`109-113.
`(17) Tooker, A.; Meng, E.; Erickson, J.; Tai, Y. C.; Pine, J. Biocompatible
`parylene neurocages. Developing a robust method for live neural network studies.
`IEEE Eng. Med. Biol. Mag. 2005, 24 (6), 30-33.
`(18) Chen, P. J.; Shih, C. Y.; Tai, Y. C. Design, fabrication and characterization
`of monolithic embedded parylene microchannels in silicon substrate. Lab Chip
`2006, 6 (6), 803-810.
`(19) Licklider, L.; Wang, X. Q.; Desai, A.; Tai, Y. C.; Lee, T. D. A
`micromachined chip-based electrospray source for mass spectrometry. Anal. Chem.
`2000, 72 (2), 367-375.
`(20) Meng, E.; Wu, S.; Tai, Y. C. Silicon couplers for microfluidic applications.
`Fresenius’ J. Anal. Chem. 2001, 371 (2), 270-275.
`(21) Xie, J.; Miao, Y.; Shih, J.; Tai, Y. C.; Lee, T. D. Microfluidic platform
`for liquid chromatography-tandem mass spectrometry analyses of complex peptide
`mixtures. Anal. Chem. 2005, 77 (21), 6947-6953.
`(22) Xie, J.; Shih, J.; Lin, Q.; Yang, B.; Tai, Y. C. Surface micromachined
`electrostatically actuated micro peristaltic pump. Lab Chip 2004, 4 (5), 495-501.
`(23) Weisenberg, B. A.; Mooradian, D. L. Hemocompatibility of materials
`used in microelectromechanical systems: platelet adhesion and morphology in
`vitro. J. Biomed. Mater. Res. 2002, 60 (2), 283-291.
`(24) Schmidt, E. M.; McIntosh, J. S.; Bak, M. J. Long-term implants of
`parylene-C coated microelectrodes. Med. Biol. Eng. Comput. 1988, 26 (1), 96-
`101.
`
`adhesion, and spreading. Protein adsorption was studied using
`bovine serum albumin (BSA) and immunoglobulin G (IgG), and
`cell adhesion and spreading were studied using NIH-3T3
`fibroblast and AML-12 hepatocyte cell lines.
`
`2. Methods and Materials
`2.1. Fabrication of Parylene-C and PDMS. Three inch silicon
`wafers were first cleaned for (cid:24)10 min using a 1:1 piranha solution
`(equal volume mixture of H2SO4 and H2O2), sufficiently rinsed with
`deionized water, nitrogen-dried, and then coated with hexameth-
`yldisilazane (HMDS). Following pretreatment, the silicon wafers
`were deposited with dichloro-di(p-xylylene) by utilizing a Labcoater
`2 PDS 2010 chemical deposition system (Specialty Coating Systems,
`Indianapolis). Inside the deposition system, dichloro-di(p-xylylene)
`is first vaporized at 150 (cid:176)C and 1 torr and then pyrolyzed at 690 (cid:176)C
`and 0.5 torr to form chloro-p-xylylenesthe monomer of parylene-
`C. A reduction in the chamber temperature causes chloro-p-xylylene
`to condense onto the wafer surfaces to form parylene-C membranes.
`Initial loading of dichloro-di(p-xylylene) onto the silicon wafers
`determines the thickness of the parylene-C membrane at a rate of
`0.5 (cid:237)m/g. With the use of the aforementioned protocol, 10 (cid:237)m thick
`parylene-C membranes were fabricated on silicon substrates.
`The PDMS substrates were fabricated by directly curing a Sylgard
`184 (Essex Chemical) elastomer in the wells of a Costar 24-well
`TC-treated cell culture microplate for nearly 2 h at 70 (cid:176)C, using a
`10:1 weight ratio of elastomer to curing agent.
