M. Klein1, H. Kassam1, M. Karver2, M. Struble2, L. Palmer2, N. Tsihlis1, S. Stupp2, B. Gavitt4, T. Pritts3, M. Kibbe1 1University Of North Carolina At Chapel Hill,General Surgery,Chapel Hill, NC, USA 2Northwestern University,Chicago, IL, USA 3University Of Cincinnati,Cincinnati, OH, USA 4United States Air Force School of Aerospace Medicine,Cincinnati, OH, USA
Introduction: Non-compressible torso hemorrhage is a leading cause of preventable death in civilian and battlefield trauma. We sought to develop a tissue factor (TF)-targeted nanofiber that can be given intravenously to slow hemorrhage until definitive bleeding control can be obtained. TF was chosen because it is only exposed to the intravascular space upon vessel disruption. Peptide amphiphile (PA) monomers that self-assemble into nanofibers were chosen as the delivery vehicle. Here, we systematically analyzed the binding interface of TF to factor VII to generate a TF-binding sequence. We hypothesize that TF-targeted nanofibers will localize to the site of bleeding in a liver hemorrhage model.
Methods: PA monomers were synthesized by solid-phase peptide synthesis, purified by high pressure liquid chromatography (HPLC), and characterized by HPLC-mass spectrometry (HPLC-MS). A TF-binding sequence SFEEARE (SFE) was added to the PA backbone. SFE-PAs (75% by weight) were co-assembled with PA backbone (20%) and a fluorescently labeled TAMRA PA (5%) to create TF-targeted nanofibers. Non-targeted PA nanofibers served as controls. Nanofiber formation was assessed via conventional transmission electron microscopy (TEM) and structure by circular dichroism (CD) spectroscopy. Male Sprague Dawley rats (250-290g) underwent a liver punch hemorrhage model in which the liver was exposed via a midline laparotomy, followed by tail vein injection of the nanofiber (2.5mg). A 12mm biopsy punch was used to injure the left lateral lobe of the liver. Shed blood was collected with pre-weighed gauze for 30 minutes and expressed as percent of total blood volume. Data are presented as mean±SEM and analyzed by ANOVA.
Results: PAs were >95% pure, as shown by HPLC-MS. Co-assemblies of PAs formed nanofibers, as shown by TEM, and displayed the expected β-sheet signal characteristic of PA nanofibers when analyzed by CD spectroscopy. Injection of the SFE-PA nanofiber demonstrated binding to the site of liver injury using fluorescent microscopy, with uninjured liver showing minimal fluorescence and control nanofibers showing minimal fluorescence. Somewhat surprisingly, given that there was no therapeutic molecule on the PA nanofiber, total blood loss for rats that received the SFE-PA nanofiber was 36% lower than sham rats (14.5±0.95% vs. 22.8±1.1%, n=6, P<0.05). Blood loss for rats injected with the non-targeted PA nanofiber was similar to sham rats (21.59±2.8%, n=6).
Conclusion: We have successfully synthesized, purified, and characterized a novel PA nanofiber that targets sites of active bleeding in a rat model of hemorrhage. Not only did our injectable TF-targeted PA nanofiber localize to the site of injury, it appears to decrease blood loss in the setting of abdominal hemorrhage. Incorporation of a therapeutic agent will make this promising technology even more effective in treatment of non-compressible torso hemorrhage.