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J. Phys. Chem. C 2007, 111, 11676-11681
Patterning of Polypeptides on a Collagen-Terminated Tissue Surface Rizaldi Sistiabudi† and Albena Ivanisevic*,†,‡ Weldon School of Biomedical Engineering, and Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907 ReceiVed: April 9, 2007; In Final Form: June 1, 2007
Collagen is a ubiquitous component of the extracellular matrix environment, and numerous studies have been devoted toward the development of collagen-based tissue scaffolds. These efforts have been primarily focused on synthetic collagenous materials made from purified collagen. In this article, we present a preliminary study toward the development of a technique that can result in a tissue-derived collagen scaffold. The tissuederived collagenous matrix was isolated from the retinal Bruch’s membrane, and dip pen nanolithography was investigated as a mean to modify the collagenous surface. Characterization experiments of the collagenous surface indicate a fairly hydrophobic surface. Minimal swelling (100 µm). We note that during the preparation steps insufficient buffer washing can result in debris from loose tissue being randomly trapped across the sample. 3.2. Collagen Swelling Study at Variable Relative Humidity Conditions. Analysis of collagen fiber diameter was performed using the accompanying Nanoscope software (Digital Instruments). To determine the diameter of the collagen fibers, a rectangle is drawn along the length of the fibers and the software performs a fast Fourier transform (FFT) of the cross section along the defined rectangle (Figure 2). The measured values of the dried fibers were consistent with several AFM studies of collagen from various sources.18-21
Figure 3. (a) Swelling of collagen fibers as a function of relative humidity. (b) High-resolution AFM contact mode images (deflection) at different humidity conditions of a randomly chosen region on the sample.
Swelling of collagen fibers was observed as a function of relative humidity. The data reported with respect to fiber diameter and percentage swelling is a result of multiple series of measurements. From the plot of fiber diameter versus relative humidity (Figure 3a), the increase in fiber diameter is estimated to be less than 7%. It is important to note that the observed increase in fiber diameter is not solely due to water absorption by the collagen fibers. The elevated humidity conditions also lead to increase in the convolution effects due to water condensation on the AFM tip. Therefore, the fiber diameters reported in Figure 3 were only extracted in order to make a qualitative comparison. The experiments shown in that figure were done to assess the relative magnitude of any surface rearrangement or drastic change in topography as a result of the increased humidity. The high-resolution images obtained at relative humidity levels of 31%, 51%, 62%, and 73% (Figure 3b) indicate negligible surface rearrangement due to increased relative humidity. If there was significant change in surface topography under the elevated humidity conditions, site-specific modification will be difficult to predict and control. 3.3. Lateral Force Microscopy Studies Using Modified AFM Tips. Lateral force microscopy (LFM) images were obtained for the same area using the three different tips. This experiment was repeated on several samples extracted from
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Figure 4. Lateral force microscopy (LFM) image of the Bruch’s membrane (BM) ICZ surface collected with (a) an unmodified tip, (b) a OHterminated tip, and (c) a CH3-terminated tip.
TABLE 1: Patterning Conditions for DPN on Clean SiO2 Surfaces clean SiO2 temp/relative humidity deposition time: drop/sub scan rate/applied load dependence tip condition
poly(glutamic acid) 23 °C/34% 5 s/10 s 6 Hz/no
polyarginine 23 °C/34% 5 s/10 s 6 Hz/no
depleted ink
depleted ink
different donor eyes, and the same results were recorded if the sample was prepared as described in the experimental procedure. We note that changes in the preservation protocol are expected to change the overall hydrophobicity of the sample. The LFM images were processed using third-order flattening from the Nanoscope software. Identical scale bars were used for the three images in Figure 4. Regions with higher friction are brighter than regions with lower friction. Qualitative observation of the images indicates an increase in friction with the use of CH3terminated tips compared to the ones collected with -OHterminated tips. One expects an increase in adhesion force when the hydrophobic CH3-terminated tip images a hydrophobic surface.22,23 The LFM images support the notion that the ICZ surface is fairly hydrophobic. We also evaluated the tissue samples using contact angle measurements. The average contact angles recorded on two randomly chosen samples were 88.8° ( 10.73° and 92.25° ( 6.45°. We observed a high degree of variability in the contact angle measurement due to nonhomogeneous nature of the samples. 3.4. Patterning of Polypeptide Ink via Dip Pen Nanolithography (DPN). 3.4.1. DPN on Inorganic Surfaces. The average contact angle values for the inorganic surfaces were (1) 9.75° ( 0.5° for clean SiO2 surface, (2) 66.83° ( 4.12° for APTES surface, and (3) 100.5° ( 1° for TMPS surface. These values were in agreement with previously reported values for well-defined monolayers.24,25 Roughness analysis was performed from the AFM height images; the rms values were 0.371 ( 0.071 nm for APTES surface and 0.434 ( 0.098 nm for TMPS surface. DPN experiments were performed under the conditions given in Tables 1 and 2. DPN of the two polypeptides occurred via diffusion on the two hydrophilic surfaces (bare SiO2 and APTES). Representative images of polypeptide patterning on the hydrophilic surfaces are given in Figure 5. The AFM images were collected following the DPN experiment using the same coated tip. The estimated line widths from friction image for the bare SiO2 surface (Figure 5) were (a) 410.155 ( 105.79 nm for poly(glutamic acid) and (b) 332.03 ( 27.62 for polyarginine. Meanwhile, the estimated
Figure 5. Representative image of polypeptide patterning via DPN on inorganic surfaces: (a) height image and (b) friction image of poly(glutamic acid) on clean a SiO2 surface.
