Facile Assembly of Cost-Effective and Locally Applicable or Injectable

Facile Assembly of Cost-Effective and Locally Applicable or Injectable Nanohemostats for Hemorrhage Control Juan Cheng,†,‡ Shibin Feng,§ Songling Han,† Xiangjun Zhang,† Yidan Chen,† Xing Zhou,†,‡ Ruibing Wang,∥ Xiaohui Li,‡ Houyuan Hu,*,§ and Jianxiang Zhang*,† †

Department of Pharmaceutics, College of Pharmacy, ‡Institute of Materia Medica, College of Pharmacy, and §Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China ∥ State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau, China S Supporting Information *

ABSTRACT: Currently, there is still unmet demand for effective and safe hemostats to control abnormal bleeding in different conditions. With the aim to develop affordable, safe, effective, easily stored, and low-cost hemostats, we developed a series of positively charged nanoparticles by a facile one-pot assembly approach. In this strategy, nanoparticles were formed by cholicacid-mediated self-assembly of polyethylenimine (PEI). Regardless of different structures of cholic acids and PEIs, well-defined nanoparticles could be successfully formed. The assembly process was dominated by multiple interactions between cholic acid and PEI, including electrostatic, hydrogen bonding, and hydrophobic forces. In vitro studies showed that assembled nanoparticles effectively induced aggregation and activation of platelets. Local application of aqueous solution containing nanoparticles assembled by different cholic acids and PEIs significantly reduced bleeding times in different rodent models including tail transection in mice as well as liver bleeding and femoral artery bleeding in rats or rabbits. Moreover, intravenous (i.v.) injection of this type of positively charged nanoparticles notably prevented bleeding in the femoral artery in rats by targeting the injured site via opsonization of nanoparticles with fibrinogen. By contrast, a control negatively charged nanoparticle showed no hemostatic activity after i.v. delivery. Also, preliminary evaluations in rats revealed a good safety profile after i.v. administration of assembled nanoparticles at a dose 4-fold higher than that used for hemostasis. These results demonstrated that cholic acid/PEI-assembled positive nanoparticles may function as cost-effective and locally applicable or injectable nanohemostats for hemorrhage control in the civilian setting and on the battlefield. KEYWORDS: self-assembly, nanoparticle, polyethylenimine, cholic acid, nanohemostat, hemostasis

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based on different hemostatic materials have been utilized to facilitate hemostasis. To this end, biologically derived materials and synthetic materials have been developed,1 such as coagulation proteins and polymeric activators or aggregators.9,10 In addition, intravenously injectable hemostatic agents like platelets and platelet substitutes have been investigated.11 Nevertheless, various limitations exist for approaches based on these hemostatic materials for controlling intraoperative and perioperative bleeding as well as uncontrolled bleeding on the battlefield. For hemostats derived from synthetic polymers,

ncontrollable and excessive bleeding is the main cause of death resulting from injuries in hospitals, emergencies, and battlefield settings.1−3 For example, about one-third of prehospital deaths are due to hemorrhage in civilian emergency conditions,4 while half of deaths are attributed to exsanguination in the military.5 Generally, hemostasis is achieved by a series of coagulatory cascades, involving the formation of a hemostatic plug by activated platelets,6 sequentially followed by the activation of multiple coagulation factors through intrinsic and/or extrinsic enzymatic pathways that can further reinforce the platelet plug.7,8 In the case of severe injuries, however, these natural clotting processes cannot rapidly and sufficiently staunch bleeding, leading to uncontrolled blood loss. Pressure dressings and absorbents © 2016 American Chemical Society

Received: June 22, 2016 Accepted: October 13, 2016 Published: October 13, 2016 9957

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Figure 1. Preparation of nanohemostats by cholic-acid-mediated self-assembly of polyethylenimine. (A) Schematic illustration of cholic-acidinduced assembly of polyethylenimine into nanoparticles. (B) Chemical structures of various cholic acids. (C) TEM images and (D) size distribution profiles of nanoparticles assembled by UDCA and branched polyethylenimine with Mw of 25 kDa (bPEI25) at varied UDCA/ bPEI25 weight ratios.