`2.2. Preparation of Surfaces. A total of eight types of surfaces
`were used in this study. Costar 24-well TC-treated cell culture
`microplates were utilized as optically clear virgin polystyrene
`substrates. Parylene-C experimental samples were prepared by
`carefully cutting the 10 (cid:237)m thick parylene-C membranes (section
`2.1) to form square-shaped pieces of (cid:24)5 mm(cid:2) 5 mm. Each cut-out
`piece of parylene-C was placed and sealed reversibly onto a PDMS-
`coated well in the microplate. Platinum glass coverslips, 18 mm (cid:2)
`18 mm in size, were used as the glass samples. Plasma-treated PDMS
`and parylene-C were obtained by treating the two polymers with air
`plasma in a Harrick PDC-001 plasma treatment chamber for 2 min.
`Protein coating to parylene-C and PDMS surfaces was performed
`by simply incubating a 5 (cid:237)g/mL fibronectin solution on the surfaces
`for 1 h.
`Each substrate was sterilized prior to the experiments. The
`sterilization of plain and plasma-treated surfaces consisted of UV
`irradiation for 30 s, followed by successive washes with 70% ethanol
`and sterile PBS, respectively. The sterilization of fibronectin-coated
`surfaces consisted of UV irradiation for 30 s followed by a 1 h
`incubation of a sterile solution of fibronectin (5 (cid:237)g/mL) on sterilized
`samples of plain PDMS and parylene-C.
`2.3. Surface Property Characterization. 2.3.1. Contact Angle
`Measurements. Contact angles were measured on static drops of
`water on different substrates by using a contact angle measurement
`system (Phoenix 300 plus, SEO) to provide information about
`hydrophobicity of the surfaces (See Table 1). The substrates were
`measured as-received or as-deposited (plain), and additional
`measurements were performed with a subset of these substrates
`(PDMS and parylene-C) that were treated with oxygen plasma and
`were coated with fibronectin. The contact angle measurements were
`
`(25) Eskin, S. G.; Armeniades, C. D.; Lie, J. T.; Trevino, L.; Kennedy, J. H.
`Growth of cultured calf aortic smooth muscle cells on cardiovascular prosthetic
`materials. J. Biomed. Mater. Res. 1976, 10 (1), 113-122.
`(26) Wright, D.; Rajalingam, B.; Karp, J.; Selvarasah, S.; Ling, Y.; Yeh, J.;
`Langer, R.; Dokmeci, M. R.; Khademhosseini, A. Reusable, reversibly sealable
`parylene membranes for cell and protein patterning. J. Biomed. Mater. Res. [Online
`early access]. DOI: 10.1002/jbm.a.31281. Published Online: Aug 29, 2007.
`http://www3.interscience.wiley.com/cgi-bin/fulltext/116310266/HTMLSTART.
`(27) Wright, D.; Rajalingam, B.; Selvarasah, S.; Dokmeci, M. R.; Khadem-
`hosseini, A. Generation of static and dynamic patterned co-cultures using
`microfabricated parylene-C stencils. Lab Chip [Online early access]. DOI: 10.1039/
`b706081e. Published Online: July 25, 2007. http://www.rsc.org/publishing/journals/
`LC/article.asp?doi)b706081e.
`(28) Demirel, M. C.; So, E.; Ritty, T. M.; Naidu, S. H.; Lakhtakia, A. Fibroblast
`cell attachment and growth on nanoengineered sculptured thin films. J. Biomed.
`Mater. Res., Part B 2007, 81 (1), 219-223.
`
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`

`11720 Langmuir, Vol. 23, No. 23, 2007
`
`Chang et al.
`
`substrates
`glass
`polystyrene
`PDMS
`
`parylene-C
`
`Table 1. Contact Angle Measurements
`contact angle (deg)
`plasma-treated
`fibronectin-coated
`
`untreated
`36.3 ( 2.6
`79.1 ( 5.9
`9.9 ( 1.1a
`105.9 ( 4.5
`99.0 ( 6.7
`73.7 ( 3.0b
`105.0 ( 10.4
`4.4 ( 2.4c
`97.2 ( 4.2
`a Measurements made immediately after PDMS was treated in oxygen
`plasma. b Measurements made after 40 min following treatment in oxygen
`plasma. c No significant change observed in measurements made
`immediately after and following 40 min after plasma treatment.
