Tuning Ligand Density on Intravenous Hemostatic Nanoparticles Dramatically Increases Survival Following Blunt Trauma

Abstract

Targeted nanoparticles are being pursued for a range of medical applications. Here, we utilized targeted nanoparticles (synthetic platelets) to halt bleeding in acute trauma. One of the major questions that arises in the field is the role of surface ligand density on targeted nanoparticles’ performance. We developed intravenous hemostatic nanoparticles (GRGDS-NP1), and previously demonstrated their ability to reduce bleeding following femoral artery injury and increase survival after lethal liver trauma in the rat. These nanoparticles are made from block copolymers, poly(lactic-co-glycolic acid)-b- poly-ι-lysine-b-poly(ethylene glycol). Surface-conjugated targeting ligand density can be tightly controlled with this system, and here we investigated the effect of varying density on hemostasis and biodistribution. We increased the targeting peptide (GRGDS) concentration 100-fold (GRGDS-NP100) and undertook an in vitro dose-response study using rotational thromboelastometry (ROTEM), finding GRGDS-NP100 hemostatic nanoparticles were efficacious at doses at least 10-fold lower than the GRGDS-NP1. These results were recapitulated in vivo, demonstrating efficacy at 8-fold lower concentration after lethal liver trauma. 1-hour survival increased to 92%, compared to a scrambled peptide control, 45% (OR=14.4, 95% CI=[1.36, 143]), a saline control, 47% (OR=13.5, 95% CI=[1.42, 125]), and GRGDS-NP1, 80% (OR=1.30, n.s.). This work demonstrates the impact of changing synthetic platelet ligand density on hemostasis, and lays the foundation for methods to determine optimal ligand concentration parameters.

Intravenously administered nanoparticles increase survival following blast trauma

“…We found that 9.3% of the injected dose of the hemostatic nanoparticles was recovered at 1 h, which is consistent with the clearance seen in our previous work (18,20). The majority of the particles (5.2% for hemostatic nanoparticles and 4.7% for control nanoparticles) were found in the lungs, a major site of bleeding for this type of injury (Fig.…”

“…PLGA-PLL-PEG-GRGDS block copolymer for the hemostatic nanoparticles and PLGA-PLL-PEG-GRADSP were synthesized using protocols described previously (18)(19)(20). In brief, the triblock polymer was synthesized using stepwise conjugate reactions.…”

“…The GRGDS was conjugated to PEG-PLGA (or the conservatively substituted GRADSP) as described previously (20). In brief, the peptide was conjugated by dissolving the PLGA-PLL-PEG (1 g) in anhydrous DMSO to a concentration of 100 mg/mL and the oligopeptide (25 mg) was dissolved in 1 mL of DMSO and added to the stirring polymer solution.…”

“…The free amine of the oligopeptide then reacted with free end of the CDI-activated PEG. The mixture reacted to 3 h, and was then transferred to dialysis tubing (SpectraPor, 2 kFa molecular weight cutoff) and dialyzed for 4 h before being snap-frozen in liquid nitrogen and lyophilized (20).…”

“…To address this need, we have developed hemostatic nanoparticles that can halt bleeding when delivered intravenously (18)(19)(20). We hypothesized that administration of these hemostatic nanoparticles could increase short-term and long-term survival following blast trauma.…”