Primary hemostasis (formation of a platelet plug) and secondary hemostasis (formation of a fibrin clot) are two simultaneous, mechanistically intertwined processes that occur upon vascular injury. Researchers in the field have sought to treat internal hemorrhage by leveraging cues specific to these processes, such as using peptides that bind activated platelets or cleaved fibrinogen to target the wound site. While such materials have been demonstrated to significantly increase survival in a variety of injury models, they are commonly designed for the exclusive purpose of treating primary or secondary hemostasis. In this work, an interactive two-component system consisting of a targeting component and a crosslinking component was developed for the treatment of internal bleeding, with the ultimate goal of amplifying platelet recruitment and mitigating plasminolysis for greater clot stability. Mixed azide/GRGDS PEG-b-PLGA nanoparticles and crosslinking nanoparticles with dibenzylcyclooctyne (DBCO) moieties were synthesized via standard conjugation techniques and ring-opening polymerization. Subsequently, the functionality of the copper-free azide-DBCO click reaction was validated by tracking nanoparticle aggregation via dynamic light scattering relative to unfunctionalized controls. Multiple azide: GRGDS ratios were screened to determine the combination most suitable for in vivo experiments, and a significant increase in both nanoparticle binding and platelet recruitment was observed relative to the nanoparticle-only system for an azide: GRGDS ratio of 1:3. This ratio was used in biodistribution and biocompatibility studies, which confirmed that the non-targeted crosslinking particles did not aggregate in the lungs or significantly skew the accumulation of the targeted nanoparticles. Following in vitro experiments to evaluate the effect of this treatment on decreasing clot degradation, this two-component system will be challenged with an injury model in mice to demonstrate its utility.

Two-component, click-functionalized hemostat for the treatment of internal hemorrhage

ACS Spring 2022

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