TABLE 2: Patterning Conditions for DPN on APTES-Terminated Surfaces APTES-terminated SiO2 temp/relative humidity deposition time: drop/sub scan rate/applied load dependence tip condition
poly(glutamic acid) 23 °C/37% 3 s/3 s 6 Hz/no
polyarginine 23 °C/36% 5 s/5 s 6 Hz/no
fresh 1/2 h dip
fresh 1/2 h dip
line widths for the APTES surface were (a) 512.69 ( 46.15 nm for poly(glutamic acid) and (b) 493.16 ( 46.15 nm for polyarginine. Our initial experiments show that the transfer rate of poly(glutamic acid) is higher than that of polyarginine on both surfaces. These experiments allowed us to optimize the tip-coating procedure and verify that one can write with a specific “pen” (tip). DPN experiments for both polypeptides on the TMPS surface failed to produce any patterns even under high relative humidity condition (80-90% relative humidity). Failure to observe features of polypeptide molecules from the coated tip to the TMPS substrate indicates the polypeptide ink did not transport on this highly hydrophobic surface. 3.4.2. DPN on the Inner Collagenous Zone of the Bruch’s Membrane. Successful DPN on the BM surface was performed under the conditions given in Table 3. AFM images were collected using the same tip following the DPN experiment. For the purpose of imaging, the scan rate was increased to 3 Hz and the deflection set point was decreased to the minimum value possible (∼0-1 nN), while still maintaining contact with the surface. The most important parameter for the transfer of polypeptide inks to the ICZ surface was the amount of force applied by the tip on the surface. From our experiments, we found that the deposition of polypeptide can occur only under high deflection set point. Loads above 200 nN were necessary for the deposition of poly(glutamic acid) and polyarginine.
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Figure 6. Representative image of polypeptide patterning via DPN on Bruch’s membrane (BM) inner collagenous zone (ICZ): (A) DPN with polypeptide ink: AFM height image of poly(glutamic acid) on the BM; (B) DPN with surfactant-added polypeptide ink: friction image of poly(glutamic acid) deposition on the BM.
TABLE 3: Patterning Conditions for DPN on the Bruch’s Membrane ICZ Surface Bruch’s membrane temp/relative humidity deposition time: drop/sub scan rate/applied load dependence tip condition
poly(glutamic acid) 23 °C/37% 30 s 0.2 Hz/yes
polyarginine 23 °C/60% 30 s 0.2 Hz/yes
fresh 1/2 h dip
fresh 1/2 h dip
Considering that damage to the surface might occur at this high set point, a separate experiment was carried out to determine the lowest set point that would induce damage to the collagenous surface. From this experiment it was determined that damage to the collagenous surface did not occur under any of the high loads applied during the patterning. We note that the conditions under which the collagenous surface was exposed and dried can greatly affect the ability to rearrange or damage the fibers on the tissue surface by applying higher contact force. We have prepared the tissue surface using a variety of different protocols and have observed in past samples that contained loosely packed collagen that one can easily cause damage by contact mode AFM imaging. We also note that the higher loads can disturb the cross-linking within and between the fibers, even though one cannot see any changes from the AFM images. Such changes can greatly influence the attachment behavior of cells to the surface. We are in the process of quantifying this effect using in vitro cell culture studies. The role of the relative humidity was less important since deposition of the polypeptides was not observed unless the necessary tip contact force was applied. Without applying loads above 200 nN the deposition of poly(glutamic acid) would not occur even when the relative humidity was increased to 8090%. With loads above 200 nN poly(glutamic acid) was deposited on the surface at ambient humidity (37%). On the other hand, the deposition of polyarginine did require an elevated relative humidity condition of at least 60%. Consistent with the result of DPN experiments on inorganic surfaces, transfer of poly(glutamic acid) is more rapid than polyarginine. Under the conditions mentioned above, the deposition of polypeptide inks occurred through molecular deposition with no diffusion of the molecules (Figure 6A). We suspected that the lack of diffusion was caused by the hydrophobicity of the BM surface. As such, Tween-20 surfactant was added into the inking solution to increase the wettability of the surface during the DPN procedure.26 Consistent with previous reports, the addition of Tween-20 enabled the deposition of the polypeptide under lower contact force. The contact force necessary to deposit
