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Collagen-Binding Peptide Interaction with Retinal Tissue Surfaces Rizaldi Sistiabudi† and Albena Ivanisevic*,†,‡ Weldon School of Biomedical Engineering and Department of Chemistry, 206 S. Martin Jischke DriVe, Purdue UniVersity, West Lafayette, Indiana 47907 ReceiVed September 19, 2007. In Final Form: January 16, 2008 One of the current challenges in treating age-related macular degeneration (AMD) is the surface modification of the retinal Bruch membrane. In this study, the collagen fibers of the inner collagenous zone of the Bruch membrane were identified as type I and type III. Subsequently, the adsorption of a collagen-binding peptide onto the inner collagenous zone surface was investigated. The collagen-binding peptide was able to bind specifically to the collagen fibers while maintaining the biological activity of the N-terminus biotin tag. These results indicate that the collagenbinding peptide may be used as an anchor to immobilize bioactive molecules on the inner collagenous zone surface of the Bruch membrane.
1. Introduction The ultimate goal of bioengineered therapy is the restoration of the patient’s physiological function. In recent years, a number of therapies have been proposed for the treatment of age-related macular degeneration (AMD).1-6 Patients suffering from AMD experience a decline in central visual function stemming from the degeneration of the macula. Early on, the degeneration of the macula is caused by changes in the retinal Bruch membrane (BM) that serves as the basement membrane of the retinal pigment epithelium layer.7 Loss of retinal pigment epithelium layer patency leads to malnourishment of the photoreceptor layer, leading to impairment of visual function. Restoration of visual function has thus been the goal for bioengineered treatments of AMD. A promising approach to recovering visual function is to restore the retinal pigment epithelium layer. For this, the implantation of donor retinal pigment epithelium cells to account for the loss of retinal pigment epithelium cells has been attempted.8-11 In animal models, direct injection of donor retinal pigment epithelium cells has been shown to repopulate damaged areas; unfortunately, such successes do not readily translate to human subjects.8 The main impediment is the limited survival of donor retinal pigment epithelium cells caused by an improper extracellular matrix (ECM) condition (i.e., diseased Bruch’s membrane). It has been shown that a diseased Bruch’s membrane is * Author to whom correspondence should be addressed. E-mail: albena@ purdue.edu. † Weldon School of Biomedical Engineering. ‡ Department of Chemistry. (1) Benner, J. D.; Sunness, J. S.; Ziegler, M. D.; Soltanian, J. Arch. Ophthalmol. 2002, 120, 586-594. (2) De Juan, E.; Loewenstein, A.; Bressler, N. M.; Alexander, J. Am. J. Ophthalmol. 1998, 125, 635-646. (3) Del Priore, L. V.; Geng, L.; Tezel, T. H. InVest. Ophthalmol. Visual Sci. 2000, 41, S858-S858. (4) Del Priore, L. V.; Ishida, O.; Johnson, E. W.; Sheng, Y.; Jacoby, D. B.; Geng, L.; Tezel, T. H.; Kaplan, H. J. InVest. Ophthalmol. Visual Sci. 2003, 44, 4044-4053. (5) Hartmann, U.; Sistani, F.; Steinhorst, U. H. Graefes Arch. Clin. Exp. Ophthalmol. 1999, 237, 940-945. (6) Lappas, A.; Weinberger, A. W. A.; Foerster, A. M.; Kube, T.; Rezai, K. A.; Kirchhof, B. Graefes Arch. Clin. Exp. Ophthalmol. 2000, 238, 631-641. (7) Zarbin, M. A. Current concepts in the pathogenesis of age-related macular degeneration. Arch. Ophthalmol. 2004, 122, 598-614. (8) Algvere, P. V.; Berglin, L.; Gouras, P.; Sheng, Y. Graefes Arch. Clin. Exp. Ophthalmol. 1994, 232, 707-716. (9) Del Priore, L. V.; Tezel, T. H. Arch. Ophthalmol. 1998, 116, 335-341. (10) Semkova, I.; Kreppel, F.; Welsandt, G.; Luther, T.; Kozlowski, J.; Janicki, H.; Kochanek, S.; Schraermeyer, U. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13090-13095. (11) Thumann, G.; Aisenbrey, S.; Schraermeyer, U.; Lafaut, B.; Esser, P.; Walter, P.; Bartz-Schmidt, K. U. Arch. Ophthalmol. 2000, 118, 1350-1355.
