Acta of Bioengineering and Biomechanics
`Vol. 11, No. 3, 2009
`
`Original Paper
`
`Interaction of parylene C with biological objects
`
`MARTA KAMIŃSKA1, WIESŁAWA OKRÓJ1, WITOLD SZYMAŃSKI1, WITOLD JAKUBOWSKI1,
`PIOTR KOMOROWSKI1, ANDRZEJ NOSAL1, HIERONIM SZYMANOWSKI1, MACIEJ GAZICKI-LIPMAN1,
`HANNA JERCZYŃSKA2, ZOFIA PAWŁOWSKA2, BOGDAN WALKOWIAK1,2,*
`
`1 Institute of Materials Science and Engineering and the Centre of Excellence NANODIAM,
`Technical University of Łódź, Poland.
`2 Department of Molecular and Medical Biophysics and the Centre of Excellence MOLMED,
`Medical University of Łódź, Poland and BioTechMed High Technology Centre, Łódź, Poland.
`
`The aim of the present work was to examine the interactions of parylene C with such selected biological objects as: blood plasma
`proteins, platelets, endothelial cells, and bacterial biofilm produced by E. coli cells. The results obtained strongly support the thesis that
`parylene C is a material worth considering for biomedical use. Parylene C coating on polished medical steel significantly reduces platelet
`adhesion to this surface. On the other hand, in the case of the surface of machined medical steel coated with parylene C, the number of
`adhered platelets is significantly higher. This also means that surface texture of substrate material is very well reproduced by parylene C
`coating and is an important factor facilitating the platelet adhesion. Adsorption of plasma proteins at parylene C surface is very effective,
`and this finding confirms a notion that cell interaction with surfaces is mediated by the adsorbed proteins. In the light of the above, a high
`susceptibility of parylene C surface to bacterial colonization is easy to explain. The results showing reduced proliferation and changes
`in endothelial cell gene expression should also be seriously analysed when parylene C is considered for the use in contact with blood
`vessels.
`
`Key words: blood–material interaction, cell adhesion, endothelial cells, plasma protein, platelets, polymer, protein adsorption, thrombogenicity
`
`1. Introduction
`
`Parylene is a generic name of a class of unusual
`polymers, the principal member of which is poly(para-
`xylylene) presented in figure 1.
`The outstanding importance of xylylene polymers
`arises from the fact that they constitute the only class
`of polymeric hydrocarbon materials that are commer-
`cially produced by a chemical vapour deposition
`(CVD) technique. The parylene process was devel-
`oped in the sixties of the twentieth century [1] and put
`on the market by the Union Carbide Corporation [2].
`A precursor compound for xylylene polymers is
`paracyclophane, a cyclic dimer, whose formula is shown
`in figure 2.
`
`Fig. 1. Structural formula of poly(para-xylelene)
`
`Fig. 2. Structural formula of paracyclophane
`
`_______________________________
`* Corresponding author: Bogdan Walkowiak, Technical University of Łódź, Stefanowskiego 1/15, 90-924 Łódź, Poland, fax:
`+48-42-6312335, e-mail: bogdan.walkowiak@umed.lodz.pl
`Received: August 17th, 2009
`Accepted for publication: October 30th, 2009
`
`Novartis Exhibit 2031.001
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`20
`
`M. KAMIŃSKA et al.
`
`Fig. 3. Schematic representation of the parylene technology and basic chemistry of the process
`
`The dimer molecule consists of two benzene rings
`connected with one another in para positions with eth-
`ylene bridges. Such a structure is subjected to a large
`molecular strain. Because of the strain energy stored in
`its molecule, paracyclophane possesses a number of
`unusual chemical properties, of which it is a relative
`ease of thermal cleavage of ethylene bonds that makes
`it so useful in the thin-film technology. Thermal de-
`composition of the dimer takes place at 650 °C and
`results in a quantitative formation of para-xylylene, an
`extremely reactive monomer species, which forms
`polymer films immediately upon its condensation on
`any surfaces. A schematic representation of the paryl-
`ene process, together with the respective chemical re-
`actions, is presented in figure 3.
`Xylylene polymers have a number of very useful
`properties, ranging from high mechanical strength and
`low friction coefficient, through superb dielectric and
`insulation characteristics to excellent barrier behaviour
`and extraordinary chemical resistance. Since the process
`is also relatively simple to handle, easy to integrate with
`other vacuum technologies, with the resulting coatings
`being characterized by uniform thickness and extraordi-
`nary penetration abilities, it is widely applied in various
`areas of life and technology. There are a number of re-
`view articles, published as entries to several encyclope-
`dia [3]–[5], that discuss these applications in detail.
