Cell Proliferation on Stereoregular isotactic-Poly(propylene oxide) as

Oct 5, 2010 - globulin (BγG), and bovine plasma fibrinogen (BPF) showed multilayered adsorption onto it-PPO. Mouse fibroblast. L929 cells adhered ont...
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Cell Proliferation on Stereoregular isotactic-Poly(propylene oxide) as a Bulk Substrate Hiroharu Ajiro and Mitsuru Akashi* Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, and The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan Received April 21, 2010; Revised Manuscript Received September 6, 2010

Stereoregular isotactic-poly(propylene oxide) (it-PPO) was investigated for use as a biomaterial surface. The conventional characteristic of nonstereoregular atactic PPO was altered to a hydrophobic solid nature (contact angle: 95.6 ( 3.8°), which resulted in the potential solid surface applications. The high crystallinity of it-PPO created both smooth and microsized, random crater-shaped surfaces using the spin coating and dip coating approaches, respectively. The results of protein adsorption with bovine serum albumin (BSA), bovine gamma globulin (BγG), and bovine plasma fibrinogen (BPF) showed multilayered adsorption onto it-PPO. Mouse fibroblast L929 cells adhered onto the it-PPO surfaces, and cultured well as compared with commercially available Cell Desk LF1 as a control surface. These unique physical characteristics of it-PPO were due to the configuration of the polymer chain backbone structure, which maintained the polyether chemical structure.

Introduction The design of the molecular structure of synthetic polymers is important for biomaterials. In general, bioinactive polymers tend to be hydrophilic and electrically neutral, possessing hydrogen bond acceptors without donor functionality.1,2 For example, Ishihara and co-workers prepared poly(2-methacryloyloxyethyl phosphorylcholine)3 as a highly biocompatible synthetic polymer4,5 bearing the biologically derived phosphoryl choline moiety. Recently, Tanaka and co-workers investigated poly(2-methoxyethyl acrylate)6 on blood compatibility7,8 and suggested that the hydration structures,9 that is, freezing bound water, played an important role in the high biocompatibilities. Various surface modifications, such as dip, spin, and cast coatings, were available; for example, the casting film on substrate using soybean oil grafted methyl methacrylate copolymer is useful.10 Among these designed synthetic polymers, poly(ethylene oxide) (PEO) has been widely employed as a bioinert synthetic polymer in the medical, cosmetic, and pharmaceutical field. It is a notable feature of PEO that it suppresses protein adsorption, which causes cell adhesion. Taking advantage of this characteristic, numerous researchers have reported the PEO modification of various material surfaces, such as biodegradable poly(lactic acid)s11 and gold particles as a bioinert metal.12 To add to their simple immobilization of the PEO polymer chain, the polymer structures have also been investigated, like the comb-type graft copolymers,13 the density of PEO brushes,14 and the encapsulation of cells by the photoinitiated polymerization of diacrylates, including PEO.15 Similarly, poly(propylene oxide) (PPO) allowed these polymer properties to expand, because it is from the same class of polyethers as PEO, with similar biocompatibility and additional hydrophilicity as PEO. It is well-known that the triblock copolymer poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) (PEO-PPO-PEO) is a nonionic polymeric surfactant and has been widely used for * To whom correspondence should be addressed. Phone: +81-6-68797356. Fax: +81-6-6879-7359. E-mail: [email protected].

