Selective Protein Adsorption and Cell Attachment to Rubbed

Chapter 26

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Selective Protein Adsorption and Cell Attachment to Rubbed Fluorinated Polyimides H . Kawakami, K. Ashiba, and Y. Okuyama Department of Applied Chemistry, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan

High-pressure mechanical rubbing was used to nanopattern a fluorinated polyimide membrane surface. Plasma protein adsorption and cell attachment to the rubbed membranes were characterized using a micro-bicinchoninic acid protein assay and phase-contrast microscopy. The morphologies of rat skin fibroblast cells attached to the rubbed membrane were threedimensional multicellular spheroids. The amount o f hydroxyproline generated on the membrane significantly increased due to rubbing. The amount of albumin adsorption on the rubbed polyimide membrane was slightly lower than that on an unrubbed membrane.

Aromatic polyimides are a class of high-performance polymers that are highly thermally stable and have high glass transition temperatures and relatively low dielectric constants. Various polyimides have become increasingly important in a variety o f technological applications, such as semiconductor devices, high-temperature adhesives, and high-performance composite materials. We have reported that polyimides containing fluorinated groups are promising materials for medical devices ( i - 5 ) . For example, polyimide hollow fibers showed not only high gas exchange (O2 transfer and C O 2 removal) but also suppression of platelet adhesion, neutrophil adhesion, and complement activation, suggesting the possibility of a novel membrane oxygenator with the advantage of both increased gas exchange and excellent biocompatibility (i-¥).

© 2004 American Chemical Society

Pinnau and Freeman; Advanced Materials for Membrane Separations ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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384 Micropatterning and nanotechnology are becoming increasingly popular for the development of improved biomaterials and medical devices. In particular, surface micro- or nano-fabrication techniques have been discovered and developed to create unique materials for regulating protein or cell functions (6). Several techniques have been investigated for creating micropatterning on surfaces, including the use of conventional photoresist lithography, photochemistry, and self-assembled monolayers. However, currently, the patterning for biomaterials and medical devices has been limited to micrometer or tenths of micrometer size features. In this paper, we report a simple method for creating nanopatterning on surfaces of biomaterials and medical devices. Mechanically rubbed polyimide membranes are widely used as alignment materials in liquid crystal displays (7). We have used high pressure rubbing to nanopattern a fluorinated polyimide membrane surface for biomaterials applications. Plasma protein adsorption and cell attachment to the rubbed polyimide membranes were evaluated using a micro-bicinchoninic acid protein assay and phase-contrast microscopy.

Experimental Materials 2, 2'-Bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6FDA) was purchased from the Japanese Hoechst Co., (Tokyo, Japan) and purified by sublimation. 2,2'-Bis(4-aminophenyl)hexafluoropropane (6FAP) was purchased from Central Glass Co., (Saitama, Japan) and recrystallized twice in ethanol solution. The fluorinated polyimide, 6FDA-6FAP, was synthesized by preparing its polyamic acid precursor and then chemically imidizing the polyamic acid as reported in the literature (5). The chemical structure of 6FDA-6FAP is presented in Scheme 1. Polyimide films were prepared by solvent casting from tetrahydrofuran solution onto a glass plate, followed by curing at 150 °C. The cast films were optically clear and were approximately 50 |i,m thick. Polystyrene (PSt) films were used as control materials.

Scheme 1. Structure of 6FDA-6FAP.


385 Rubbed Polyimide Membrane Nanopatterning was performed by mechanically rubbing the surface of the polyimide membranes using a constant velocity rotating cylinder covered with a rubbing cloth. The cylinder velocity was 7.5 mm/s, and the pressure added to the cylinder was 6.0x10 Pa. 4

Protein Adsorption Bovine serum albumin (BSA) was purchased from Nacalai Tesque, Inc., (Tokyo, Japan). B S A was used without farther purification. PBS (pH=7.4) was purchased from the Yatoron Co. (Tokyo, Japan). The polyimide membranes were rinsed with deionized water and ethanol prior to use. The amount of B S A adsorbed to the membrane surface from 1.0 mg/ml B S A solution was evaluated by a micro-bicinchoninic acid protein assay (Pierce, Inc., Micro BCA™ Protein Assay) at 36.5 °C in PBS as reported in the literature (4). The B S A adsorbed on the membrane was removed by rinsing with 1% sodium dodecyl sulphate. Then, the amount of B S A in the rinse solution was determined by Micro BCA™ Protein Assay at 562 nm using a spectrophotometer (Ubest-55, J A S C O , Tokyo, Japan).