`Among many potential applications of various
`xylylene polymers, a chlorinated parylene, known as
`Parylene C, is a promising candidate for metallic im-
`plant coatings separating an implant body from the
`surrounding tissues. Unfortunately, little is known
`about its biocompatibility, although some papers sug-
`gest its good hemo- or thrombocompatibility [6], [7].
`Any implant introduced into the human body will
`rapidly interact with body fluids, triggering the first
`and very important response. Proteins present in these
`fluids adsorb on the surface of the implant and, de-
`
`pending on its hydrophobic properties, a very thin
`protein film is created. This initial contact is responsi-
`ble for the further history of the implant interaction
`with the surrounding tissues. Implants designed for
`tissue integration should exhibit a high affinity to
`proteins of body fluids, since these proteins mediate
`further cell–surface interactions. Those materials that
`are intended for a contact with blood should exhibit
`quite different properties. On one hand, adhesion of
`platelets to the implant surface should be excluded
`because of the possibility of thrombosis. On the other
`one, however, both adhesion and growth of endothe-
`lial cells, lining blood vessels, are welcome. This
`situation makes it difficult to design and to produce an
`appropriate implant surface.
`The above difficulties are additionally deepened by
`the susceptibility of artificial surfaces to bacterial colo-
`nization, also mediated by the adsorbed proteins. Once
`a microbial biofilm is formed, it is extremely difficult
`to be overcome, which very often causes health prob-
`lems. Tissue–surface interactions on a molecular level
`and a possible risk resulting from implant presence in
`the human body are described by WALKOWIAK [8].
`The aim of the present work was to examine the in-
`teractions of parylene C, coating medical steel substrates,
`with such selected biological objects as: blood plasma
`proteins, platelets, endothelial cells and E. coli cells.
`
`2. Materials and methods
`
`The samples for study were prepared as follows:
`round bar (8 mm in diameter) of commercially avail-
`able stainless steel (AISI 316 L) was cut into 3-mm
`thick discs. The samples obtained by means of ma-
`chining or polishing procedures were then coated with
`parylene. The coating procedures were carried out in
`
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`
`

`

`Interaction of parylene C with biological objects
`
`21
`
`a self-designed parylene deposition reactor [9] based
`on the process presented schematically in figure 3.
`Before each coating procedure, substrates were put in
`the reactor’s deposition chamber and a carefully
`weighed amount of the dimer was placed in the sub-
`limer. For one μm of the coating thickness we need
`approximately 1 g of the dimer. After the evacuation
`of the reactor down to the pressure of 1 Pa, the py-
`rolysis oven was heated up to 650 °C. When the oven
`reached the desired temperature, another heater,
`heated up to 150 °C, was inserted onto the sublimer.
`This initiated the deposition process, which was later
`carried out up to a complete expenditure of the dimer.
`Medical steel was coated with parylene powder parti-
`cles (ca. 1-mm mean diameter) which was carried out
`in a tumbler rotating reactor.
`Hydrophobicity of the surfaces studied was esti-
`mated by the measurement of a contact angle of a drop
`of deionized water. The values of the contact angle
`were determined with the use of commonly available
`software Image J.
`For testing the plasma protein adsorption under
`flow conditions, parylene C was deposited onto com-
`mercially available glass plates of the sensor pre-
`coated with gold (SIA kit Au, BIACore AB, Uppsala,
`Sweden). For the proper operation of the employed
`BiaCore X instrument, the thickness of parylene layer
`must not exceed 20 nm.
`Adsorption of blood plasma proteins at parylene C
`surface, under flow conditions, was measured with
`BiaCore X system (BIACore AB, Uppsala, Sweden).
`The prepared sensor was subjected to a routine test
`of sensitivity [10]. Next, the sensor was brought to
`a contact with flowing diluted blood plasma proteins.
`Changes in the mass of adsorbed proteins were pro-
`portional to the surface plasmon resonance (SPR)
`signal. For experiments we used human blood plasma
`diluted 1000 times, and the flow of plasma proteins
`was changed in the range between 10 and 100 μl/min.
`As a reference surface, pure gold film was used.
`Interaction of parylene C surface with platelets
`was studied by a standard method developed in our
`laboratory [11]. Blood used for experiments, accepted
`by the Bioethical Committee of the Medical Univer-
`sity of Łódź, was collected from healthy volunteers.
`The donors have not been treated with any antiplatelet
`drugs for at least two weeks prior to the examination.
