PLGA (Resomer 503H) was purchased from Evonik Industries. Poly-ι-lysine (500–4000 Da MW) and PEG (~4600 Da MW) were purchased from Sigma Aldrich. All reagents were ACS grade and were purchased from Fisher Scientific. PLGA-PLL-PEG coblock polymer was made using standard bioconjugation techniques as previously described.^{16, 17, 38, 39}

PLGA-PLL-PEG (1 g) was dissolved in anhydrous DMSO to a concentration of 100 mg/ml. Oligopeptides (25 mg GRGDS or GRADSP) was dissolved in 1 ml DMSO and added to the stirring polymer solution. This was reacted for 3 hours, and then transferred to dialysis tubing (SpectraPor 2 kDa MWCO). Dialysis water was changed every half hour for 4 hours with Type I D.I. water. The product was then snap-frozen in liquid nitrogen and lyophilized for 2–5 days.

The resulting quadblock copolymer PLGA-PLL-PEG-GRGDS was then dissolved to a concentration of 20 mg/ml in acetonitrile (120 mg/6 ml). This solution was added dropwise to a stirring volume of PBS. Precipitated nanoparticles form as the water-miscible solvent dissipates. Particles were collected using a coacervate precipitation method. Briefly, one mass equivalent of dry poly(acrylic acid) was added to the stirring particle suspension. 15ml of 1% w/v pAA was then added slowly to the stirring suspension until flocculation occurs. After 5 minutes, the flocculated particles were collected by centrifugation at 500g, and rinsed 3 times with 1% pAA (centrifuging @ 500 g, 2m, 4C between rinses). On the final rinse, particles were resuspended with D.I. water, snap-frozen and lyophilized for 2–5 days. Particles were resuspended in PBS and briefly sonicated at 4W to a total energy of 50 J using a probe sonicator (VCX-130, Sonics & Materials, Inc.) prior to use.

As previously demonstrated, successful conjugation of PLL, PEG and peptide ligands was confirmed using UV-spectroscopy, 1H-NMR and amino acid analysis HPLC (BioRad, Varian and Shimadzu respectively).¹⁶ Nanoparticles from 5 independent syntheses batches were characterized for size distribution using dynamic light scattering (90Plus, Brookhaven Instruments Corporation) and zeta potential (Zeta Pals, Brookhaven Instruments Corporation). Reported figures from DLS are given as number average. Scanning electron microscopy was performed to visualize particle morphology (Hitachi S4500).

Amino acid analysis (AAA) was used to quantify the GRGDS peptide conjugation to the triblock polymer PLGA-PLL-PEG. The outcome arg:lys ratio was used to measure this relative conjugation efficiency. Briefly, a 5 mg aliquot of polymer was hydrolyzed for 24 h in a hydrolysis/derivitization workstation (Eldex Laboratories, Inc., Napa, CA). The hyrolysate was then neutralized with a redrying solution (ethanol: water: triethylamine in a 2:2:1 ratio) and derivitized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, using the Water’s AccQ-Tag system. These samples were run on an HPLC (Shimadzu, with Water’s PicoTag Column) and measured using a fluorescence detector. Standard addition of known quantities of arg and lys to hydrolyzed samples was used to correct for polymer hydrolysate background.

All animal studies were performed in accordance with the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC). Sprague Dawley rats (225–275g, Charles River) were anesthetized with intraperitoneal ketamine/xylazine (90:10 mg/kg, respectively), and injured according to the previously established liver injury model.^{16, 40–43} In brief, after confirmation of complete anesthesia, the abdomen was accessed and the medial lobe of the liver was marked with an arch radius 1.3 cm from the suprahepatic vena cava using a handheld cautery device. Once marked, the tail vein was catheterized with a saline-flushed 24G × 3/4″ Excel Safelet Catheter. The medial liver lobe was then resected along the marked lines to create the injury. Treatments were administered via tail vein catheter immediately after injury and included saline, scrambled particles, and functionalized particles. All particle treatments were resuspended in a 0.5 ml PBS carrier solution.

