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Preparation protein-resistant surfaceson bio-stainless steel by microwave cold plasm

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Abstract:Microwave cold plasma surface modification technique was investigated to enhance biocompatibility of bio-stainless steeltype 316L by resisting proteins attach to surfaces. Based on high resolution XPS and ATR-FTIR spectrum, this coating was athin poly (ethylene glycol)-like film deposited in a microwave cold-plasma of tetraglyme conditions and built up mainly of—CH2—CH2—O—linkages on surfaces, and oxygen and carbon formed covalent bond on the interfacial layer between metal andorganic films. Plasma protein was adsorbed onto control and stainless steel surfaces, and the relative adsorbed amount of pro-teins on the surfaces waevaluated by ESCA. The adsorbed protein on modified-surface decreased rapidly compared with controland exhibited particularly effective in preventing protein adsorption.
  Key words:stainless steel; microwave cold-plasma; preparation; film; protein adsorption
  1 Introduction
Stainless steel type 316L is a material widely used for medical implant purposes, for example, for artificialhips. The first use of stainless implant in modern medicine is dated back to 1926[1]. E.W. Haey Groves used18-8 stainless screws in fixation of femoral neck fracture. Following this landmark event several advances havebeen made in the use of materials in the human body[2]. Biocompatibility is essential to successful biomedical de-vices. One of the main problems of using biomaterials has been surface-induced thrombus formation, which is
initiated by the adsorption of certain plasma proteins[3]. More importantly, the adsorbed protein layer can fur-ther mediate additional biological responses, like cell attachment and activation, and can create unpredicted per-turbations to device operation. When evaluating the biocompatibility of the 316L, both of these must be consid-ered[1~3]. Much concern exists over both these issues in the case of 316L. Dissolution of Fe ions and the possi-bility of inducing allergic and toxic effects associated with the biological properties of Fe is the greatest prob-lem[4]. Surface-induced thrombus formation which is initiated by the adsorption of certain plasma proteins is re-lated to Ferelease ranges from excellent to poor in the body. Some plasma-polymerize coatings have better cor-rosionresistancandbiocompatibility, which have been used to improve the corrosion resistance and corre-sponding stables of 316L. Non-specific protein adsorption is surface reactions, surface modification of stainlesssteel is one technique to control protein adsorption[3,4]. A promising approach is based on poly (ethylene gly-col), or PEG. PEG has been used most widely for surface modification because of its unique properties such ashydrophilicity, flexibility, high exclusion volume in water[5]. PEG is also non-toxic and FDA-approved for use
in biotechnology and consumer applications. Because PEG lacks the mechanical integrity and strength, it is usedas a coating rather than as a structural material. To a large extent, the success of PEG surface modification is afunction of surface coverage of ethylene oxide moieties[6]. Therefore, the choice of coating technique can have adirect impact on the coating performance.In this study, we were present a surface modification technique to produce stainless steel surfaces that showlow levels of protein adsorption. Electron cyclotron resonance (ECR) microwave cold-plasma polymerization oftetraglyme (pp4G) (CH3—O—(CH2—CH2—O)4—CH3) is one of the effective methods because of its rapidand uniform creation of active radicals without altering the bulk properties of the biomaterials. It is a gas-phase,thin-film deposition process. This process was capable of producing thin solid PEG-like coatings that offer uni-m surface coverage and protein resistance. The chemistry of deposited coatings were characterized by X-rayphotoelectron spectroscopy (XPS) and attenuated total reflection-Fourier transform IR (ATR-FTIR) spectral a-nalysis. Plasma protein was adsorbed on to stainless steel and then evaluated by XPS analysis, the modified sur-faces were capable of significantly reducing protein adsorption.
2 Experimental
2.1 Sample preparation
The material used in this work was a medical stainless steel type 316L in sheet form (0.80mm in thickness)produced by Lanzhou Seemine SMA Co. Ltd in the flat annealed condition. Chips of 1cm2of surface area werecut from the sheet and washed in a hot acetone detergent, methyl alcohol (AR, Tianjin Bodi Chemical Co.,Ltd) for 30min, rinsed five times in de-ionized water by ultrasonic clean method. Tetraglyme (AR, ShanghaiReagent Factory) was degassed before use but was not subjected to any purification procedure.