(PLGA)-b-poly(L-lysine)-b-polyethylene glycol nanoparticles that can bind to activated platelets and, therefore, are able to halve bleeding time after intravenous (i.v.) administration in a femoral artery injury model in rats.23 The effectiveness of this nanohemostat was also confirmed in other bleeding models such as blunt trauma and blast trauma.24−27 Okamura et al. constructed dodecapeptide HHLGGAKQAGDV conjugated free-standing PLGA nanosheets or albumin particles that could recognize the active form of glycoprotein IIb/IIIa, thereby displaying increased adhesive rate with activated platelets.28,29 Nanoparticles, with platelet-like functions such as vascular injury site-directed margination, site-specific adhesion, and amplification of injury site-specific aggregation, were designed and evaluated, which could accumulate at the wound site and reduce bleeding time in a mouse model.30 In addition to nanoparticles, fibrous nanomaterials were found to be effective hemostatic agents. A nanofiber self-assembled by a peptide RADA16-I can halt bleeding in a number of injury models when it was applied topically.31 Also, enhanced clotting in both normal and heparin-treated rats with a lateral liver incision was realized by injectable and nanofibrous hemostats.12 More recently, it was found that tissue factor (TF)-targeted nanofibers assembled by an amphiphilic peptide covalently conjugated with a TF-binding sequence may notably reduce blood loss in a punch-induced liver hemorrhage model in rats.32 By covalently conjugating a fibrin-binding peptide onto a watersoluble polymer, Chan et al. synthesized a fibrin cross-linking

limited efficacy, toxic byproducts, possible tissue necrosis, and local irritation are major issues restricting their broad applications.12 On the other hand, biologically derived hemostats suffer from drawbacks of immunogenicity and batch-to-batch variability. As for platelets, the short shelf life, stringent storage requirements, graft-versus-host disease, alloimmunization, and transfusion-related acute lung injuries are main concerns for their clinical applications.13 Furthermore, currently, few hemostats are available for cessation of internal bleeding or hemorrhage at less accessible injury sites in civilian and military traumas.14,15 In addition, effective techniques are desperately required for the treatment of bleeding that cannot be halted by external compression.16 Consequently, there is a still unmet need for discovering strategies or hemostats for effective and safe hemostasis in the hospital and on the battlefield.17 Recent development in nanotechnology offers opportunities for designing and creation of hemostats. Nanomaterials with tunable biophysicochemical properties (such as size, shape, flexibility, and surface biology) have been examined for hemostasis in various settings.1,16−18 For example, Arg-GlyAsp (RGD) peptide-modified liposomes were found to be able to target and bind to activated platelets,19,20 while liposomes with surface decorated by collagen- and von Willebrand factor (vWF)-binding peptides combined with a fibrinogen-mimetic peptide showed high hemostatic efficacy in a mouse tail amputation model.21,22 On the other hand, Lavik’s group developed RGD-functionalized poly(lactic-co-glycolic acid) 9958

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Figure 2. Molecular modeling and experimental characterization of interactions between UDCA and bPEI. (A−F) Computational simulation of UDCA−bPEI interactions. (A) Two-dimensional and (B) three-dimensional images showing the lowest energy conformation of the UDCA−bPEI complex. In all images, gray, red, and dark blue denote carbon, oxygen, and nitrogen atoms, respectively, and light gray indicates polar hydrogen atoms that can form H-bonds. The interaction and lipophilic sites on the illustrated surface were rendered and colored by the Connolly method built in MOE. Binding energy contributed by various forces including (C) electrostatic force, (D) Hbonding, and (E) van der Waals force. (F) Total binding energy. (G) Characterization of UDCA/bPEI25 forces by Fourier transform infrared spectroscopy. (H) 1H NMR spectra of UDCA, bPEI25, or UDCA/bPEI25 mixture in DMSO-d6.

poly(amidoamine) dendrimers were able to induce fibrinogen aggregation.37 This effect was considered to be mediated by electrostatic interactions between the positively charged dendrimer surface and negatively charged fibrinogen domains because the isoelectric point of fibrinogen is about pH 5.5.38 Therefore, we hypothesize that positively charged nanoparticles may be potential nanohemostats by initiating the aggregation of platelets and fibrinogen. To demonstrate our hypothesis and develop effective and translational nanohemostats, we constructed well-structured positive nanoparticles that can be easily assembled by various cholic acids and polyethylenimines (PEIs). In addition to examination of cholic acid/PEI interactions and characterization of physicochemical properties of assembled nanoparticles, in vitro studies were conducted to investigate the effects of constructed nanoparticles on platelets.