The rats were allowed to bleed for 1 hour or until death, as confirmed by lack of both breathing and a palpable heartbeat. Before measuring blood loss, all rats were injected with a 1 ml lethal dose of sodium pentobarbital. The abdomen was then reopened and blood collected with pre-weighed gauze. The clot adherent to the liver was collected last as this usually caused additionally bleeding to occur. The resected liver was weighed and fixed in 10% buffered formalin solution. Remaining liver, kidney, spleen and lungs were harvested and similarly preserved in 10% buffered formalin.

Liver, kidney, spleen, lung and adherent clots were harvested and lyophilized for the biodistribution assay. The dry weight of the whole organ was recorded and 100–200 mg of dry tissue was homogenized (Precellys 24) and incubated overnight in acetonitrile at 37 C. This dissolved any nanoparticles present in the tissue and left the C6 in the organic solvent solution. Tubes were then centrifuged at 15,000 g for 10 minutes to remove solid matter and supernatant was tested on the HPLC. Mobile phase was 80% acetonitrile, and 20% aqueous (8% acetic acid). Stationary phase was a Waters Symmetry C18 Column, 100Å, 5 μm, 3.9 mm × 150 mm with fluorescence detection (450/490 nm ex/em). Based on the known C6 loading and dosage, data is represented as percent (%) of particles injected.

Tissue samples from the left lobe of the liver (uninjured), medial lobe (injured) with adherent clot, lung, kidney, and spleen were fixed in formalin, soaked overnight in sucrose, frozen and cryosectioned to 20-μm thickness. Sections were imaged with an inverted fluorescence microscope (Zeiss Axio Observer.Z1). The DsRed filter was used to image tissue background fluorescence as a reference channel since staining with VectaShield DAPI, or H&E displaced nanoparticles from the tissue.

Coagulation assays, using Sprague Dawley rat blood, were performed using the ROTEM’s NATEM test in the presence of either saline, GRGDS-NPs, or scrambled GRADSP-NPs as previously described.¹⁶ The outcomes we considered include the standard ROTEM parameters clotting time (CT), clot formation time (CFT), the sum of the two (CT+CFT), and maximum clot firmness (MCF). CT is defined as the time from the start of the assay until the initial clotting is detected (thickness = 2mm). CFT is defined as the time between clot initiation until a clot thickness of 20 mm is detected. MCF is defined as the maximum thickness (in mm) that a clot reaches during the duration of the test. The dosing study was performed blood samples from 10 rats, starting with the highest concentration of particles (20 mg/ml, n=5), and titrating downward (2 mg/ml, n=7; 0.2 mg/ml, n=9; 0.02 mg/ml, n=9) until no effect was observed (0.002 mg/ml, n=3). Each sample was run in triplicate on the ROTEM, normalized to a saline control, and averaged for each rat.

ANOVA with ad-hoc Tukey comparisons was used to analyze blood loss and ROTEM data (Minitab). 1-hour survival was analyzed with a binomial logistic regression with chi-squared tests between odds-ratios (SAS), and survival curves with a log-rank (Mantel-Cox) test. Quantification of histology was analyzed with two sample t-tests with Welch’s correction.

Polymer nanoparticle characteristics were the same as previously described (Table 1, Figure 1)¹⁶, except for the higher conjugation efficiency of RGD peptide to the activated PEG groups (Figure 2). This was accomplished primarily by performing the peptide conjugation before nanosphere formation and reacting in organic phase (anhydrous DMSO) rather than aqueous to form what we term the quadblock polymer (PLGA-b-p(lys)-b-PEG-b-GRGDS). After nanoparticle formation, peptide loading levels were measured with amino acid analysis and the arginine to lysine ratio was determined to obtain the percentage of polymer chains with the GRGDS peptide. Nanoparticles made from the quadblock polymer had 2 orders of magnitude more GRGDS than the nanoparticles made from triblock polymer used in the hemostatic nanoparticles tested previously (Arg:Lys ratio 4.35*10⁻³ compared to 0.428).¹⁶ The high-RGD-loaded nanoparticles based on the quadblock polymer are referred to as GRGDS-NP100 and Scrambled-NP100, while the previously used triblock nanoparticles are referred to as GRGDS-NP1 and Scrambled-NP1.

