Research ArticleBlood clotting

Intravenous Hemostat: Nanotechnology to Halt Bleeding

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Science Translational Medicine  16 Dec 2009:
Vol. 1, Issue 11, pp. 11ra22
DOI: 10.1126/scitranslmed.3000397

Abstract

Blood loss is the major cause of death in both civilian and battlefield traumas. Methods to staunch bleeding include pressure dressings and absorbent materials. For example, QuikClot effectively halts bleeding by absorbing large quantities of fluid and concentrating platelets to augment clotting, but these treatments are limited 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 developed synthetic platelets based on Arg-Gly-Asp functionalized nanoparticles, which halve bleeding time after intravenous administration in a rat model of major trauma. The effects of these synthetic platelets surpass other treatments, including recombinant factor VIIa, which is used clinically for uncontrolled bleeding. Synthetic platelets were cleared within 24 hours at a dose of 20 mg/ml, and no complications were seen out to 7 days after infusion, the longest time point studied. These synthetic platelets may be useful for early intervention in trauma and demonstrate the role that nanotechnology can have in addressing unmet medical needs.

Introduction

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

Alternatives to topical dressings include allogeneic platelet transfusions, 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 allogeneic platelets can cause graft-versus-host disease, alloimmunization, and transfusion-associated lung injuries (6). Recombinant factors, including activated factor VII (NovoSeven), can augment hemostasis, but immunogenic and thromboembolic complications are unavoidable risks (7). Nonetheless, NovoSeven is used in trauma and surgical situations where bleeding cannot otherwise be controlled (8). Nonplatelet alternative 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 toxicity, thrombosis, and limited efficacy restrict the clinical utility of these products (5).

Polymer engineering has opened up new possibilities for controlling bleeding. For example, a self-assembling peptide rapidly halts bleeding in a number of injury models when applied topically (10), functionalized liposomes (11) and micrometer-sized sheets (12) interact with activated platelets in vitro, and a block copolymer of hemoglobin and fibrinogen acts as an oxygen carrier and maintains normal bleeding times at high concentrations where typical oxygen carriers lead to hemodilution and long bleeding times (13). We have taken a different approach and have designed synthetic platelets based on functionalized nanoparticles that bind to activated platelets to augment clotting in a safe, localized manner. This system leverages existing biological processes by providing a nanostructure that binds to activated platelets and enhances their rate of aggregation, which stops bleeding. They are made from polymers that are already used in the medical device and pharmaceutical industries, and a strong track record of safety makes them well suited for clinical translation.

We tested the synthetic platelets in vitro to optimize their binding efficiency to activated platelets and in vivo in a rat major femoral artery injury model to determine their efficacy in promoting hemostasis and their biodistribution. The synthetic platelets halved bleeding time after intravenous administration in the femoral artery injury model and performed significantly better than activated recombinant factor VIIa (rFVIIa), which is now being used in the clinic for uncontrolled bleeding.

Results

Design and synthesis of synthetic platelets

Our synthetic platelets consist of poly(lactic-co-glycolic acid)-poly-l-lysine (PLGA-PLL) block copolymer cores to which we conjugated polyethylene glycol (PEG) arms terminated with RGD functionalities (Fig. 1A). 1H nuclear magnetic resonance (1H NMR) demonstrated successful conjugation of PEG to PLGA-PLL (fig. S1C). Nanospheres were fabricated with a single emulsion solvent evaporation technique (14), which resulted in core diameters of ~170 nm by scanning electron microscopy (SEM) (Fig. 1, B and D). After fabrication, nanospheres were analyzed with 1H-NMR to confirm that the conjugated PEG was present. The subsequent conjugation of RGD to PLGA-PLL-PEG nanospheres was then confirmed with amino acid analysis (Fig. 1C). Amino acid analysis was also used for ascertaining the RGD conjugation for the PLGA-PLL-PEG-RGD polymer used for in vitro characterization (fig. S2A). RGD conjugation efficiency was independent of both the PEG molecular weight and peptide sequence [that is, RGD versus GRGDS (Gly-Arg-Gly-Asp-Ser)] (Fig. 1C and fig. S2A). Synthetic platelets have an average RGD peptide content of 3.3 ± 1.1 μmol/g (mean ± SD) (Fig. 1C), which corresponds to a conjugation efficiency of 16.2 ± 5.9% (mean ± SD) (~600 RGD moieties per synthetic platelet). The coupling agent used to attach the peptide, carbonyldiimidazole, is hydrolyzable in water and thus can be quenched during the aqueous peptide coupling, leading to the modest efficiency. One could augment the efficiency by repeated activation and coupling reactions or the use of an alternative agent, but the efficiencies here were effective in our subsequent studies. Although cores are ~170 nm in diameter for all of the preparations (Fig. 1D), the hydrodynamic diameter of the spheres, determined by dynamic light scattering (DLS), increased with increasing PEG molecular size (Fig. 1D). The SEM and DLS results suggest that a surface enrichment of PEG arms exists, and in a hydrated environment, PEG arms extend to create a PEG corona on the nanosphere surface (15). On the basis of these results, we concluded that, under hydrated conditions, the surface proximity of the conjugated RGD peptide varies as a function of PEG molecular size.