micellization.16 Since it was reported that PEO-PPO-PEO coated surfaces attenuated protein adsorption,17 it has been applied as a biomaterial in several areas, including cell encapsulation,18 cell transfection,19 bacterial adhesion,20 and as a release vehicle.21 At present, a great deal of information is available on polyether as a biomaterial. However, oxidative degradation is a problem for the polyether backbone, and it becomes difficult to use for longer-term applications. To avoid potentially degraded polyether linkages, alternative chemical structures have been utilized, such as poly(2-methacryloyloxyethyl phosphorylcholine),3-5 carboxybetaine polymers,22 and poly(2-methyl-2oxazoline).1 Another possible approach might be the use of the stereoregular polymer backbone, while keeping the same chemical structures. It is noteworthy that the different stereoregularities of synthetic polymers generate varied chemical and physical characteristics, even though they originated from the same monomer source. Actually, this is the reason why such a great deal of effort has been expended in the polymerization catalyst field. It is well-known that the melting point of syndiotactic (st-)polystyrene23 is higher than that of isotactic (it-)24 and atactic (at-)polystyrenes and that the hydrophilicities of it-poly(methyl methacrylate)25 and st-poly(methyl methacrylate)26 were different. Most importantly, stereoregular polymers tend to show high crystallinity. In fact, diverse stereoregular poly(lactide-bε-caprolactone)s were prepared by four different polymerization catalysts, resulting in some control of their biodegradabilities.27 Motivated by the aforementioned improvements in their macromolecular nature through configuration control, we became interested in polyether as a well-examined biomaterial, with the expectation that its high crystallinity would protect it from the oxidative degradation and thus it may have novel uses as a solidstate surface in aqueous media. We have focused on PPO because it is structurally impossible for PEO to adopt stereoregularity. The propylene oxide (PO) monomer contains (S)-PO and (R)PO; therefore, the optical resolution of PO was previously necessary to obtain it-PPO. Furthermore, the conventional

10.1021/bm100926p  2010 American Chemical Society Published on Web 10/05/2010

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Figure 1. Chemical structures of PPO and the illustration of coating with at-PPO (a) and it-PPO (b). The solid it-PPO could stably cover substrate disk, while conventional at-PPO could not cover it because of the liquid nature.

polymerization method28 involved the elimination of the methyl proton, and that resulted in a low molecular weight and typically liquid PPO (Figure 1a). On the other hand, the recent development of polymerization catalysts enabled the usage of it-PPO as a bulk material (Figure 1b). In 2005, Coates and co-workers discovered cobalt-based catalysts for the highly isotactic selective polymerization of racemic PO.29-32 In this study, high molecular weight and highly stereoregular it-PPO (mm > 99)29 were investigated for the solid-water interface as a novel biomaterial source, by contact angle, protein adsorption, and cell proliferation as indices.

Experimental Section Materials. PO (Tokyo Chemical Industry Co., Ltd., Japan) was distilled with calcium hydride before use. it-PPO (Mn ) 74000, Mw/ Mn ) 4.07; mm > 99) was synthesized according to the previously reported method.29 The number average molecular weights and their distribution were measured by gel permeation chromatography (GPC; Tosoh System HLC-8120GPC) with PMMA standards at 40 °C. Two commercial columns (TSKgel SuperH4000 and TSKgel GMHXL) were connected in series and tetrahydrofuran was used as an eluent. it-PPO was then coated onto a plastic disk (Cell Desk LF1, Sumitomo Bakelite Co., Ltd.) in two ways using a chloroform solution at 10 mg/mL: a spin coating and a dip coating. Spin coating was achieved by the dropping it-PPO solution to the spinning plastic disk with 80 µL at 4000 rpm for 30 s (Spincoater 1H-D7, Mikasa Co., Ltd.). Dip coating was carried out by the dipping a plastic disk into the 10 mL of it-PPO solution (20, 10, and 5 mg/mL) for several seconds, then it was taken out from the solution to vaporize chloroform at room temperature. Both of the it-PPO coated disks were dried under vacuum before use. Contact Angles. Static contact angles were measured using an automatic contact angle meter apparatus (Drop Master 100, Kyowa Interface Science, Co. Ltd., Japan) at room temperature. A total of 0.5 µL of ultrapure water was introduced using a micro syringe onto each it-PPO coated disk after drying in a vacuum. Laser Scanning Microscope. Three dimensional images were observed by Violet Laser Scanning Microscope (VK-9700, Keyence Co. Ltd., Japan), using each it-PPO coated disk after drying in a vacuum. Protein Uptake. Protein uptake was examined using bovine serum albumin (BSA), bovine gamma globulin (BγG), and bovine plasma fibrinogen (BPF; Sigma, St. Louis, U.S.A.). Protein concentrations were adjusted to 9.0, 2.0, and 0.6 mg/mL in phosphate buffer saline (PBS). it-PPO coated disk was incubated in each protein solution (900 µL; pH 7.4) for 4 h at 37 °C. After rinsing with PBS, adsorbed proteins were removed by 1 wt % of n-sodium dodecyl sulfate (SDS; 1000 µL) for 4 h. Proteins recovered from disks were evaluated by a Micro BCA kit (Pierce, IL, U.S.A.), using BSA standard with SDS.33 Cell Proliferation. Mouse fibroblast (L-929) cells were purchased from the RIKEN cell bank (Saitama, Japan) and routinely cultured in

