Cell Engineering Biointerface Focusing on Cytocompatibility Using

Anomalous phospholipid polymers with an isomeric oligo(lactic acid) segment were ... In this study, we focused on the biological responses such as pro...
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Biomacromolecules 2005, 6, 1797-1802

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Cell Engineering Biointerface Focusing on Cytocompatibility Using Phospholipid Polymer with an Isomeric Oligo(lactic acid) Segment Junji Watanabe and Kazuhiko Ishihara* Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received February 25, 2005

Initial contact between a biological environment and a biomaterial ultimately decides the in vivo performance. Therefore, the fabrication of a delicate biointerface is important because it can be utilized as a platform for novel biomaterials. For the preparation of advanced biomedical devices such as biochips, nanoparticles, and cell engineering devices, the surface properties may be modified by the design of polymeric biomaterials. Anomalous phospholipid polymers with an isomeric oligo(lactic acid) segment were designed and evaluated as a biointerface. The phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine was easily copolymerized with isomeric oligo(lactic acid) macromonomers, and the obtained polymer could easily form thin coating membranes as biointerfaces. The oligo(lactic acid) involves three kinds of isomers: DL-, D-, and L-forms. The favorable characteristic on the surface provides regulation of cell-material interactions on the biointerface. The oligo(lactic acid) segment could form hydrophobic domains, which were considered to be located on the interface, to enhance protein adsorption and cell adhesion. The most favorable characteristics on the biointerface were dual functions of cytocompatibility by the phospholipid polymer and cell adhesion property by the oligo(lactic acid) segment. In this study, we focused on the biological responses such as protein adsorption and cell adhesion by change in the oligo(lactic acid) component. The cell viability on the confluent stage was evaluated in terms of metabolic activity. Introduction The design of a biointerface between the biological environment and material is an important regulator of the biomaterial performance. Particularly, the biointerface plays an important role on cell engineering in the field of microfabrication.1,2 Conventional industrial materials, for example, polyester, polystyrene, and polyolefin, are well-known for preparation of disposable products aiming at biomedical research. However, nobody has paid much attention to the material information in terms of cytocompatibility. Though the conventional materials are routinely used for the biomedical research, severe issues concerning about the cell stress, which was received from the materials surface, were clearly reported.3 To improve the surface property, surface modification with excellent biocompatible polymers by simply dip-coating would be a promising technique to provide an excellent biointerface, cell adhesion, and cell activity in terms of metabolism, on the conventional polymer surface. A series of phospholipid polymers, whose major component is 2-methacryloyloxyethyl phosphorylcholine (MPC), showed excellent biological properties at the biointerface.3-8 The phospholipid polymers could easily form a thin-layered membrane by dip-coating on every material surface such as metals, glass, and polymers, onto which the phosphorylcho* To whom correspondence should be addressed. Tel: +81-3-5841-7124. Fax: +81-3-5841-8647. E-mail: [email protected].

line groups self-assembled.4-6 The most significant property is the higher free water content on the phospholipid polymer surface.7,9,10 Therefore, protein adsorption induced by hydrophobic interaction between the phospholipid polymer surface and proteins was suppressed. Furthermore, cell adhesion via the protein adsorption layer was clearly inhibited, and their details were previously reported.11 So, we considered that when cell adherent component and/or bioactive molecules are incorporated into the phospholipid polymer, it provides a good surface environment for cell adhesion. We have reported bioconjugate phospholipid polymers with active ester groups.12-14 The bioactive molecules such as proteins and RGDS peptide were easily modified in the phospholipid polymer backbone via the active ester linkage. As a result, many kinds of the bioactive molecules were incorporated for specific cell adhesion. In this study, we prepared anomalous phospholipid polymeric materials, suggesting that the interaction between biomolecules and surface can be modified. A novel macromonomer with an oligo(lactic acid) segment was designed for the cell adherent component.15,16 The oligo(lactic acid) has three kind of isomers: DL-form (racemic compound), L-form, and D-form. The oligomer shows different crystallinities that were used to regulate the surface property. The next favorable characteristics of the polymers are cytocompatibility by the phospholipid polymer surface. In this study, general cellular response, nonspecific cell adhesion, and protein adsorption was discussed using the phospholipid

10.1021/bm050138f CCC: $30.25 © 2005 American Chemical Society Published on Web 04/02/2005

