Modulating Nanoparticle Size to Understand Factors Affecting Hemostatic Efficacy and Maximize Survival in a Lethal Inferior Vena Cava Injury Model

Abstract

Intravenous nanoparticle hemostats offer a potentially attractive approach to promote hemostasis, in particular for inaccessible wounds such as noncompressible torso hemorrhage (NCTH). In this work, particle size was tuned over a range of <100–500 nm, and its effect on nanoparticle–platelet interactions was systematically assessed using in vitro and in vivo experiments. Smaller particles bound a larger percentage of platelets per mass of particle delivered, while larger particles resulted in higher particle accumulation on a surface of platelets and collagen. Intermediate particles led to the greatest platelet content in platelet–nanoparticle aggregates, indicating that they may be able to recruit more platelets to the wound. In biodistribution studies, smaller and intermediate nanoparticles exhibited longer circulation lifetimes, while larger nanoparticles resulted in higher pulmonary accumulation. The particles were then challenged in a 2 h lethal inferior vena cava (IVC) puncture model, where intermediate nanoparticles significantly increased both survival and injury-specific targeting relative to saline and unfunctionalized particle controls. An increase in survival in the second hour was likewise observed in the smaller nanoparticles relative to saline controls, though no significant increase in survival was observed in the larger nanoparticle size. In conjunction with prior in vitro and in vivo experiments, these results suggest that platelet content in aggregates and extended nanoparticle circulation lifetimes are instrumental to enhancing hemostasis. Ultimately, this study elucidates the role of particle size in platelet–particle interactions, which can be a useful tool for engineering the performance of particulate hemostats and improving the design of these materials.

Engineering a Two‐Component Hemostat for the Treatment of Internal Bleeding through Wound‐Targeted Crosslinking

“…These polymers were used to generate nanoparticles (NPs) of approximately 180 nm, as previous studies had demonstrated that this size was optimal for the recruitment of activated platelets and accumulation at the injury site. [ 17 ] With the exception of pure azide NPs (only used in kinetic studies), all sizes fell within the range of 140–220 nm. The size of the nanoparticle crosslinker, also within the range of 140–220 nm, was chosen to match its biodistribution and pharmacokinetic properties to the targeting component, maximizing the timeframe over which the two components would coexist within the bloodstream.…”

“…Characterization Methods: All synthesized materials were characterized as previously described. [17] In brief, polymers were characterized via nuclear magnetic resonance (NMR) on a Bruker Advance III DPX 400 spectrometer in deuterated DMSO at a concentration of 5 mg mL −1 . The ratio of lactide to glycolide was quantified via lactide protons (≈5.2 ppm) and glycolide protons (≈4.8 ppm), and the molecular weight of the polymer was calculated using the PEG macroinitiator as a standard (≈3.5 ppm).…”

“…The two-component system proposed herein provides a potential solution to these points. By leveraging increased accumulation of activated-platelet-targeted materials at the wound site-a phenomenon widely reported in literature [17,41] -this system applies the concept of the critical gelation concentration to achieve wound-targeted clot strengthening without increased lung accumulation or systemic toxicity. Its targeting mechanism through activated platelets and bioorthogonal crosslinking through azide-DBCO reactions mitigate the effect of fibrin depletion in severe hemorrhage, as demonstrated through enhanced platelet recruitment and overall coagulation potential even in fully hemodiluted environments.…”

“…Poly(ethylene oxide)-b-Poly(d,l-lactide-co-glycolide)-Cyanine 7 (PEG-PLGA-Cy7): PEG-PLGA-Cy7 was synthesized as previously described [17] -a summary of the protocol has been provided below. In brief, one equivalent of Cyanine 7 free acid was added to PEG-b-PLGA-OH (synthesized using a methoxy-PEG macroinitiator) and dissolved in DCM with 0.4 equivalent of 4-dimethylaminopyridine (DMAP).…”

“…Platelet Flow Cytometry: Flow cytometry was conducted using a FACS LSR II instrument (BD Biosciences) with FACSDIVA software and 405, 488, 561, and 640 nm lasers in a modified protocol from the literature. [17] In brief, a diluted solution of activated PRP was prepared by combining 85 μL of FACS buffer (0.9 wt % saline + 5% bovine serum albumin), 15 μL of 100 × 10 −3 m calcium chloride, 100 μL of thrombin at 100 U mL −1 , 150 μL of 15 × 10 −3 m GPRP-NH 2 , and 150 μL PRP. Inactivated PRP was prepared similarly by substituting thrombin and calcium chloride with FACS buffer.…”