the surfactant-added polypeptide inks was between 80 and 100 nN with relative humidity of 40% (Figure 6B). 4. Discussion and Conclusions Removal of the RPE basal lamina layer from the retinal BM revealed the collagenous surface of the ICZ. Under elevated humidity conditions, this surface undergoes negligible rearrangement with fiber swelling less than 7%. As such, this surface is a potential candidate for DPN which often necessitates elevated relative humidity conditions for biomolecules.15 Further investigation of this tissue-derived collagen surface showed a relatively hydrophobic surface. This conclusion was derived from the analysis of LFM images and contact angle measurements. The nature of the ICZ surface poses two main challenges for polypeptide patterning via DPN. First is the surface roughness; analysis from the AFM height images revealed a highly rough surface with significant variability (rms value 75.91 ( 26 nm). Investigation by others has shown that surface roughness has a significant effect on the pull-off force of the AFM tip.27 Therefore, the high variability in surface roughness may result in variations of molecular transport rates across the surface. The second challenge is the hydrophobicity of the ICZ surface which creates a barrier toward diffusion and transport of the polypeptide ink onto the surface. As observed in the DPN experiments on inorganic surfaces, both poly(glutamic acid) and polyarginine diffuse freely with high transport rate on the more hydrophilic surfaces (clean SiO2 and APTES). Lines having feature widths of 300-500 nm were created within seconds of tip-to-surface contact. Meanwhile, deposition of molecules did not occur on the hydrophobic TMPS surface. On the collagen surface, deposition of the polypeptide ink could be achieved by adjusting the DPN conditions. An important parameter is the contact force between the tip and the surface. The addition of Tween-20 surfactant into the ink solution increased the wettability of the BM surface and allowed us to use lower loads during the patterning procedure. Another important parameter is the relative humidity and the solubility of the ink. In general, a higher relative humidity condition translates to an increase in the width of the water meniscus which facilitates the transfer of molecules onto the surface.12 Meanwhile, the higher solubility of polyarginine in water may explain the lower transport rate in comparison to that of poly(glutamic acid). L-Arginine is 22 times more soluble in water than L-glutamic acid.28 Having a higher propensity to stay in the water meniscus, polyarginine required a higher driving force for molecular deposition.
Polypeptides on a Collagen-Terminated Surface In summary, we have successfully shown the patterning of polypeptides onto a tissue-derived collagenous surface and discussed the parameters that can influence the success of the DPN experiment. Understanding the important parameters is the first step toward the utilization of DPN for surface modification of tissue-derived collagenous surfaces. An ongoing work in our laboratory is to use peptide molecules with specificity toward the collagen fibers. We are in the process of optimizing the pattering parameters of these inks and plan to use the results to test the utility of DPN-generated scaffolds in cell culture. Acknowledgment. The authors thank the veterinary staff at the Weldon School of Biomedical Engineering for help with harvesting the donor eyes. References and Notes (1) Matsushita, O.; Jung, C. M.; Minami, J.; Katayama, S.; Nishi, N.; Okabe, A. A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. J. Biol. Chem. 1998, 273 (6), 3643-3648. (2) Marshall, J.; Hussain, A. A.; Starita, C.; Moore, D. J.; Patmore, A. L. Aging and Bruch’s Membrane. In The Retinal Pigment Epithelium; Marmor, M. E., Wolfensberger, T. J., Eds.; Oxford University Press: New York, 1998; pp 669-692. (3) Chen, M. C.; Liang, H. F.; Chiu, Y. L.; Chang, Y.; Wei, H. J.; Sung, H. W. A novel drug-eluting stent spray-coated with multi-layers of collagen and sirolimus. J. Controlled Release 2005, 108 (1), 178-189. (4) McDaniel, D. P.; Shaw, G. A.; Elliott, J. T.; Bhadriraju, K.; Meuse, C.; Chung, K. H.; Plant, A. L. The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype. Biophys. J. 2007, 92 (5), 17591769. (5) Tezel, T. H.; DelPriore, L. V.; Kaplan, H. J. Ability of different layers of human Bruch’s membrane to support reattachment of human retinal pigment epithelium. InVest. Ophthalmol. Visual Sci. 1997, 38 (4), 43934393. (6) Duan, X. D.; McLaughlin, C.; Griffith, M.; Sheardown, H. Biofunctionalization of collagen for improved biological response: scaffolds for corneal tissue engineering. Biomaterials 2007, 28 (1), 78-88. (7) De Souza, S. J.; Brentani, R. Collagen binding site in collagenase can be determined using the concept of sense-antisense peptide interactions. J. Biol. Chem. 1992, 267 (19), 13763-13767. (8) Wang, A.; Mo, X.; Chen, C. S.; Yu, S. M. Facile modification of collagen directed by collagen mimetic peptides. J. Am. Chem. Soc. 2005, 127, 4130-4131. (9) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. “Dip-pen” nanolithography. Science 1999, 283, 661-663. (10) Manandhar, P.; Jang, J.; Schatz, G. C.; Ratner, M. A.; Hong, S. Anomalous surface diffusion in nanoscale direct deposition processes. Phys. ReV. Lett. 2003, 90, 115505.