Cell Culture Rat skin fibroblast (FR) cells were cultured in flasks containing E M E M and alpha-MEM supplemented with 10% F B S at 37°C in a 5% CQz incubator. The cells were seeded at l x l O cells/well in 96-well plates and incubated for 24 h at 37°C. The cells were observed by phase-contrast microscopy (Nikon, E C L P I S E TE-300, Tokyo, Japan) and their function was determined based on the collagen production as measured by a specific assay for hydroxyproline. 4

Analysis of Hydroxyproline The assay was performed as reported in the literature (9). The absorption was measured using a spectrophotometer (Ubest-55, J A S C O , Tokyo, Japan) operating at 560 nm.

Surface Analysis Contact angle measurements for water were performed on dry membranes using a contact angle measurement device (Kyowa Co. Elma GI, Tokyo, Japan).


386 A F M Analysis The surface morphology of the membranes was visualized using an atomic force microscope (AFM: Seiko SPI3700, Tokyo, Japan) operating in air at room temperature (10). The cantilevers (Seiko SN-AF01), with a spring constant of 0.021 N/m, were microfabricated from silicon nitride. The surface was continuously imaged in feedback mode with a scan area of 500 nm x 500 nm and a constant scan speed of 2 Hz. The surface roughness profile was analyzed using a parameter, R , which is the arithmetic average of departures of the roughness profile from die mean line. The force-distance curves were obtained by moving the cantilever vertically at a speed of approximately 70 nm/s over a maximum travel height of 700 nm. The tip radius was 20 nm. a

Results and Discussion Characteristics of Rubbed Polyimide Membranes The fluorinated polyimide, 6FDA-6FAP, had a molecular weight of 9.2 x 10 and a polydispersity index of 1.2. Therefore, a high molecular weight polyimide with a narrow molecular weight distribution was prepared by chemical imidization. Based on H-NMR spectroscopy, the degree of imidization of 6FDA-6FAP was complete. The polyimide membranes were prepared by a solvent-casting method and were cured at 150°C for 15 hours. The amount of residual solvent in the membrane was measured using thermogravimetric analysis (Seiko, TG/FDTA300, Tokyo Japan). These measurements indicated that the curing step completely removed the solvent. Figure 1 presents A F M images of the top surfaces of two 6FDA-6FAP membranes. The surface area imaged in these pictures is 500 x 500 nm. The surface morphology on the rubbed membrane is nanopatterned. The width between peaks was approximately 100 nm and the height of each peak was 2-3 nm. In contrast, the surface of the unrubbed polyimide membrane was essentially smooth. To elucidate the surface properties of the rubbed 6FDA-6FAP membrane, we measured the contact angle of water and the adhesion forces on the membrane surface. As shown in Table I, there was no difference in the contact angle between the rubbed and unrubbed membranes. The level of adhesion between the cantilever tip and the polyimide surface was determinedfromforcedistance experiments on the top and bottom surfaces of the membranes. The silicon nitride tip used in this study was hydrophilic in nature. Interestingly, the adhesion forces on the top and bottom surfaces of the rubbed membrane were different; they exhibited values of 8.5±0.3 and 4.4±0.3 nN, respectively. One explanation for this finding may be the increased hydrophilic nature of the top surface of the rubbed membrane and the increased hydrophobic nature of the bottom surface. 5

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387

Unrubbed 6FDA-6 F A P

Rubbed 6FDA-6FAP Figure 1. AFM images of top surface of 6FDA-6FAP membranes.


388

Table I. Water contact angle and adhesion force on 6FDA- 6FAP surfaces Polyimide Rubbed 6FDA-6FAP Unrubbed 6FDA-6FAP

