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Templating r-Helical Poly(L-lysine)/Polyanion Complexes by Nanostructured Uniaxially Oriented Ultrathin Polyethylene Films Thomas F. Keller,† Martin M€uller,‡ Wuye Ouyang,‡ Jian-Tao Zhang,† and Klaus D. Jandt*,† †
Institute of Materials Science & Technology (IMT), Chair in Materials Science, Friedrich-Schiller-University Jena, L€ obdergraben 32, D-07743 Jena, Germany, and ‡Leibniz Institute of Polymer Research Dresden, Department of Polyelectrolytes and Dispersions, Hohe Strasse 6, D-01069 Dresden, Germany Received July 14, 2010. Revised Manuscript Received September 10, 2010
We report a templating effect of uniaxially oriented melt-drawn polyethylene (MD-PE) films on R-helical poly(L-lysine)/ poly(styrenesulfonate) (R-PLL/PSS) complexes deposited by the layer-by-layer (LBL) method. The melt-drawing process induced an MD-PE fiber texture consisting of nanoscale lamellar crystals embedded in amorphous regions on the MD-PE film surface whereby the common crystallographic c axis is the PE molecular chain direction parallel to the uniaxial melt-drawing direction. The MD-PE film and the R-PLL/PSS deposit were analyzed by atomic force microscopy (AFM) and in situ attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) using polarized light as a complementary method. Both methods revealed that R-PLL/PSS complexes adsorbed at the MD-PE surface were anisotropic and preferentially oriented perpendicular to the crystallographic c direction of the MD-PE film. Quantitatively, from AFM image analysis and ATR-FTIR dichroism of the amide II band of the R-PLL, mean cone opening angles of 12-18° for both rodlike R-PLL and the anisotropic R-PLL/PSS complexes with respect to the PE lamellae width direction were obtained. A model for the preferred alignment of R-PLL along the protruding PE lamellae is discussed, which is based on possible hydrophobic driving forces for the minimization of surface free energy at molecular and supermolecular topographic steps of the PE surface followed by electrostatic interactions between the interconnecting PSS and the R-PLL during layer-by-layer adsorption. This study elucidates the requirements and mechanisms involved in orienting biomolecules and may open up a path for designing templates to induce directed protein adsorption and cell growth by oriented polypeptide- or protein-modified PE surfaces.
Introduction Surface-biomolecule interactions are crucial for the successful performance of biomaterials1 because their surface is spontaneously covered by a protein layer mediating essentially all subsequent biological reactions.2-4 Besides chemical surface functionalization,5-7 the introduction of specific surface topographies has recently attracted attention,8,9 as for example with increasing surface roughness the amount of adsorbed protein may increase beyond what can be attributed to the sole increase in surface area.10 Apart from the total quantity of adsorbed biomolecules, we are interested in how nanoscale surface topography can influence their conformation, mutual molecular arrangement, and orientation and alignment, which together trigger many further cell reactions.8,11,12 So far, it is known that steps of ∼0.3 nm are sufficient to locally *Corresponding author. Tel: þ49 3641 947730. Fax: þ49 3641 947732. E-mail:
[email protected]. (1) Jandt, K. D. Adv. Eng. Mater. 2007, 9, 1035–1050. (2) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28–60. (3) Kasemo, B. Surf. Sci. 2002, 500, 656–677. (4) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233–244. (5) Thevenot, P.; Hu, W. J.; Tang, L. P. Curr. Top. Med. Chem. 2008, 8, 270–280. (6) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 8168–8173. (7) Jeyachandran, Y. L.; Mielezarski, E.; Rai, B.; Mielczarski, J. A. Langmuir 2009, 25, 11614–11620. (8) Lord, M. S.; Foss, M.; Besenbacher, F. Nano Today 2010, 5, 66–78. (9) Dalby, M. J.; Riehle, M. O.; Johnstone, H.; Affrossman, S.; Curtis, A. S. G. Cell Biol. Int. 2004, 28, 229–236. (10) Rechendorff, K.; Hovgaard, M. B.; Foss, M.; Zhdanov, V. P.; Besenbacher, F. Langmuir 2006, 22, 10885–10888. (11) Berglin, M.; Pinori, E.; Sellborn, A.; Andersson, M.; Hulander, M.; Elwing, H. Langmuir 2009, 25, 5602–5608. (12) Engel, M. F. M.; Visser, A. J. W. G.; van Mierlo, C. P. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11316–11321. (13) Gettens, R. T. T.; Bai, Z. J.; Gilbert, J. L. J. Biomed. Mater. Res. A 2005, 72, 246–257.