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Watanabe and Ishihara

Scheme 1. Chemical Structure of Phospholipid Polymer Containing an Isomeric Oligo(lactic acid) Segment

polymer-coated surface. Particularly, the number of adhered cells and cytocompatibility in terms of metabolic activity were focused for the preparation of the biointerface to regulate the surface response between the biomaterial and natural components. Materials and Method Materials. D,L-Lactide was kindly supplied by Musashino Chemical Laboratory (Tokyo, Japan) and was recrystallized from ethyl acetate. D-Lactide and L-lactide (medical grade) were kindly supplied by Dainippon Ink and Chemicals, Inc. (Tokyo, Japan), and used without further purification. n-Butyl methacrylate (BMA, Wako Pure Chemical Co., Ltd., Osaka, Japan) and 2-isocyanate ethyl methacrylate (IEMA, Showa Denko Co., Tokyo, Japan) were distilled at a reduced pressure, and the following fractions were used (BMA, 50 °C/20 mmHg; IEMA, 60 °C/2.5 mmHg). n-Dodecanol, stannous octoate, and dibutyltin dilaurate were purchased from Wako Pure Chemical Co., Ltd., and used without further purification. MPC was synthesized and purified by a method previously reported.17 Other reagents were commercially available and used as received. Preparation of Phospholipid Polymer Membranes. The synthesis of macromonomers containing an oligo(lactic acid) segment were described in previous reports.15-16 The preparative process consisted two steps: (i) ring-opening polymerization of lactide, which is a cyclic dimer of lactic acid, at the terminal hydroxyl group of n-dodecanol, and (ii) methacrylation of the terminal hydroxyl group in the oligo(lactic acid) via urethane bond formation with IEMA. The phospholipid polymers with three kinds of oligo(lactic acid) graft segments were synthesized by copolymerization of MPC, BMA, and the corresponding macromonomers (Scheme 1). The obtained phospholipid polymer was dissolved in chloroform, with a concentration of 1 wt %. To prepare the thin polymer membrane, poly(ethylene terephthalate) (PET) film (cell culture grade, Wako Pure Chemical Co., Ltd.) was used as a commercially available substrate. The phospholipid polymer membrane was prepared by dip-coating, was dried in vacuo (before contacting water), and was immersed in distilled water for surface equilibration (after contacting water). Physical Property on the Phospholipid Polymer Surface. The surface properties were characterized in terms of molecular mobility. The static contact angle (SCA) by water was measured by using an automatic contact angle meter apparatus (CA-W, Kyowa Interface Science Co., Ltd., Saitama, Japan) at 25 °C. The droplet (10 µL) of pure water was added to the membrane by using a microsyringe. In these surface characterizations, two kind of samples were prepared: dry condition (before contacting water) and wet condition (after contacting water).

Evaluation of Bioresponse on Phospholipid Polymer Surface. Bioresponse was evaluated in terms of protein adsorption, cell adhesion, and metabolic activity on adherent cells. Bovine plasma fibrinogen (F-8630, SIGMA Chemical Co., MO) was used for the protein adsorption. The concentration was adjusted to 3 mg/mL. At first, the membranes were equilibrated by immersion into phosphate buffer saline (PBS) overnight and then incubated in each protein solution (pH 7.4) for 3 h. After rinsing with PBS, the adsorbed proteins were completely removed by 1 wt % of n-sodium dodecyl sulfate (SDS). The concentration of recovered proteins in the SDS solution was determined by using Micro BCA kit (Pierce, #23235, IL). The polymer-coated membranes were set into a 24-well plate (Falcon 353047, Becton Dickinson and Company, NJ) with a silicon ring and sterilized by UV irradiation. The membranes were first equilibrated by PBS (pH 7.1). Mouse fibroblast (L-929) cell (RCB 1451, RIKEN cell bank, Saitama, Japan) was used and was routinely cultured in Eagle’s minimum essential medium (Nissui, Tokyo, Japan), supplemented with 5% calf serum (Gibco, NY) at 37 °C in a 5% CO2 incubator. After treatment with 0.25% trypsinethylenediaminetetraacetic acid (Gibco), the cell density was adjusted to 6 × 103 cells/mL, and 1 mL of the cell suspension was seeded into each well. After a given time, the number of adhered cells on the membrane was evaluated using lactate dehydrogenase test Wako (Wako Pure Chemical Co., Ltd.) after treatment with 0.01 wt % Triton X-100. The metabolic activity was evaluated by fluorescence indicator, Alamar Blue assay kit (Biosource, CA). After 2 days cell culture, 50 µL of Alamar Blue was added to the each well without dilution. The metabolic activity was measured by using multiplate reader (1420 ARVOmx-2, Perkin-Elmer Japan, Tokyo, Japan) after 4 and 8 h incubations. Results and Discussion Phospholipid Polymers with an Oligo(lactic acid) Segment. Three kinds of macromonomers containing D-, L-, and DL-form oligo(lactic acid) segments were obtained. The molecular weight of the macromonomer was estimated by gel permeation chromatography (GPC; polystyrene standard; eluent, chloroform) and was to be about 4000 (L-form), 3700 (D-form), and 7900 (DL-form). The repeating number of the oligo(lactic acid) segment was calculated by using 72 Da of the monomer unit; 51 (L-form), 47 (D-form), and 105 (DLform) were obtained. On the other hand, 1H NMR measurement of the macromonomers was carried out using chloroform-d as an alternative method. The degrees of polymerization in the oligo(lactic acid) segment were 23 (L-form), 27 (Dform), and 40 (DL-form). The estimation of the oligo(lactic acid) segment using 1H NMR was roughly half of the results of GPC. In the case of GPC, the macromonomer would be