2.2 Plasma polymerization of pp4G
The apparatus for used preparation plasma polymerization coatings in this study was an electron cyclotronresonance (ECR) microwave cold plasma system. With the frequency 2.45GHz, power 130kW in 1.5ms pulsewas focused by dielectric lens into the vacuum chamber[7]. All plasma treatment were preceded by evacuationand purging of stainless steel, vapor, and gas supply lines with, and oxygen-exposure of the reaction chamberto a 30min oxygen-plasma environment, tetraglyme vapor was introduced to the chamber at a flow rate 1.3ml/s. The plasma power is first maintained at 80W for 2minutes at 350mT, than reduces to deposit the functionalPEG-like coating. It is maintained at 15W for one minute, then at 10W for another one minute for a total plas-ma-on time of 4minutes. All treatments are carried out under a room temperature in this study. Additional de-tails of the monomer preparation and the deposition process will be discussed elsewhere[8]. At the end of theplasma reactions, the samples are aseptically removed from the reactor and stores in clean, sterile petri dishesuntil analytical evaluations were initiated.
2.3 XPS analysis
X-ray photoelectron spectroscopy (XPS) is a simple excitation process that makes peak shift analysis easierto interpret for binding state investigations. In this research, the XPS measurements were performed at roomtemperature (25℃) samples with a XSAM800KRATOS instrument system employing monochromatic Mg Kαradiation at 300W and 20mA. A take-off angle of 55°was used in all sample analysis. Survey scans were meas-ured for atomic species on the surfaces over a binding energy range of 0~1000eV using a pass energy of 89.45eVat 1eV/step. Lower pass energy (17.90eV at 0.1eV/step) was employed in multiplex scan.
2.4 ATR-FTIR analysis
Nicolet Impact 420 Fourier transform infrared (FTIR) was used to characterize the compositions for theplasma-deposited coatings. FT-IR spectra were recorded using ATR mode from layers deposited on stainlesssteel surfaces. The differential spectra of plasma polymer were recorded by subtracting the ATR spectrum ofthe stainless steel substrate.
2.5 Plasma protein adsorption experiment
Plasma protein (Wuhan Tianhui Bioengineering Co., Ltd.) was diluted with phosphate-buffered saline(PBS) to make a 1% solution. The undeposited and oxygen/pp4G plasma deposited stainless steel hydrated inPBS five times at 37℃was placed in contact with the above solution at the same temperature for 30min. Thesamples were washed with PBS, after incubation, and then washed with purified water to remove unadsorbedproteins. After vacuum drying, the change in protein adsorption of the undeposited and oxygen/pp4G plasma
deposited samples were investigated by XPS.
3 Results and discussions
3.1 Surface chemistry characterization
Oxygen pp4G cold-plasma treatment significantly modified the stainless steel surface structure and charac-teristics. The surface chemistry of undeposited and oxygen / pp4G plasma deposited stainless steel was exam-ined using XPS in Fig 1 (a) and (b). Fig 1 (b) shows a survey scan of a coating on stainless steel, the carbon-
oxygen ratio of the coating is 2.2, and it is also comparable to the C-to-O ratio of PEG, which is 2.Fig 2 (1) and (2) shows the carbon 1s high-resolution scan of an undeposited and a oxygen/pp4G plasmaFig 1 XPS survey scan spectra of (a)undeposited and (b) oxygen /
pp4G plasma deposited stain-less steeldeposited stainless steel. From Fig 2 (2), four new peaks of 1.30eV
peak width were used in the peak fitting process. The dominant carbonfunctional group is the ether carbon (C—O) group at 286.4eV (68.4%). C—C linkages were not detected in the surface layers of pp4Gplasma treated stainless steel. However, the resulting macromolecularlayers contained significant amounts of C O (287.9eV, 14.8%),
O C O (289. 7eV, 13. 4%) and O—CO—O (291. 6eV, 3. 4%)functionalities compared with undeposited (Fig 2 (1))[8]. These spectra
show that the majority of the oxygen does not bond to metal to form ox-ide on undeposited samples, while the majority of the oxygen does bondto carbon to form significant amounts of C—O and C O on oxygen/pp4G plasma treated stainless steel surfaces[7], oxygen and carbon form-ing covalent bond in the interfacial layer of metal and PEG-like coatings. Thus it is clear that the surface oxida-tion was occurred by oxygen atom, and additionally new functional groups from oxygen and carbon for hydro-philic surfaces were generated. It is considered that oxygen species activated are in the role of surface oxidation,affecting surface property. Based on the XPS analysis, it is concluded that the coating retained its PEG-likecharacteristics after the plasma deposition process. These findings support our initial hypothesis that the forma-tion of plasma-generated PEG and active species (e.g., free-radical sites) rendered a surface recombination
