Tuning ligand density on intravenous hemostatic nanoparticles dramatically increases survival following blunt trauma

Abstract

Targeted nanoparticles are being pursued for a range of medical applications. Here we utilized targeted nanoparticles (synthetic platelets) to halt bleeding in acute trauma. One of the major questions that arises in the field is the role of surface ligand density in targeted nanoparticles' performance. We developed intravenous hemostatic nanoparticles (GRGDS-NP1) and previously demonstrated their ability to reduce bleeding following femoral artery injury and increase survival after lethal liver trauma in the rat. These nanoparticles are made from block copolymers, poly(lactic-co-glycolic acid)-b-poly L-lysine-b-poly(ethylene glycol). Surface-conjugated targeting ligand density can be tightly controlled with this system, and here we investigated the effect of varying density on hemostasis and biodistribution. We increased the targeting peptide (GRGDS) concentration 100-fold (GRGDS-NP100) and undertook an in vitro dose-response study using rotational thromboelastometry, finding that GRGDS-NP100 hemostatic nanoparticles were efficacious at doses at least 10 times lower than the GRGDS-NP1. These results were recapitulated in vivo, demonstrating efficacy at eight-fold lower concentration after lethal liver trauma. 1 h survival increased to 92% compared with a scrambled peptide control, 45% (OR = 14.4, 95% CI = [1.36, 143]), a saline control, 47% (OR = 13.5, 95% CI = [1.42, 125]), and GRGDS-NP1, 80% (OR = 1.30, n.s.). This work demonstrates the impact of changing synthetic platelet ligand density on hemostasis and lays the foundation for methods to determine optimal ligand concentration parameters.

Figure 1

SCANNING ELECTRON MICROSCOPY. Image of nanoparticles under SEM (Hitachi S4500).

Figure 2

AMINO ACID ANALYSIS. Peptide conjugation efficiency Arg:Lys ratio. Peptide conjugation levels are approximately 100-fold higher when the conjugation reaction is performed in DMSO instead of aqueous phase. This leads to the nomenclature, NP100 and NP1 for the organic and aqueous phase polymers respectively. Error bars denote SEM.

Figure 3

IN VITRO DOSE RESPONSE GRGDS-NP100. a–b) Total clotting time (CT+CFT) and maximum clot firmness (MCF) dose-responses, recapitulated the in vivo response observed: high doses adversely impact clotting parameters (increase CT+CFT; decrease MCF). This is observed until dosing down to 0.02–0.2 mg/ml. C–D) 0.25 mg/ml was then further tested directly against a scrambled-NP100 control (n=6). CT+CFT was reduced compared to saline (p=0.0346), with no significant impact on MCF. Dotted lines denote normalization to the saline-treated controls values. Error bars denote SEM.

Figure 4

IN VIVO DOSE RESPONSE GRGDS-NP100. Dose response with GRGDS-NP100 in rat liver injury model (n=3 for pilot study). b) Percentage of animals surviving to 1-hour is reduced in the 40 mg/kg and 20 mg/kg groups, but increased in the 5 mg/kg dose. b) Blood loss is significantly reduced in the 5 mg/kg dose, and not significantly changed with either 40 mg/kg or 20 mg/kg doses compared to the saline control. Error bars denote SEM.

Figure 5

LIVER INJURY RESULTS NP100. Rat medial liver injury model at 5 mg/kg dose. a) 1-hour (endpoint) survival was increased to 92%, compared to a scrambled peptide control, 45% (OR=14.4, 95% CI=[1.36, 143]), a saline control, 47% (OR=13.5, 95% CI=[1.42, 125]), and GRGDS-NP1, 80% (OR=1.30, n.s.). Survival curves display increased survival with GRGDS-NP100 compared to the scrambled and saline groups, log-rank (Mantel-Cox) test, p=0.0362. b) Blood loss was significantly reduced in the GRGDS-NP100 group compared to saline (p=0.0067). Error bars denote SEM.

Figure 6

BIODISTRIBUTION (HPLC ASSAY). An assay for fluorescent C6 was performed using HPLC. a) There is a large proportion of nanoparticles in the lungs for the targeted GRGDS group (~50%). The liver accumulates 7.5–10.5% of the injected dose (GRGDS, scrambled respectively), while ~11% becomes entrapped in the adherent clot found in the abdominal cavity post-mortem, regardless of the peptide group. Less than 1% is found in the kidney and spleen, and the particles are rapidly cleared from the blood plasma, with only 2% remaining in circulation at the end of the 1-hour experiment. b) However, due to 8x lower dose with NP100, there are fewer nanoparticles by mass in the lungs compared to the NP1. Error bars denote SEM.

Figure 7

HISTOLOGY AND QUANTIFICATION. Histology was performed on the kidneys, uninjured left lobe of the liver, lungs, and injured medial lobe of the liver with adherent clot intact. Sections are 20 μm, and were not stained to prevent displacement of the nanoparticles. Quantification of the particles is measured in triplicate for n=3 rats per group, and represented as pixels/mm². a) Kidneys show no significant differences between treatment groups, b) Uninjured liver (left lobe), contains higher density of particles in the scrambled group compared to GRGDS (p=0.0467). c) Lungs show a larger proportion of particles accumulating in the GRGDS group compared to scrambled, d) Clot adherent to remaining liver. Green = coumarin-6 (C6) loaded hemostatic nanoparticles; Red = Tissue background fluorescence (DsRed filter) used as reference channel. While the concentration of particles in the adherent clot is equal between groups, the particles in the GRGDS group appear as clusters, while the scrambled particles appear evenly dispersed. Error bars denote SEM.