Cyclo(RGDfK) Functionalized Spider Silk Cell Scaffolds: Significantly Improved Performance in Just One Click.

Recombinant spider silk has the potential to provide a new generation of biomaterial scaffolds as a result of its degree of biocompatibility and lack of immunogenicity. These recombinant biomaterials are, however, reported to exhibit poor cellular adhesion which limits their potential for use in applications such as tissue engineering and regenerative medicine. In this study, a simple chemical functionalization approach is described that specifically addresses this issue and significantly improves the adhesion of human mesenchymal stem cells (CiMSCs) to a recombinant spider silk biomaterial. This utilizes copper-catalyzed or strain-promoted azide-alkyne cycloaddition (CuAAC/SPAAC) "click" chemistry to covalently attach cyclo(RGDfK) peptides to the azide group of l-azidohomoalanine, a methionine analogue previously site specifically incorporated into the primary sequence of a thioredoxin (TRX)-tagged silk fusion protein, TRX-4RepCT, to give TRX3Aha -4RepCT3Aha . This method is used to produce cyclo(RGDfK) functionalized films and macroscopic fibers. Over 24 h, cyclo(RGDfK) functionalized TRX3Aha -4RepCT3Aha  films and 4RepCT3Aha  fibers display significantly improved performance in CiMSC culture, yielding far greater cell numbers than the controls. This approach circumvents the previously observed lack of cell adhesion, thus allowing spider silk derived biomaterials to be used where such adhesion is critical, in tissue engineering, regenerative medicine and wound healing.

Synthesis of folic acid functionalized gold nanoclusters for targeting folate receptor-positive cells.

We report on the synthesis of water-soluble gold nanoclusters capped with polyethylene glycol (PEG)-based ligands and further functionalized with folic acid for specific cellular uptake. The dihydrolipoic acid-PEG-based ligands terminated with -OMe, -NH2 and -COOH functional groups are produced and used for surface passivation of Au nanoclusters (NCs) with diameters <2 nm. The produced sub 2 nm Au NCs possess long-shelf life and are stable in physiologically relevant environments (temperature and pH), are paramagnetic and biocompatible. The paramagnetism of Au NCs in solution is also reported. The functional groups on the capping ligands are used for direct conjugation of targeting molecules onto Au NCs without the need for post synthesis modification. Folic acid (FA) is attached via an amide group and effectively target cells expressing the folate receptor. The combination of targeting ability, biocompatibility and paramagnetism in FA-functionalized Au NCs is of relevance for their exploitation in nanomedicine for targeted imaging.

Synthesis and Biological Activity of Peptide α-Ketoamide Derivatives as Proteasome Inhibitors.

Proteasome activity affects cell cycle progression as well as the immune response, and it is largely recognized as an attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudodi/tripeptides bearing at the C-terminal position different α-ketoamide moieties as pharmacophoric units for the interaction with the catalytic threonine residue that sustains the proteolytic action of the proteasome. Among these, we identified the 1-naphthyl derivative 13c as a potent and selective inhibitor of the ß5 subunit of the 20S proteasome, exhibiting nanomolar potency in vitro (ß5 IC50 = 7 nM, ß1 IC50 = 60 µM, ß2 IC50 > 100 µM). Furthermore, it significantly inhibited proliferation and induced apoptosis of the human colorectal carcinoma cell line HCT116.

Accelerated 19F·MRI Detection of Matrix Metalloproteinase-2/-9 through Responsive Deactivation of Paramagnetic Relaxation Enhancement.

Paramagnetic gadolinium ions (GdIII), complexed within DOTA-based chelates, have become useful tools to increase the magnetic resonance imaging (MRI) contrast in tissues of interest. Recently, "on/off" probes serving as 19F·MRI biosensors for target enzymes have emerged that utilize the increase in transverse (T 2 ∗ or T 2) relaxation times upon cleavage of the paramagnetic GdIII centre. Molecular 19F·MRI has the advantage of high specificity due to the lack of background signal but suffers from low signal intensity that leads to low spatial resolution and long recording times. In this work, an "on/off" probe concept is introduced that utilizes responsive deactivation of paramagnetic relaxation enhancement (PRE) to generate 19F longitudinal (T 1) relaxation contrast for accelerated molecular MRI. The probe concept is applied to matrix metalloproteinases (MMPs), a class of enzymes linked with many inflammatory diseases and cancer that modify bioactive extracellular substrates. The presence of these biomarkers in extracellular space makes MMPs an accessible target for responsive PRE deactivation probes. Responsive PRE deactivation in a 19F biosensor probe, selective for MMP-2 and MMP-9, is shown to enable molecular MRI contrast at significantly reduced experimental times compared to previous methods. PRE deactivation was caused by MMP through cleavage of a protease substrate that served as a linker between the fluorine-containing moiety and a paramagnetic GdIII-bound DOTA complex. Ultrashort echo time (UTE) MRI and, alternatively, short echo times in standard gradient echo (GE) MRI were employed to cope with the fast 19F transverse relaxation of the PRE active probe in its "on-state." Upon responsive PRE deactivation, the 19F·MRI signal from the "off-state" probe diminished, thereby indicating the presence of the target enzyme through the associated negative MRI contrast. Null point 1H·MRI, obtainable within a short time course, was employed to identify false-positive 19F·MRI responses caused by dilution of the contrast agent.