Discovery of Heparin Mimetic, Potent, and Selective Inhibitors of Human Clotting Factor XIIIa.

Factor XIIIa (FXIIIa) is a cysteine transglutaminase that catalyzes the last step in the coagulation process. An anion-binding site inhibition of FXIIIa is a paradigm-shifting strategy that may offer key advantages of controlled inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We previously reported a flavonoid trimer-based allosteric inhibitor of FXIIIa with moderate potency and selectivity. To further advance this approach, we evaluated a series of 27 variably sulfonated heparin mimetics against human FXIIIa. Only 13 molecules exhibited inhibitory activity at the highest concentration tested with IC50 values of 2-286 µM. Specifically, inhibitor 16 demonstrated an IC50 value of 2.4 ± 0.5 µM in a bisubstrate, fluorescence-based trans-glutamination assay. It also demonstrated a significant selectivity over other clotting factors including thrombin, factor Xa, and factor XIa as well as other cysteine enzymes including papain and tissue transglutaminase 2. Inhibitor 16 did not affect the viability of three human cell lines at a concentration that is 5-fold its FXIIIa-IC50. The molecule had a very weak effect on the activated partial thromboplastin time of human plasma at a concentration of >700 µM, further supporting its functional selectivity. Importantly, molecule 16 inhibited FXIIIa-mediated polymerization of fibrin(ogen) in a concentration-dependent manner as shown by the gel electrophoresis experiment. Michaelis-Menten kinetics revealed that the molecule competes with the Gln-donor protein substrate, i.e., dimethylcasein, but not with the Lys-donor small substrate, i.e., dansylcadaverine. Molecular modeling studies revealed that this type of molecule likely binds to an anion-binding site comprising the basic amino acids of Lys54, Lys61, Lys73, Lys156, and Arg244 among others. Overall, our work puts forward a new anion-binding site, selective, nontoxic, sulfonated heparin mimetic FXIIIa inhibitor 16 for further development as an effective and safer anticoagulant.

Ultra-low intensity light pulses for large cargo delivery into hard-to-transfect cells using an rGO mixed PDMS microtip device.

Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.