intravenous hemostat: nanotechnology to halt bleeding

DOI: 10.1126/scitranslmed.3000397, 11ra22 (2009);1 Sci Transl Med, et al.James P. Bertram

Intravenous Hemostat: Nanotechnology to Halt Bleeding

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R E S EARCH ART I C L E

BLOOD CLOTT ING

Intravenous Hemostat: Nanotechnology to Halt BleedingJames P. Bertram,1 Cicely A. Williams,2 Rebecca Robinson,1

Steven S. Segal,3,4 Nolan T. Flynn,5 Erin B. Lavik6*
(Published 16 December 2009; Volume 1 Issue 11 11ra22)
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Blood loss is the major cause of death in both civilian and battlefield traumas. Methods to staunch bleedinginclude pressure dressings and absorbent materials. For example, QuikClot effectively halts bleeding by ab-sorbing large quantities of fluid and concentrating platelets to augment clotting, but these treatments arelimited to compressible and exposed wounds. An ideal treatment would halt bleeding only at the injury site,be stable at room temperature, be administered easily, and work effectively for internal injuries. We have de-veloped synthetic platelets based on Arg-Gly-Asp functionalized nanoparticles, which halve bleeding time afterintravenous administration in a rat model of major trauma. The effects of these synthetic platelets surpass othertreatments, including recombinant factor VIIa, which is used clinically for uncontrolled bleeding. Synthetic plate-lets were cleared within 24 hours at a dose of 20 mg/ml, and no complications were seen out to 7 days afterinfusion, the longest time point studied. These synthetic platelets may be useful for early intervention in traumaand demonstrate the role that nanotechnology can have in addressing unmet medical needs.

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INTRODUCTION

Traumatic injury is the leading cause of death for individuals betweenthe ages of 5 and 44 years (1), and blood loss is the major factor inboth civilian and battlefield trauma (2, 3). After injury, the cessation ofbleeding, or hemostasis, is established through a series of coagulatoryevents, including platelet activation. With severe injuries, however,these processes are insufficient and uncontrolled bleeding results.Although immediate intervention is one of the most effective meansof minimizing mortality associated with severe trauma (4), pressuredressings and absorbents are now the only hemostats available forfield administration.

Alternatives to topical dressings include allogeneic platelet trans-fusions, clotting factors, and platelet substitutes, but limited efficacy,immunogenicity, and thrombosis have stalled their application (5).Administration of allogeneic platelets can halt bleeding. However,platelets have a short shelf life, and administration of allogeneicplatelets can cause graft-versus-host disease, alloimmunization, andtransfusion-associated lung injuries (6). Recombinant factors, includ-ing activated factor VII (NovoSeven), can augment hemostasis, butimmunogenic and thromboembolic complications are unavoidablerisks (7). Nonetheless, NovoSeven is used in trauma and surgical situa-tions where bleeding cannot otherwise be controlled (8). Nonplateletalternative coagulants including red blood cells modified with the Arg-Gly-Asp (RGD) sequence, fibrinogen-coated albumin microcapsules,and liposomal systems have been studied as coagulants (9), but tox-icity, thrombosis, and limited efficacy restrict the clinical utility ofthese products (5).

1Department of Biomedical Engineering, Yale University, 55 Prospect Street, MaloneEngineering Center, New Haven, CT 06511, USA. 2Interdepartmental NeuroscienceProgram, Yale University, New Haven, CT 06511, USA. 3Department of Medical Phar-macology and Physiology, University of Missouri School of Medicine, Columbia, MO65212, USA. 4Dalton Cardiovascular Research Center, Columbia, MO 65211, USA.5Department of Chemistry, Wellesley College, Wellesley, MA 02481, USA. 6Departmentof Biomedical Engineering, Case Western Reserve University, Room 309, WickendenBuilding, 10900 Euclid Avenue, Cleveland, OH 44106-7207, USA.*To whom correspondence should be addressed. E-mail: [email protected]

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Polymer engineering has opened up new possibilities for control-ling bleeding. For example, a self-assembling peptide rapidly haltsbleeding in a number of injury models when applied topically (10),functionalized liposomes (11) and micrometer-sized sheets (12) inter-act with activated platelets in vitro, and a block copolymer of hemo-globin and fibrinogen acts as an oxygen carrier and maintains normalbleeding times at high concentrations where typical oxygen carrierslead to hemodilution and long bleeding times (13). We have taken adifferent approach and have designed synthetic platelets based onfunctionalized nanoparticles that bind to activated platelets to augmentclotting in a safe, localized manner. This system leverages existingbiological processes by providing a nanostructure that binds to acti-vated platelets and enhances their rate of aggregation, which stopsbleeding. They are made from polymers that are already used inthe medical device and pharmaceutical industries, and a strong trackrecord of safety makes them well suited for clinical translation.