Fig. 1

Design, synthesis, and characterization of synthetic platelets. (A) Schematic of synthetic platelet composed of PLGA-PLL core with PEG arms terminated with the RGD moiety. (B) SEM micrograph of synthetic platelets. Scale bar, 1 μm. (C) Lysine and peptide concentrations of synthetic platelets as determined by amino acid analysis. Conjugation efficiency was defined as the peptide-to-lysine ratio multiplied by 100. (D) Diameter of PLGA-PLL core and PLGA-PLL-PEG nanospheres as determined by SEM and DLS. SEM diameter based on n = 80. Data are expressed as mean ± SD.

In vitro validation and optimization of synthetic platelets

The surface proximity of the RGD functionality affects interactions with activated platelets (16). To determine the optimal PEG arm length, 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 the role of the components of the synthetic platelets on platelet adhesion and aggregation. Other traditional in vitro assays, such as the aggregometer, could not be used with our synthetic platelets because the nanoparticles led to substantial scattering in the system, which produced noise that masked the results. The in vitro platelet aggregation 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-chloromethylfluorescein diacetate (CMFDA)–labeled platelets and the aggregation stimulant adenosine diphosphate (ADP), we examined the effects of the PEG length and RGD functionality on platelet aggregation and adhesion (Fig. 2, B and C). Platelet aggregation increased with increased PEG length, with PEG 4600 compounds leading to greater adhesion and aggregation than PEG 1500 compounds for all RGD variants studied. This is consistent with previous observations (17).

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 μm. (D) Quantification of platelet adhesion. Area of fluorescence represents area of platelet aggregation (n = 3). Data are expressed as mean ± SEM. *P < 0.05 versus PEG 4600 alone.

We found that activated platelet adhesion increased as we added flanking amino acids around the RGD sequence, with the GRGDS formulations leading to the greatest adhesion (Fig. 2D). Control experiments verified that PEG alone, and the conservative substitute peptide 4600-GRADSP (Gly-Arg-Ala-Asp-Ser-Pro), yielded the same values as the PLGA-only group, inducing only minimal adhesion and aggregation.

For clinical utility, it is critical that the activated platelets bind specifically to the synthetic platelets, as nonspecific binding or induced platelet activation could lead to adverse concomitant thrombotic events, including embolism and stroke. We found that nonactivated platelets did not bind to any of the PLGA-PLL-PEG-RGD polymers tested (fig. S3C). Furthermore, platelets did not activate without the addition of ADP. The polymers did not induce platelet adhesion, even with agitation, except when ADP was added (fig. S3D). As a simple test of our system, we added synthetic platelets to platelet-rich plasma (PRP) (fig. S3). Even with prolonged agitation (10 min), synthetic platelets did not induce endogenous platelet aggregation. This is in contrast to PRP added to the collagen-coated control wells. Even in the absence of ADP, endogenous platelets aggregated and adhered to the collagen (fig. S2B). We only saw adhesion and coagulation with the synthetic platelets when a proaggregatory stimulus (ADP) was added. The materials used to fabricate synthetic platelets did not activate endogenous platelets, and nonactivated platelets did not bind, which suggests that the materials are unlikely to induce activation or nonspecific platelet binding on their own.

