pubs.acs.org/Langmuir © 2010 American Chemical Society
Characterization of Conformational Adsorbate Changes on a Tissue-Derived Substrate Using Fourier Transform Infrared Spectroscopy Marcus A. Kramer,† Benjamin Andrews,‡ Daniel L. Hugar,† Arjun Jaitli,† Seij B. Larsen,† Benjamin P. Kline,† Kristin N. McEllen,† Namita Agrawal,† Si Min Su,† Sandhya A. Dammu,† Ryan M. Kammeyer,† and Albena Ivanisevic*,†,§ †
Weldon School of Biomedical Engineering, ‡Department of Biochemistry, and §Department of Chemistry, Purdue University, West Lafayette, Indiana, United States Received July 1, 2010
Fourier transform infrared (FT-IR) spectroscopy is utilized to observe adsorbate interactions with a tissue-derived collagen scaffold extracted from the Bruch’s membrane of pig eyes. The characterization includes conformational changes in isoleucine, polyisoleucine, collagen-binding peptide, RGD-tagged collagen-binding peptide, and laminin after adsorption onto the substrate. Isotopically labeled isoleucine is further utilized to understand changes in the biomolecular structure upon binding to a tissue-derived surface. The adsorbates associated with the collagen scaffold predominately through hydrophobic interactions and hydrogen bonding. The results of this study can be used to improve our understanding of surface chemistry changes during the engineering of biomimetic scaffolds before and after biomolecule adsorption.
Introduction A number of research studies have focused on the use of Fourier transform infrared (FT-IR) spectroscopy to characterize biological materials. To this end, FT-IR spectroscopy has been used to map tissues,1,2 monitor cell fate,3 determine protein structure,4 track metabolites in plasma,5 and characterize the effects that the cell and substrate have on each other.6 In addition, a large effort is directed toward utilizing FT-IR as a chemical imaging tool for pathology.7 One common characteristic among FT-IR studies is that they use a window material for mounting the specimen.4 These window materials do not represent a natural substrate for the cells; therefore, broader conclusions as to the cells’ response in a natural environment cannot be made. The strong sensitivity of FT-IR spectroscopy to changes in protein structure makes it an ideal technique for the characterization of conformational variations that might occur after adsorbates are used to modify tissues. Despite the great capabilities and usefulness of this spectroscopic tool, it is rarely used to understand changes in eye tissues. In particular, the Bruch’s membrane (BM) *Corresponding author. E-mail:
[email protected]. (1) Chiriboga, L.; Xie, P.; Yee, H.; Vigorita, V.; Zarou, D.; Zakim, D.; Diem, M. Infrared spectroscopy of human tissue. I. Differentiation and maturation of epithelial cells in the human cervix. Biospectroscopy 1998, 4, 47–53. (2) Fournier, F.; Guo, R.; Gardner, E. M.; Donaldson, P. M.; Loeffeld, C.; Gould, I. R.; Willison, K. R.; Klug, D. R. Biological and biomedical applications of two-dimensional vibrational spectroscopy: proteomics, imaging, and structural analysis. Acc. Chem. Res. 2009, 42, 1322–1331. (3) Gault, N.; Rigaud, O.; Poncy, J. L.; Lefaix, J. L. Infrared microspectroscopy study of gamma-irradiated and H2O2-treated human cells. Int. J. Radiat. Biol. 2005, 81, 767–779. (4) Barth, A. Infrared spectroscopy of proteins. Biochim. Biophys. Acta, Bioenerg. 2007, 1767, 1073–1101. (5) Petibois, C.; Gionnet, K.; Goncalves, M.; Perromat, A.; Moenner, M.; Deleris, G. Analytical performances of FT-IR spectrometry and imaging for concentration measurements within biological fluids, cells, and tissues. Analyst 2006, 131, 640–647. (6) Meade, A. D.; Lyng, F. M.; Knief, P.; Byrne, H. J. Growth substrate induced functional changes elucidated by FTIR and Raman spectroscopy in in-vitro cultured human keratinocytes. Anal. Bioanal. Chem. 2007, 387, 1717–1728. (7) Bhargava, R. Towards a practical Fourier transform infrared chemical imaging protocol for cancer histopathology. Anal. Bioanal. Chem. 2007, 389, 1155–1169.