We tested the synthetic platelets in vitro to optimize their bindingefficiency to activated platelets and in vivo in a rat major femoralartery injury model to determine their efficacy in promoting hemo-stasis and their biodistribution. The synthetic platelets halved bleed-ing time after intravenous administration in the femoral artery injurymodel and performed significantly better than activated recombinantfactor VIIa (rFVIIa), which is now being used in the clinic for un-controlled bleeding.

RESULTS

Design and synthesis of synthetic plateletsOur synthetic platelets consist of poly(lactic-co-glycolic acid)-poly-L-lysine (PLGA-PLL) block copolymer cores to which we conjugatedpolyethylene glycol (PEG) arms terminated with RGD functionalities(Fig. 1A). 1H nuclear magnetic resonance (1H NMR) demonstratedsuccessful conjugation of PEG to PLGA-PLL (fig. S1C). Nanosphereswere fabricated with a single emulsion solvent evaporation technique(14), which resulted in core diameters of ∼170 nm by scanning electronmicroscopy (SEM) (Fig. 1, B and D). After fabrication, nanospheres

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were analyzed with 1H-NMR to confirm that the conjugatedPEG was present. The subsequent conjugation of RGD toPLGA-PLL-PEG nanospheres was then confirmed withamino acid analysis (Fig. 1C). Amino acid analysis was alsoused for ascertaining the RGD conjugation for the PLGA-PLL-PEG-RGD polymer used for in vitro characterization(fig. S2A). RGD conjugation efficiency was independent ofboth the PEG molecular weight and peptide sequence [thatis, RGD versus GRGDS (Gly-Arg-Gly-Asp-Ser)] (Fig. 1C andfig. S2A). Synthetic platelets have an average RGD peptidecontent of 3.3 ± 1.1 mmol/g (mean ± SD) (Fig. 1C), which cor-responds to a conjugation efficiency of 16.2 ± 5.9% (mean ±SD) (∼600 RGD moieties per synthetic platelet). The cou-pling agent used to attach the peptide, carbonyldiimidazole,is hydrolyzable in water and thus can be quenched duringthe aqueous peptide coupling, leading to the modest effi-ciency. One could augment the efficiency by repeated acti-vation and coupling reactions or the use of an alternativeagent, but the efficiencies here were effective in our subse-quent studies. Although cores are ∼170 nm in diameter forall of the preparations (Fig. 1D), the hydrodynamic diam-

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eter of the spheres, determined by dynamic light scattering (DLS),increased with increasing PEG molecular size (Fig. 1D). The SEMand DLS results suggest that a surface enrichment of PEG armsexists, and in a hydrated environment, PEG arms extend to createa PEG corona on the nanosphere surface (15). On the basis of theseresults, we concluded that, under hydrated conditions, the surfaceproximity of the conjugated RGD peptide varies as a function ofPEG molecular size.

In vitro validation and optimization of synthetic plateletsThe surface proximity of the RGD functionality affects interactionswith activated platelets (16). To determine the optimal PEG armlength, as well as to identify the most appropriate peptide sequence,we adapted an in vitro platelet aggregation and adhesion assay (Fig.2A) (16). The in vitro assay provided a simple means to assess therole of the components of the synthetic platelets on platelet adhe-sion and aggregation. Other traditional in vitro assays, such as theaggregometer, could not be used with our synthetic platelets becausethe nanoparticles led to substantial scattering in the system, whichproduced noise that masked the results. The in vitro platelet aggrega-tion and adhesion assay was validated with collagen controls (fig. S2B).We coated the surface of a 96-well plate with variants of the PLGA-PLL-PEG-RGD polymer (fig. S1A). Using 5-chloromethylfluoresceindiacetate (CMFDA)–labeled platelets and the aggregation stimulantadenosine diphosphate (ADP), we examined the effects of the PEGlength and RGD functionality on platelet aggregation and adhesion(Fig. 2, B and C). Platelet aggregation increased with increased PEGlength, with PEG 4600 compounds leading to greater adhesion andaggregation than PEG 1500 compounds for all RGD variants studied.This is consistent with previous observations (17).

We found that activated platelet adhesion increased as we addedflanking amino acids around the RGD sequence, with the GRGDSformulations leading to the greatest adhesion (Fig. 2D). Control ex-periments verified that PEG alone, and the conservative substitutepeptide 4600-GRADSP (Gly-Arg-Ala-Asp-Ser-Pro), yielded the samevalues as the PLGA-only group, inducing only minimal adhesionand aggregation.