`
`Table 2. Surface Roughness Measurements
`substrate
`roughness (nm)
`1.6 ( 0.6
`1.2 ( 0.2
`19.3 ( 6.3
`19.3 ( 5.4
`29.0 ( 11.5
`2.2 ( 0.6
`0.4 ( 0.1
`3.2 ( 0.6
`
`glass
`polystyrene
`parylene-C
`plasma-treated parylene-C
`fibronectin-coated parylene-C
`PDMS
`plasma-treated PDMS
`fibronectin-coated PDMS
`
`performed by dispensing deionized water drops (5-10 (cid:237)L) on each
`substrate with a micropipette (Ted Pella Inc.). Each data point
`represents an average of >10 independent measurements.
`2.3.2. Surface Roughness Measurements. Surface roughness values
`of four different substrates (glass, polystyrene, PDMS, and parylene-
`C) as received were measured with atomic force microscopy (AFM)
`(Q-Scope 250, Quesant Instrument Corporation) using noncontact
`mode with a cantilever tip (NCS 16, Quesant). Scan areas of 50 (cid:237)m
`(cid:2) 50 (cid:237)m were randomly selected on the substrates. To obtain the
`surface roughness values from an as-deposited thin (10 (cid:237)m) parylene
`membrane, we first peeled the parylene off the silicon wafer and
`then placed it on top of a robust substrate (1 mm thick PDMS slab).
`Afterward, we performed the AFM measurements. To obtain the
`surface roughness of the surface-treated parylene-C, we applied
`surface treatments (O2 plasma treatment or fibronectin coating) on
`the parylene surface mounted on a PDMS slab and performed AFM
`surface roughness measurements. Three independent measurements
`from 5 (cid:237)m (cid:2) 5 (cid:237)m squares of each surface were performed and
`averaged. Roughness values (mean) acquired from various samples
`corresponding to the variations in surface heights are summarized
`in Table 2.
`2.4. Protein Adsorption Measurements. Protein adsorption was
`characterized by incubating 50 (cid:237)g/mL of fluorescein isothiocyanate
`(FITC)-conjugated BSA (Sigma-Aldrich) and 100 (cid:237)g/mL of FITC-
`conjugated IgG (Sigma-Aldrich) on each substrate for 1 h. The
`substrates were encased in aluminum foil to prevent photodegradation
`of the FITC. Following incubation, the substrates were rinsed with
`deionized water and imaged using a fluorescent microscope (Nikon
`TE 2000) with a constant exposure time of 500 ms. Emitted
`fluorescence was then measured using ImageJ pixel brightness
`analysis tool (National Institutes of Health, U.S.A.). The average
`pixel brightness of each image is an indirect measurement of the
`protein adsorption onto the substrates. Control substrates were also
`used to eliminate the effect of autofluorescence from the substrates.
`2.5. Cell Culture. NIH-3T3 fibroblasts were cultured in Dul-
`becco’s modification of Eagle medium (DMEM) (Invitrogen)
`supplemented with 10% fetal bovine serum (Atlanta Biologicals)
`and 1% penicillin-streptomycin (MediaTech). AML-12 hepatocytes
`were preserved in 44.5% DMEM and 44.5% Ham’s F12 media
`(Invitrogen) supplemented with 10% FBS and 1% penicillin-
`streptomycin (Sigma-Aldrich). The cells were maintained under
`humid conditions, at 37 (cid:176)C, and in a 5% CO 2 atmosphere.
`2.6. Preparation of Samples for Cell Adhesion. NIH-3T3
`fibroblasts and AML-12 hepatocytes were trypsinized and resus-
`pended in their respective media to form a 5 (cid:2) 104 cells/mL stock
`solution. A volume of 2 mL of this stock solution was then
`
`incubated on each of the substrates for 6 h. Each well of the Costar
`24-well microplate has a base area of 283.5 mm2. This corresponds
`to a loading density of (cid:24)353 cells/mm2. Next, the substrates were
`rinsed twice with 1(cid:2) phosphate-buffered saline (Invitrogen), and
`cells adhering to the substrates were then fixed using 4% parafor-
`maldehyde (Sigma-Aldrich) and permeabilized with 0.2% Triton
`X-100 (Sigma) for 10 and 5 min, respectively.