J. Phys. Chem. C, Vol. 111, No. 31, 2007 11681 (11) Jang, J. Y.; Hong, S. H.; Schatz, G. C.; Ratner, M. A. Self-assembly of ink molecules in dip-pen nanolithography: a diffusion model. J. Chem. Phys. 2001, 115, 2721-2729. (12) Hampton, J. R.; Dameron, A. A.; Weiss, P. S. Double-ink dip-pen nanolithography studies elucidate molecular transport. J. Am. Chem. Soc. 2006, 128, 1648-1653. (13) Rozhok, S.; Piner, R.; Mirkin, C. A. Dip-pen nanolithography: what controls ink transport. J. Phys. Chem. B 2003, 107, 751-757. (14) Rozhok, S.; Sun, P.; Piner, R.; Lieberman, M.; Mirkin, C. A. AFM study of water meniscus formation between an AFM tip and NaCl substrate. J. Phys. Chem. B 2004, 108 (23), 7814-7819. (15) Cho, Y.; Ivanisevic, A. SiOx surfaces with lithographic features composed of a TAT peptide. J. Phys. Chem. B 2004, 108, 15223-15228. (16) Del Priore, L. V.; Tezel, T. H. Reattachment rate of human retinal pigment epithelium to layers of human Bruch’s membrane. Arch. Ophthalmol. 1998, 116 (3), 335-341. (17) Kurth, D. G.; Bein, T. Surface reactions on thin layers of silane coupling agents. Langmuir 1993, 9, 2965-2973. (18) Denis, F. A.; Pallandre, A.; Nysten, B.; Jonas, A. M.; DupontGillain, C. C. Alignment and assembly of adsorbed collagen molecules induced by anisotropic chemical nanopatterns. Small 2005, 1 (10), 984991. (19) Wilson, D. L.; Martin, R.; Hong, S.; Cronin-Golomb, M.; Mirkin, C. A.; Kaplan, D. L. Surface organization and nanopatterning of collagen by dip-pen nanolithography. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (24), 13660-13664. (20) Graham, J. S.; Vomund, A. N.; Phillips, C. L.; Grandbois, M. Structural changes in human type I collagen fibrils investigated by force spectroscopy. Exp. Cell Res. 2004, 299 (2), 335-342. (21) Cisneros, D. A.; Hung, C.; Franz, C. A.; Muller, D. J. Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy. J. Struct. Biol. 2006, 154 (3), 232-245. (22) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Functional group imaging by chemical force microscopy. Science 1994, 265, 2071-2074. (23) Noy, A.; Frisbie, C. D.; Rozsnyai, L. F.; Wrighton, M. S.; Lieber, C. M. Chemical force microscopy: exploiting chemically-modified tips to quantify adhesion, friction, and functional-group distributions in molecular assemblies. J. Am. Chem. Soc. 1995, 117 (30), 7943-7951. (24) Ulman, A. Formation and structure of self-assembled monolayers. Chem. ReV. 1996, 96, 1533-1554. (25) Xiao, S. J.; Textor, M.; Spencer, N. D.; Sigrist, H. Covalent attachment of cell-adhesive, (Arg-Gly-Asp)-containing peptides to titanium surfaces. Langmuir 1998, 14 (19), 5507-5516. (26) Jung, H.; Dalal, C. K.; Kuntz, S.; Shah, R.; Collier, C. P. Surfactant activated dip-pen nanolithography. Nano Lett. 2004, 4, 2171-2177. (27) Jang, J. Y.; Ratner, M. A.; Schatz, G. C. Atomic-scale roughness effect on capillary force in atomic force microscopy. J. Phys. Chem. B 2006, 110 (2), 659-662. (28) Jin, X. Z.; Chao, K. C. Solubility of four amino acids in water and of four pairs of amino acids in their water solutions. J. Chem. Eng. Data 1992, 37, 199-203.
