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We report on the synthesis and properties of a photoactivatable caged RGD peptide and its application for phototriggering integrin and cell binding to surfaces. We analyzed in detail (i) the differences in the integrin binding affinity of the caged and uncaged forms via quartz crystal microbalance (QCM) studies, (ii) the efficiency and yield of the photolytic uncaging reaction, (iii) the biocompatibility of the photolysis byproducts and irradiation conditions and (iv) the possibility of site, temporal and density control of integrin binding and, therefore, human cell attachment, (v) the possibility of in situ generation of cell patterns and cell gradients by controlling the exposure. These studies provide a clear picture of the potential and limitations of caged ligands for integrin-mediated cell adhesion and demonstrate the application of this approach to control and study other types of cell interactions and response.

Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product.