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induce the directed adsorption of fibrinogen, such as on the surface of highly ordered pyrolytic graphite (HOPG).13,14 F-actin protein filaments align along 1- to 2-nm-high steps; however, they adsorb randomly at steps of 3 to 4 nm heights.15 Nanostructures with vertical surface features of ∼20 nm result in a maximum amount of adsorbed fibronectin and collagen type IV at poly(lactic-co-glycolic acid) (PLGA) polymer surfaces,16 whereas high-aspect-ratio structures in the submicrometer range reduce the amount of adsorbed fibrinogen on PLGA surfaces as compared to that on flat films.17 A smart approach to designing nanometer-scale domains of chemical or topographic contrast utilizes the self-assembly of block copolymers by microphase separation. At such surfaces, adsorbed proteins may align into nanoarrays.18,19 Here, we used the crystallization-driven self-assembly of semicrystalline polyethylene (PE) to create highly oriented nanometer-scale surface patterns.20 Different grades of PE are widely applied or are in development as biomaterials such as for sutures,21 ligament replacement,22 cochlear implants,23 composites (14) Reichert, J.; Wei, G.; Jandt, K. D. Adv. Eng. Mater. 2009, 11, B177–B181. (15) Galli, C.; Coen, M. C.; Hauert, R.; Katanaev, V. L.; Groning, P.; Schlapbach, L. Colloids Surf., B 2002, 26, 255–267. (16) Carpenter, J.; Khang, D.; Webster, T. J. Nanotechnology 2008, 19, 505103. (17) Koh, L. B.; Rodriguez, I.; Venkatraman, S. S. Biomaterials 2010, 31, 1533– 1545. (18) George, P. A.; Donose, B. C.; Cooper-White, J. J. Biomaterials 2009, 30, 2449–2456. (19) Matsusaki, M.; Omichi, M.; Kadowaki, K.; Kim, B. H.; Kim, S. O.; Maruyama, I.; et al. Chem. Commun. 2010, 46, 1911–1913. (20) Keller, T.; Grosch, M.; Jandt, K. D. Macromolecules 2007, 40, 5812–5819. (21) Nishimura, K.; Mori, R.; Miyamoto, W.; Uchio, Y. Clin. Biomech. 2009, 24, 403–406. (22) Wintermantel, E.; Ha, S.-W. Medizintechnik: Life Science Engineering; Springer: Berlin, 2009. (23) Schindler, R. A.; Gladstone, H. B.; Scott, N.; Hradek, G. T.; Williams, H.; Shah, S. B. Am. J. Otol. 1995, 16, 304–309.
Published on Web 11/23/2010
DOI: 10.1021/la102811v
18893
Article
for skull implants,24 and syringes, tubes, and packaging.22 The ultrahigh-molecular-weight polyethylene (UHMW-PE) grade is used in endoprosthetic devices,25,26 where protein layers adsorbed from the synovial fluid on the UHMW-PE surface are known to lubricate the articulation under uniaxial compressive load during a combined rolling and gliding motion.27 In this framework, it is particularly interesting to obtain quantitative information on the orientational state of proteins adsorbed on PE and its dependence on the PE surface nanostructure. R-helical homopolypeptides may serve as model systems for R-helical-rich proteins such as human serum albumin (HSA), fibrinogen,28-30 and R-helical protein segments. The R-helical content of the secondary structure of bovine serum albumin (BSA, the bovine analogue) amounts, for example, to approximately 54%.31 Because an R-helical conformation of the poly(L-lysine) (PLL) polypeptide can be easily induced by several environmental parameters such as temperature,32 solvent,33 and specific salt ions34 or by controlling the pH,33 PLL is an interesting candidate for investigating such directed surface-biomolecule interactions on textured surfaces. PLL is a cationic polyelectrolyte (PEL) that can be consecutively adsorbed with polyanions to form polyelectrolyte multilayers (PEM) using the layer-by-layer (LBL) technique.35 PEM consisting of R-helical PLL (R-PLL)/ polyanion complexes can be oriented onto textured silicon substrates.36 The R-helical conformation of PLL, a sufficiently high molecular weight, and the presence of scratched grooves were identified by atomic force microscopy (AFM) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) as requirements for inducing orientational effects.37 A texture and molecular chain orientation can be introduced in high-density PE (HD-PE)38-41 and UHMW-PE20 by the meltdrawing technique, resulting in semicrystalline, highly oriented, ultrathin melt-drawn PE (MD-PE) films, which may serve as nanostructured substrates for investigating the adsorption of proteins or