`2.7. Visualization and Imaging of Adhered Cells. To count the
`number of adhered cells on each surface, images of fluorescently
`labeled nuclei were collected using a fluorescent microscope (Nikon
`Eclipse TE 2000). Three pictures per well and three wells per substrate
`were analyzed and counted using ImageJ software.
`2.8. Visualization and Imaging of Cells for Estimating Shape
`Factors. To analyze cell spreading on various surfaces, data was
`collected from at least 70 adhered cells per sample. To effectively
`analyze cell shape, the dimensionless shape factor, S, was used to
`compare the spreading of cells. It is computed as
`
`S ) 4(cid:240)A/P2
`
`where A is the area occupied by the cell and P is the perimeter of
`the cell. A shape factor of 1 corresponds to a perfect circle, whereas
`a shape factor of 0 represents a line. Cell shape factors were computed
`utilizing the calibration and measurement features of the SPOT
`Imaging Software.
`
`3. Results and Discussion
`We evaluated the surface properties of parylene-C stencils in
`comparison with other commonly used biomedical materials,
`such as PDMS, glass, and polystyrene. In addition, we analyzed
`the effect of two common surface treatments, oxygen plasma
`and protein coating on these substrates. The surfaces were
`characterized for their hydrophobicity and roughness as well as
`for protein adsorption, cell adhesion, and cell morphology.
`Particular attention was paid to the differences between parylene-C
`and PDMS, due to their emerging applications in biomedical
`microfabrication.
`3.1. Surface Analysis. Surface hydrophobicity and surface
`roughness are important factors in cell adhesion and the resulting
`cellular morphology.14,29-31 In addition, hydrophobicity has also
`been shown to affect protein adsorption.32-35 Therefore, it is
`important to evaluate these properties in parylene-C membranes
`to understand the interaction of mammalian cells with these
`substrates. To assess the hydrophobicity of the surfaces, we
`measured contact angles of as-deposited and treated parylene-C
`surfaces and compared the values to control surfaces (Table 1).
`The substrates varied greatly in their water contact angles, from
`(cid:24)36(cid:176)
`for glass to (cid:24)111(cid:176)
`for PDMS. As-deposited parylene-C
`and plain PDMS were both hydrophobic as they exhibited contact
`angles of (cid:24)100(cid:176), which is consistent with our previous study. 36
`
`(29) MacDonald, D. E.; Rapuano, B. E.; Deo, N.; Stranick, M.; Somasundaran,
`P.; Boskey, A. L. Thermal and chemical modification of titanium-aluminum-
`vanadium implant materials: effects on surface properties, glycoprotein adsorption,
`and MG63 cell attachment. Biomaterials 2004, 25 (16), 3135-3146.
`(30) Miller, D. C.; Thapa, A.; Haberstroh, K. M.; Webster, T. J. Endothelial
`and vascular smooth muscle cell function on poly(lactic-co-glycolic acid) with
`nano-structured surface features. Biomaterials 2004, 25 (1), 53-61.
`(31) Lee, J. H.; Lee, H. B. A wettability gradient as a tool to study protein
`adsorption and cell adhesion on polymer surfaces. J. Biomater. Sci., Polym. Ed.
`1993, 4 (5), 467-481.
`(32) Toworfe, G. K.; Composto, R. J.; Adams, C. S.; Shapiro, I. M.; Ducheyne,
`P. Fibronectin adsorption on surface-activated poly(dimethylsiloxane) and its
`effect on cellular function. J. Biomed. Mater. Res., Part A 2004, 71 (3), 449-461.
`(33) Harnett, E. M.; Alderman, J.; Wood, T. The surface energy of various
`biomaterials coated with adhesion molecules used in cell culture. Colloids Surf.,
`B 2007, 55 (1), 90-97.
`(34) Warkentin, P.; Walivaara, B.; Lundstrom, I.; Tengvall, P. Differential
`surface binding of albumin, immunoglobulin G and fibrinogen. Biomaterials
`1994, 15 (10), 786-795.
`(35) Absolom, D. R.; Zingg, W.; Neumann, A. W. Protein adsorption to polymer
`particles: role of surface properties. J. Biomed. Mater. Res. 1987, 21 (2), 161-
`171.