Figure 2. Photos of commercialized cell culture disk (Cell Desk LF 1; (a)), it-PPO dip-coated plastic disk (b), and the microscope observation of the surface of it-PPO dip-coated disk (c); it-PPO dipcoated plastic disk with 20 (d), 10 (e), and 5 mg/mL (f); and it-PPO spin-coated plastic disk with 20 mg/mL at 500 rpm (g), 20 mg/mL at 1000 rpm (h), 20 mg/mL at 4000 rpm (i), 10 mg/mL at 500 rpm (j), 10 mg/mL at 1000 rpm (k), 10 mg/mL at 4000 rpm (l), 5 mg/mL at 500 rpm (m), 5 mg/mL at 1000 rpm (n), and 5 mg/mL at 4000 rpm (o).

Eagle’s minimum essential medium (E-MEM, code 05900, Nissui, Tokyo, Japan) containing 10% fetal bovine serum, 0.2 mol % L-glutamine, and sodium hydrogen carbonate at 37 °C under a 5% CO2 atmosphere. After treatment with 0.25% trypsin, the cell density was adjusted to 1.0 × 106 cells/mL, and 5.0 × 104 cells were seeded onto each it-coated disk (Cell Desk LF1: diameter 1.35 cm and surface area 1.43 cm2). Cells were cultured for 8 days, and the E-MEM was changed every other day. Disks were transferred to another well for rinsing with 700 µL of PBS solution, and were dipped into a 200 µL aliquot of a 0.25% trypsin solution for 1 min. Samples were combined with 500 µL of fresh E-MEM, and cells were collected by centrifugation. Cell pellets were dispersed with a 200 µL aliquot of a E-MEM solution, and one-tenth of the aliquot was removed and mixed with 20 µL of trypan blue to count the number of cells using a hemocytometer.

Results and Discussion It has been reported that it-PPO showed high crystallinity, with a melting point (Tm) around 67 °C,29 whereas commercially available at-PPO is usually a liquid and cannot be utilized as a bulk substrate in aqueous circumstances. Thus, it-PPO is considered a solid biomaterial on the interface within a living

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Figure 3. Laser scanning microscope profiles. Photo of an it- PPO spin-coated disk with 10 mg/mL at 4000 rpm (a) and a dip-coated disk with 10 mg/mL (b). Black bar indicates 50 µm. Three-dimensional profiles of an it-PPO spin-coated disk with 10 mg/mL at 4000 rpm (c) and a dip-coated disk with 10 mg/mL (d).

Ajiro and Akashi

Figure 5. FT-IR/ATR spectra of Cell Desk LF1 (a), bulk it-PPO (b), it-PPO dip-coated disk with 10 mg/mL (c), it-PPO spin-coated disk with 10 mg/mL at 4000 rpm (d), and the decomposed it-PPO after 4 h in air at 100 °C (e). Intensity comparison of it-PPO dip-coated disk with 20 (f), 10 (g), and 5 mg/mL (h) and spin-coated disk with 10 mg/mL at 500 (i), 1000 (j), and 4000 rpm (k), due to the intensity at 668 cm -1 as a standard.

Figure 4. Static contact angles on it-PPO spin coated disk with 10 mg/mL at 4000 rpm (a), it-PPO dip coated disk with 10 mg/mL (b), and commercialized LF1 disk as control (c) (n ) 3).