In vivo: Femoral artery injury model

The goal of this work was to develop a safe synthetic platelet with hemostatic efficacy in vivo. We tested our synthetic platelets in a rat model of a major femoral artery injury (Fig. 3A) (18). The injury leads to blood spurting in a continuous stream from the injury site (Fig. 3A) and is easily visualized. We investigated three doses (10, 20, and 40 mg/ml) of the synthetic platelets (fig. S4A). We found that 10 mg/ml had no effect on bleeding time and 40 mg/ml produced cardiopulmonary complications in some animals, presenting as elevated heart rate and gasping. Therefore, we focused on determining the optimal particle characteristics with intravenous administration at 20 mg/ml.

Fig. 3

In vivo performance of synthetic platelets: hemostatic efficacy. (A) Femoral artery injury model used for all studies. Arrow points to injury site and the blood spurting from the injured vessel. (B) Bleeding times after intravenous administration of PEG 1500 synthetic platelets at 20 mg/ml (n = 5). Data are presented as percentage of no injection mean value ± SEM. **P < 0.01, ***P < 0.001 versus PEG 1500 alone. (C) Bleeding times after intravenous administration of PEG 4600 synthetic platelets at 20 mg/ml and rFVIIa at 100 μg/kg (n = 5). Data are presented as percentage of no injection mean value ± SEM. *P < 0.05, ***P < 0.001 versus saline; #P < 0.05 versus rFVIIa. (D) Bleeding times comparing synthetic platelet administration (4600-GRGDS) to synthetic platelets stored in a lyophilized state at room temperature for 2 weeks (4600-GRGDS**RT) (n = 5). Data are presented as percentage of no injection mean value ± SEM. ***P < 0.001 versus PEG 4600 alone. (E) Bleeding times when treatments were administered after injury. Bleeding times represented as a percentage of no injection bleeding time values (255 ± 12 s). Synthetic platelets were administered at 20 mg/ml. Data are presented as mean ± SEM (n = 5). **P < 0.01 versus saline injection; #P < 0.01 versus no injection. (F) SEM micrograph of clot excised from injured artery after synthetic platelet administration (4600-GRGDS). Arrow points to synthetic platelets intimately associated with clot and connecting fibrin mesh. The large (5 μm) spheres are blood cells. Scale bar, 1 μm.

The PEG 1500 nanospheres with the GRGDS peptide led to the greatest reduction in bleeding time (Fig. 3B), paralleling the in vitro findings. Also paralleling the in vitro findings was the fact that there was a greater reduction in bleeding time with the RGD-functionalized PEG 4600 spheres relative to their PEG 1500 counterparts (Fig. 3C); 4600-GRGDS nanospheres led to significantly shorter bleeding times relative to the 1500-GRGDS nanospheres (n = 5, P < 0.05).

The injury alone with no injection gave a baseline bleeding time of 240 ± 15 s (mean ± SEM, n = 5). Injection of the 4600-GRGDS synthetic platelets halved the bleeding time [131 ± 11 s (mean ± SEM, n = 5)] (Fig. 3C). To validate our synthetic platelets as a hemostatic agent, we compared bleeding times after synthetic platelet administration to bleeding times after the injection of rFVIIa. Recombinant factor VIIa has proven clinically useful in the treatment of surgery- and trauma-associated bleeding (19) and is the current standard of care for uncontrolled bleeding (8). Although a bolus injection of rFVIIa (100 μg/kg; the recommended dose is 90 to 100 μg/kg) significantly reduced bleeding times [187 ± 16 s (mean ± SEM, n = 5)], 4600-GRGDS synthetic platelets reduced bleeding time ~25% more than did rFVIIa (Fig. 3C). Furthermore, 4600-GRGDS synthetic platelets stored in a lyophilized state at room temperature for 2 weeks maintained their hemostatic properties with no reduction in efficacy (Fig. 3D).

In these studies, the agents were administered intravenously before the injury. We chose this approach initially because the bleeding times in this injury without treatment (240 ± 15 s) are close to the time it took to inject the synthetic platelets or controls (180 s). However, for the synthetic platelets to be clinically effective, they need to work when administered after an injury. Therefore, we tested the synthetic platelets in the same rat femoral artery injury model, but we administered the synthetic platelets (20 mg/ml, 0.5 ml injected) or one of the controls over a period of 20 s. Administration of saline alone (0.5 ml) or the unfunctionalized PEG 4600 nanospheres (20 mg/ml, 0.5 ml administered) increased bleeding time relative to the no injection control group. This is likely the result of systemic blood dilution due to the rapid bolus administration of fluid, an important concern when administering fluids and clotting factors intravenously after injury (20). Nonetheless, even with this diluting effect, the 4600-GRGDS synthetic platelets significantly reduced bleeding time by ~23% relative to no injection and by 31% relative to saline injection (Fig. 3E).