`The investigated surfaces were immersed in the whole
`citrated blood at 37 °C for one hour. Blood was con-
`stantly kept in motion by end-to-end mixing. There-
`after, the samples were rinsed twice in 0.1 M phos-
`phate buffer, pH 7.4. The fixing procedure was carried
`out with glutaraldehyde and sample dehydration was
`
`achieved with ethanol used in an increasing concen-
`tration. Finally, the surface was sputtered with a thin
`layer of gold. Quantitative analysis of SEM images,
`obtained from thirty randomly selected areas, was
`carried out for every sample.
`Endothelial immortalized cell line EA.hy 926 was
`used for the experiment [12]. Cells were grown, in the
`presence of medical steel powder particles both coated
`with parylene or not coated, to 80% of confluence.
`For a control, cell culture without any powder parti-
`cles was employed. Total RNA was then isolated from
`the cells. The purity and quality of the RNA obtained
`were evaluated by electrophoresis in 1.5% agarose gel.
`As the next step, cRNA was obtained in PCR reaction,
`and then it was amplified and labelled with biotin-16-
`dUTP. The synthesized molecular probes (biotin-
`cRNA) were used for hybridization (12 hours) with
`oliogonucleotide fragments immobilized on commer-
`cially available arrays (SuperArray). Chemiluminescent
`reaction was triggered by adding CDP-Star (1,2-
`dioxetane) and recorded by means of autoradiography.
`Gene expression was determined by the spot darkness
`analysis on X-ray film (Kodak) by using Image Scan-
`nerII and ImageMaster 2D software (Amersham Bio-
`tech.). For proliferation experiment the cells were grown
`on the parylene C surface coating the disc of medical
`steel substrate. Proliferation and viability of the cells
`were tested with bis-benzimide (live cells) and propid-
`ium iodide (dead cells) fluorescence probes [13].
`E. coli cells were cultured on the surface of paryl-
`ene C coating a medical steel substrate. The samples
`were incubated for 24 h in a medium containing E.
`coli cells (DH5α strain) at 37 °C under stationary or
`flow conditions. An electromagnetic stirrer set at 150
`or 350 rpm forced the rotational flow. After incuba-
`tion, sample surfaces were extensively washed with
`deionised water and labelled by immersion in 10 ml of
`a fluorescent dyes solution. The solution contained
`two fluorescent dyes, bis-benzimide and propidium
`iodide, which made the visualization of both live and
`dead cells possible [14].
`Both F-Snedecor’s test and unpaired Student’s
`t-test or alternatively nonparametric ANOVA test
`were used for statistical analysis of the results. The
`values of p < 0.05 were considered as significant.
`
`3. Results
`
`Independently of the way of preparing medical
`steel samples, parylene C coatings increase their sur-
`face hydrophobicity (figure 4).
`
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`

`

`22
`
`M. KAMIŃSKA et al.
`
`Fig. 4. Photographs of the drop of deionised water (20 μl)
`on the surfaces examined. The measured values of contact angle
`are presented next to the photographs
`
`Fig. 5. Example of sensograms obtained
`for blood plasma proteins
`flowing by parylene C surface
`
`Fig. 6. Platelets adhered to the surfaces studied.
`A and C – parylene C coating samples of polished and machined
`medical steel, respectively, B and D – polished and machined
`medical steel, respectively
`
`Protein adsorption at parylene C surface was as-
`sessed at different flow rates, i.e. 10, 25, 50, and
`100 μl/min. It is evident that an increase in shear
`stress, with increasing flow rate, caused a decrease in
`the amount of adsorbed proteins (figure 5).
`Table 1 summarizes the results of measurements of
`blood plasma proteins adsorption on parylene C and
`on control gold surfaces.
`One-hour contact of the samples tested with citrated
`whole blood resulted in adhesion of numerous platelets
`to the surfaces. Only single platelet adhesion, without
`forming any aggregates, and all three forms of platelet
`activation were observed for all the samples (figure 6).
`The highest and the lowest number of adhered
`platelets were found respectively on the machined
`
`Table 1. Comparison of mass change on the sensor surfaces as a function of the flow rate. Shear stress was calculated
`for regular cuboid channel, and amount of adsorbed proteins was estimated from the following approximation: 1 R.U. ~ 1 pg/mm2
`Gold
`Parylene C
`Gold
`Parylene C
`Gold
`Parylene C
`Gold
`Parylene C
`10
`25
`50
`100
`0.74
`1.92
`3.83
`7.39
`
`259.4
`+10.3
`0.26
`
`110.3
`+12.2
`0.11
`
`204.9
`+56.7
`0.20
`
`Flow (μl/min)
`Shear stress (Pa)
`Mass change (R.U.)