Biomacromolecules, Vol. 6, No. 3, 2005 1799

Cell Engineering Biointerfaces Table 1. Synthetic Results of Phospholipid Polymersa monomer unit compositionb (mol %) in feed

in polymer

abb.

MPC

BMA

macromonomer

MPC

BMA

macromonomer

chiralityc

yield (%)

PMBLA5 PMBLA10 PMBLA30 PMBLA50 PMBLLA10 PMBLLA30 PMBDLA10 PMBDLA30

5 5 5 5 5 5 5 5

90 85 75 65 85 75 85 75

5 10 20 30 10 20 10 20

10 13 20 16 16 19 16 23

85 77 51 31 68 53 72 51

5 10 29 53 16 28 12 26

DL-

40 46 63 53 55 60 69 64

DLDLDLLLDD-

a Preparative condition: [Monomer] ) 0.5 mol/L, [azobisisobutyronitrile] ) 2.5 mmol/L, 60 °C, 24 h. b Determined by 1H NMR. c Oligo(lactic acid) segment.

aggregated in chloroform. Therefore, the molecular weight was overestimated. From this, the short repeating unit (2040) of the isomeric lactic acid segment was incorporated into the macromonomers. Table 1 shows synthetic results of the phospholipid polymers. The names of the polymers were abbreviated PMBLA, PMBLLA, and PMBDLA, which contained DL-, L-, and D-forms of the oligo(lactic acid) segment, respectively. The monomer unit in the polymers was a roughly quantitative manner. In this study, we changed the macromonomer unit composition to regulate cell adhesion and protein adsorption. On the other hand, the MPC unit incorporated was 10-20 mol %, though only 5 mol % was fed in the polymerization. Chloroform-d was used as a solvent for 1H NMR measurement. The phosphorylcholine group was associated under a nonpolar solvent such as chloroform.18,19 Therefore, the mobility of the phosphorylcholine group was strongly restricted, so the proton signal was obtained as a broad signal. The broad signal could not show the exact proton ratio, which was calculated using integral curve; therefore, overestimation of the MPC unit would have occurred. The reactivity of the macromonomer was better, because the monomer unit composition in the polymer was in good relation to the feed ratio. In this polymerization, the role of BMA was to function as a spacer to polymerize MPC and the macromonomer. The BMA molecule is relatively smaller than MPC and the macromonomer; particularly, the n-butyl group is smaller than the phosphorylcholine group and the oligomer. Therefore, the oligo(lactic acid) segment was randomly incorporated into the polymer backbone as a graft chain, so the oligo(lactic acid) segment could spontaneously form the domain structure by coating. Surface Property on Phospholipid Polymer Membranes. The thickness of the coating membrane would be below 20 nm by dip-coating, which was estimated in our previous paper.15,16 In this study, the polymer-coated surface was analyzed at a dry condition (before contacting water) and at a wet condition (after contacting water). For the preparation of the sample in the wet condition, the coating membrane was immersed into distilled water overnight and the membrane was then freeze-dried. In this process, molecular conformations on the polymer surface were fixed, thus, maintaining the same polymer conformation as under the wet state. The oligo(lactic acid) segment is a well-known

