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

Abstract

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, mPEG 750 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 mPEG 750 UHRA-8 and 32 in mPEG 750 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.

Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design

“…Putative cationic binding group structures described in the literature were screened for their amine pK a values, charge spacing, and linker chemistry, which may affect their flexibility. Embedding such charge switching cationic binding groups within a biocompatible scaffold known to prevent non-specific interactions resulted in a library of polycations with low charge density in their unbound state, particularly as compared to other polycation inhibitors of polyP 25 , 32 , 33 , 63 . When compared to larger polyanionic macromolecules with lower charge density, the unique features of the MPI’s scaffold serve to improve biocompatibility, presumably (at least in part) by limiting nonspecific interactions through the steric repulsion created by the PEG corona (brush layer) on the surface of the MPI.…”

“…For this assay, we followed a recent report from our group 63 . Detailed procedure is given in the supplementary methods.…”

Inhibitors of Polyphosphate and Neutrophil Extracellular Traps

“…58 Termed UHRAs (for universal heparin reversal agents), they were designed with cationic-binding groups based on tertiary amines (reasoning that they would be less toxic than primary amines) within the core of a dendritic polymer, shielded from nonspecific interactions with blood components using a protective short-chain layer of polyethylene glycol. We subsequently reported that certain members of this UHRA family are potent polyP inhibitors, and in fact two of these compounds (UHRA-9 and UHRA-10) were potent polyP blockers in vitro 59 and protected against experimentally induced thrombosis in vivo in mice. 60 These agents had significantly less bleeding risk compared with a therapeutically equivalent dose of heparin, but nonetheless at high doses they were mildly anticoagulant in vitro and caused some bleeding sideeffects in vivo via an unknown mechanism.…”

Polyphosphate as an antithrombotic target and hemostatic agent

“…12,151 Thus, the design of potent polycationic PolyP inhibitors with good biocompatibility remains challenging. 152 Smith et al studied a series of polycationic polymers as PolyP inhibitors, and discovered that low molecular weight polyethyleneimine (LMW-PEI) exhibited the highest efficiency in inactivating the procoagulant effect of PolyP in vitro. 12 In a thrombotic mouse model, all tested PolyP inhibitors prolonged the time of vascular occlusion.…”

“…160 For instance, conjugating PAMAM dendrimers and polyethyleneimine with PEG-750 achieved enhanced biocompatibility, leading to sterically shielded PolyP inhibitors. 152 However, the above-mentioned polycations all exhibit certain toxicity and bleeding risks. In 2023, Jayachandran and his colleagues reported a design concept towards inhibiting PolyP, termed macromolecular polyanion inhibitors (MPI) (Fig.…”