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`

`Biocompatibility of Parylene-C
`
`Langmuir, Vol. 23, No. 23, 2007 11721
`
`Figure 1. Adsorption of FITC-BSA (A) and FITC-IgG (B) onto each of the substrates. (A) Adsorption of FITC-BSA onto parylene-C
`and PDMS exceeds that on glass and polystyrene. Surface modifications of parylene-C and PDMS show a negative effect on FITC-BSA
`adsorption. (B) FITC-IgG adsorbs onto glass, parylene-C, and PDMS in a comparable manner. Adsorption onto polystyrene is the highest.
`Surface modification of parylene-C and PDMS reduces their affinity for FITC-IgG. In general, plasma treatment and fibronectin coating
`the two polymers reduces their ability to adsorb proteins. The / indicates p < 0.05.
`
`Furthermore, fibronectin-coated parylene-C and PDMS were also
`hydrophobic (contact angles of (cid:24)100(cid:176)). This hydrophobic
`property of fibronectin-coated PDMS is confirmed by results
`obtained by other groups.32 Even though there has not been
`investigation in the past on fibronectin-coated parylene-C, it is
`logical to expect it to be hydrophobic. Because fibronectin
`coatings have no electron donor components and have low surface
`energy,33 materials coated with fibronectin would not form
`hydrogen bonds with water molecules, so they would become
`hydrophobic. Furthermore, air plasma treatment reduced the
`contact angle of both parylene-C and PDMS substrates to less
`
`(36) Selvarasah, S.; Chao, S. H.; Chen, C. L.; Mao, D.; Hopwood, J.; Ryley,
`S.; Sridhar, S.; Khademhosseini, A.; Busnaina, A.; Dokmeci, M. R. A high aspect
`ratio, flexible, transparent and low-cost parylene-C shadow mask technology for
`micro patterning applications. Presented at the 14th International Conference on
`Solid-State Sensors, Actuators and Microsystems, Lyon, France, June 10-14,
`2007; 533-536.
`
`than 10(cid:176). This finding agrees well with the previous findings that
`the formation of hydroxyl groups from the O2 plasma treatment
`process significantly increases the hydrophilicity of surfaces.8
`One of the main drawbacks of using PDMS for fluidic devices
`is that the plasma-induced hydrophilicity of the PDMS surfaces
`is short term.7 In many applications involving fluidics and cells,
`the ability to generate substrates that remain hydrophilic may be
`beneficial. To compare the stability of plasma-treated surfaces,
`we measured the contact angles of plasma-treated parylene-C
`and PDMS surfaces immediately and 40 min after plasma
`treatment. It was observed that although the plasma treatment
`initially decreased the contact angle values, the hydrophilicity
`of a PDMS substrate deteriorated rapidly (Table 1). This is due
`to the viscoelastic properties of PDMS, in which the surface
`molecules “turn over” with time exposing non-plasma-treated
`molecules of the PDMS on its surface. On the other hand, the
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`11722 Langmuir, Vol. 23, No. 23, 2007
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`Chang et al.
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`Figure 2. Adhesion of (A) NIH-3T3 fibroblasts and (B) AML-12 hepatocytes on the various substrates. (A) The cells do not adhere to
`as-deposited parylene-C and plain PDMS. Furthermore, plasma treatment and fibronectin coating of the two polymers increase their adhesiveness
`to NIH-3T3 cells. (B) Similar trends are exhibited by AML-12 adhesion to the various substrates. The / indicates p < 0.05.
`
`contact angle for plasma-treated parylene-C did not change
`significantly after 40 min. The fact that the plasma-treated parylene
`surface stays hydrophilic for longer periods could be advantageous
`for various biological applications. We next examined the surface
`roughness of the parylene-C and PDMS substrates by using AFM.