Table 2. Results of SCA and the Mobility Factora SCA by water (deg)

abb. PMBLA5 PMBLA10 PMBLA30 PMBLA50 PMBLLA10 PMBLLA30 PMBDLA10 PMBDLA30 PET

before contacting after contacting water water (dry condition, θD) (wet condition, θW) 85.4 ( 1.2 88.9 ( 1.1 82.5 ( 1.1 81.3 ( 1.6 77.6 ( 0.8 79.4 ( 1.0 78.4 ( 1.4 77.6 ( 1.0 82.4 ( 1.8

75.8 ( 2.3 71.8 ( 1.1 74.8 ( 1.8 74.0 ( 0.9 70.0 ( 2.3 68.3 ( 3.0 68.1 ( 1.3 63.9 ( 2.1 76.4 ( 1.4

mobility factorb 0.112 ( 0.015 0.192 ( 0.003 0.093 ( 0.010 0.089 ( 0.008 0.098 ( 0.020 0.140 ( 0.027 0.131 ( 0.001 0.177 ( 0.017 0.073 ( 0.004

a N ) 10, mean ( S.D. b Calculated by the following equation: mobility factor ) (θD - θW)/θW.

biodegradable polymer at physiological conditions. We estimated the degree of hydrolysis by using 1H NMR. The phospholipid polymer with the oligo(lactic acid) segment was dissolved in CDCl3 with CF3COOD. The degradation condition was homogeneous and accelerated using acidic catalyst. We compared the chemical shift regarding methine proton, showing 5.2 ppm in oligomer and 4.1 ppm in lactic acid monomer. From this, the oligo(lactic acid) segment was stable for 1 month. And also, the surface wettability was estimated by using SCA measurement. On the phospholipid polymer-coated surface, the wettability did not significantly change through 2 weeks under the physiological conditions. These preliminary results indicated that the surface property regarding the oligo(lactic acid) segment was stable within 1 week. The SCA was measured using a polymer-coated surface under both dry and wet conditions (Table 2). Table 2 shows surface wettability on the phospholipid polymer surfaces. In the case of dry conditions, the contact angle changed from 77 to 85°, and the SCA on the racemic oligo(lactic acid) (PMBLA) was higher than that of isomeric oligomers (PMBLLA and PMBDLA). After the water contact and freeze-dried treatment, the contact angle decreased by roughly 10-17°. The change in surface wettability was discussed by the definition of the calculated parameter, the mobility factor. The parameter was calculated using the following equation: mobility factor ) (θD - θW)/θD

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Figure 1. Calculation results of the mobility factor on the phospholipid polymer-coated surface. N ) 10, mean value and S.D. are indicated.

Here, θD and θW represent SCA under the dry and wet conditions, respectively (Figure 1). Originally, the SCA of the phospholipid polymer and PET surface roughly showed 85° under the dry condition, indicating similar hydrophobicities on the surface. In the case of the phospholipid polymers, the SCA decreased after contacting water by the rearrangement of the phosphorylcholine group; therefore, the obtained mobility factor was 0.1-0.2. The PET substrate showed a quite low value of the mobility factor, indicating no significant chain rearrangement. The relationship between macromonomer composition and the isomeric type of the oligo(lactic acid) segment was not clear. However, the mobility factor on the phospholipid polymers with the enantiomeric oligo(lactic acid) segment was higher than that of the racemic form, except for PMBLA10. In the case of the racemic form, the oligo(DL-lactic acid) segment could not form a crystalline region; on the other hand, oligo(Dlactic acid) and oligo(L-lactic acid) segments could spontaneously form crystalline structure, which consists of hydrophobic [oligo(lactic acid)] and hydrophilic (phospholipid polar group) domains. The formation of crystalline structure was observed by wide-angle X-ray diffraction and was previously reported.15 Taking these results into account, the surface property was clearly different from the isomers of the oligo(lactic acid) segment, though chemical structure and chemical composition were quite similar. Fibrinogen Adsorption on Phospholipid Polymer Membranes. Fibrinogen is one of the major proteins in whole blood. In the case of biomaterial surface characterization, the relationship between fibrinogen adsorption and cell adhesion shows good correlation.20 Therefore, fibrinogen adsorption would be an important key parameter to discuss to elucidate the surface property of biomaterials, which was newly designed. Figure 2 shows the amount of fibrinogen adsorbed on the phospholipid polymers with the racemic oligo(lactic acid) segment. In the case of PMBLA5, fibrinogen adsorption was not detected using the Micro BCA kit. The adsorbed fibrinogen clearly increased with the macromonomer composition in the phospholipid polymers. The maximum fibrinogen adsorption (1.2 µg/cm2) was observed on PMBLA50, providing similar value to the PET substrate. The calculated value for the fibrinogen adsorption was reported as a monolayer; side-on adsorption (0.18 µg/cm2) and end-on adsorption (1.7 µg/cm2), assuming that the shape of fibrinogen is a rugby ball.21 Therefore, we could evaluate