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For clinical utility, it is critical that the activated platelets bind spe-cifically to the synthetic platelets, as nonspecific binding or inducedplatelet activation could lead to adverse concomitant thromboticevents, including embolism and stroke. We found that nonactivatedplatelets did not bind to any of the PLGA-PLL-PEG-RGD polymerstested (fig. S3C). Furthermore, platelets did not activate without theaddition of ADP. The polymers did not induce platelet adhesion,even with agitation, except when ADP was added (fig. S3D). As asimple test of our system, we added synthetic platelets to platelet-richplasma (PRP) (fig. S3). Even with prolonged agitation (10 min), syn-thetic platelets did not induce endogenous platelet aggregation. Thisis in contrast to PRP added to the collagen-coated control wells. Evenin the absence of ADP, endogenous platelets aggregated and adheredto the collagen (fig. S2B). We only saw adhesion and coagulationwith the synthetic platelets when a proaggregatory stimulus (ADP)was added. The materials used to fabricate synthetic platelets did notactivate endogenous platelets, and nonactivated platelets did not bind,which suggests that the materials are unlikely to induce activation ornonspecific platelet binding on their own.

In vivo: Femoral artery injury modelThe goal of this work was to develop a safe synthetic platelet withhemostatic efficacy in vivo. We tested our synthetic platelets in arat model of a major femoral artery injury (Fig. 3A) (18). The in-jury leads to blood spurting in a continuous stream from the injurysite (Fig. 3A) and is easily visualized. We investigated three doses(10, 20, and 40 mg/ml) of the synthetic platelets (fig. S4A). Wefound that 10 mg/ml had no effect on bleeding time and 40 mg/mlproduced cardiopulmonary complications in some animals, present-ing as elevated heart rate and gasping. Therefore, we focused ondetermining the optimal particle characteristics with intravenous ad-ministration at 20 mg/ml.

The PEG 1500 nanospheres with the GRGDS peptide led to thegreatest reduction in bleeding time (Fig. 3B), paralleling the in vitrofindings. Also paralleling the in vitro findings was the fact that therewas a greater reduction in bleeding time with the RGD-functionalizedPEG 4600 spheres relative to their PEG 1500 counterparts (Fig. 3C);

Fig. 1. Design, synthesis, and characterization of synthetic platelets. (A) Schematicof synthetic platelet composed of PLGA-PLL core with PEG arms terminated with the
RGD moiety. (B) SEM micrograph of synthetic platelets. Scale bar, 1 mm. (C) Lysineand peptide concentrations of synthetic platelets as determined by amino acid anal-ysis. Conjugation efficiency was defined as the peptide-to-lysine ratio multiplied by100. (D) Diameter of PLGA-PLL core and PLGA-PLL-PEG nanospheres as determinedby SEM and DLS. SEM diameter based on n = 80. Data are expressed as mean ± SD.



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4600-GRGDS nanospheres led to significantly shorter bleeding timesrelative to the 1500-GRGDS nanospheres (n = 5, P < 0.05).

The injury alone with no injection gave a baseline bleeding timeof 240 ± 15 s (mean ± SEM, n = 5). Injection of the 4600-GRGDSsynthetic platelets halved the bleeding time [131 ± 11 s (mean ± SEM,n = 5)] (Fig. 3C). To validate our synthetic platelets as a hemostaticagent, we compared bleeding times after synthetic platelet adminis-tration to bleeding times after the injection of rFVIIa. Recombinantfactor VIIa has proven clinically useful in the treatment of surgery-and trauma-associated bleeding (19) and is the current standard ofcare for uncontrolled bleeding (8). Although a bolus injection ofrFVIIa (100 mg/kg; the recommended dose is 90 to 100 mg/kg) sig-nificantly reduced bleeding times [187 ± 16 s (mean ± SEM, n = 5)],4600-GRGDS synthetic platelets reduced bleeding time ∼25% morethan did rFVIIa (Fig. 3C). Furthermore, 4600-GRGDS synthetic plate-lets stored in a lyophilized state at room temperature for 2 weeks main-tained their hemostatic properties with no reduction in efficacy (Fig. 3D).

In these studies, the agents were administered intravenouslybefore the injury. We chose this approach initially because the bleed-ing times in this injury without treatment (240 ± 15 s) are close tothe time it took to inject the synthetic platelets or controls (180 s).However, for the synthetic platelets to be clinically effective, theyneed to work when administered after an injury. Therefore, we testedthe synthetic platelets in the same rat femoral artery injury model, butwe administered the synthetic platelets (20 mg/ml, 0.5 ml injected) orone of the controls over a period of 20 s. Administration of salinealone (0.5 ml) or the unfunctionalized PEG 4600 nanospheres (20mg/ml, 0.5 ml administered) increased bleeding time relative to theno injection control group. This is likely the result of systemicblood dilution due to the rapid bolus administration of fluid, animportant concern when administering fluids and clotting factorsintravenously after injury (20). Nonetheless, even with this dilutingeffect, the 4600-GRGDS synthetic platelets significantly reducedbleeding time by ∼23% relative to no injection and by 31% relativeto saline injection (Fig. 3E).