not conducive toward cellular attachment, thereby causing apoptosis of the donor cells.12 A number of studies have been devoted to the creation of a suitable scaffold for donor cells. Examples include (a) a synthetic polymer scaffold from polyL-lactic acid (PLLA) and poly-co-glycolic acid (PLGA);13 (b) a surface-modified anterior lens capsule;5,14 and (c) a surfacemodified BM through treatment with detergent followed by the deposition of ECM protein.3,15 Despite these efforts, there is still an unmet need to make scaffolds that can produce a better environment for the survival of retinal pigment epithelium cells. In this letter, we report an approach to modify the surface of the Bruch membrane. The Bruch membrane consists of five different layers, among which are two collagenous layers known as the inner collagenous zone and the outer collagenous zone.16 The proposed approach to modify the Bruch membrane surface is by exposing the inner collagenous zone and immobilizing biomolecules onto the surface through binding to collagen. We have previously shown that the inner collagenous zone surface is suitable for lithographic patterning with biomolecules.17 Here, we investigate a way to modify the surface through specific biochemical interactions. First, we further our understanding of the inner collagenous zone surface by identifying the types of collagen fibers that are exposed on the very top surface layer. The identification of the type of exposed collagen fibers was carried out using antibodies against collagen. Subsequently, we investigated the binding of a certain collagen-binding peptide. The collagen-binding peptide can serve as the anchor for the immobilization of biomolecules onto the inner collagenous zone surface. Designing methods for the immobilization of biomolecules onto the inner collagenous zone layer is important because it can enable us to re-engineer a tissue surface composed of moieties that can enhance the attachment and survival of retinal pigment epithelium cells. We hypothesize that, by exposing the inner collagenous zone surface and utilizing the collagen-binding (12) Tezel, T. H.; DelPriore, L. V.; Kaplan, H. J. InVest. Ophthalmol. Visual Sci. 1997, 38, 4393-4393. (13) Lavik, E. B.; Klassen, H.; Warfvinge, K.; Langer, R.; Young, M. J. Biomaterials 2005, 26, 3187-3196. (14) Nicolini, J.; Kiilgaard, J. F.; Wiencke, A. K.; Heegaard, S.; Scherfig, E.; Prause, J. U.; La Cour, M. Acta Ophthalmol. Scand. 2000, 78, 527-531. (15) Tezel, T. H.; Del Priore, L. V.; Kaplan, H. J. InVest. Ophthalmol. Visual Sci. 2004, 45, 3337-3348. (16) 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. (17) Sistiabudi, R.; Ivanisevic, A. J. Phys. Chem. C 2007, 111, 11676-11681.