Watanabe and Ishihara

Figure 2. Fibrinogen adsorption on the phospholipid polymer surface with a racemic oligo(lactic acid) segment. Mean value and standard deviation are indicated (N ) 3). Dotted lines indicate the fibrinogen adsorption value calculated as side-on (0.18 µg/cm2) and end-on (1.7 µg/cm2) adsorption. N.D.: not detected.

Figure 3. Fibrinogen adsorption on the phospholipid polymer surface with an enantiomeric oligo(lactic acid) segment. Mean value and standard deviation are indicated (N ) 3). Dotted lines indicate the fibrinogen adsorption value calculated as side-on (0.18 µg/cm2) and end-on (1.7 µg/cm2) adsorption.

that the protein adsorption layer was a monolayer or a multilayer. It is considered that fibrinogen was attached on the surface as a monolayer. From this evaluation, the phospholipid polymer surface with racemic oligo(DL-lactic acid) segments provided a fibrinogen adsorption property with good correlation to their composition of the macromonomer. We have examined the fibrinogen adsorption using the phospholipid polymer with an enantiomeric oligo(lactic acid) segment (Figure 3). In the case of enantiomers, the fibrinogen adsorption increased with the macromonomer composition, and the amount of adsorption was higher than that of the racemic form, even if the macromonomer content was only 10 mol % in the polymer. The higher fibrinogen adsorption would be correlated with their domain structure, which was formed by the oligo(lactic acid) segment. In the case of enantiomers, oligo(lactic acid) segment, L-form and D-form, was spontaneously forming a hydrophobic domain, which was composed of a crystalline region. In this case, the hydrophobic and hydrophilic regions are clearly separated; thus, the mobility factor was higher than that of the racemic form. Though the surface showed hydrophilic property based on the phosphorylcholine group, the hydrophobic region formed by the oligo(lactic acid) segment was located on the surface for the protein adsorption site. Thus, it is considered that the surface would show dual functions, excellent biological compatibility and protein adsorption, so the surface would be utilized for a smart biointerface for cell engineering. On the other hand, the phospholipid polymer containing

Cell Engineering Biointerfaces

Biomacromolecules, Vol. 6, No. 3, 2005 1801 Table 3. Number of Adhered Cells after 5 Days of Cell Culturea abb.

cell number (cells/cm2)

PMBLA5 PMBLA10 PMBLA30 PMBLA50 PMBLLA10 PMBLLA30 PMBDLA10 PMBDLA30 PET

b 11 000 ( 200 29 200 ( 400 23 300 ( 1200 39 300 ( 3700 55 500 ( 5300 50 500 ( 4800 49 500 ( 4700 40 300 ( 3800

a N ) 3, mean ( S.D. b The number of adhered cells on PMBLA5 was below 500 cells/cm2.

Figure 4. Fibroblast cell adhesion on the phospholipid polymer surface with a racemic oligo(lactic acid) segment: white bar, 6 h; gray bar, 24 h; and black bar, 48 h. Asterisk means below 500 cells/cm2. N ) 3, mean value and S.D. are indicated.