Langmuir 2010, 26(23), 18083–18088
extracted from eye tissues provides a unique opportunity for FT-IR studies because it is contained in one of the few naturally existing 2-D tissues. In the tissue, the BM is directly below a clearly defined retinal pigment epithelial cell (RPE) sheet. These two layers are extremely important in maintaining the homestasis of the retina and manifest symptoms of disease.8 A number of histological studies have been published to understand changes in the tissue with disease.9 The RPE has been studied for its responses to a variety of substrates and cell-specific factors. Beyond this, the BM has been modified on both tissue-wide10 and micrometer length scales11,12 with amino acids, peptides, and/or proteins. Many of the published studies have focused on understanding the pathology of the tissue and have explored the refurbishment of the existing tissue with soluble biomolecules.8 Modifications to refurbish the tissue have been principally performed on dried tissues with no ideal methodology for adsorbate structural analysis because of the complex nature of the substrate.10-12 On the basis of reviews of the current literature,13 we have identified the need to examine these structural changes that might occur when biomolecules are adsorbed onto eye tissues such as the BM. In this study, we assess the feasibility of using FT-IR to map the presence of biomolecules on eye tissue samples. Sections of BM were isolated, cleaned, and prepared from a number of pig donors. (8) Tezel, T. H.; Bora, N. S.; Kaplan, H. J. Pathogenesis of age-related macular degeneration. Trends Mol. Med. 2004, 10, 417–420. (9) Miura, Y.; Klettner, A.; Noelle, B.; Hasselbach, H.; Roider, J. Change of morphological and functional characteristics of retinal pigment epithelium cells during cultivation of retinal pigment epithelium-choroid perfusion tissue culture. Ophthalmic Res. 2010, 43, 122–133. (10) Tezel, T. H.; Del Priore, L. V.; Kaplan, H. J. Reengineering of aged Bruch’s membrane to enhance retinal pigment epithelium repopulation. Invest. Ophthalmol. Visual Sci. 2004, 45, 3337–3348. (11) Kramer, M. A.; Park, H. C.; Ivanisevic, A. Dip-pen nanolithography on SiOx and tissue-derived substrates: comparison with multiple biological inks. Scanning 2010, 32, 30–34. (12) Sistiabudi, R.; Ivanisevic, A. Dip-pen nanolithography of bioactive peptides on collagen-terminated retinal membrane. Adv. Mater. 2008, 20, 3678–3681. (13) Cai, H.; Shin, M. C.; Tezel, T. H.; Kaplan, H. J.; DelPriore, L. V. Use of iris pigment epithelium to replace retinal pigment epithelium in age-related macular degeneration. Arch. Ophthalmol. 2006, 124, 1276–1285.
Published on Web 11/10/2010
DOI: 10.1021/la1038766
18083
Article
Kramer et al.
Several biomolecules were adsorbed on the tissue surface: isoleucine, polyisoleucine, collagen-binding peptide (CQDSETRTFY), RGD-tagged collagen-binding peptide (RGDSCQDETRTFY), and laminin. Isoleucine was chosen as the molecule of interest because of its simple structure and lack of nitrogen in its side chain. This eliminates any potential peak overlap during 15N labeling of the isoleucine (Supporting Information). The heavier nitrogen isotope is expected to cause a shift in the characteristic frequencies of its bonds and can be used to detect the presence of the functional groups of isoleucine adsorbed on a tissue surface. Polyisoleucine was investigated in order to understand changes associated with secondary structure. The collagen-binding peptide sequence and laminin have been previously utilized to enhance the cell adhesion properties of the BM.10,14 Furthermore, the binding specificity of the collagen-binding sequences to the BM was verified. However, monitoring changes in adsorbate conformation on tissue surfaces can help rationalize and tune cell adhesion behavior, which is critically important when one tries to engineer biomimetic scaffolds. We present our results and a discussion focused on understanding changes in the structure of the biomolecules upon adsorption onto eye tissues.
Materials and Methods The tissue (inner collagen layer, ICL, of the BM) was prepared via a slight modification to a published protocol15 and was harvested from porcine eyes by carefully cutting circumferentially along the ora serrata. Next, the vitreous gel and retina were removed from the Bruch’s membrane. The remaining posterior portion of the eye was transected twice to leave four equal sections. Each section was placed in 0.05 M ammonium hydroxide for 20 min with and without sonication and briefly rinsed in TRIS (Aldrich, pH 7.5, 150 mM) buffer. Finally, the membranes were placed BM down on a hydrophilic polycarbonate membrane (Whatman) for 5 min. Subsequently, the BM was carefully separated from the sclera by removing the sparse connections between the two. The BM was then dried (ICL up) overnight on a nuclepore polyester membrane (Whatman) and removed the following day for examination. This creates a thin free-standing substrate for FT-IR without the need to utilize a microtome to produce a thin tissue slice. Atomic force microscopy has previously been used to show that one produces a surface terminated on collagen fibers using this method.11 Dried tissue samples were modified with multiple molecular inks physisorbed to the tissue surface using 0.5 μL drops. Small drop sizes do not change the overall structure of the collagen because of wetting drying cycles and are ideal for characterizing the adsorbate. The solutions were composed of isoleucine (Sigma, 40 mg/mL), polyisoleucine (Sigma,