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Participation of synthetic plateletsin the clotWe next determined whether synthetic plateletswere present in the clots. We excised the injuredvessel segments and performed SEM. Syntheticplatelets were intimately associated with the fibrinmesh (Fig. 3F) and distributed throughout the cloton the luminal, interior surface of the femoral ar-tery (fig. S4B).

Biodistribution of synthetic plateletsFor synthetic platelets to be therapeutically vi-able, they must be effectively cleared when notparticipating in clot formation at the site of injury.To test this parameter, we labeled synthetic plate-lets (4600-GRGDS) by encapsulating coumarin 6(C6), a fluorochrome commonly used for biodis-tribution studies (21). We examined the biodis-tribution and clearance of C6-labeled syntheticplatelets up to 7 days after intravenous injectionwith fluorescence spectroscopy. Less than 0.5% ofthe C6 fluorochrome label was released from thenanospheres after 24 hours, and ∼1.5% was re-

leased after 7 days (Fig. 4A), indicating that virtually all of the C6fluorescence was associated with synthetic platelets. A characteristicdistribution of synthetic platelets was observed consistent with intra-venous nanosphere administration (Fig. 4B) (22). Within 5 min ofinjection, 68.3% of the injected particles were found in the liver,16.1% were in the blood, and minimal accumulations were seen inthe kidneys, lungs, and spleen (<3.6%, 2.2%, and 5.2%, respective-ly). The sensitivity of our biodistribution analysis is 0.2 ng of C6 permilligram of tissue and 3.3 ng of C6 per milliliter of plasma. At 3and 7 days after injection, no C6 was detected, suggesting that allsynthetic platelets had been cleared from circulation. Furthermore,no adverse effects were seen in any of the animals at all time points.We also examined the biodistribution of synthetic platelets imme-diately after a femoral artery injury and 1 hour after injury (Fig. 4, Cand D). For both time points, tissue distribution was similar for theinjured and uninjured animals (equivalent to 10-min and 1-hour timepoints in uninjured animals). This suggests that even after a severeinjury and with circulating activated platelets, the unbound syntheticplatelets are effectively cleared within 24 hours.

Quantification of synthetic platelets within the clotWe were able to both visualize and quantify the C6-labeled syntheticplatelets in the clot after injury. Cross sections show that the syn-thetic platelets (green) were distributed throughout the clot (Fig.5A), consistent with the SEM findings (Fig. 3F and fig. S4B). Wequantified the amount of synthetic platelets within the clots withhigh-performance liquid chromatography (HPLC) and comparedthese findings to the distribution of C6-labeled PEG 4600 nano-spheres with no functionalities. Although PEG 4600 nanosphereswere entrapped in the clots after injury, clots from animals that re-ceived synthetic platelets had more than double the number of par-ticles relative to PEG 4600 nanospheres [3.9 ± 0.4 and 1.8 ± 0.2 ngof C6, respectively (mean ± SEM, n = 5)] (Fig. 5B). However, PEG4600 nanospheres had no significant effect on bleeding time (Fig.3C), suggesting that the synthetic platelets interact preferentially

Fig. 2. In vitro characterization of the interactions of polymers with activated platelets. (A)Schematic of in vitro assay for quantifying platelet adhesion to polymers. Adhesion of CMFDA-
labeled platelets to polymers after agitation and addition of ADP. PPP, platelet-poor plasma.(B and C) Appearance of assay with PEG 4600 (B) and 4600-GRGDS (C). Scale bars, 500 mm.(D) Quantification of platelet adhesion. Area of fluorescence represents area of plateletaggregation (n = 3). Data are expressed as mean ± SEM. *P < 0.05 versus PEG 4600 alone.
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with activated platelets as a result of RGD functionalization andthereby actively halt bleeding (as opposed to indirectly inducingplatelet flocculation).

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DISCUSSION
The ultimate goal of this work was to design and optimize a syn-thetic platelet that is stable at room temperature, safely administeredintravenously, and able to halt bleeding. Through our in vitro studies,we found that nanospheres functionalized with PEG arms with amolecular size of 4600 daltons and the GRGDS moiety led to thegreatest platelet adhesion and aggregation. These trends were re-peated in vivo in the rat femoral artery injury model.

It has been well established that the incorporation of the RGDmoiety can influence cellular interactions with biomaterials (23),and the proximity of the moiety to the biomaterial surface is a crit-ical parameter in these interactions (16, 24). By having a longerPEG spacer between the PLGA surface and the RGD moiety, thereis a greater probability that the RGD functionality is available tobind with the activated platelets. Beyond a certain PEG arm length,specifically a molecular size of 5000 daltons (15), the PEG moleculehas enough repeat units to have a strong probability of folding overand shielding the functional region.