Participation of synthetic platelets in the clot

We next determined whether synthetic platelets were present in the clots. We excised the injured vessel segments and performed SEM. Synthetic platelets were intimately associated with the fibrin mesh (Fig. 3F) and distributed throughout the clot on the luminal, interior surface of the femoral artery (fig. S4B).

Biodistribution of synthetic platelets

For synthetic platelets to be therapeutically viable, they must be effectively cleared when not participating in clot formation at the site of injury. To test this parameter, we labeled synthetic platelets (4600-GRGDS) by encapsulating coumarin 6 (C6), a fluorochrome commonly used for biodistribution studies (21). We examined the biodistribution and clearance of C6-labeled synthetic platelets up to 7 days after intravenous injection with fluorescence spectroscopy. Less than 0.5% of the C6 fluorochrome label was released from the nanospheres after 24 hours, and ~1.5% was released after 7 days (Fig. 4A), indicating that virtually all of the C6 fluorescence was associated with synthetic platelets. A characteristic distribution of synthetic platelets was observed consistent with intravenous nanosphere administration (Fig. 4B) (22). Within 5 min of injection, 68.3% of the injected particles were found in the liver, 16.1% were in the blood, and minimal accumulations were seen in the kidneys, lungs, and spleen (<3.6%, 2.2%, and 5.2%, respectively). The sensitivity of our biodistribution analysis is 0.2 ng of C6 per milligram of tissue and 3.3 ng of C6 per milliliter of plasma. At 3 and 7 days after injection, no C6 was detected, suggesting that all synthetic 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 immediately after a femoral artery injury and 1 hour after injury (Fig. 4, C and D). For both time points, tissue distribution was similar for the injured and uninjured animals (equivalent to 10-min and 1-hour time points in uninjured animals). This suggests that even after a severe injury and with circulating activated platelets, the unbound synthetic platelets are effectively cleared within 24 hours.

Fig. 4

Biodistribution of 4600-GRGDS synthetic platelets. (A) In vitro evaluation of cumulative C6 released from C6-loaded synthetic platelets over 7 days. Data are presented as mean ± SD (n = 3). (B) Biodistribution of 4600-GRGDS synthetic platelets. No fluorescence was detected at 3- and 7-day time points after injection (n = 3). Data are expressed as mean ± SEM. (C) Biodistribution of 4600-GRGDS synthetic platelets (20 mg/ml injection dose) immediately after femoral artery injury (n = 3). Because synthetic platelets are allowed to circulate for 5 min before injury, and the injury bleeds for ~3 min, this time is compared to 10-min biodistribution with no injury. Data are presented as mean ± SEM. (D) Biodistribution of 4600-GRGDS synthetic platelets 1 hour after femoral artery 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’s saline solution.

Quantification of synthetic platelets within the clot

We were able to both visualize and quantify the C6-labeled synthetic platelets in the clot after injury. Cross sections show that the synthetic platelets (green) were distributed throughout the clot (Fig. 5A), consistent with the SEM findings (Fig. 3F and fig. S4B). We quantified the amount of synthetic platelets within the clots with high-performance liquid chromatography (HPLC) and compared these findings to the distribution of C6-labeled PEG 4600 nanospheres with no functionalities. Although PEG 4600 nanospheres were entrapped in the clots after injury, clots from animals that received synthetic platelets had more than double the number of particles relative to PEG 4600 nanospheres [3.9 ± 0.4 and 1.8 ± 0.2 ng of C6, respectively (mean ± SEM, n = 5)] (Fig. 5B). However, PEG 4600 nanospheres had no significant effect on bleeding time (Fig. 3C), suggesting that the synthetic platelets interact preferentially with activated platelets as a result of RGD functionalization and thereby actively halt bleeding (as opposed to indirectly inducing platelet flocculation).

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 muscle and endothelial cells; green, C6 from synthetic platelets within clot. Scale bar, 100 μm. (B) HPLC quantification of clot-associated C6 after injury (n = 5). Data are expressed as mean ± SEM. **P < 0.01.

