Introduction: Severe trauma is the leading cause of death for individuals between the ages of 1 and 44, and subsequent hemorrhaging is the most common cause of post-traumatic mortality [1]. Currently, few clinical methods exist that are available and effective at treating internal hemorrhaging. Hemostatic nanoparticles have been investigated as a novel clinical treatment, and have been observed to significantly reduce bleeding in rat femoral artery injury models [2]. However, these polymeric nanoparticles have had limited success in porcine liver injury models, where immune responses, such as Complement Activation-Related Pseudo-Allergy (CARPA) have been observed. These immune responses must be identified in order to be mitigated to advance the potential of hemostatic nanoparticles as a treatment for human beings.
Materials and Methods: PLGA-PLL-PEG-cRGD nanoparticles were synthesized using a nanoprecipitation method [3].
Subsequent characterization confirmed that these nanoparticles were small in size (~500 nm) with a neutral zeta potential (-3 mV to 3 mV) and approximately 10% poly(ethylene glycol) chain density (confirmed via Nuclear Magnetic Resonance). These nanoparticles were administered in 25 milligram, 60 milligram, and 100 milligram doses intravenously into porcine patients 5 minutes after the left lobe of the liver was resected. To identify immune responses in these liver injury models, blood loss and vitals surgery data was recorded and correlated with C3 and C3a complement activation assays which were conducted using plasma samples from 17 treated porcine patients [4]. Subsequent statistical and graphical analyses, including the Pearson’s Product-Moment Correlation Coefficient (PPMCC) and a four-parameter logistic regression suggested the presence of immune responses to some of these treatments.
Results and Discussion: C3 and C3a complement activation assays observed negative immune responses in the majority (9) of the 17 treated patients. The relationship between C3 and C3a was confirmed using the Pearson’s Product-Moment Correlation Coefficient, which observed a positive relationship between the concentration of C3 and the time-of-death of the patient (Pearson’s Coefficient of 0.596). Additionally, the change in C3 concentration was observed to correlate negatively with blood loss following administration of hemostatic nanoparticles (Pearson’s Coefficient of -0.569). These observed statistical relationships between C3 and C3a concentrations, blood loss, and time-of-death seem to confirm a negative immune response to hemostatic nanoparticle treatments. Furthermore, this immune response was observed regardless of the size of the administered dose. However, the complement activation assays observed changes on the scale of nanograms per milliliter, which coupled with unexpected decreases in C3a concentrations suggests that additional immunoassays, such as the C5 ELISA Kit, may be needed to verify the sensitivity of the C3 and C3a complement activation assays.
Conclusions: The potential of a hemostatic agent that reduces or halts bleeding following a traumatic injury has substantial applications in both the battlefield and civilian life. The effectiveness of such a treatment relies heavily on its ability to reduce bleeding with few to no harmful side effects. Because the hematology of humans and pigs are comparable, identifying immune responses to hemostatic nanoparticles in porcine patients is an important step in refining the effectiveness of hemostatic nanoparticles in porcine models. Although hemostatic nanoparticles appear to reduce bleeding in these porcine patients, this study identified immune responses that may be limiting the effectiveness of these treatments. Future work focused on optimizing the poly(ethylene glycol) chain density and zeta potential of these nanoparticles to minimize immune responses in vitro and in vivo offer opportunities to maximize the effectiveness of hemostatic nanoparticles in porcine injury models.
Case Alumni Association; Support of Undergraduate Research and Creative Endeavors (SOURCE) at Case Western Reserve University; Department of Biomedical Engineering, Case Western Reserve University
References:
[1] D. S. Kauvar, “Impact of hemorrhage on trauma outcome: an overview of epidemiology, clinical presentations, and therapeutic considerations,” J. Trauma, 2006, vol. 60, pp. 3-11
[2] A. J. Shoffstall, “Intravenous hemostatic nanoparticles increase survival following blunt trauma injury,” Biomacromolecules, 2014, vol. 13, pp. 3850-3857
[3] M. Lashof-Sullivan, “Intravenous Hemostats: Challenges in Translation to Patients,” Nanoscale, 2013, vol. 5, pp. 10719-10728
[4] J. P. Bertram, “Intravenous hemostat: Nanotechnology to halt bleeding,” Sci Transl Med, 2009, vol. 11, pp. 11-22