Figure 6. Protein adsorption of BSA, BγG, and BPF on it-PPO spincoated disks (a), it-PPO dip-coated disks (b), and Cell Desk LF 1 as a control (c) (n ) 3).

organism, and it was expected that oxidative degradation could be prevented moderately under physiological conditions around 37 °C. Actually, the crystalline of stereoregular poly(L-lactide) deposited on a substrate decomposed much slower than the amorphous form.34 Consequently, the spin coating and dip coating of it-PPO onto a plastic substrate were selected as surface modifications, and a plastic disk without any coating was employed as a control in this study. The appearance of the disk spin-coated with it-PPO looked similar to the transparent state of the control disk (Figure 2a). However, the dip coating from the chloroform solution resulted in a white, cloudy surface (Figure 2b). When observed by microscopy, the asperity on the surface was confirmed with many irregular craters, probably due to the rapid crystallization of it-PPO on the surface under humid circumstances with microsized water droplets (Figure 2c). Interestingly, humidity on the surface of amphiphilic block copolymers has been reported as an effective approach for the creation of a microsized honeycomb pattern.35 It was concluded that similar phenomenon appeared in the case of the dip coating process, and the resultant random geometry could affect cell morphology. Therefore, it was also used in this study, together with the smooth surface from spin coating. To investigate the microstructure of the surface, laser scanning microscope observation was achieved (Figure 3), because the clear images were not observed with atomic force microscope (AFM) observation (Supporting Information, Figure S1). Rela-

tively smooth surface was obtained by spin coating method (Figure 3c), however, some crater-shaped structures with around 6 µm height were observed from dip-coated disks (Figure 3d). This structural size was varied from 10 to 50 µm, depending on the it-PPO concentration (Supporting Information, Figure S2). The water contact angles of it-PPO dip- and spin-coated disks averaged 95.6 ( 3.8° and 82.6 ( 2.2°, although the Cell Desk LF1 was 69.9 ( 1.8° (Figure 4). It is known that the surface that contact angle is around 70° provides a good cell adhesion,36 and thus it-PPO surface provided an unexpected hydrophobic characteristic, suggesting that the ether linkages were buried inside the molecule and that a regular configuration of methyl groups were responsible for the hydrophobic nature. FT-IR/ATR spectra were employed for the analysis of surface coverage (Figure 5). The bulk it-PPO shows the C-H stretching vibration of methyl group from 2971 to 2908 cm-1, and the C-O-C stretching vibration of the ether group around 1100 cm-1 (Figure 5b).37 The same peaks appeared in both spectra of dip- and spin-coated disks (Figure 5c,d), in spite of the different intensities. The amount of coating it-PPO was dependent on the concentrations and speed of spin coating, and the surface was not fully covered. To estimate the amount of coated it-PPO, spectral intensities of the methyl group were compared (Figure 5f-k) using the peak appearing around 668 cm-1 in the bare Cell Desk LF1 as standard (Figure 5a). On the other hand, the carbonyl stretching peak at 1730 cm-1

Stereoregular isotactic-Poly(propylene oxide)

Figure 7. Cell numbers of mouse fibroblast L929 during culture after every 2 days on it-PPO spin coating (a), it-PPO dip coating (b), and Cell Desk LF 1 (c) (n ) 3). Statistically significant differences (*p < 0.01) were obtained using a two-sample t test for each comparison. NS ) no significant difference.

appeared when it-PPO was partially decomposed.38 After the screening of coating conditions with laser scanning microscope and FT-IR/ATR spectra, we decided to use it-PPO dip-coated disks with 10 mg/mL and spin-coated disks with 10 mg/mL at 4000 rpm for the cell proliferation test. Figure 6 depicts protein adsorption using BSA, BγG, and BPF, for both dip- and spin-coated disks, respectively (n ) 3). The amount of BSA adsorbed by the dip- and spin-coated disks was 8.10 µg/cm2 and 7.17 µg/cm2, respectively. There was no significant difference due to the preparation method of the itPPO coating, and the same tendencies were recognized in the cases of BγG (dip coating, 1.99 µg/cm2; spin coating, 2.34 µg/