Discussion

The ultimate goal of this work was to design and optimize a synthetic platelet that is stable at room temperature, safely administered intravenously, and able to halt bleeding. Through our in vitro studies, we found that nanospheres functionalized with PEG arms with a molecular size of 4600 daltons and the GRGDS moiety led to the greatest platelet adhesion and aggregation. These trends were repeated in vivo in the rat femoral artery injury model.

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

Activated platelets bind to RGD moieties through specific ligand-receptor interactions between RGD and surface receptors expressed on activated platelets (25). More specifically, RGD interacts with the activated platelet receptors glycoprotein IIb-IIIa and integrin αvβ3 (16). We hypothesize that the RGD moiety, and the GRGDS moiety in particular, interacts with the activated platelets through these receptors to promote adhesion between the polymers and the platelets. Flanking amino acids to the RGD motif produce a more active binding conformation (26). This increased bioactivity in turn influences integrin affinity for the RGD moiety (27) and increases cellular attachment (17). These features explain why the GRGDS peptide demonstrates the greatest binding and adhesion of the activated platelets. Longer, more specific sequences have higher affinities for activated platelets than does the GRGDS peptide. However, the length of the peptide can have marked effects on its temperature stability (28) as well as production costs. Increasing the number of amino acid residues increases the time for synthesis, decreases yield, and increases the difficulty of purification (29). At the laboratory scale, the addition of two amino acids, from a 5–amino acid sequence to a 7–amino acid sequence, can increase the cost by an order of magnitude.

Our results show that the RGD-functionalized polymer interacts specifically with activated platelets. In the in vitro studies, the PEG alone and conservative substitute peptide, 4600-GRADSP, groups behaved like the PLGA-only group, inducing only minimal adhesion and aggregation. Thus, neither the polymer alone nor the polymer with a substitute peptide induces adhesion and aggregation, whereas adhesion and aggregation are seen with all of the specific RGD variants. This indicates that the activated platelets’ affinity for the GRGDS moiety increases platelet adhesion to the polymer. This implies that the binding of activated platelets to the functionalized PLGA-PLL-PEG is specific. Similar findings have been reported with cell attachment assays for other cell types (24).

Nonfunctionalized nanoparticles can activate platelets and alter the coagulation cascade (30). It is hypothesized that the surface charge on the particles, their aggregation in water, and their shape are important parameters influencing platelet activation and potentially thrombus formation in vivo (30). Thus, it is important for safety to establish what impact the components of our synthetic platelets have on coagulation. The cores of the synthetic platelets are degradable polyester nanoparticles, and degradable polyester nanoparticles have been shown to induce clot formation (31). However, PEGylation of the particles markedly reduces this effect. During normal clot formation, PEGylated nanoparticles do not alter clotting behavior (31). This lack of alteration in the coagulation cascade with PEGylated 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, increasing their circulation time (32), which allows the particles to reach the site of injury before being cleared.

Ease of administration, stability, nonimmunogenicity, and hemostatic efficacy without pathological thrombogenicity are required properties of an intravenously administered hemostatic agent and were critical design requirements of the synthetic platelets. Each of the materials used in our synthesis—PLGA, PEG, and the RGD moiety—has been approved in other devices by the Food and Drug Administration (3335). The choice of PEGylated nanoparticles facilitated the synthetic platelets’ administration and residence in the circulation (36). By using a small synthetic peptide sequence (RGD) rather than a protein, problems with immunogenicity and stability are minimized. Furthermore, the lower cost of smaller active peptide sequences makes them more amenable to translation than large protein counterparts. The track record of the materials and design for safety, coupled with lack of platelet activation and rapid clearance exhibited here, suggests that the synthetic platelets will be safe. The significant improvement in halting bleeding relative to established treatments (rFVIIa) demonstrates that these synthetic platelets are a strong candidate for translation into not only the clinic but also a field medic’s bag. These results provide compelling evidence that these synthetic platelets have the potential to stop bleeding and fundamentally change trauma care.