`
`Amount of proteins
`(ng/mm2)
`Significance
`
`335.9
`+60.8
`0.34
`
`1186.5
`+62.9
`1.19
`
`181.3
`+22.5
`0.18
`
`342.8
`+32.1
`0.34
`
`139.8
`+19.9
`0.14
`
`S (p < 0.003)
`
`S (p < 0.03)
`
`S (p < 0.02)
`
`NS
`
`Surface
`
`Table 2. Mean number of adhered platelets per surface unit
`Number of
`Significance
`adhered
`platelets per
`surface unit
`1.82+0.18
`Polished medical steel
`0.89 + 0.09
`Polished medical steel coated with parylene C
`2.45+0.23
`Machined medical steel
`Machined medical steel coated with parylene C 3.07+0.24
`
`S (p < 0.05)
`
`NS
`
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`
`

`

`Interaction of parylene C with biological objects
`
`23
`
`medical steel and on the polished medical steel sur-
`faces coated with parylene C. A detailed analysis of
`the data collected indicates that parylene C deposited
`onto polished medical steel significantly reduces the
`platelet adhesion (table 2).
`The presence of parylene C powder particles
`caused any measurable changes neither in endothelial
`cells proliferation nor in their viability, but the culture
`of the cells on the parylene C surface significantly
`reduced cells proliferation and increased their mortal-
`ity (figure 7 and table 3).
`
`Fig. 7. EA.hy 926 cells grown on the surfaces studied
`were labelled with with bis-benzimide and propidium iodide
`
`Table 3. Endothelial cells proliferation and mortality assessed for
`cells cultured on parylene surface. As the control surface a standard
`COSTAR multi-well cell culture plate was employed. Each sample
`was subjected to fluorescence microscopy examination,
`and six randomly selected areas were documented and processed
`
`Surface
`
`Control
`Parylene C
`Significance
`
`Number of
`independent
`readings
`6
`6
`
`Mean number
`of living cells
`
`Mean number
`of dead cells
`
`5 + 1
`682 + 38
`33 + 5
`77 + 52
`S (p < 0.0001) S (p < 0.0001)
`
`The culture of endothelial cells in the presence of
`parylene C coating medical steel substrate resulted in
`a high level of changes in expression of genes respon-
`sible for cell cycle and apoptosis path. It mainly con-
`cerns cyclins, cyclin-dependent kinases and genes re-
`sponsible for apoptosis. Changes occur also in genes,
`which are responsible for cell proliferation and growth.
`The gene expression changes were detected for both
`materials, i.e. medical steel and parylene C, but paryl-
`ene C caused more changes and they were more pro-
`found (see figures 8 and 9 and table 4).
`
`Table 4. Summary of changes in gene expression observed with cell cycle and apoptosis microarrays.
`Position of the particular gene on the microarray is presented in parentheses
`
`Gene type
`
`S phase and DNA replication
`
`G2 phase and G2/M
`transition
`G1 phase and G1/S transition
`
`Cell cycle checkpoint
`and cell cycle arrest
`Regulation of the cell cycle
`
`Negative regulation
`of the cell cycle
`M phase
`
`p53 and DNA damage
`response
`TNF receptor family
`
`Death domain family
`Bcl-2 family
`
`IAP family
`
`Caspase family
`Anti-apoptosis
`
`Parylene coating polished medical steel
`
`Polished medical steel
`OligoGEArray® Human Cell Cycle microarray
`(gene position on the cell cycle microarray)
`overexpression (89)
`overexpression (89, 113)
`suppression (109, 113)
`suppression (85, 109)
`overexpression (5, 26)
`overexpression (5, 12, 26, 81)
`suppression (4)
`suppression (4)
`suppression (36)
`overexpression (36)
`suppression (23)
`overexpression (16, 36, 50, 55, 56, 57)
`suppression (8, 27, 62, 63, 82, 83, 96, 112)
`overexpression
`(17, 33, 35, 41, 42, 60, 67, 75, 94, 95)
`suppression (2, 22, 24, 28, 48, 51, 66, 70, 90)
`overexpression (13)
`suppression (112)
`suppression (98)
`
`overexpression (16, 55, 56)
`suppression (8, 36, 50, 57, 62, 82, 83, 112)
`overexpression (17, 33, 35, 42, 60, 67)
`suppression (2, 22, 24, 28, 41, 48, 51, 69, 75, 90, 94, 95)
`
`overexpression (13)
`suppression (112)
`
`OligoGEArray® Human Apoptosis microarray
`(gene position on the apoptosis microarray)
`overexpression (3, 67)
`overexpression (3, 67)
`
`overexpression (84)
`suppression (80, 87)
`overexpression (66, 108)
`overexpression (34, 73)
`suppression (13)
`overexpression (28)
`suppression (25)
`suppression (50)
`suppression (70)
`
`overexpression (84)
`suppression (80, 87)
`overexpression (66, 108)
`overexpression (73)
`suppression (13)
`suppression (25)
`
`suppression (50)
`suppression (70)
`
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`Regeneron v. Novartis, IPR2021-00816
`
`

`

`24
`
`M. KAMIŃSKA et al.