A dosing study with anticoagulated whole rat blood was performed to titrate the optimal dose of the NP100 nanoparticles (Figure 3). Rotational thromboelastometery (ROTEM) was used to determine clotting time (CT), clot formation time (CFT), and maximum clot firmness (MCF). Each sample consisted of 300 μl of anticoagulated blood, 20 μl of a particle dosing solution, and 20 μl of CaCl solution to replace the calcium in the blood and initiation coagulation. Previously, using the GRGDS-NP1 particles, we found that a particle dosing concentration of 2.5 mg/ml (blood concentration = 147 μg/ml) reduced clotting time in this in vitro model. When testing the GRGDS-NP100 particles at this same concentration, we observed a trend of increased total clotting time (CT+CFT), and a decrease in MCF, demonstrating an anticoagulant-like effect (Figure 3a). A dose response was then performed to titrate down to the optimal dose for the GRGDS-NP100 particles (Figure 3a–b). We found that the optimal dose was at least 10-fold lower, between 0.02–0.25 mg/ml (blood concentration = 1.2–14.7 μg/ml). Further testing 0.25 mg/ml at this concentration yielded a reduced total clotting time (CT+CFT, p=0.0346 versus saline), with no adverse impact on MCF (Figure 3c–d). There was no significant difference between the scrambled and GRGDS groups.

Previous experiments with the low-peptide conjugated nanoparticles (GRGDS-NP1) at 20 mg/ml concentration in a 0.5 ml carrier solution (40mg/kg) led to increased 1 hour survival (80% compared to 47% saline control) in a model of lethal liver trauma.¹⁶ When this experiment was repeated with high-peptide conjugated nanoparticles (GRGDS-NP100) at the same dose concentration (40 mg/kg) and ½ dose (20 mg/kg), survival time was drastically reduced from a mean time of 43 minutes (saline) to 28 minutes and 34 minutes, respectively, suggesting an adverse effect on the injury model. The effects of NP100 particles appeared to be harmful until dosing down to 5 mg/kg, at which, 1 hour survival increased to 100% for the pilot study with n=3 animals (Figure 4a). Blood loss was also significantly reduced compared to the saline control (p=0.0115, Figure 4b).

We then scaled up the study (n=13) at this new dosage, 5 mg/kg. 1-hour survival was increased to 92.3% compared to a scrambled peptide control 45% (OR=14.4, 95% CI=[1.36, 143], power=0.836) a saline control 47% (OR=13.5, 95% CI=[1.42, 125], power=0.888) and the previously reported hemostatic nanoparticles 80% (OR=1.3, n.s., Figure 5a). Blood loss, as measured by collecting intra-abdominal blood with pre-weighed gauze, was significantly decreased from a mean of 26.0 ml/kg (saline) to 19.25 ml/kg (GRGDS-NP100, p=0.0067, Figure 5b).

Compared to the previous liver injury study¹⁶, where 40 mg/kg of GRGDS-NP1 particles were injected, the present study only used 5 mg/kg of GRGDS-NP100 particles, 1/8 the previous mass. We find that similar proportions of the injected dose are found in the tissues, with the majority of particles being cleared through the liver (7.5–10.5%) or becoming entrapped in the clot (11%) or lungs (2–46%) as measured using an HPLC fluorescence assay for C6 (Figure 6). 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.

Since biodistribution within anisotropic organs can have a heterogeneous distribution, a histological investigation was conducted, looking at the kidney, capillary beds of the deep lung, the uninjured left lobe of the liver, and the adherent clot attached to the injured medial lobe of the liver (Figure 7). We found that while the proportion of particles accumulating in the clot was similar between the GRGDS and scrambled particles (by HPLC), the GRGDS particles appeared in clusters rather than individual satellite particles (by histology), suggesting that they may be actively participating in platelet aggregation (Figure 7d). The number of particles found within the clot (11% injected dose), while a small mass, represents a large number of particles, 9.2×10⁹, or a number equivalent to ~50% of the total circulating platelets in a 250 g rat (assuming: nanoparticle density, 1.3 g/cm3; rat blood volume, 68.6 ml/kg; normal rat platelet concentration 1.180×10⁹/ml)^{44, 45}

The distribution of particles to the capillary beds of the lung was significantly smaller than expected for the GRGDS group (Figure 7b), suggesting that the large quantity of particles found in the GRGDS-NP100 group, by the HPLC assay of the whole lung tissue, is not collected in the capillary beds, and must be accumulating in higher order branches.