Materials and Methods

Materials

PLGA 503H (Resomer 503H, 50:50 lactic to glycolic acid ratio, and a number-average molecular size of ~25 kD) was from Boehringer Ingelheim. H signifies PLGA terminated with a carboxylic acid group. Poly(ɛ-carbobenzoxy-l-lysine) (molecular size, ~1000 daltons) was from Sigma. PEG (molecular size, ~1500 and 4600 daltons) was from Acros Organics and Sigma, respectively. RGD peptide sequences were from EMD Biosciences. Peptide sequences include RGD, RGDS, GRGDS, and GRADSP. Collagen I (rat tail) was from BD Biosciences. Deuterated dimethyl sulfoxide (DMSO) was from Cambridge Isotope Laboratories. Poly(vinyl alcohol) (88 mol% hydrolyzed) was purchased from Polysciences. CMFDA was from Molecular Probes. Recombinant factor VIIa was from Innovative Research. Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) was from Vector Laboratories. All other chemicals were American Chemical Society reagent grade, and other materials were used as received from Sigma.

Methods

Polymer synthesis and characterization. Detailed methods are included in the Supplementary Material.

Nanosphere fabrication and characterization. Detailed methods are included in the Supplementary Material.

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

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

In vitro characterization of PLGA-PLL-PEG-RGD polymer

Ninety-six–well plates were coated with PLGA-PLL-PEG-RGD polymer to examine interactions between our polymers and platelets. Briefly, 5 mg of polymer was dissolved in 1.0 ml of trifluoroethanol (TFE). One hundred microliters of polymer solution was added to each well of a 96-well plate. By allowing the TFE to evaporate, we were able to effectively coat the well with our polymer (37). Wells were then washed three times with phosphate-buffered saline (PBS). After the PBS rinse, 100 μl of PRP with CMFDA fluorescently labeled platelets (5 × 108 per milliliter) was added to each well. This was followed by the addition of 10 μl of 100 μM ADP as a proaggregatory stimulus or PBS as a control. Immediately after ADP or PBS addition, the 96-well plate was agitated for 1 min on an orbital shaker (Barnstead International) at 180 rpm (16). After a 3-min equilibration, plasma and nonaggregated or adhered platelets were gently extracted, and entire wells were imaged from the bottom with a 4× objective at 490-nm excitation and 525-nm emission (Olympus IX71 fluorescent microscope). Area of fluorescence was then quantified to elucidate the differences in platelet adherence or aggregation (38). Experiments were performed in triplicate.

Examination of synthetic platelets in vitro

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

In vitro release of C6 from 4600-GRGDS nanospheres

The release of C6 from the 4600-GRGDS nanospheres was investigated. Briefly, 5 mg of C6-labeled 4600-GRGDS nanospheres was reconstituted with 1.0 ml of PBS in a 1.5-ml Eppendorf tubes. Mixtures were then incubated at 37°C on a rotating shaker. At specific time points (1 and 5 hours and 1, 3, and 7 days), the mixture was centrifuged and the supernatant was collected. An equal volume of PBS was then added to replace the withdrawn supernatant, and the nanospheres were resuspended and returned to the shaker. Extracted supernatants were freeze-dried and reconstituted in 1.0 ml of DMSO. Samples were then analyzed at 444-nm excitation and 538-nm emission (SpectraMax M5 spectrophotometer, Molecular Devices) for C6 content. A C6 standard curve in DMSO was established with sensitivity to 10 ng/ml.

Surgical preparation for femoral artery injury

Male Sprague-Dawley rats (about 180 to 200 g), obtained from Charles River Laboratories, were used in accordance with procedures approved by Animal Care and Use Committees of Yale University and follow the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were initially anesthetized with an intraperitoneal injection of ketamine-xylazine and placed in a supine position on a heat pad. Body temperature was maintained at 37°C. An incision was made from the abdomen to the knee on the left hindlimb. After exposure of the femoral vein, polyethylene tubing (PE 10) was used as a catheter and inserted into the femoral vein. Sutures secured the catheter, the cavity was closed, and the skin was sutured. The cannulated vein was later used for the intravenous 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 artery was then isolated from the surrounding connective tissue by placing a small piece of aluminum foil between the vessel and the underlying tissue. 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-min equilibration period, the synthetic platelets or control treatment was administered intravenously through the cannulated femoral vein over 3 min.

