Design And Synthesis Of A Potent Bioactive Peptide To Dual-Promote Cell Adhesion

Cell-matrix interaction is a central fundamental topic in studies of biomaterials and regenerative medicine. Living organism can be considered as a complex of cells and extracellular matrix (ECM). It is much meaningful for promoting cell adhesion and fabricating a mimic ECM through surface modification. Thus linker reagents which could bind to a substrate and meanwhile significantly enhance cell adhesion are key chemicals in modification of biomaterials. Cell adhesion is usually the first event in cellular response to biomaterials. There are basically two ways for cell adhesion, specific and non-specific cell adhesion.RGD (arginine-glycine-aspartic acid) is a sequence of fibronectin in ECM. The agent containing cyclo(-RGDfK-) (f:D-phenylalanine, K:lysine) is so far the most efficient chemical to trigger specific cell adhesion, and widely use for surface modification in studies of biomaterials and cells. Nevertheless, cell adhesion on a substrate modified by this chemical is still not much significantly better than on a very popular and commercialized tissue culture plates (TCPs). So, a more potent chemical is called for, and more insight of the pertinent design principle is required.The thyme of this thesis is examination of the relationship between the specific and non-specific adhesion, design and synthesis of a potent peptide. The main achievements are summarized as follows:1. Design and synthesis of a series of hybrid peptides. New peptide-containing linkers combining both specific and nonspecific cell adhesion moieties were designed and synthesized through solid-phase peptide synthesis (SPPS) with an approach of 9-fluorenylmethoxycarbonyl (Fmoc) methodology. The combined linker is composed of three parts, a cyclopeptide containing RGD to trigger specific cell adhesion via RGD-integrin conjugation, a linear oligopeptide to enhance early nonspecific cell adhesion due to the charge effect, and a thiol end ready to react with noble metals such as gold. The middle part acts also as a spacer of cyclo-RGD, which is necessary for an efficient conjugation of a ligand to its receptor in cell membrane. The peptides backbones were synthesized, side-chain prolonged and end-group functionalized directly on the CTC resin, which simplify the synthesis and purification.2. Confirmation of the bioactivity of the peptides and finding of cooperativity between specific and nonspecific cell adhesions.A self assembled monolayer (SAM) was form by using the synthesized linkers to modify gold surface, and then cells were cultured and observed. The goal linker containing cyclic (-RGDfK-) ligand and linear triplet Lys, RGD-K, led to excellent cell spreading and abundant cytoskeleton. The cooperativity effect between nonspecific and specific cell adhesion was revealed.3. Preparation of pertinent hydrogels. We generated a cell-adhesion contrast of peptide modified nano-patterned poly(ethylene glycol) (PEG) hydrogel surface by using our synthesized peptides. We investigate the adhesion of NIH/3T3 and MC3T3-E1 on this patterned hydrogel, and confirmed the potency of RGD-K again. We also tried several methods to improve the mechanic properties of PEG hydrogels.This methodology also offers a much helpful technical platform to study cell-biomaterial interactions. The cyclic-linear hybrid linker of RGD-K might be very useful in surface modification of biomaterials for tissue engineering and regenerative medicine, and also in drug or gene delivery carriers as an efficient targeting group. Hence, the present work has shed light into cell adhesion types and their interplay, and we believe that the design strategy offers possibility for further developing a series of novel chemicals for medical applications as well as fundamental researches.