`
`4. Discussion
`
`The results presented strongly support the thesis
`that parylene C is worth considering for biomedical
`use. It is also worth noting that parylene C coating on
`polished medical steel significantly reduces platelet
`adhesion to this surface. On the other hand, in the case
`of the surface of machined medical steel coated with
`parylene C, the extent of platelet adhesion is signifi-
`cantly higher. This also means that parylene coating
`very well reproduces the surface texture of substrate
`material and is an important factor improving the
`platelet adhesion. Adsorption of plasma proteins at
`parylene C surface is very effective, and this finding
`confirms that the proteins adsorbed mediate cell inter-
`action with surfaces. In the light of the above, a high
`susceptibility of parylene C surface to bacterial colo-
`nization is easy to explain.
`Some authors reported low or none cytotoxicity of
`parylene used for coating of implantable devices [15],
`[16]. In our opinion, the presence of parylene C in the
`culture media affected neither the proliferation of endo-
`thelial cells nor viability, but even then numerous
`changes in gene expression were observed (figures 8 and
`9 and table 4). In contrast to the above, our attempt to
`culture endothelial cells directly on the parylene C sur-
`face resulted in strongly reduced cell proliferation and
`accelerated cell death (figure 7 and table 3). These ob-
`servations are compatible with in vitro observation of
`a high number of fibroblasts around the parylene C
`coated device and only a moderate number of these cells
`found on the surface [17]. Another report [18] describes
`deeply inhibited proliferation of neuronal cells on paryl-
`ene C, but not on the other forms of parylene.
`Summing up, the application of parylene C coating
`onto well-polished medical steel significantly im-
`proves the thrombocompatibility of the devices pro-
`duced. On the other hand, a long contact time of par-
`ylene C surface with human body facilitates a growth
`of microbial biofilm and introduces a potential risk of
`health complication. Although currently it is difficult
`to interpret them unequivocally, changes in gene ex-
`pression should be considered with caution. A poten-
`
`Fig. 8. An example of OligoGEArray® Cell Cycle Human
`microarray
`
`Fig. 9. An example of OligoGEArray® Apoptosis Human
`microarray
`
`The results obtained for both stationary and flow
`conditions showed that the parylene C surface was
`very susceptible to E. coli colonization, much more
`than medical steel surface, and the toxicity of the
`bacteria on this material was low and comparable to
`that of medical steel (figure 10 and table 5).
`
`A
`
`B
`
`Fig. 10. E. coli cells found on surfaces of parylene C (A)
`and medical steel (B) under stationary conditions
`
`Surface
`
`Polished medical steel
`Polished medical steel
`coated with parylene
`Significance
`
`Table 5. Bacterial biofilm formation on surfaces examined
`Surface occupied by E. coli cells (per cent of total surface)
`Live/death test
`Per cent of live
`Laminar flow
`Turbulent flow
`Stationary conditions
`E. coli cells
`(150 rpm)
`(350 rpm)
`91.07+35.77
`1.35+0.31
`0.83+0.34
`0.02+0.01
`3.55+0.62
`3.22+0.62
`0.65+0.17
`91.04+23.84
`NS (p > 0.8 )
`S (p < 0.004)
`S (p < 0.002)
`S (p < 0.02)
`
`Novartis Exhibit 2031.006
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Interaction of parylene C with biological objects
`
`25
`
`tial risk resulting from such changes should seriously
`be taken into account and biocompatibility tests with
`the use of molecular biology techniques should rou-
`tinely be performed for any type of a new biomaterial.
`
`Acknowledgements
`
`This work was supported by projects N205 072 31/3208 and
`05/WK/P01/0001/SPB-PSS/2008.
`
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`Novartis Exhibit 2031.007
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