Influence of Steric Shield on Biocompatibility and Antithrombotic Activity of Dendritic Polyphosphate Inhibitor

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP's prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths (mPEG-750 versus mPEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with mPEG-750 brush layer, mPEG₇₅₀ UHRA-10, showed superior biocompatibility compared to its mPEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in mPEG₇₅₀ UHRA-8 and 32 in mPEG₇₅₀ UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the mPEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.