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Controlling Nonspecific Protein Interactions in Silicon Biomicrosystems with Nanostructured Poly(ethylene glycol) Films Sadhana Sharma,†,§ Ketul C. Popat,†,§ and Tejal A. Desai*,†,‡ Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, and Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215 Received June 19, 2002. In Final Form: July 30, 2002 Protein interaction with surfaces is one of the most important criteria for the selection of implantable biomaterials as it can mediate further cellular interaction and therefore influence biocompatibility of the surfaces. An ideal biomaterial would adsorb no protein, which is practically impossible to achieve. Here we have demonstrated that control of protein adsorption can be achieved by modifying silicon surfaces with a biocompatible polymer like poly(ethylene glycol) (PEG). Two different methods to modify the silicon surface were used. Two model proteins, namely, fibrinogen and bovine serum albumin, were used to study the adsorption on PEG-modified surfaces. The adsorbed protein was characterized using fluorescence microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy.
Introduction Inorganic materials such as silicon are gaining acceptance for use in implantable microdevices. Silicon biomicrodevices are currently being used as implants that can record from, sense, stimulate, and deliver to biological systems. For instance, micromachined neural prostheses, drug delivery micropumps/microneedles, immunoisolation biocapsules, and retinal implants have been shown to function successfully in biological systems.1-6 Although silicon-based microdevices have shown promising results in the laboratory, their clinical use is greatly limited due to the inability to effectively interface with the biological milieu, in a nonimmunogenic and stable manner. Despite chemical and mechanical stability of these materials in a biological environment, several studies on silicon implants have shown the formation of a thin, avascular, and fibrous capsule around the implant.1-3 Such a structure may impede the transport of molecules diffusing from microvasculature to the implant, thus limiting the long-term functioning of these microsystems. This is because the silicon surface in water is negatively charged at neutral pH. When exposed to air or water, it develops a native oxide layer with surface silanol groups. These silanol groups are ionizable in water, which results in a negative charge on the silicon surface at physiological pH levels. A charged surface will create a streaming potential in the fluid flow and thus may promote protein adsorption, that is, biofouling. * Corresponding author. Mailing address: Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215. Tel: 617-358-3054. Fax: 617-353-6676. Email:
[email protected]. † University of Illinois at Chicago. ‡ Boston University. § Both authors contributed equally to this work. (1) Turner, J.; Shain, W. Exp. Neurol. 1999, 156, 33-49. (2) Edell, D.; Toi, V.; McNeil, V.; Clark, L. IEEE Trans. Biomed. Eng. 1992, 39 (6), 635-643. (3) Schmidt, S.; Horch, K.; Normann, R. J. Biomed. Mater. Res. 1993, 27 (11), 1393-1399. (4) Desai, T.; Chu, W.; Tu, J.; Beattie, G.; Hayek, A.; Ferrari, M. Biotechnol. Bioeng. 1998, 57, 118-120. (5) Desai, T.; Tu, J.; Rasi, G.; Boroni, P.; Ferrari, M. Biomed. Microdevices 1999, 1 (2), 131-141. (6) Chow, A.; Peachey, N. Ophthalmic Res. 1999, 31 (3), 246.
Poly(ethylene glycol) (PEG), a water soluble, nontoxic, and nonimmunogenic polymer, serves as an excellent coating material since it is compatible with biological systems and has been shown to reduce protein adsorption and cell adhesion on synthetic surfaces.7 The protein nature of PEG is mainly attributed to its hydrophilicity, steric stabilization force, and chain mobility effect. The two main contributions to this repulsive force are the excluded volume component and the mixing interaction component. When protein molecules approach the PEGcoupled surfaces, the available volume for each polymer segment is reduced, and consequently a repulsive force is developed due to loss of conformational entropy of the PEG chains. Also, the number of available conformations of the PEG segments is reduced owing to their compression or interpenetration of the protein chains generating an osmotic repulsive force.8 In this letter, we have used two types of nanostructured PEG films to study their interactions with proteins: one by coupling PEG-silane in the solution phase and another by vapor deposition of ethylene oxide to grow PEG on a surface. We call these PEG films “nanostructured” as these films have thicknesses in the lower nanometer range and hence are suitable for nano- and microdevices. We used the PEG-silane coupling procedure for the surface modification of silicon membranes with pores of nanometer dimensions. These membranes are currently being investigated in our laboratory for drug delivery applications and pancreatic islet immunoisolation applications.9,10 The PEG films formed by this method were very thin (20 ( 0.93 Å).11,12 Such lower thickness values were desired due to the use of these PEG films for nanoporous membranes. Some biosensors involve more complicated patterns such (7) Zhang, M.; Desai, T.; Ferrari, M. Biomaterials 1998, 19, 953960. (8) Lee, J.; Lee, H.; Andrade, J. Prog. Polym. Sci. 1995, 20, 10431079. (9) Leoni, L.; Boiarski, A.; Desai, T. Biomed. Microdevices 2002, 4 (2), 131-139. (10) Desai, T.; Hansford, D.; Leoni, L.; Essenpreis, M.; Ferrari, M. Biosens. Bioelecton. 2000, 15, 453-462. (11) Sharma, S.; Johnson, R. W.; Desai, T. A. In Second Annual International IEEE-EMBS Special Topic Conference in Microtechnologies in Medicine and Biology Proceedings; Dittmer, A., Beebe, D., Eds.; The Printing House: Stoughton, WI, 2002; pp 41-45.
