Trauma is the leading cause of death for people ages 1-44, with blood loss comprising 60-70% of mortality in the absence of lethal CNS or cardiac injury. Immediate intervention is critical to improving chances of survival. While there are several products to control bleeding for external and compressible wounds including pressure dressings, tourniquets or topical materials (e.g. QuikClot, HemCon), there are no products that can be administered in the field for internal bleeding. There is a tremendous unmet need for a hemostatic agent to address internal bleeding in the field. We have developed hemostatic nanoparticles (GRGDS-NPs) that reduce bleeding times by ~50% in a rat femoral artery injury model. Here, we investigated their impact on survival following administration in a lethal liver resection injury in rats. Administration of these hemostatic nanoparticles reduced blood loss following the liver injury and dramatically and significantly increased 1-hour survival from 40 and 47% in controls (inactive nanoparticles and saline, respectively) to 80%. Furthermore, we saw no complications following administration of these nanoparticles. We further characterized the nanoparticles’ effect on clotting time (CT) and maximum clot firmness (MCF) using rotational thromboelastometry (ROTEM), a clinical measurement of whole-blood coagulation. Clotting time is significantly reduced, with no change in MCF. Administration of these hemostatic nanoparticles after massive trauma may help staunch bleeding and improve survival in the critical window following injury, and this could fundamentally change trauma care.

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.

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels (“synthetic extracellular matrix” or “synthetic ECM”). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.