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cm2) and BPF (dip coating, 0.64 µg/cm2; spin coating, 0.29 µg/ cm2), although it is unclear why BSA and BγG adsorbed so strongly. However, the estimated amounts indicated larger values due to a monolayered adsorption, because it was calculated from protein sizes of 0.9, 1.8, and 1.7 µg/cm2 for BSA, BγG, and BPF, respectively.39 Thus, the multilayered adsorption of these proteins would seem to be the case for this it-PPO hydrophobic surface, which is in good agreement with the contact angles. Next, we studied it-PPO as a substrate for cell proliferation, using the commercially available Cell Desk LF 1 as a control. The results of the cell culture with mouse fibroblast L929 cells are summarized in Figure 7, and the cell morphologies are shown in Figure 8. The cell numbers on the it-PPO dip- and spin-coated disks were evaluated after 2 days of incubation in Figure 4, resulting in both smaller numbers than the control surface. The dip-coated surface also held fewer cells than the spin-coated disk. It could be considered that both hydrophobic and rough factors were simultaneously reflected on these surfaces. Interestingly, it looks as if some of the cells on the it-PPO dip-coated disk would fit to the cratered shapes (Figure 8b), whereas cells on the it-PPO spin-coated disk looked similar to the control (Figure 8a,c). Recently, it has been reported that a spatially isolated cell tends to proliferate poorly,40 and thus, the same effect was initially observed. However, the number of cells after 4 days in vitro lost their significant differences. Moreover, the cells on the it-PPO dip-coated disks proliferated more than those on the it-PPO spin-coated disk after 4 and 8 days (Figure 7), suggesting that the walls of these random craters about 6 µm was not enough to separate each cell. In fact, it was impossible to recognize the cell fitting to the cratered shapes (Figure 8e and 8h).

Figure 8. Cell morphology after 2 days on it-PPO spin coating (a), it-PPO dip coating (b), Cell Desk LF 1 (c); after 4 days on it-PPO spin coating (d), it-PPO dip coating (e), Cell Desk LF 1 (f); and after 6 days on it-PPO spin coating (g), it-PPO dip coating (h), Cell Desk LF 1 (i). Black bar indicates 50 µm.

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A confluent state was observed after 6 days for the control, although the relatively slow proliferation on the it-PPO dipcoated disk did not reach confluence. Meanwhile, it is notable that almost the same cell numbers were observed on the itPPO dip-coated disks after 8 days (Figure 7), suggesting that the surface roughness delayed the cell proliferation. Furthermore, limited cytotoxicity was observed (Supporting Information, Figure S4). The aforementioned demonstration showed the characterization of it-PPO that may be useful as a biomaterial when the stereoregularity was controlled with high molecular weight.

Conclusion it-PPO was evaluated for its biocompatibility by contact angles, protein adsorption, and cell proliferation. The stereoregularity of PPO resulted in a solid material and a dramatic change on the surface hydrophilicity, resulting in a contact angle of 95.6 ( 3.8°. This bulk feature of it-PPO also affected protein adsorption in multilayers, and mouse fibroblast cells adhered well onto the surface of it-PPO-coated disks. It is suggested that the hydration ability of polyether created this novel hydrophobic surface, while maintaining the same chemical structure with a different configuration of the polymer chain backbone. Acknowledgment. The authors are grateful to Dr. M. Matsusaki for the measurement of three-dimensional images with a laser scanning microscope. This study was partially supported by a Grant-in-Aid for Young Scientists (B; 21750220) and Tokuyama Science Foundation. We thank Drs. T. Kida, M. Matsusaki, J. Watanabe, T. Akagi, and Prof. Geoffrey W. Coates for their helpful discussions. Supporting Information Available. AFM image, photos of contact angles, cytotoxicity test, GPC chart of it-PPO, IR spectra of it-PPO coated disks. This material is available free of charge via the Internet at http://pubs.acs.org.

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