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as enclosed microchannels. The solution phase surface modification technique is not appropriate for the micro/ nanoscale channels in these sensors. Due to enclosed micron/nanoscale size features on the surface, properties such as viscosity and surface tension of the solution injected for surface modification become extremely important. The liquid may clog the channel, forming lumps and aggregates. Thus, a vapor deposition technique may be more efficient in coating closed features since it can more effectively form uniform and conformal films. Therefore, we have developed a solvent-free vapor deposition technique to modify silicon surfaces by growing PEG using ethylene oxide and a weak Lewis acid as a catalyst.13 Materials and Method Silicon surfaces (1 cm × 1 cm) were modified with PEG, in the solution phase for a 10 mM PEG concentration for 1 h immobilization time and in the vapor phase for a 40 mmol/cm2 ethylene oxide concentration for 4 h.11-13 To investigate the protein interactions of these surfaces, fibrinogen and albumin adsorption on these surfaces was studied. Clean and dry, PEGmodified and unmodified silicon surfaces were transferred into wells of standard 24-well plates (14 mm across internal diameter). For X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM) analysis, 1000 µL of a 0.5 mg/mL fibrinogen solution in phosphate-buffered saline (PBS) (pH 7.4) was added to the samples. For albumin adsorption studies using ellipsometry, a 0.5 mg/mL albumin solution in PBS (pH 7.4) was also used. Adsorption was allowed to proceed in an incubator (5% CO2) for 2 h at 37 °C. These conditions (pH 7.4, 5% CO2, 37 °C) were chosen as they are very similar to physiologic conditions. Upon completion of adsorption, the samples were thoroughly washed three times with deionized water for removal of nonadsorbed proteins and buffer salts. The samples were dried with nitrogen, and their surface composition was determined using XPS for fibrinogen adsorption. The amount of protein adsorbed on the surface was calculated from the value of the thickness of the protein layer measured using ellipsometry. The protein layer thickness was determined by subtracting the thickness of the background PEG film and the oxide layer. The amount of adsorbed protein (in ng cm-2) was determined from thickness data using the method of Stenberg and Nygren.14 Furthermore, the surface topography of fibrinogen-adsorbed PEG-modified surfaces was investigated with AFM as cell adhesion is primarily mediated by proteins. The roughness of the surface was determined by measuring roughness parameters (Rrms). Rrms is defined as the root-mean-square (rms) average of the height (z) taken from the mean data plane, expressed as
Rrms )
x
1
N
∑z N
2 i
i)1
For fluorescence analysis, a 2 mg/mL FITC-labeled bovine serum albumin (BSA) solution was added to the samples. The fluorescence images were taken using a CCD camera attached to a microscope.
Results and Discussion The overall objective of this work was to create hydrophilic and biocompatible PEG-modified surfaces suitable for microsystems. The choice of the model proteins, viz., fibrinogen and albumin, was prompted by the fact that these proteins are the two major constituents of blood (12) Sharma, S.; Johnson, R. W.; Desai, T. A. In Thin Films: Preparation, Characterization and Applications; Soriaga, M. P., Stickney, J., Bottomley, L. A., Kim, Y., Eds., ACS Symposium Series; Kluwer Academic/Plenum Publishing: New York, 2002; pp 326-333. (13) Popat, K.; Johnson, R.; Desai, T. J. Assoc. Lab. Auto. 2002, 7 (3), 66-68. (14) Stenberg, M.; Nygren, H. J. Phys. 1983, C-10, 83-86.