polypeptides. The structural units of UHMW-PE and HD-PE are therefore similar (i.e., on the molecular scale, the CH2 repeat units and on the nanoscale, typical lamellar crystals protruding approximately a few nanometers out of the surrounding amorphous matrix of tie molecules). Polycaprolactone (PCL) crystallizes heteroepitactically on oriented MD-PE substrates with their chain axis aligned parallel to the HD-PE chain direction,42 indicating that under suitable conditions on the (24) Zhang, Y.; Tanner, K. E. J. Mater. Sci.: Mater. Med. 2008, 19, 761–766. (25) Kurtz, S. M.; Pruitt, L.; Jewett, C. W.; Crawford, R. P.; Crane, D. J.; Edidin, A. A. Biomaterials 1998, 19, 1989–2003. (26) Taddei, P.; Affatato, S.; Fagnano, C.; Toni, A. Biomacromolecules 2006, 7, 1912–1920. (27) Gibson, D. S.; Rooney, M. E. Proteomics: Clin. Appl. 2007, 1, 889–899. (28) Peters, T. Adv. Protein Chem. 1985, 37, 161–245. (29) Haeberlin, A., Ed. Human Protein Data; VCH: Weinheim, Germany, 1995. (30) Putnam, F. W. The Plasma Proteins. Academic Press: New York, 1975. (31) Kinsella, J. E.; Whitehead, D. M. Adv. Food. Nutr. Res. 1989, 33, 343. (32) Susi, H.; Timasheff, S. N.; Stevens, L. J. Biol. Chem. 1967, 242, 5460. (33) M€uller, M.; Buchet, R.; Fringeli, U. P. J. Phys. Chem. 1996, 100, 10810– 10825. (34) Sugai, S.; Ebert, G. Adv. Colloid Interface Sci. 1986, 24, 247–282. (35) Decher, G. Science 1997, 277, 1232–1237. (36) M€uller, M. Biomacromolecules 2001, 2, 262–269. (37) M€uller, M.; Ouyang, W.; Kessler, B. Int. J. Polym. Anal. Charact. 2007, 12, 35–45. (38) Petermann, J.; Gohil, R. M. J. Mater. Sci. 1979, 14, 2260–2264. (39) Jandt, K. D.; Buhk, M.; Petermann, J.; Eng, L. M.; Fuchs, H. Polym. Bull. 1991, 27, 101–107. (40) Jandt, K. D.; Buhk, M.; Miles, M. J.; Petermann, J. Polymer 1994, 35, 2458– 2462. (41) Jandt, K. D.; Mc Master, J.; Miles, M. J.; Petermann, J. Macromolecules 1993, 26, 6552–6556. (42) Yan, C.; Li, H. H.; Zhang, J. M.; Ozaki, Y.; Shen, D. Y.; Yan, D. D.; et al. Macromolecules 2006, 39, 8041–8048.
18894 DOI: 10.1021/la102811v
Keller et al.
surface of oriented PE other synthetic macromolecular chains can be oriented. However, the surface-directed adsorption of biomolecules, such as polypeptides on textured polymer surfaces, has not been reported so far to the best of our knowledge. Here we report for the first time that an anisotropic adsorption of biomolecules, such as PLL, can be achieved on textured PE surfaces provided that the PE molecular chains and crystalline lamellae are themselves highly oriented. The LBL technique was used to adsorb a three-layered complex of R-PLL and the much smaller polyanion poly(styrenesulfonate) (PSS) that interconnects the R-PLL rods at the MD-PE film surface. The anisotropy of PE lamellae and R-PLL rods complexed by PSS was quantified by AFM and compared with the mean PE and PLL molecular chain orientation determined by polarizationdependent Fourier-transformed infrared spectroscopy in ATR and transmission (TRANS-FTIR) geometry. Consecutively depositing both PLL and PSS by the LBL technique increases the total adsorbed amount of PLL as compared to a single deposition step of PLL because in the latter case the adsorbed amount is limited by the self-repulsion of the positively charged PLL. In other words, applying the LBL technique permits a more accurate spectroscopic analysis of the orientational state of the R-PLL rods simply by their higher adsorbed amount. Oriented polypeptide layers, such as the R-PLL/PSS complexes adsorbed at highly textured MD-PE surfaces, could be used in the future as a template to induce specific cellular orientational arrangements or to serve as model to optimize the protein lubrication of unidirectional motions in endoprosthetic devices.
Experiments and Methods Sample Preparation. PE Film Substrates. As previously described,20,40 MD-PE films were prepared as follows. HD-PE pellets (Sigma-Aldrich, Schnelldorf, Germany) were dissolved in xylene (synthesis grade, Merck KGaA, Darmstadt, Germany) to obtain a 1 wt % polymer solution, which was heated to 120 °C. Subsequently, a few droplets were placed onto a heated glass plate on top of a precision heating plate kept at a temperature of 125 °C. After evaporation of the solvent, an ultrathin, highly oriented polymer film (thickness