10.1021/la703561d CCC: $40.75 © 2008 American Chemical Society Published on Web 02/07/2008
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peptide as an anchor, we can controllably modify the biochemical and surface properties of the Bruch membrane. 2. Materials and Methods 2.1. Preparation of the Bruch Membrane Surface. All samples used in this study were terminated on the inner collagenous zone. This part of the Bruch membrane is located beneath the basement membrane of the retinal pigment epithelial cells.16 In all cases, we removed the retinal pigment epithelium basement membrane in order to expose the collagenous layer of interest. All tissue samples were harvested from porcine eyes immediately after surgery or after being stored in a -80 °C refrigerator. The inner collagenous zone surfaces were prepared as previously published.17 The exposed inner collagenous zone surfaces were imaged using atomic force microscopy (AFM). The model was Multimode SPM from Veeco Instruments, Santa Barbara, CA. Images were obtained in contact mode using silicon nitride contact mode tips (k ) 0.05 N/m, Veeco Instruments, Santa Barbara, CA). 2.2. Identification of Exposed Collagen Type Using Labeled Antibodies. The following antibodies were used to investigate the types of exposed collagen on the surface: (a) monoclonal mouse anti-collagen I antibody was purchased from Sigma (C2456); (b) monoclonal mouse anti-collagen III antibody was purchased from Upstate/Millipore (MAB 3392); (c) monoclonal mouse anti-human CTFG/CCN2 C-terminal peptide antibody was a kind gift from Dr. Alyssa Panitch; and (d) sheep anti-mouse IgG (whole molecule) F(ab′)2 fragment-Cy3 antibody was purchased from Sigma (C2182). A working dilution of 1:1000 in 50 mM Tris buffer at pH 7.5 was used to make solutions of anti-collagen I, anti-collagen III, and anti-mouse antibodies. A working dilution of 1:100 in buffer was used to make a solution of anti-human CTFG/CCN2 C-terminal peptide antibody. Three inner collagenous zone sample surfaces were incubated in anti-collagen I solution, and another three samples were incubated in anti-collagen III solution. Samples were left to incubate for 24 h at 4 °C. As negative controls, two samples were incubated in buffer containing no antibodies (blank control), and another two samples were incubated in a solution of anti-human CTFG/CCN2 C-terminal peptide antibody (mouse antibody control). All samples were then rinsed copiously with buffer and incubated in Cy3-labeled anti-mouse antibody solution for another 24 h at 4 °C. Finally, the samples were rinsed well and stored in buffer. The use of a blank control was meant to show that the Cy3labeled anti-mouse antibodies were specifically binding to the mouseproduced antibodies (anti-collagen I and anti-collagen III). Meanwhile, the use of a mouse antibody control was meant to show that the anti-collagen antibodies that were produced in mouse were binding specifically to the collagen fibers on the Bruch membrane surface. The samples were investigated using an Olympus IX81/FV1000 laser scanning confocal microscope (Olympus America Inc., Center Valley, PA). Images were collected and analyzed using the accompanying Fluoview software. Three random spots were chosen on each sample, and the average fluorescence intensities over the whole field of view were recorded. The average fluorescence intensities of each sample were then statistically compared using a two-sample t test performed by the Minitab software. 2.3. Adsorption Study of a Collagen-Binding Peptide. The amino acid sequence of the collagen-binding peptide was CQDSETRTFY (Cys-Gln-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr). An Nterminus biotin-labeled collagen-binding peptide was purchased from GenScript Corp. (Piscataway, NJ). Colloidal gold (20 nm) conjugated with streptavidin (cat. no. 15843) was purchased from Ted Pella Inc. (Redding, CA). Cy3-labeled streptavidin solution (S6402) was purchased from Sigma. A collagen-binding peptide-biotin solution was made by dissolving 2 mg of collagen-binding peptide-biotin in 40 mL of 50 mM Tris buffer containing 150 mM NaCl at pH 7.5 to obtain a final concentration of 50 µg/mL. Inner collagenous zone samples (n ) 6) were incubated in collagen-binding peptide-biotin solution overnight under gentle shaking at room temperature. To wash
Figure 1. Schematics of collagen-binding peptide adsorption studies: (A) fluorescence study of group 1 and (B) FESEM and AFM studies of group 2.