Activated platelets bind to RGD moieties through specific ligand-receptor interactions between RGD and surface receptors expressedon activated platelets (25). More specifically, RGD interacts with theactivated platelet receptors glycoprotein IIb-IIIa and integrin avb3(16). We hypothesize that the RGD moiety, and the GRGDS moietyin particular, interacts with the activated platelets through these re-

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ceptors to promote adhesion between the polymers and the platelets.Flanking amino acids to the RGD motif produce a more activebinding conformation (26). This increased bioactivity in turn influ-ences integrin affinity for the RGD moiety (27) and increases cellularattachment (17). These features explain why the GRGDS peptidedemonstrates the greatest binding and adhesion of the activatedplatelets. Longer, more specific sequences have higher affinities foractivated platelets than does the GRGDS peptide. However, thelength of the peptide can have marked effects on its temperature sta-bility (28) as well as production costs. Increasing the number ofamino acid residues increases the time for synthesis, decreases yield,and increases the difficulty of purification (29). At the laboratoryscale, the addition of two amino acids, from a 5–amino acid se-quence to a 7–amino acid sequence, can increase the cost by an orderof magnitude.

Our results show that the RGD-functionalized polymer interactsspecifically with activated platelets. In the in vitro studies, the PEGalone and conservative substitute peptide, 4600-GRADSP, groupsbehaved like the PLGA-only group, inducing only minimal adhesionand aggregation. Thus, neither the polymer alone nor the polymerwith a substitute peptide induces adhesion and aggregation, whereasadhesion and aggregation are seen with all of the specific RGD var-iants. This indicates that the activated platelets’ affinity for theGRGDS moiety increases platelet adhesion to the polymer. This im-plies that the binding of activated platelets to the functionalizedPLGA-PLL-PEG is specific. Similar findings have been reported withcell attachment assays for other cell types (24).

Nonfunctionalized nanoparticles can activate platelets and alterthe coagulation cascade (30). It is hypothesized that the surfacecharge on the particles, their aggregation in water, and their shape

Fig. 3. In vivo performance of syntheticplatelets: hemostatic efficacy. (A) Femoralartery injury model used for all studies.Arrow points to injury site and the bloodspurting from the injured vessel. (B) Bleed-ing times after intravenous administration ofPEG 1500 synthetic platelets at 20 mg/ml(n = 5). Data are presented as percentageof no injection mean value ± SEM. **P <0.01, ***P < 0.001 versus PEG 1500 alone.(C) Bleeding times after intravenous ad-ministration of PEG 4600 synthetic plate-lets at 20 mg/ml and rFVIIa at 100 mg/kg(n = 5). Data are presented as percentageof no injection mean value ± SEM. *P <0.05, ***P < 0.001 versus saline; #P < 0.05versus rFVIIa. (D) Bleeding times compar-ing synthetic platelet administration (4600-GRGDS) to synthetic platelets stored in alyophilized state at room temperature for2 weeks (4600-GRGDS**RT) (n = 5). Dataare presented as percentage of no injectionmean value ± SEM. ***P < 0.001 versus PEG4600 alone. (E) Bleeding times when treat-ments were administered after injury. Bleed-
ing times represented as a percentage of no injection bleeding timevalues (255 ± 12 s). Synthetic platelets were administered at 20 mg/ml.Data are presented as mean ± SEM (n = 5). **P < 0.01 versus salineinjection; #P < 0.01 versus no injection. (F) SEM micrograph of clot ex-
cised from injured artery after synthetic platelet administration (4600-GRGDS). Arrow points to synthetic platelets intimately associated withclot and connecting fibrin mesh. The large (5 mm) spheres are blood cells.Scale bar, 1 mm.

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are important parameters influencing platelet activation and poten-tially thrombus formation in vivo (30). Thus, it is important forsafety to establish what impact the components of our syntheticplatelets have on coagulation. The cores of the synthetic plateletsare degradable polyester nanoparticles, and degradable polyesternanoparticles have been shown to induce clot formation (31). How-ever, PEGylation of the particles markedly reduces this effect. Duringnormal clot formation, PEGylated nanoparticles do not alter clottingbehavior (31). This lack of alteration in the coagulation cascade withPEGylated nanoparticles is attributed to the hydrophilic PEG corona,which reduces the surface adsorption of plasma proteins and lipids.The PEG corona also acts as a shield for nanoparticles, increasingtheir circulation time (32), which allows the particles to reach thesite of injury before being cleared.