Figure 5. Fibroblast cell adhesion on phospholipid polymer surface with an enantiomeric oligo(lactic acid) segment: white bar, 6 h; gray bar, 24 h; and black bar, 48 h. N ) 3, mean value and S.D. are indicated.

amorphous oligo(lactic acid) showed a low mobility factor, indicating more hydrophobic surface in comparison with enantiomers. However, fibrinogen did not adsorb on the surface; particularly, the fibrinogen adsorption was quite low below 30 mol % of the macromonomer composition. In PMBLA5, -10, and -30, the property by phosphorylcholine group, which provides an excellent biocompatible surface, was dominant in comparison with protein adsorption. Cell Adhesion and Its Biological Activity. Cell adhesion using fibroblast cells was carried out for the purpose of preliminary characterization on the phospholipid polymer surface. Generally, a protein adsorption layer is necessary for the cell adhesion; then, fibroblast was utilized to evaluate cell-surface interaction. The cell suspension was adjusted to 6 × 103 cells/mL. The cell density reached to monolayer confluence on the polystyrene dish (4.0 × 104 cells/cm2) after 5 days of cell culture. In this study, the number of adhered cells was evaluated after 6 h, 1 day, and 2 days (Figures 4 and 5). In addition, the number of adhered cells was compared with the time to reach confluence (5 days of cell culture; Table 3). In the case of PMBLA5, cell adhesion was not observed (Figure 4, below 500 cells/cm2). And the cells on the PMBLA5 could not grow, even with 5 days of cell culture (Table 3). The number of adhered cells did not significantly increase with cell culture time on the PMBLA10 and PMBLA30. On the other hand, the PMBLA50 showed good time dependence on the adhered cells. This result

indicated that there is little protein adsorption from cell culture media for cell adhesion. The number of adhered cells increased with the higher macromonomer composition, showing good correlation with the amount of fibrinogen adsorption. After 5 days, the fibroblast proliferated over 4.0 × 104 cells/cm2 on the PET substrate, whose cell number reached monolayer confluence. On the other hand, the time to reach confluence on the PMBLA50 would be 6 days of cell culture, because it was roughly 2.3 × 104 cells/cm2 for 5 days. The cell proliferation on the phospholipid polymer with the racemic oligo(lactic acid) segment was correlated with higher macromonomer compositions. Figure 5 shows the number of adhered cells on the enantiomeric form. No significant difference was observed among these phospholipid polymer surfaces. This result indicated that the surface properties were similar in the cell adhesion, even if there were some differences: mobility factor and fibrinogen adsorption. Particularly, PMBLLA10 showed anomalous behavior; the amount of fibrinogen was only 0.6 µg/cm2, which was roughly half the value in comparison with PMBDLA30 and PET. However, the number of adhered cells was over 4.0 × 104 cells/cm2 after 5 days of cell culture. The surface formed by PMBLLA10 showed good cell adhesion and cell proliferation, even though fibrinogen adsorption was clearly low. We assumed that the surface would be a good interface for cell engineering because lower protein adsorption means an effect on the phosphorylcholine-assembled surface, which showed an excellent biologically compatible interface. In this study, the surface formed by PMBLLA10 would be a good candidate with dual functions: cell adhesion property and cytocompatibility. The cytocompatibility was examined in terms of metabolic activity. The metabolic activity was estimated using Alamar Blue reagent, which contains a redox indicator changing its color of fluorescence intensity, synchronizing metabolic activity. The fluorescence reagent is generally used for the quantitative detection of the cell viability.22 The Alamar Blue reagent is highly fluorescent; the oxidized form of Alamar Blue is a dark blue color, and then the color turns red after reduction into the cells. Figure 6 shows the cell viability by the phospholipid polymer surface using Alamar Blue reagent. The cell viability was examined after 2 days of cell culture, and change in fluorescence intensity (λEm, 560 nm; λEx, 590 nm) was measured by microplate reader after addition of the Alamar Blue reagent (4 and 8 h). The viability was

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Figure 6. Cell viability on phopholipid polymer surface after addition of Alamar Blue reagent; 4 h (top bar) and 8 h (bottom bar). Dotted line means viability of PET substrate. N ) 3, mean value and S.D. are indicated.