mechanism of pp4G origin molecular fragments different from that traditional oxidized surfaces. It also shouldbe noted that free-radical species trapped in the nascent macromolecular networks could initiate intense ex situoxidation reactions under open laboratory conditions.Fig 2 XPS C1s high resolution spectra of (1) undeposited and (2) oxygen/pp4G plasma deposited stainless steel
3.2 ATR-FTIR results
Figure 3 shows the influence of the deposition time on the patternsof the differential ATR-FTIR spectra of PEG-like macromolecular struc-tures by plasma generated. These results show the formation of the mac-romolecular networks is influenced by the plasma reaction time. It can beobserved that the IR diagram of macromolecular exhibits two strong ab-sorptions at 1500~1300cm-1, which are characteristic C—O and C—Hvibrations. PEG-like polymeric structures are easily recognizable due tothe intense absorption at the 1500~1300cm-1wave number. It also canbe noticed that PEG-like structures exhibit a weak C O (1750cm-1)vibration absorption. These results might be explained by a more intensefragmentation and surface recombination mechanisms and consequentlyby the presence of significant ex situ free-radical-mediated post-plasmaoxidation processes. The existence of a more intense 1750cm-1absorp-tion in the plasma-polymer spectra allows us to suggest that these macromolecular structures have a partbranched and/or crosslinked nature.
3.3 Plasma protein adsorption analysis
Plasma protein was adsorbed onto undeposited and deposited surfaces of samples, and the relative adsorbed777YANG Jun, et al:Preparation protein-resistant surfaces on bio-stainless steel by microwave cold plasmaunt of proteins on the surfaces was evaluated by XPS. The nitrogen peak area from the peptide bonds wasused as an indicator of surface protein adsorption. Fig 4 shows XPS C1s spectra of undeposited and deposited
samples surfaces after plasma protein adsorption. The atomic percentcalculated from this result and its ratio are shown in Table 1.Table1XPS of control and plasma treated surfaces after plasma proteinadsorption
N-C=O/C-ON Ratiob
Control 82.082 12.279 5.639 0.943
Treated 64.088 35.910 0.002 0.021
  a:Analyzed from survey scan spectra   b: Analyzed from C1s spectra
The plasma deposited surfaces showed much less protein adsorptioncompared with undeposited samples, indicating the large amount of pro-tein adsorption on the control surface. It may be explained that this isdue to the hydrophobic interaction of protein molecules with the hydro-phobic stainless steel surface. From these study results, it was foundthat a PEG-like structure has biocompatible properties, because its low interfacial free energy, hydrophilicity,high surface mobility, and steric stabilization effect.
4 Conclusions
It has been observed that pp4G microwave cold plasma environment is suitable for the deposition of PEG-like structures on stainless steel surfaces. XPS and ATR-FTIR investigations indicate that the deposited PEG-like coatings are built up mainly of—CH2—CH2—O—linkages. The C O based functionalities present inthe coatings are related to ex situ post-plasma oxidation mechanisms. These reactions are mediated probably byplasma-generated free-radicals. Plasma protein was adsorbed onto control and stainless steel surfaces, and the
relative adsorbed amount of proteins on the surfaces was evaluated by XPS analysis. The adsorbed protein onmodified-surface decreased rapidly compared with undeposited. From these studies, it can be concluded that aPEG-like structure has biocompatible properties.
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[2] Park J B, Bronzino J D. Biomaterials: Principals and applications [M]. Boca Raton:CRC Press, 2002. 529-798.
[3] Oh H K, Young C N, Ki D P. [J]. J Appl Polym Sci, 1999, 71: 631-641.
[4] Ratner B D, Hoffman A S. Thin Films, Grafts, and Coatings[A]. Ratner B D, Hoffman A S, Schoen F J,et al. BiomaterialsScience, an Introduction to Materials in Medicine[C]. San Diego: Academic Press, 1996.105-110.
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