Engineering Low Volume Resuscitants for the Prehospital Care of Severe Hemorrhagic Shock.

Globally, traumatic injury is a leading cause of suffering and death. The ability to curtail damage and ensure survival after major injury requires a time-sensitive response balancing organ perfusion, blood loss, and portability, underscoring the need for novel therapies for the prehospital environment. Currently, there are few options available for damage control resuscitation (DCR) of trauma victims. We hypothesize that synthetic polymers, which are tunable, portable, and stable under austere conditions, can be developed as effective injectable therapies for trauma medicine. In this work, we design injectable polymers for use as low volume resuscitants (LVRs). Using RAFT polymerization, we evaluate the effect of polymer size, architecture, and chemical composition upon both blood coagulation and resuscitation in a rat hemorrhagic shock model. Our therapy is evaluated against a clinically used colloid resuscitant, Hextend. We demonstrate that a radiant star poly(glycerol monomethacrylate) polymer did not interfere with coagulation while successfully correcting metabolic deficit and resuscitating animals from hemorrhagic shock to the desired mean arterial pressure range for DCR - correcting a 60% total blood volume (TBV) loss when given at only 10% TBV. This highly portable and non-coagulopathic resuscitant has profound potential for application in trauma medicine.

ENGINEERED INTRAVENOUS THERAPIES FOR TRAUMA.

Trauma leading to severe hemorrhage and shock on average kills patients within 3 to 6 hours after injury. With average prehospital transport times reaching 1-6 hours in low- to middle-income countries, stopping the bleeding and reversing hemorrhagic shock is vital. First-generation intravenous hemostats rely on traditional drug delivery platforms, such as self-assembling systems, fabricated nanoparticles, and soluble polymers due to their active targeting, biodistribution, and safety. We discuss some challenges translating these therapies to patients, as very few have successfully made it through preclinical evaluation in large-animals, and none have translated to the clinic. Finally, we discuss the physiology of hemorrhagic shock, highlight a new low volume resuscitant (LVR) PEG-20k, and end with considerations for the rational design of LVRs.

Leukocyte activation primes fibrinogen for proteolysis by mitochondrial oxidative stress.

Critical illness leads to rapid fibrinogen consumption, hyperfibrinolysis, and coagulopathy that exacerbates bleeding and increases mortality. Immune cell activation and inflammation are associated with coagulopathy after injury but play an undetermined role. We performed high dimensional immunophenotyping and single-cell imaging flow cytometry to investigate for a pathophysiological mechanism governing the effects of leukocyte-associated inflammation on fibrinogen function. Fibrinogen was oxidized early, followed by its degradation after 3 hours of lipopolysaccharides (LPS)-induced sterile inflammation in a rat model in vivo. Fibrinogen incubated with human leukocytes activated by TNFα was similarly oxidized, and later proteolyzed after 3 hours in vitro. TNFα induced mitochondrial superoxide generation from neutrophils and monocytes, myeloperoxidase (MPO)-derived reactive oxygen species (ROS) from neutrophils, and nitric oxide from lymphocytes and monocytes. Inhibition of mitochondrial superoxide prevented oxidative modification and proteolysis of fibrinogen, whereas inhibition of MPO attenuated only fibrinogen proteolysis. Quenching of both mitochondrial superoxide and MPO-derived ROS prevented coagulopathy better than tranexamic acid. Collectively, these findings indicate that neutrophil and monocyte mitochondrial superoxide generation can rapidly oxidize fibrinogen as a priming step for fibrinogen proteolysis and coagulopathy during inflammation.

Optimizing the Polymer Chemistry and Synthesis Method of PolySTAT, an Injectable Hemostat.

There is a lack of prehospital hemostatic agents, especially for noncompressible hemorrhage. We previously reported PolySTAT, a unimeric, injectable hemostatic agent, that physically cross-links fibrin to strengthen clots. In this work, we sought to improve the water-solubility and synthesis yield of PolySTAT to increase the likelihood of clinical translation, reduce cost, and facilitate future mass production. First, we focused on side-chain engineering of the carrier polymer backbone to improve water-solubility. We found that substitution of the 2-hydroxyethyl methacrylate (HEMA) monomer with glycerol monomethacrylate (GmMA) significantly improved the water-solubility of PolySTAT without compromising efficacy. Both materials increased clot firmness and decreased lysis as measured by rotational thromboelastometry (ROTEM). Additionally, we confirmed the in vivo activity of GmMA-based PolySTAT by improving rat survival in a femoral artery bleed model. Second, to reduce waste, we evaluated PolySTAT synthesis via direct polymerization of peptide monomers. Methacrylamide and methacrylate peptide-monomers were synthesized and polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. This approach markedly improved the yield of PolySTAT synthesis while maintaining its biological activity in ROTEM. This work demonstrates the flexibility of PolySTAT to a variety of comonomers and synthetic routes and establishes direct RAFT polymerization of peptide monomers as a potential route of mass production.

Formulation of thrombin-inhibiting hydrogels via self-assembly of ionic peptides with peptide-modified polymers.

Cell therapy for spinal cord injuries offers the possibility of replacing lost cells after trauma to the central nervous system (CNS). In preclinical studies, synthetic hydrogels are often co-delivered to the injury site to support survival and integration of the transplanted cells. These hydrogels ideally mimic the mechanical and biochemical features of a healthy CNS extracellular matrix while also providing the possibility of localized drug delivery to promote healing. In this work, we synthesize peptide-functionalized polymers that contain both a peptide sequence for incorporation into self-assembled peptide hydrogels along with bioactive peptides that inhibit scar formation. We demonstrate that peptide hydrogels formulated with the peptide-functionalized polymers possess similar mechanical properties (soft and shear-thinning) as peptide-only hydrogels. Small angle neutron scattering analysis reveals that polymer-containing hydrogels possess larger inhomogeneous domains but small-scale features such as mesh size remain the same as peptide-only hydrogels. We further confirm that the integrated hydrogels containing bioactive peptides exhibit thrombin inhibition activity, which has previously shown to reduce scar formation in vivo. Finally, while the survival of encapsulated cells was poor, cells cultured on the hydrogels exhibited good viability. Overall, the described composite hydrogels formed from self-assembling peptides and peptide-modified polymers are promising, user-friendly materials for CNS applications in regeneration.