polymer that showed effective hemostatic activities in both in vitro tests and a rat model of trauma after i.v. administration.33 Whereas recent progress in this field has unambiguously demonstrated the effectiveness and advantages of nanomaterialderived hemostats for bleeding control in diverse settings, considerable challenges remain for the development of affordable, safe, effective, easily storable, conveniently useful, and low-cost hemostats.1,17,34 Complicated manufacturing methods, high production cost, poor scalability, and limited capacity of regulatory approval are major issues hindering clinical translation of currently investigated hemostats.16 Previously, it was found that positively charged molecules including protamine, polybrene, and polylysine could induce the aggregation of blood platelets, which was not influenced by inhibitors of platelet metabolism.35,36 Additionally, cationic 9959

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Figure 3. In vitro aggregation and activation of platelets induced by UDCA/bPEI25 nanoparticles. (A,B) Phase contrast micrographs and (C,D) SEM images showing platelets treated by normal saline (A,C) or UDCA/bPEI25 nanoparticles (B,D). The arrows indicate aggregated platelets. Aggregation cureves of (E) platelets and (F,G) quantitative results after various treatments. (H) Confocal laser scanning microscopy images of UDCA/Cy5-bPEI25 nanoparticle-induced platelet aggregation. The formation of large, tight aggregates composed of platelets could be clearly observed. (I) Secretion of granules or platelet microparticles after treatment with UDCA/Cy5-bPEI25 nanoparticles. Platelets were stained with FITC-labeled anti-CD61 antibody (green), and bPEI25 was labeled with Cy5 (red).

one-pot assembly strategy may be employed to fabricate nanoparticles (Figure 1A) using PEI and various cholic acids (Figure 1B). Initially, ursodeoxycholic acid (UDCA) was utilized as a hydrophobic component, while branched PEI with Mw of 25 kDa (bPEI25) served as a hydrophilic polymer. Figure 1C shows transmission electron microscopy (TEM) images of nanoparticles assembled by UDCA/bPEI25 at the weight ratio varying from 1:1, 2:1, 3:1, to 4:1. Independent of various weight ratios, well-defined spherical nanoparticles were formed. Determination by dynamic light scattering (DLS) revealed relative narrow size distribution for all the obtained

Furthermore, we interrogated hemostatic efficacy of this type of nanoparticles in various rodent models including tail amputation, liver hemorrhage, and femoral artery bleeding, after topical application or i.v. injection.

RESULTS AND DISCUSSION Nanoparticles Formed by Ursodeoxycholic-Acid-Directed Self-Assembly of Polyethylenimine. According to our previous studies, nanoparticles with different sizes can be efficiently assembled by small-molecule-mediated self-assembly of hydrophilic polymers.39−43 Herein, we found that a similar 9960

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documented, platelets are negatively charged due to the presence of sialic acid.44 Furthermore, anionic phospholipids such as phosphatidylserine may be transported from the inner to the outer platelet membrane surface upon platelet activation.45 Accordingly, we speculate that positively charged UDCA/bPEI25 nanoparticles may induce platelet aggregation by Coulomb force. Observation by optical microscopy indicated considerable platelet aggregation when UDCA/ bPEI25 nanoparticles were added into platelet-rich plasma, which was distinctly different from the saline-treated group (Figure 3A,B). This was further affirmed by imaging with scanning electron microscopy (SEM, Figure 3C,D). We then determined the platelet aggregation curve by a resistance method, which is based on the fact that resistance is increased upon the aggregation of platelets. Whole blood was employed in this study, while adenosine diphosphate (ADP), an agonist for platelet aggregation,46 was used as a positive control. As illustrated in Figure 3E, addition of either ADP or UDCA/bPEI25 nanoparticles in whole blood caused significant platelet aggregation in time and dose response patterns. Generally, platelet aggregation is composed of different stages, including a lag phase, primary aggregation, release of various molecular contents and granules, and secondary aggregation.47 The primary aggregation is relatively loose, while more dense structure may be formed in the secondary aggregation, resulting from agglomeration of a large number of platelets. Accordingly, increased resistance concurs with enhanced aggregation. Of note, the aggregation induced by nanoparticles at 0.5 mg/mL was more obvious than that treated with 100 μM ADP. Further analysis of amplitude and slope of the aggregation curve revealed that UDCA/bPEI25 nanoparticles displayed an aggregation rate significantly higher than that of ADP at examined doses (Figure 3F,G). To learn more details regarding UDCA/bPEI25 nanoparticle-induced platelet aggregation, fluorescence imaging was performed using nanoparticles assembled by UDCA and Cy5-labeled bPEI25 (Cy5-bPEI25, Figure S3). Platelets were stained with FITC-labeled anti-CD61 antibody because CD61 (a cell surface protein) is a selective marker of platelets. Treatment of platelet suspensions with UDCA/Cy5-bPEI25 nanoparticles resulted in significantly aggregated and amplified fluorescence signals (Figure 3H). Furthermore, the colocalization of Cy5 fluorescence (red) with that of FITC (green) could be clearly observed, indicating the presence of both nanoparticles and platelets in the formed aggregates. In addition to platelet aggregation, we could visualize a large number of CD61-positive particles with smaller size compared to platelets (Figure 3I). These small particles could also be observed in the SEM image (Figure 3D, areas indicated by black arrows). This may be attributed to platelet degranulation, which can lead to release of platelet-derived microparticles.48 Because granule secretion by platelets is generally related to platelet activation,48,49 these results suggested that UDCA/bPEI25 nanoparticles may activate platelets. While the inactivated platelets are in a crisp discoid shape, they may change to a more amorphous form with pseudopodia upon activation.18 This was additionally supported by a SEM image of nanoparticle-treated platelets (Figure S4). Collectively, UDCA/bPEI25 nanoparticles can induce platelet aggregation and activate platelets. These effects were realized by electrostatic force-mediated agglomeration of free platelets, which was followed by platelet activation that can further amplify Coulomb force via exposure of negative