Figure 2. Representative AFM image of the sample surface collected in contact mode: (A) deflection AFM image and (B) 3-D topography image. unbound collagen-binding peptide-biotin, the samples were rinsed and incubated in the buffer solution under gentle shacking at room temperature. The wash procedure was performed twice, each time with a 10 min incubation period. Following the wash procedure, the samples were separated into two groups, each containing three samples. In addition, two samples were incubated in buffer containing no collagen-binding peptide-biotin to serve as negative controls. To verify the binding of collagen-binding peptide to the inner collagenous zone surface, the first group (n ) 3) and control samples (n ) 2) were incubated in a 1 to 100 solution of Cy3-labeled streptavidin in buffer. The incubation period for these samples was 24 h at 4 °C. Following incubation, the samples were rinsed copiously with- and stored in- buffer. The samples were evaluated using an Olympus IX81/FV1000 laser scanning confocal microscope, Olympus America Inc. (Center Valley, PA). Three random spots were chosen from each sample and images were collected using the accompanying Fluoview software. A schematic of this study is shown in Figure 1A. To further investigate the binding of collagen-binding peptide on the inner collagenous zone surface, the second group (n ) 3) was incubated in 20 mM Tris buffer containing 154 mM NaCl at pH 8.2 for 10 min (gold buffer). Subsequently, these samples were incubated in 5 mL gold buffer solution containing 300 µL streptavidin-gold. The incubation period was 24 h at room temperature under gentle shaking. Following incubation, the samples were rinsed copiously with gold buffer and left placed in a covered petri-dish to dry. These samples were investigated using FEI NOVA nanoSEM highresolution filed emission scanning electron microscope (FESEM) and AFM (Multimode SPM, Veeco Instruments, Santa Barbara, CA) in tapping mode. A schematic of this study is shown in Figure 1B.
3. Results 3.1. Preparation of the Inner Collagenous Zone. The topography of a typical sample used in these studies is shown in Figure 2A. In agreement with our previously published results, we observed numerous randomly oriented collagen fibers.17 Small areas where the fibers were aligned and tightly packed were also
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Figure 4. Box plot of the average fluorescence intensities, where symbols *, ´, ‡, and • indicate statistical significance with an R value of 0.05. Accordingly, statistical significance is achieved for (1) collagen I vs blank control, (2) collagen I vs mouse antibody control, (3) collagen III vs blank control, and (4) collagen III vs mouse antibody control.
Figure 3. Fluorescent images of labeled antibodies adsorbed onto the inner collagenous zone surface.
recorded. The quality of the collagen exposure was maintained over large areas for all samples used in the adsorption studies. Because of the drying procedure carried out in air, the samples showed a lot of curvature (Figure 2B). The periodicity of the collagen fibers and the sample roughness were in agreement with the literature.17 The quality of the samples and removal of excess small pieces of tissue were highly dependent on sufficient washing with buffer. With respect to contact mode AFM imaging, a separate experiment was carried out to determine the lowest set point that would induce damage on the collagenous surface. From this experiment, it was determined that damage to the collagenous surface did not occur even under loads much higher than the one used for imaging. 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 a 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. 3.2. Identification of Exposed Collagen Type Using Labeled Antibodies. On the basis of literature reports, there are three types of collagen in the inner collagenous zone: (1) collagen type I; (2) collagen type III; and (3) collagen type V. We focused our efforts on collagen types I and III because they are the major fibrillar collagens.18 Representative confocal microscopy images are shown in Figure 3. The statistical analysis performed by Minitab software resulted in the following p values: (1) p ) 0.037 for collagen I versus blank control; (2) p ) 0.025 for collagen I versus mouse antibody control; (3) p ) 0.004 for collagen III versus blank control; (4) p ) 0.002 for collagen III versus mouse antibody control; and (5) p ) 0.999 for collagen (18) Richard-Blum, S.; Ruggiero, F.; Van der Rest, M. Top. Curr. Chem. 2005, 247, 35-84.
Figure 5. Representative fluorescent images of the collagen-binding peptide labeled with Cy3 on the inner collagenous zone surface.