examined using only PMBLA50, PMBLLA10, PMBLLA30, PMBDLA10, PMBDLA30, and PET, whose cell numbers were almost the same (5.0 × 103 cells/cm2) after 2 days of cell culture, because cell viability was strongly influenced by the cell density. From Figure 6, the selected phospholipid polymer surface showed excellent cell viability in comparison with the PET substrate. The enhanced viability was roughly 1.5 times for both 4 and 8 h of incubation. In the case of PMBLLA10, the cell viability was at a significantly high level, even though fibrinogen adsorption was lower. There is not a significant difference between the racemic form and the enantiomeric form. It is considered that higher cell viability on the phospholipid polymer surface was based on the phosphorylcholine-assembled surface. Taking these bioresponses into account, the phospholipid polymer surface with an oligo(lactic acid) segment provided dual functions: cytocompatibility on metabolic activity and regulation of cell adhesion and protein adsorption. The anomalous surface would be a promising candidate for a cell engineering biointerface. Further surface analysis in terms of domain structure is now in progress and will be reported soon. Conclusions The novel phospholipid polymers composed of MPC, BMA, and oligo(lactic acid) macromonomers were synthesized as a cytocompatible polymer biointerface for cell

Watanabe and Ishihara

engineering. The mobility of the monomer segment on the coating membranes was confirmed by SCA measurement. Spontaneous rearrangement based on the MPC unit was observed on the phospholipid polymer, and the surface holds adequate hydrophobicity for cell adhesion. The phospholipid polymer interface provides specific surface properties for biological response. By changing the monomer composition, we found an anomalous biointerface using the phospholipid polymer with the oligo(lactic acid) segment; a higher cell number and cell viability were prepared, though the amount of fibrinogen adsorption was quite low. The specific biointerface provided dual functions: cytocompatibility and cell adhesion ability. By using the phospholipid polymer material, specific surface modification can be realized for the preparation of advanced medical devices. References and Notes (1) Cheng, X.; Wang, Y.; Hanein, Y.; Bo¨hringer, K. F.; Ratner, B. D. J. Biomed. Mater. Res. 2004, 70A, 159. (2) Biran, I.; Walt, D. R. Anal. Chem. 2002, 74, 3046. (3) Sawada, S.; Sasaki, S.; Iwasaki, Y.; Nakabayashi, N.; Ishihara, K. J. Biomed. Mater. Res. 2003, 64A, 411. (4) Whelan, D. M.; Giessen, W. J.; Krabbendam, S. C.; Vliet, E. A.; Verdouw, P. D.; Serruys, P. W.; Beusekom, H. M. M. Heart 2000, 83, 338. (5) Oki, A.; Adachi, S.; Takamura, Y.; Ishihara, K.; Ogawa, H.; Ogawa, Y.; Ichiki, T.; Horiike, Y. Electrophoresis 2001, 22, 341. (6) Yoneyama, T.; Sugihara, K.; Ishihara, K.; Iwasaki, Y.; Nakabayashi, N. Biomaterials 2002, 23, 1445. (7) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323. (8) Miyamoto, D.; Watanabe, J.; Ishihara, K. Biomaterials 2004, 25, 71. (9) Kitano, H.; Sudo, K.; Ichikawa, K.; Ide, M.; Ishihara, K. J. Phys. Chem. B 2000, 104, 11425. (10) Kitano, H.; Imai, M.; Mori, T.; Gommei-Ide, M.; Yokoyama, Y.; Ishihara, K. Langmuir 2003, 19, 10260. (11) Ishihara, K.; Ishikawa, E.; Watanabe, A.; Iwasaki, Y.; Kurita, K.; Nakabayashi, N. J. Biomater. Sci., Polym. Ed. 1999, 10, 1047. (12) Konno, T.; Watanabe, J.; Ishihara, K. Biomacromolecules 2004, 5, 342. (13) Takei, K.; Konno, T.; Watanabe, J.; Ishihara, K. Biomacromolecules 2004, 5, 858. (14) Park, J.-W.; Kurosawa, S.; Watanabe, J.; Ishihara, K. Anal. Chem. 2004, 76, 2649. (15) Watanabe, J.; Eriguchi, T.; Ishihara, K. Biomacromolecules 2002, 3, 1109. (16) Watanabe, J.; Ishihara, K. Artif. Organs 2003, 27, 242. (17) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355. (18) Miyazawa, K.; Winnik, F. M. Macromolecules 2002, 35, 2440. (19) Watanabe, J.; Ishihara, K. Chem. Lett. 2003, 32, 192. (20) Matsuda, T. J. Jpn. Soc. Biomat. 1994, 12, 187. (21) Baskin, A.; Lyman, D. J. J. Biomed. Mater. Res. 1980, 14, 393. (22) Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parandoosh, Z. J. Immunol. Methods 1997, 204, 205.

BM050138F