Figure 1. (a) Area (%) of the C-N peak in C1s high-resolution XPS spectra. (b) Ellipsometric measurement of protein adsorbed on unmodified and PEG-coupled silicon surfaces. Table 1. Elemental Surface Composition of Protein Adsorbed on Unmodified and PEG-Modified Surfaces clean silicon PEG solution phase PEG vapor phase
O, %
C, %
Si, %
N, %
23.93 17.14 19.56
52.66 49.88 48.42
11.25 25.85 24.76
12.16 7.14 7.23
plasma and play a very important role in device rejection. More specifically, platelets are recruited through adsorbed fibrinogen during fibrotic response.15 Also, these proteins have very different molecular sizes (fibrinogen, 60 × 60 × 450 Å; albumin, 40 × 40 × 140 Å)16 and, therefore, are useful for testing the fouling characteristics of microdevices. Studies have shown that bare silicon-based (e.g., silica, quartz, silicon) surfaces show greater fibrinogen/ albumin adsorption7,17,18 as compared to PEG-modified surfaces. XPS analysis confirmed that fibrinogen adsorption was less on modified surfaces as compared to unmodified surfaces. Table 1 shows the elemental composition of protein-adsorbed surfaces. High-resolution spectra show a sharp increase in the Si2p (100 eV) peak but a significant decrease in the C1s (285 eV) and N1s (410 eV) peaks for protein adsorption on PEG-modified silicon, which is reflected in the elemental surface composition. The overall C1s peak in the XPS spectra consists of C-C, C-O, and (15) Biomaterials Science. A introduction to materials in medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, CA, 1996. (16) Malmsten, M.; Lassen, B. In Proteins at interfaces II; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington, DC, 1995; pp 228-238. (17) Malmsten, M.; Emoto, K.; Van Alstine, J. M. J Colloid Interface Sci. 1998, 202, 507-517. (18) Sofia, S. J.; Premnath, V.; Merill, E. W. Macromolecules 1998, 31, 5059-5070.
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Figure 2. Fluorescence images for albumin adsorption on (a) clean silicon, (b) solution phase PEG, and (c) vapor-deposited PEG on silicon surfaces.
C-N peaks. Protein adsorption is characterized by the percent of the C-N peak in the overall C1s peak. Changes in the amount of protein adsorption will change the percent of the C-N peak in the overall C1s peak. Figure 1a shows the percent of C-N bond in the overall C1s peak for protein adsorbed on PEG-modified and unmodified silicon surfaces. A decrease in the percent of C-N bond compared to that of the unmodified surface indicates that there is a considerable decrease in protein adsorption on PEGmodified surfaces. This inference is further supported by ellipsometric data (Figure 1b). We observe a significant decrease in both albumin and fibrinogen adsorption on solution-coupled and vapor-deposited PEG surfaces as compared to that on bare silicon. FITC-labeled BSA adsorbed surfaces were observed under the fluorescence microscope (Figure 2). In our case, we define fluorescence intensity as the amount of green color on the surface since we are using FITC (green color) labeled protein. One hundred percent fluorescence intensity means that the surface is totally green, whereas 0% fluorescence intensity means that the surface is totally black. This intensity can be directly correlated with the amount of protein adsorbed on the surface. Much lower fluorescence intensity is shown by PEG-modified silicon as compared to unmodified silicon (Figure 3). This clearly suggests less protein adsorbed on the PEG-modified surface compared to the unmodified surface. To investigate the topography of proteins on PEGmodified surfaces, AFM analysis of these surfaces was carried out. Figure 4 shows 2D, 3D, and section plots for
Figure 3. Percentage fluorescence intensity of FITC-labeled BSA adsorbed on clean and PEG-modified silicon surfaces.
500 nm size scans of protein adsorbed on unmodified and PEG-modified silicon. More irregularities in protein adsorption on the PEG-modified surface can be attributed to the hydrophilic and protein-repelling nature of the PEG component in this silane. This is also supported by the rms roughness parameters (Figure 5). Irregularly defined and nonuniformly distributed peaks are seen in the 3D image and section plot of protein on PEG-modified surfaces compared to data for unmodified surfaces. A closer examination of the section plots of unmodified and PEGmodified surfaces indicates that protein is evenly ad-
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Figure 4. 2D and 3D AFM images of 500 nm scans for fibrinogen adsorption on surfaces: (a) clean silicon, (b) solution phase PEG, and (c) vapor-deposited PEG; (1) 500 nm 2D image, (2) 500 nm 3D image, and (3) 500 nm section plot.
Conclusions In the present research effort, we investigated the efficacy of solution-coupled and vapor-deposited nanostructured PEG films in controlling nonspecific protein interactions. An extensive analysis of protein adsorption on unmodified and PEG-modified silicon surfaces using XPS, ellipsometry, AFM, and fluorescence microscopy indicated that these PEG films are very efficient in reducing protein adsorption. Therefore, we expect them to show reduced cell adhesion and perform favorably in actual biological environments.
Figure 5. The rms roughness parameters for fibrinogenadsorbed unmodified and PEG-modified silicon surfaces.
sorbed on unmodified surfaces (due to formation of a charged surface in an aqueous physiological environment) as compared to random adsorption on PEG-modified surfaces.
Acknowledgment. Portions of this work were funded by the Whitaker Foundation, the National Science Foundation (ECS-9820829), and a National Science Foundation Career Award (BES-9983840). The authors sincerely appreciate Dr. Robert W. Johnson of Abbott Laboratories, Abbott Park, IL, for providing XPS and AFM facilities. LA026097F