nanoparticles (Figure 1D and Figure S1). The average diameter was 171 ± 3, 221 ± 7, 195 ± 2, and 191 ± 2 nm for nanoparticles at the UDCA/bPEI25 weight ratio of 1:1, 2:1, 3:1, and 4:1, respectively (Figure S1A). This suggested that the size of assembled nanoparticles was initially increased with an increase in UDCA feeding, which was then decreased when the UDCA content was enhanced to a certain degree. Measurement of ζ-potential indicated that all of the assembled nanoparticles had positive surface charges (Figure S1B). Nevertheless, the ζ-potential values of various nanoparticles were comparable. We then interrogated interactions dominating assembly of UDCA/bPEI. First, a molecular simulation approach was employed, with UDCA/bPEI as a model system. The AutoDock program was used to estimate the docking energy and intermolecular energy of the assembly system containing water, UDCA, and bPEI. Because the binding sites in bPEI were not defined, the blind docking was implemented to the whole polymer chain and UDCA. Figure 2A,B shows 2D and 3D images of the UDCA−bPEI complex with the lowest energy. After molecular docking, the lowest energy conformation of bPEI displayed a big cavity formed by the backbone and branched chain of bPEI, into which UDCA was closely included via electrostatic, hydrogen bonding (H-bonding), and van der Waals forces. The intermolecular energy contributed by different forces, including electrostatic force, H-bonding, and van der Waals binding (VWB, including hydrophobic interactions), was examined. Among different molecular pairs, UDCA−bPEI exhibited the strongest electrostatic force (Figure 2C). In the case of H-bonding and VWB, the relatively higher interaction energy was found for UDCA−UDCA and UDCA− bPEI (Figure 2D,E). Estimation of the total intermolecular binding energy revealed the largest value for UDCA−bPEI, which was followed by that of UDCA−UDCA, UDCA−water, bPEI−water, and bPEI−bPEI (Figure 2F). These results suggested that multiple intermolecular interactions between UDCA and bPEI can conquer other forces dominating crystallization of UDCA molecules. Subsequently, interactions between UDCA and bPEI were experimentally characterized. According to Fourier transform infrared (FTIR) spectra, the stretching vibration at 1710 cm−1 due to UDCA carbonyl was shifted to 1580 cm−1, concomitant with notably reduced intensity (Figure 2G). In addition, an absorption band at ∼2820 cm−1 from alkyl moieties of bPEI25 was considerably attenuated (Figure S2). This implied the existence of H-bonding and hydrophobic forces between UDCA and bPEI25. Further examination was performed by 1 H NMR spectroscopy. In addition to disappearance of the proton signal at 11.9 ppm due to carboxyl acid, proton signals from two hydroxyl groups and methylene adjacent to carboxyl of UDCA were significantly attenuated, and broadened peaks were found for other protons (Figure 2H). This result revealed the presence of electrostatic and hydrophobic forces in the UDCA/bPEI25 system. Moreover, the 1H NMR spectrum of UDCA/bPEI25 mixture also showed their strong interactions even in DMSO. Taken together, both molecular simulation and experimental results unambiguously demonstrated that there are multiple interactions between UDCA and bPEI25, including electrostatic, H-bonding, and hydrophobic forces, and their synergistic effects contribute to the assembly of UDCA/bPEI25 into well-shaped nanoparticles. Assembled UDCA/bPEI25 Nanoparticle-Induced Activation and Aggregation of Platelets. As it is well9961

