Photocontrolled cell adhesion on a surface functionalized with a caged arginine-glycine-aspartate peptide.

Contact between cells and the extracellular matrix (ECM) is necessary for cell adhesion, growth, and migration, and is mediated by cell-surface receptors. Binding between the ECM and the receptor results in adhesion of the cell to the ECM and the spreading of cells, and is critical for cell survival in vivo and in vitro. Adherence to the surface of the culture dish is a prerequisite for successful cultivation of most cell lines. It is difficult to study binding and the subsequent signaling pathways kinetically because binding and cell adhesion occur spontaneously in culture dishes. Herein we describe a technique for manipulating cell adhesion by using a photoresponsive culture dish. The method we developed involves an RGD (arginineglycine-aspartate) peptide, which has been identified as a major integrin ligand motif in ECMs such as fibronectin and laminin, and has been used to modify biomaterials to enhance cell adhesion. We prepared a caged RGD peptide, the sequence of which (YAVTGRGDSPASS) is the longest conserved sequence in vertebrates ranging from teleosts to mammals but containing a nitrobenzyl group as a cage. The 2-nitrobenzyl group was introduced at the amide bond between the Gly and Arg residues because this site is critical for biological activity. The photoresponsive culture dish (Figure 1) was prepared by modifying a commercially available culture dish coated with poly-l-lysine (PLL) by using a bifunctional cross-linked polyethylene glycol (PEG) and the caged RGD peptide. We used HeLa cells to study adhesion to the photoresponsive culture dish because they are typical of adhesive cells. The cells were plated and preincubated for 30 minutes and the dish then rinsed with phosphate-buffered saline (PBS). The cells, which did not adhere to the dish, were readily removed by gentle replacement of the culture medium with PBS (0 min in Figure S2 in the Supporting Information), thus showing that the photoresponsive culture dish was inactive towards cell adhesion when the peptide was in the caged form. Irradiation of the photoresponsive culture dish with UV light converted it into a cell-adhesive state (see Figure S2 in the Supporting Information). The cells remained on the UVirradiated dish after rinsing, but had spread and were flat, namely, the cells were adherent. The number of adherent cells correlated with the duration of irradiation of the dish. The photoresponsiveness of the dish can be ascribed completely to the nitrobenzyl moiety, which can be removed by UV irradiation (Figure 1). HeLa cells adhered to the culture dishes coated with the RGD peptide, but did not adhere to culture dishes coated with PLL or a DGR peptide (see Figure S3 in the Supporting Information). There was no change in the adhesive properties of the culture dishes coated with the intact RGD peptide, PLL, or PEG upon irradiation with UV light. These control experiments show that the intact RGD peptidic moiety is responsible for cell adhesion in this system and that conversion of the photoresponsive culture dish into an adhesive state is caused by the release of the nitrobenzyl group from the cage. The correlation between the number of adherent cells and the duration of UV irradiation of the plate was not linear (see Figure S2 in the Supporting Information), which suggests that a threshold density of RGD moieties may be required for adhesion. Photolithography is an important component of tissue engineering and screening based on cell chips. Spatially restricted cell adhesion was achieved by limiting the area of UV irradiation in the photoresponsive culture dish (Figure 2). Figure 1. Photochemical reaction of a caged RGD peptide attached to a culture dish. The caged RGD peptide was linked to poly-l-lysine (depicted as a wavy line).