I versus collagen III. The p value for a test is the conditional probability of obtaining a test statistic at least as extreme as the one that actually occurred. In practice, the p value is often compared to the desired significance level (R value). If the p value is less than the significance level, then the test is statistically significant for the particular significance level. Therefore, using an R value of 0.05 confirmed the presence of collagen types I and III (because the p value is less than the R value). However, one cannot be certain whether one type predominates (because the p value is greater than the R value). A box plot of the average fluorescence intensities is given in Figure 4. 3.3. Adsorption Study of a Collagen-Binding Peptide. The collagen-binding peptide sequence (CQDSETRTFY) used in this study was derived from the collagen-binding fragment of fibronectin.19 This sequence was chosen because it is relatively short and has yet to be studied as an anchoring molecule on a collagen surface.20,21 Representative fluorescent images from (19) Korah, R.; Boots, M.; Wieder, R. Cancer Res. 2004, 64, 5414-4522.
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Figure 6. FESEM images of the collagen-binding peptide labeled with gold nanoparticles on the inner collagenous zone surface.
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To further investigate the binding specificity of the collagenbinding peptide on the inner collagenous zone surface, gold labeling was used to enable high-resolution images to be taken by FESEM and AFM. The FESEM images, as shown in Figure 6, confirmed the sparse distribution of the collagen-binding peptide on the surface. However, because FESEM images were taken without coating the surface with a conductive layer the topography of the inner collagenous zone surface is poorly resolved. Using the AFM, high-resolution images of the collagen-binding peptide labeled with gold nanoparticles were collected while maintaining a good topographical image of the inner collagenous zone surface. AFM images are shown in Figure 7 and indicate that the collagen-binding peptide binds to the groove of the collagen fibers. Overall, the combined results of fluorescent and gold-nanoparticle labeling support the notion that the collagenbinding peptide is binding specifically to the collagen fibers on the inner collagenous zone surface.
4. Discussion and Conclusion
Figure 7. Amplitude images collected with tapping mode AFM: (1) collagen-binding peptide labeled with gold nanoparticles on the inner collagenous zone surface (A and B) and (2) inner collagenous zone surface prior to modification (C and D).
samples in group 1 are shown in Figure 5. Qualitative comparisons between treated and control samples indicate binding between the collagen-binding peptide and the inner collagenous zone surface. The fluorescent images did not show uniform binding across the entire field of view. Instead, it seems that the collagenbinding peptide is binding to specific locations over the surface. (20) Tye, C. E.; Hunter, G. K.; Goldberg, H. A. J. Biol. Chem. 2005, 280, 13487-13492. (21) Wang, A.; Mo, X.; Chen, C. S.; Yu, S. M. J. Am. Chem. Soc. 2005, 127, 4130-4131.
AFM images of the exposed inner collagenous zone surface confirmed the presence of well-defined collagen fibers that withstand the drying process of the membrane. Furthermore, the ability of antibodies to collagen types I and III to bind to the surface indicates that the collagen fibers retain their biological activity. The presence of collagen types I and III on the inner collagenous zone surface is in agreement with the bulk composition of the inner collagenous zone.16 Results of collagen-binding peptide binding studies support the notion that this sequence is specific for binding to collagen fibers on the inner collagenous zone surface. Additionally, the biotin label placed on the N-terminus of the peptide also retains its biological capability to bind to streptavidin after surface adsorption. Hence, the chosen peptide sequence may serve as an anchor to modify the surface of the inner collagenous zone. It is noted that given the specificity of the collagen-binding peptide it does not bind uniformly across the entire inner collagenous zone surface. However, as observed on the high-resolution images, a sufficient amount of collagen-binding peptides can be observed over an area of hundreds of micrometers (corresponding to a typical cell size). Therefore, specific cellular responses may be elicited through surface modification of the inner collagenous zone using the collagen-binding peptide as an anchor. Ongoing work in our laboratory is underway to quantify the binding kinetics of the collagen-binding peptide. Acknowledgment. We thank the veterinary staff at the Weldon School of Biomedical Engineering for help with harvesting the donor eyes. LA703561D
