J. D. Greenwood1, S. A. Egner2, B. Ledford1, G. Steinl1, L. Yu1, M. R. Karver2, T. Pritts3, L. C. Palmer2, N. Tsihlis1, S. I. Stupp2, M. R. Kibbe1 1University Of Virginia, Charlottesville, VA, USA 2Northwestern University, Chicago, IL, USA 3University Of Cincinnati, Cincinnati, OH, USA
Introduction:
Hemorrhage is the leading cause of potentially preventable death for both military and civilian trauma. Though new devices and topical hemostatic agents have been developed for compressible hemorrhage, non-compressible torso hemorrhage remains a major problem. This research aims to develop an injectable delivery vehicle that rapidly and specifically targets sites of active hemorrhage. We hypothesize that self-assembling peptide amphiphile (PA) nanofibers bearing FVII-derived peptide sequences will target tissue factor (TF) at sites of injury.
Methods:
PA molecules and peptides were synthesized using solid-phase peptide synthesis and purified via high-performance liquid chromatography (HPLC) with purity assessed by LC-mass spectrometry (LCMS). TF binding affinity of 12 different peptide sequences was assessed via microscale thermophoresis (MST). PAs were co-assembled using either 0 mol % or 5 mol % targeted PA. Nanofiber affinity for TF on disturbed endothelium was assessed using HUVECs under flow on collagen IV-coated slides via a microfluidic pump system. After 5 days, cells were exposed to TNFα (2 nM) for 4 hours. 30 minutes after nanofibers were added fluorescence was quantified on a confocal microscope. In vivo targeting experiments were performed using a rat liver punch biopsy model of severe hemorrhage in male Sprague Dawley rats (250-350 g, n=6 per group, UVA IACUC# 4376-12-21). Hemodynamics were monitored invasively and blood loss was measured using pre-weighed gauze. After 30 minutes, organs were harvested for fluorescence quantification using a LagoX in vivo imaging system.
Results:
PA molecules and peptides were >95% pure by LCMS. MST showed that “ERTF” peptide bound to TF with a Kd of 4.8 ±1.1 μM, the highest affinity of all peptides tested. In vitro, ERTF-targeted nanofibers bound to HUVECs significantly more than non-targeted control nanofibers (2.35±0.18 vs. 0.98±0.15% imaged channel area, P< 0.001). In vivo, a similar trend was seen in the rat liver hemorrhage model, where imaging demonstrated that targeted ERTF-PA nanofibers had superior localization to the injured liver vs. non-targeted control nanofibers (18.4 vs. 11.6 relative fluorescence units, P= 0.003). Mean blood loss was not significantly affected by the use of targeted or non-targeted nanofibers compared to sham controls (26.4, 32.1, and 30.8% total blood volume, respectively). Recovery of blood pressure to pre-injury baseline was not affected by treatment with nanofibers compared to sham controls and all animals survived to the 30-minute post-injury time point.
Conclusion:
TF-targeted PA nanofibers preferentially accumulate at sites of hemorrhage in a rat liver injury model of severe hemorrhage. Future work will include assessing PA safety and biocompatibility, as well as incorporating antifibrinolytics that directly inhibit plasmin and therapeutics that augment platelet aggregation/adhesion to sites of injury.