°[) 85 84

Adhesion force (nN) top bottom 8.5±0.3 4.4±0.3 7.9±0.1 7.9±0.1

Cell Attachment to Rubbed Polyimide Membranes Figure 2 shows phase contrast micrographs of FR cells on 6FDA-6FAP membranes. Interestingly, the morphologies of FR cells attached to the rubbed 6FDA-6FAP membrane were three-dimensional multicellular spheroids. On the other hand, the cells on the unrubbed 6FDA-6FAP membrane were a twodimensional monolayer. Recently, study of spheroids of normal cells has attracted much attention, since spheroids show functional similarities to tissues and organs, unlike the conventional two-dimensional monolayer culture of cells (11). Tissues and organs w vivo are made from cells of many types, which are systematically and functionally combined together. Therefore, the spheroid may be an important approaches for regenerating tissue or organs. The nanopatterning formed on the rubbed 6FDA-6FAP membrane induces the attachment of highly aggregated cells and generation of three-dimensional multicellular spheroids. Conventional methods of generating spheroid morphologies include the spontaneous aggregation of cells on a non-adhesive substrate such as as agarose or polyhydroxyethylmethacrylate-coated plastics or tbe spontaneous detachment of cellsfroma semi-adhesive surface such as proteoglycan or positively charged polystyrene. Both methods significantly limit the cell species from which spheroids can be made. In contrast, the rubbing method described in this article is an extremely simple method for forming multicellular spheroids, and it should be applicable to a wide variety of cell species. In cell culture systems, efficient recovery of cells from the culturing substrate is very important to maintain the cell function. In general, confluent cells in substrates were treated with trypsin and detached from the substrate. However, trypsin treatment destroys two-dimensional monolayer culture of cells formed on the substrate and damages cell membranes by hydrolyzing various membrane-associated proteins. In this study, spheroids formed on the rubbed 6FDA-6FAP membrane were easily detached from the substrate and were recovered when treated with EDTA. The detachment process of the spheroids from the rubbed 6FDA-6FAP membrane is illustrated in Figure 3. Figure 4 shows the cell function of FR. The function was determined based on collagen production measured by a specific assay for hydroxyproline. The amount of hydroxyproline generated on the rubbed 6FDA-6FAP membrane was approximately 10-fold higher than that generated on the unrubbed membrane.


389

Unrubbed 6FDA-6FAP

5 tun Rubbed 6FDA-6FAP Figure 2.Phase contrast microscopy images of FR cells cultured on 6FDA6FAP surfaces.


Figure 3. Detachment process of cells from polymer membrane (a) rubbed 6FDA-6FAP surface; (b) tissue culture polystyrene surface. Pinnau and Freeman; Advanced Materials for Membrane Separations ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

391 Therefore, the interactions between cells, due to the formation of spheroids, were strong enough to facilitate the differentiated functions of F R cells.

Protein Adsorption Figure 5 presents the amount o f B S A adsorption on 6FDA-6FAP membranes. The B S A adsorption measurements were determined using a micro-bicinchoninic acid protein ( B C A ) assay. We have already reported that fluorinated polyimides show excellent biocompatibility and suppress plasma protein adsorption (i-5). Actually, in this study, 6FDA-6FAP also suppressed B S A adsorption relative to PSt; the amount of B S A adsorbed onto 6FDA-6FAP was 2-fold smaller than that on PSt. The amount of B S A adsorption on the rubbed 6FDA-6FAP membrane decreased slightly relative to that adsorbed on the unrubbed membrane. In general, more protein is adsorbed on a hydrophobic surface than on a hydrophilic surface. We speculate that the nano-ordered hydrophilic and hydrophobic patterns formed at the top and bottom surfaces of the rubbed 6FDA-6FAP membrane may facilitate B S A adsorption. However, these arguments require validation by evaluating the direct interaction between the rubbed polyimide membrane and plasma protein.

Conclusions The purpose of this study was to demonstrate a simple method for creating nanopatterning on biomaterial and medical device surfaces. We prepared nanopatterned fluorinated polyimide surfaces using a conventional, highpressure mechanical rubbing method. The plasma protein adsorption and cell attachment to the rubbed polyimide membranes were evaluated in vitro using a micro-bicinchoninic acid protein assay and phase-contrast microscopy. Interestingly, morphologies of F R cells attached to the rubbed 6FDA-6FAP membrane were three-dimensional multicellular spheroids, while the cells on the unrubbed membrane were two-dimensional monolayers. In addition, the amount of hydroxyproline generated on the rubbed 6FDA-6FAP membrane was about 10-fold higher than that generated on the unrubbed membrane. Therefore, the interactions between cells, due to the formation of spheroids, were strong enough to facilitate the differentiated functions of F R cells. On the other hand, the amount of B S A adsorption on the rubbed 6FDA-6FAP membrane decreased slightly relative to that on the unrubbed membrane. In previous papers, we reported that fluorinated polyimides were promising biomaterials with excellent biocompatibility. The results obtained in this study indicate that the rubbed 6FDA-6FAP membrane may be a novel nanopatterned biomaterial for regulating protein or cell functions.


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Selective Protein Adsorption and Cell Attachment to Rubbed

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