Ease of administration, stability, nonimmunogenicity, and hemo-static efficacy without pathological thrombogenicity are required prop-erties of an intravenously administered hemostatic agent and werecritical design requirements of the synthetic platelets. Each of thematerials used in our synthesis—PLGA, PEG, and the RGD moiety—has been approved in other devices by the Food and Drug Admin-istration (33–35). The choice of PEGylated nanoparticles facilitatedthe synthetic platelets’ administration and residence in the circulation(36). By using a small synthetic peptide sequence (RGD) rather than aprotein, problems with immunogenicity and stability are minimized.Furthermore, the lower cost of smaller active peptide sequences makesthem more amenable to translation than large protein counterparts.

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The track record of the materials and design for safety, coupled withlack of platelet activation and rapid clearance exhibited here, suggeststhat the synthetic platelets will be safe. The significant improve-ment in halting bleeding relative to established treatments (rFVIIa)demonstrates that these synthetic platelets are a strong candidatefor translation into not only the clinic but also a field medic’s bag.These results provide compelling evidence that these synthetic plate-lets have the potential to stop bleeding and fundamentally changetrauma care.

MATERIALS AND METHODS

MaterialsPLGA 503H (Resomer 503H, 50:50 lactic to glycolic acid ratio, anda number-average molecular size of ∼25 kD) was from BoehringerIngelheim. H signifies PLGA terminated with a carboxylic acid group.Poly(e-carbobenzoxy-L-lysine) (molecular size, ∼1000 daltons) wasfrom Sigma. PEG (molecular size, ∼1500 and 4600 daltons) was fromAcros Organics and Sigma, respectively. RGD peptide sequences werefrom EMD Biosciences. Peptide sequences include RGD, RGDS,GRGDS, and GRADSP. Collagen I (rat tail) was from BD Bio-sciences. Deuterated dimethyl sulfoxide (DMSO) was from Cam-bridge Isotope Laboratories. Poly(vinyl alcohol) (88 mol% hydrolyzed)was purchased from Polysciences. CMFDA was from MolecularProbes. Recombinant factor VIIa was from Innovative Research.Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) was fromVector Laboratories. All other chemicals were American ChemicalSociety reagent grade, and other materials were used as receivedfrom Sigma.

MethodsPolymer synthesis and characterization. Detailed methods are

included in the Supplementary Material.Nanosphere fabrication and characterization. Detailed meth-

ods are included in the Supplementary Material.

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Fig. 4. Biodistribution of 4600-GRGDS synthetic platelets. (A) In vitroevaluation of cumulative C6 released from C6-loaded synthetic platelets
over 7 days. Data are presented as mean ± SD (n = 3). (B) Biodistributionof 4600-GRGDS synthetic platelets. No fluorescence was detected at 3-and 7-day time points after injection (n = 3). Data are expressed asmean ± SEM. (C) Biodistribution of 4600-GRGDS synthetic platelets (20mg/ml injection dose) immediately after femoral artery injury (n = 3).Because synthetic platelets are allowed to circulate for 5 min before in-jury, and the injury bleeds for ∼3 min, this time is compared to 10-minbiodistribution with no injury. Data are presented as mean ± SEM. (D)Biodistribution of 4600-GRGDS synthetic platelets 1 hour after femoralartery injury (n = 3). Data are presented as mean ± SEM. In all cases,synthetic platelets were administered at 20 mg/ml in 0.5 ml of Ringer’ssaline solution.
Fig. 5. C6 visualization and quantification of synthetic platelets. (A)Cross section of clot after femoral artery injury and injection of C6-
labeled synthetic platelets. Blue, DAPI-labeled nuclei of smooth muscleand endothelial cells; green, C6 from synthetic platelets within clot.Scale bar, 100 mm. (B) HPLC quantification of clot-associated C6 afterinjury (n = 5). Data are expressed as mean ± SEM. **P < 0.01.



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Fluorescent labeling of rat platelets. Detailed methods are in-cluded in the Supplementary Material.

Validation of in vitro assay. Detailed methods are included inthe Supplementary Material.

In vitro characterization of PLGA-PLL-PEG-RGD polymerNinety-six–well plates were coated with PLGA-PLL-PEG-RGDpolymer to examine interactions between our polymers and plate-lets. Briefly, 5 mg of polymer was dissolved in 1.0 ml of trifluor-oethanol (TFE). One hundred microliters of polymer solutionwas added to each well of a 96-well plate. By allowing the TFE toevaporate, we were able to effectively coat the well with our polymer(37). Wells were then washed three times with phosphate-bufferedsaline (PBS). After the PBS rinse, 100 ml of PRP with CMFDA fluo-rescently labeled platelets (5 × 108 per milliliter) was added to eachwell. This was followed by the addition of 10 ml of 100 mM ADP as aproaggregatory stimulus or PBS as a control. Immediately after ADPor PBS addition, the 96-well plate was agitated for 1 min on an or-bital shaker (Barnstead International) at 180 rpm (16). After a 3-minequilibration, plasma and nonaggregated or adhered platelets weregently extracted, and entire wells were imaged from the bottom witha 4× objective at 490-nm excitation and 525-nm emission (OlympusIX71 fluorescent microscope). Area of fluorescence was then quan-tified to elucidate the differences in platelet adherence or aggregation(38). Experiments were performed in triplicate.

