Modified Oligopeptide And Its Application In Molecular Imaging And Supramolecular Hydrogel

Caspases guide cell apoptosis, a major form of cell death. Many toxic agents, including most of the anticancer drugs, induce apoptosis. Thus, factors influencing apoptotic cell death may contribute to the outcome of cancer therapy. Caspase-3(Casp3), an important protease of caspase family, is tightly associated with cell apoptosis. An oligopeptide sequence, Asp-Glu-Val-Asp (DEVD), is the specific substrate for Caspase-3cleavage. To sensing Caspase-3activity in vitro and in cells and exploring the regulation on the fate of cells, fluorescent nanoprobes and hydrogelators based on DEVD were designed to targeting Caspase-3.FRET is a mechanism describing distance-dependent energy-transfer between donor molecule and accepter molecule. The energy-transfer efficiency is dependent on the donor-acceptor distance. Using gold nanoparticles (AuNPs) and DABCYL as quenchers, and an oligopeptide sequence, Asp-Glu-Val-Asp-Gly-Gly-Gly (DEVDGGG) as spacer which has a calculated length of3.3nm, we rationally designed two Caspase-3activatable nanoprobes based on FRET induced quenching: FITC-DEVDGGG-AuNP (nanoprobe1) and FITC-DEVDGGG-EDA-DABCYL (nanoprobe2). Solid Phase Peptide Synthesis (SPPS) was applied to synthesize the spacer which was covalently linked to FITC and the quencher through carboxylic ammonia condensation etc. Nanoprobe1and nanoprobe2have quenching efficiencies of98.6%and98.1%respectively. Cleavage of the probes at the site of DEVD↓GGG by Caspase-3was validated with transmission electron microscopy (TEM) observation, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization mass spectrum (MALDI-MS) analyses. Nanoprobe2was successfully applied to sensing Caspase-3activity in apoptotic cell lysate and directly imaging Caspase-3activity in apoptotic cells. Thus, our new Caspase-3activatable probes, based on fluorophore-quencher system, hold promise for assessing the chemotherapeutic effect of cancer treatment.Supramolecular hydrogels were widely chosen as the scaffolds to culture cells in three dimensions due to their biocompatibilities and biodegradabilities. We envisioned that hydrogels formed with DEVD-based molecular hydrogelators would probably exhibit better property for3D cell culture than other supramolecular hydrogels because the DEVD-based hydrogelators might interact with Caspase-3in cells and minimize cellular apoptosis induction which is very crucial for parental cells’passage on the scaffolds. Thus we report the rational design of a DEVD-based heptapeptide hydrogelator1(Acetyl-Asp-Glu-Val-Asp-Gly-Gly-Gly-EDA-Fmoc), and its isomeric hydrogelator2(Acetyl-Asp-Glu-Asp-Val-Gly-Gly-Gly-EDA-Fmoc) with a DEDV-based heptapeptide sequence. Both heptapeptidic derivatives could self-assemble into nanofibers to form supramolecular hydro gels:self-assembly of1in water results in flexuous, long nanofibers to form supramolecular hydrogel I with higher mechanical strength than that of hydrogel Ⅱ which is composed of rigid, short nanofibers of2. In vitro enzymatic analyses by HPLC and ESI-MS indicated that1is susceptive to Caspase-3while2is not. In contrast to the49%o f viability of HepG2cells incubated with2at400μM for3days,86%of HepG2cells treated with1at the same conditions survived.3-(4,5-dimethylthiazol-2-yl)2,5diphenyl tetrazolium bromide (MTT) and Western blot analyses indicated that DEDV-based hydrogelator2induces cell death via apoptotic pathway while the DEVD-based hydrogelator1minimizes cellular apoptosis induction. Structural optimization of1to form hydrogel at physiological condition may promote its application in potential3D cell culture and tissue engineering.