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Figure 4. Hemostatic effects of UDCA/bPEI25 nanoparticles in different bleeding models. (A) Schematic showing the surgical procedures of a liver bleeding model in rats. The red line indicates the injured site in the left lobe. (B) Digital photos of liver bleeding at various stages before and after treatment with saline (control) or nanoparticles assembled by UDCA/bPEI25 at a weight ratio of 2:1. (C) Liver bleeding times after treatment with nanoparticles assembled by UDCA/bPEI25 at various weight ratios. (D) Liver bleeding times of rats treated by various concentrations of UDCA/bPEI25 nanoparticles at a UDCA/bPEI25 weight ratio of 2:1. (E) Schematic illustration of the surgical procedures for establishment of a femoral artery bleeding model in rats by puncture with a 26-gauge needle. (F) Pictures indicating femoral artery bleeding before and after treatment. (G,H) Bleeding times of the injured femoral artery after treatment with UDCA/bPEI25 nanoparticles containing different contents of UDCA (G) or at various doses (H). For the dose-dependent study, UDCA/bPEI25 nanoparticles assembled at a weight ratio of 2:1 were used. Data are mean ± SE (n = 3) of independent experiments; **p < 0.01, ***p < 0.001.

platelets.35−37,50 Assembled UDCA/bPEI25 nanoparticles should exert their activity through the similar mechanism due to the existence of a high positive charge density on the particle surface as indicated by their ζ-potential values (Figure S1B). Absorption of Serum Proteins on UDCA/bPEI25 Nanoparticles. It was found that different proteins will be

components from activated platelets, thereby eliciting the coagulation cascade. Our finding is consistent with previous reports that cationic molecules (such as protamine, polybrene, and polylysine) can induce platelet aggregation by binding to the negatively charged platelet membrane, reducing the negative surface charge, and forming bridges between adjacent 9962

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Figure 5. Comparison of the hemostatic activity of UDCA/bPEI25 nanoparticles with a negatively charged PS NP and a clinically used gelatin sponge in different bleeding models. (A) TEM image and (B) size distribution profile of PS NPs. Hemostatic effects of different formulations in a (C) tail amputation model in mice, (D) liver bleeding model in rats, and (E) femoral artery bleeding model in rats. The bleeding times in a (F) liver injury model and (G) femoral artery injury model in rabbits after treatment with different hemostatic agents. Data are mean ± SE (n = 3) of independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.

In Vivo Hemostasis by Assembled UDCA/bPEI25 Nanoparticles. Based on the above findings, in vivo hemostatic effects of UDCA/bPEI25 nanoparticles were examined in different bleeding models. A mouse tail bleeding model was first adopted (Figure S6A).21,54 Nanoparticles assembled with UDCA/bPEI25 at various weight ratios were examined. After tail amputation, the average bleeding time for mice treated with saline was 741 s. Whereas treatment of the transected site in aqueous solution containing bPEI alone at 2.5 mg/mL notably reduced the bleeding time to 402 s, which was additionally shortened when an aqueous solution containing different UDCA/bPEI25 nanoparticles at the same concentration of 2.5 mg/mL was applied (Figure S6B). Of note, nanoparticles with a high content of UDCA exhibited a more significant hemostatic effect, with the average bleeding time of 379, 250, 205, and 162 s for nanoparticles at UDCA/bPEI25 of 1:1, 2:1, 3:1, and 4:1, respectively. In addition to the content of UDCA, the concentration of nanoparticles had a profound

rapidly absorbed on nanoparticles upon incubation with human plasma.51 Thus, the formed plasma protein corona has profound effects on hemolysis, platelet activation, and tissue distribution of nanoparticles. Accordingly, we identified and quantified protein components on UDCA/bPEI25 nanoparticles by a label-free quantitative liquid chromatography mass spectrometry (LC-MS). After 5 min of incubation of nanoparticles with freshly collected rat plasma, 16 proteins were determined on nanoparticles (Figure S5). Among them, fibrinogen was found to be the most abundant protein absorbed on UDCA/bPEI25 nanoparticles, which was followed by albumin. This result is agrees with the previous finding that cationic dendrimers can initiate blood clot formation,37 which was mediated by electrostatic interactions with carboxyl groups on the fibrinogen surface.52 Since fibrinogen is closely related with thrombus formation,53 its absorption on nanoparticles is beneficial for hemostasis. 9963