Examination of synthetic platelets in vitroFor qualitative examination, a variation of the in vitro assay was used.Instead of coating the well with polymer, synthetic platelets in PBS(10 ml of 1500-RGDS synthetic platelets at 20 mg/ml) were added towells containing PRP (100 ml). This was followed by the addition of10 ml of 100 mM ADP or PBS. Wells with PBS were agitated for upto 10 min, whereas wells with ADP were agitated for only 1 min.After agitation, wells were examined for opaque aggregates of endog-enous or synthetic platelets. The wells were then photographed.

In vitro release of C6 from 4600-GRGDS nanospheresThe release of C6 from the 4600-GRGDS nanospheres was inves-tigated. Briefly, 5 mg of C6-labeled 4600-GRGDS nanospheres wasreconstituted with 1.0 ml of PBS in a 1.5-ml Eppendorf tubes. Mix-tures were then incubated at 37°C on a rotating shaker. At specifictime points (1 and 5 hours and 1, 3, and 7 days), the mixture wascentrifuged and the supernatant was collected. An equal volume ofPBS was then added to replace the withdrawn supernatant, and thenanospheres were resuspended and returned to the shaker. Extractedsupernatants were freeze-dried and reconstituted in 1.0 ml ofDMSO. Samples were then analyzed at 444-nm excitation and538-nm emission (SpectraMax M5 spectrophotometer, MolecularDevices) for C6 content. A C6 standard curve in DMSO wasestablished with sensitivity to 10 ng/ml.

Surgical preparation for femoral artery injuryMale Sprague-Dawley rats (about 180 to 200 g), obtained fromCharles River Laboratories, were used in accordance with proce-dures approved by Animal Care and Use Committees of Yale Uni-versity and follow the National Institutes of Health Guide for theCare and Use of Laboratory Animals. Rats were initially anesthe-tized with an intraperitoneal injection of ketamine-xylazine and

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placed in a supine position on a heat pad. Body temperature wasmaintained at 37°C. An incision was made from the abdomen tothe knee on the left hindlimb. After exposure of the femoral vein,polyethylene tubing (PE 10) was used as a catheter and inserted intothe femoral vein. Sutures secured the catheter, the cavity was closed,and the skin was sutured. The cannulated vein was later used for theintravenous administration of anesthetics and treatment groups.

After cannulation, a similar incision was made on the right hindlimb,and the femoral vessels were exposed. A portion of the femoral arterywas then isolated from the surrounding connective tissue by placing asmall piece of aluminum foil between the vessel and the underlyingtissue. Once the vessel was isolated, the cavity was irrigated (5 ml/min)with 0.9% NaCl irrigation fluid (Braun Medical) at 37°C. After a 10-minequilibration period, the synthetic platelets or control treatment wasadministered intravenously through the cannulated femoral vein over3 min.

In vivo analysis of hemostasisMale Sprague-Dawley rats (about 180 to 200 g), obtained fromCharles River Laboratories, were used in accordance with proce-dures approved by Animal Care and Use Committees of Yale Uni-versity. Treatment groups included a sham (injury alone), vehicle(saline) alone, rFVIIa (100 mg/kg), PLGA-PLL-PEG (unfunctionalizednanospheres), or PLGA-PLL-PEG-RGD nanospheres (synthetic plate-lets) at 20 mg/ml. All treatments (excluding sham group) were in0.5-ml vehicle solution. The surgeon performing the injury wasblinded to the treatment groups. Anesthetized rats were given anintravenous injection via femoral vein cannula, and the syntheticplatelets or control were allowed to circulate for 5 min. After circula-tion, we induced an injury to the femoral artery (18). Briefly, a trans-verse cut made with microscissors encompassing one-third of thevessel circumference resulted in the extravasation of blood. Time re-quired for bleeding to cease for at least 10 s was recorded as thebleeding time. Experiments included five rats per group.

Administration of synthetic platelets after injuryA variation to the described artery injury model included the intra-venous administration of synthetic platelets immediately after injury.For postinjury injections, treatments [saline, PEG 4600 (20 mg/ml),or 4600-GRGDS (20 mg/ml)] were administered over a 20-s intervalimmediately after an injury to the femoral artery.