`As shown in Table 2, as-deposited and treated parylene-C
`substrates were significantly rougher compared to other substrates,
`including glass, PDMS, and polystyrene. Fibronectin-treated
`parylene-C surfaces had the highest roughness values of (cid:24)30
`nm. The higher surface roughness values of parylene-C may be
`due to the irregularities in the deposition process, which were
`further increased with fibronectin molecules adsorbed onto the
`surface. On the other hand, PDMS, glass, and polystyrene were
`much smoother with surface roughness values of <3 nm. An
`increase in surface roughness enhances the protein adsorption
`level, since there is more available surface area for proteins to
`attach.29 When there are more proteins adsorbed onto the surface,
`
`more integrin receptors on the cells will bind to the proteins and,
`therefore, mediate the attachment of cells.29
`
`3.2. Protein Adsorption. To generate substrates that are
`favorable for cell adhesion, a routine procedure is to coat a layer
`of adhesive proteins on the substrates. To measure protein
`adsorption properties of parylene-C relative to other substrates,
`we incubated each sample with fluorescently labeled BSA and
`IgG. First observation we made was that significantly more BSA
`and IgG adsorbed to polystyrene relative to glass. This finding
`is consistent with other studies that BSA and IgG adsorb more
`onto highly hydrophobic surfaces like polystyrene, compared to
`relatively hydrophilic surfaces like glass.14,29,31,33 Similarly, BSA
`adsorption level on plain PDMS and as-deposited parylene-C
`were 3 times higher relative to glass (Figure 1A). On the other
`hand, IgG adsorption levels on plain PDMS and as-deposited
`parylene-C were similar to adsorption on glass. We believe that
`
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`

`Biocompatibility of Parylene-C
`
`Langmuir, Vol. 23, No. 23, 2007 11723
`
`Figure 3. Dimensionless cell shape factor measurements for (A) NIH-3T3 fibroblasts and (B) AML-12 hepatocytes cultured on various
`substrates. (A) NIH-3T3 cells exhibit greater spreading on fibronectin-coated parylene-C and PDMS, as compared to the other substrates.
`(B) Due to nonaxial spreading, shape factor was not an adequate measurement of AML-12 proliferation on the surfaces, and ANOVA was
`not conducted on this data. The / indicates p < 0.05.
`
`this discrepancy is caused by the intrinsic difference in the
`structure of two proteins.
`In addition, we analyzed the effects of plasma treatment and
`initial protein coating on IgG and BSA adsorption. Plasma
`treatment is routinely used to increase the surface hydrophilicity
`of materials, such as PDMS and polystyrene, and can be used
`to modify the surface of parylene-C substrates (Table 1). In our
`studies, plasma treatment of parylene-C and PDMS increased
`the hydrophilicity of the surfaces and reduced the adhesion of
`both BSA (Figure 1A) and IgG (Figure 1B). This is because of
`increased hydrogen bonding between the surface and water
`molecules, which displaces the weak electrostatic interaction
`and hydrophobic interactions between serum proteins and the
`surface.33 In addition, fibronectin coatings, which improve cellular
`adhesion on biomaterials, could also be used to minimize the
`subsequent adsorption of BSA and IgG. This can be explained
`
`by the fact that the adsorption of the first layer of protein results
`in the creation of a thermodynamically stable interface of water
`molecules coupled with the hydrophilic regions of the adsorbed
`protein layer.34 This phenomenon is commonly used in immu-
`noassays, in which an adsorbed layer of protein is applied to
`minimize background adsorption of the antibody to the substrate.34
`Thus, our results indicate that as-deposited parylene-C has
`high BSA and IgG adsorption, while surface treatments on
`parylene-C can be used to decrease levels of adsorption of these
`proteins. The ability to modify the level of protein adsorption
`on the parylene-C substrates is of potential value for various
`biomedical applications and microfabrication techniques.
`3.3. Cell Adhesion and Spreading. To evaluate the cyto-
`compatibility of parylene-C substrates relative to other materials,
`we analyzed the adhesion of and spreading of fibroblast (NIH-
`3T3) and hepatocyte (AML-12) cell lines. In these experiments,
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`11724 Langmuir, Vol. 23, No. 23, 2007
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`Chang et al.
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`Figure 4. Micrographs of NIH-3T3 fibroblasts (A) and AML-12 hepatocytes (B) on various surfaces. The insets contain images which have
`been cropped and magnified for optimal viewing. Scale bar ) 100 (cid:237)m.
`
`cells were seeded on various surfaces and incubated for 6 h, and
`the adherent cells were counted and measur