In vivo analysis of hemostasis

Male Sprague-Dawley rats (about 180 to 200 g), obtained from Charles River Laboratories, were used in accordance with procedures approved by Animal Care and Use Committees of Yale University. Treatment groups included a sham (injury alone), vehicle (saline) alone, rFVIIa (100 μg/kg), PLGA-PLL-PEG (unfunctionalized nanospheres), or PLGA-PLL-PEG-RGD nanospheres (synthetic platelets) at 20 mg/ml. All treatments (excluding sham group) were in 0.5-ml vehicle solution. The surgeon performing the injury was blinded to the treatment groups. Anesthetized rats were given an intravenous injection via femoral vein cannula, and the synthetic platelets or control were allowed to circulate for 5 min. After circulation, we induced an injury to the femoral artery (18). Briefly, a transverse cut made with microscissors encompassing one-third of the vessel circumference resulted in the extravasation of blood. Time required for bleeding to cease for at least 10 s was recorded as the bleeding time. Experiments included five rats per group.

Administration of synthetic platelets after injury

A variation to the described artery injury model included the intravenous 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 interval immediately after an injury to the femoral artery.

Biodistribution

RGD nanospheres were fabricated as described in the Supplementary Material, with the addition of C6 to the dichloromethane (0.5%, w/v). The biodistribution of the RGD nanospheres was examined after intravenous injection. A 0.5-ml injection (20 mg/ml) of C6-labeled 4600-GRGDS nanospheres was administered via tail vein injection. Biodistribution was examined at 5 and 10 min, 1 hour, and 1, 3, and 7 days after injection. At each time point, animals were killed and blood, lungs, liver, kidneys, and spleen were collected. Blood was centrifuged (180g for 10 min), and 1.0 ml of plasma was extracted. Plasma and organs were then freeze-dried for 3 days, and dry organ mass was then determined.

To determine organ C6 content, 50 mg of dry organ was homogenized (Precellys 24 Tissue Homogenizer, Bertin Technologies) in 1.0 ml of DMSO. The homogenates were covered and incubated at 37°C for 6 hours to ensure nanosphere or C6 dissolution. Homogenates were then centrifuged, and 200 μl of samples was extracted and analyzed for C6 content. Samples were analyzed at 444-nm excitation and 538-nm emission (SpectraMax M5 spectrophotometer) for C6 content. A C6 standard curve in DMSO was established with sensitivity to tissue (0.2 ng/mg) and plasma (3.3 ng/ml). Organs without C6 were analyzed 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 also examined after the thrombogenic injury to the femoral artery. Nanospheres were injected intravenously through the femoral vein cannula. Organs were extracted 1 hour after or immediately after bleeding had stopped. 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 associated with the clot after injury. Two groups were analyzed in the femoral artery injury: PEG 4600 and 4600-GRGDS nanospheres. Five rats were used in each group. After injury and the cessation of bleeding, the clot was excised and immersed in acetonitrile overnight. Samples were centrifuged and C6 content was determined with reversed-phase HPLC (Shimadzu Scientific Instruments) with a fluorescence detector and a Nova-Pak C18, 4 μm, 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 flow rate of 1.0 ml/min. A standard curve for C6 (excitation, 450 nm; emission, 490 nm; retention time, ~3.1 min) was established in acetonitrile with a sensitivity limit of 0.25 ng/ml.

Clot visualization

For visualization of C6-labeled nanospheres associated within clots after thrombogenic injury, injured vessel segments containing clots were excised and fixed in 10% formalin overnight. After fixation, clots were either mounted for visualization via SEM or embedded in optimal cutting temperature compound. Embedded clots were then cryosectioned to 15-μm cross sections and mounted with Vectashield with DAPI. Cross sections were visualized with a Zeiss Axiovert 200 microscope (Carl Zeiss).

Statistical analysis

Data were analyzed with a one-way analysis of variance followed by the Student-Newman-Keuls test for determining differences between groups. Differences were accepted as statistically significant with P < 0.05. Student’s t test was used for clot-associated C6 comparison and in vitro assay validation with collagen.

Supplementary Material

www.sciencetranslationalmedicine.org/cgi/content/full/1/11/11ra22/DC1

Materials and Methods

Fig. 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

Footnotes

References and Notes

  1. 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 Scientist Training Program training grant 5T32GM07025 (C.A.W.), Richard and Gail Siegal and Carol Sirot (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, and wrote the paper. C.A.W., R.R., and N.T.F. assisted in performing experiments and edited the paper. S.S.S. provided expertise and training in vascular injury techniques and analysis and 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.
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