BiodistributionRGD nanospheres were fabricated as described in the SupplementaryMaterial, with the addition of C6 to the dichloromethane (0.5%, w/v).The biodistribution of the RGD nanospheres was examined after in-travenous injection. A 0.5-ml injection (20 mg/ml) of C6-labeled4600-GRGDS nanospheres was administered via tail vein injection.Biodistribution was examined at 5 and 10 min, 1 hour, and 1, 3, and7 days after injection. At each time point, animals were killed andblood, lungs, liver, kidneys, and spleen were collected. Blood wascentrifuged (180g for 10 min), and 1.0 ml of plasma was extracted.Plasma and organs were then freeze-dried for 3 days, and dry organmass was then determined.

To determine organ C6 content, 50 mg of dry organ was ho-mogenized (Precellys 24 Tissue Homogenizer, Bertin Technologies)in 1.0 ml of DMSO. The homogenates were covered and incubatedat 37°C for 6 hours to ensure nanosphere or C6 dissolution. Homog-




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enates were then centrifuged, and 200 ml of samples was extracted andanalyzed for C6 content. Samples were analyzed at 444-nm excitationand 538-nm emission (SpectraMax M5 spectrophotometer) for C6content. A C6 standard curve in DMSO was established with sensitivityto tissue (0.2 ng/mg) and plasma (3.3 ng/ml). Organs without C6 wereanalyzed at the same wavelengths to establish background fluorescence.Experiments were performed in triplicate for each time point.

Biodistribution of C6-labeled 4600-GRGDS nanospheres was alsoexamined after the thrombogenic injury to the femoral artery. Nano-spheres were injected intravenously through the femoral vein cannula.Organs were extracted 1 hour after or immediately after bleeding hadstopped. Tissue was processed and C6 was quantified as described.Experiments were performed in triplicate at each time point.

Further analysis included the quantification of C6 associatedwith the clot after injury. Two groups were analyzed in the femoralartery injury: PEG 4600 and 4600-GRGDS nanospheres. Five ratswere used in each group. After injury and the cessation of bleeding,the clot was excised and immersed in acetonitrile overnight. Sampleswere centrifuged and C6 content was determined with reversed-phase HPLC (Shimadzu Scientific Instruments) with a fluorescencedetector and a Nova-Pak C18, 4 mm, 3.9 mm × 150 mm column(Waters). Mobile phase was prepared as described by Eley et al. (21)and consisted of acetonitrile–acetic acid (8%) (80:20, v/v) with a flowrate of 1.0 ml/min. A standard curve for C6 (excitation, 450 nm;emission, 490 nm; retention time, ∼3.1 min) was established in ace-tonitrile with a sensitivity limit of 0.25 ng/ml.

Clot visualizationFor visualization of C6-labeled nanospheres associated within clotsafter thrombogenic injury, injured vessel segments containing clotswere excised and fixed in 10% formalin overnight. After fixation,clots were either mounted for visualization via SEM or embeddedin optimal cutting temperature compound. Embedded clots were thencryosectioned to 15-mm cross sections and mounted with Vectashieldwith DAPI. Cross sections were visualized with a Zeiss Axiovert 200microscope (Carl Zeiss).

Statistical analysisData were analyzed with a one-way analysis of variance followed bythe Student-Newman-Keuls test for determining differences betweengroups. Differences were accepted as statistically significant with P <0.05. Student’s t test was used for clot-associated C6 comparison andin vitro assay validation with collagen.

SUPPLEMENTARY MATERIALwww.sciencetranslationalmedicine.org/cgi/content/full/1/11/11ra22/DC1Materials and MethodsFig. S1. Polymer synthesis and characterization.Fig. S2. Polymer characterization and in vitro assay with collagen.Fig. S3. In vitro analysis of PRP with synthetic platelets.Fig. S4. In vivo analysis of bleeding times for different concentrations of synthetic platelets.References

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39. Funding: Coulter Foundation (Early Career Award to E.B.L.) and Richard and Gail Siegal (J.P.B.),NIH Neuroengineering training grant T90-DK070068 (J.P.B. and R.R.), NIH Medical ScientistTraining Program training grant 5T32GM07025 (C.A.W.), Richard and Gail Siegal and CarolSirot (C.A.W. and R.R.), and NIH grants HL41026 and HL56786 (S.S.S.).Author contributions: J.P.B. designed and performed experiments, analyzed results, andwrote the paper. C.A.W., R.R., and N.T.F. assisted in performing experiments and editedthe paper. S.S.S. provided expertise and training in vascular injury techniques and analysisand edited the paper. E.B.L. designed the approach and experiments, provided funding,assisted in the analysis of results, and edited the paper.Competing interests: J.P.B. and E.B.L. are inventors on a filed patent regarding this work.

Submitted 18 September 2009Accepted 18 November 2009Published 16 December 200910.1126/scitranslmed.3000397

Citation: J. P. Bertram, C. A. Williams, R. Robinson, S. S. Segal, N. T. Flynn, E. B. Lavik,Intravenous hemostat: Nanotechnology to halt bleeding. Sci. Transl. Med. 1, 11ra22 (2009).

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