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Figure 6. Prevention of bleeding in the femoral artery in rats by i.v. injected UDCA/bPEI25 nanohemostats. (A) Bleeding times of the injured femoral artery after treatment with UDCA/bPEI25 nanoparticles at a weight ratio of 2:1. (B) Hemostasis by i.v. injected UDCA/bPEI25 nanoparticles at various doses. (C) Comparison of the hemostatic activity of different formulations at the same dose of 6.0 mg/kg. (D) Timedependent distribution (the top panel) and excretion kinetics (the bottom panel) of UDCA/Cy7.5-bPEI25 nanoparticles in blood after i.v. injection. (E) Accumulation of UDCA/Cy7.5-bPEI25 nanoparticles in representative major organs 3 h after i.v. administration. (F) Localization of UDCA/Cy5-bPEI25 nanoparticles at the injured site in the femoral artery after i.v. injection. (G) Typical hematological parameters of rats after treatment with i.v. injection of UDCA/bPEI25 nanoparticles (UDCA/bPEI25 NP) at 6.0 mg/kg. RBC, red blood cell; WBC, white blood cell; PLT, platelet; HGB, hemoglobin. The red windows indicate the normal levels of different parameters. Data are mean ± SE (n = 4) of independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.

aqueous solution containing bPEI25 or various UDCA/bPEI25 nanoparticles at the same concentration of 2.5 mg/mL (Figure 4B,C). Compared to bPEI25 alone, assembled nanoparticles showed strikingly potentiated hemostatic effects, with the exception of nanoparticles at UDCA/bPEI25 of 1:1 (Figure 4C). Likewise, the hemostatic potency was significantly enhanced when nanoparticles with higher contents of UDCA were utilized. For nanohemostats assembled at the UDCA/ bPEI25 weight ratio of 0:1, 1:1, 2:1, 3:1, and 4:1, the bleeding time was 132, 121, 78, 64, and 38 s, respectively. Also, increasing nanoparticle concentration may afford increased hemostatic effects. For the nanohemostat assembled by UDCA/bPEI25 at a weight ratio of 2:1, the bleeding time

influence on their hemostatic capability. In the case of nanoparticles with a UDCA/bPEI25 weight ratio of 2:1, the bleeding time varied from 305, 245, to 187 s when the nanoparticle concentration was increased from 1.0, 2.0, to 4.0 mg/mL (Figure S6C). A liver bleeding model was then used to interrogate the hemostatic capability of UDCA/bPEI25 nanoparticles. The bleeding model was established in rats according to the surgical procedure illustrated in Figure 4A.31 When the injured site with transverse cut was treated with 300 μL of saline, considerable bleeding could be observed (Figure 4B), with the bleeding time of 165 s (Figure 4C). By contrast, the bleeding time was remarkably reduced by treatment with the same volume of 9964

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was slightly more potent than that of the gelatin sponge, although no significant differences were found between them. Accordingly, hemostatic efficacy of UDCA/bPEI25 nanoparticles is independent of bleeding models in different animals. Prevention of Femoral Artery Bleeding by Intravenously Injected UDCA/bPEI25 Nanoparticles. Since different nanohemostats could effectively prevent bleeding after i.v. injection,16,17,21,23,24,26,27 we also examined whether UDCA/bPEI25 nanoparticles can be utilized as injectable nanohemostats to stop bleeding. To this end, a femoral artery bleeding model in rats was employed. Nanoparticles assembled by UDCA/bPEI25 at various weight ratios were i.v. injected via tail vein at 6.0 mg/kg. After circulation for 5 min, an injury was induced in the femoral artery by puncture with a 26-gauge needle. Quantitative measurement indicated that the bleeding time was reduced from 160 to 113 s by pretreatment with bPEI25 alone at 6.0 mg/kg. By contrast, the average bleeding time was 88 s for assembled nanoparticles at a UDCA/bPEI25 weight ratio of 2:1 (Figure 6A). Moreover, increasing the concentration of nanohemostats resulted in significantly shorter bleeding times when compared with the saline control (Figure 6B). Consequently, these findings demonstrated that nanoparticles assembled by UDCA/bPEI25 can also function as injectable nanohemostats to reduce the bleeding time at injured sites. In a separate study, we compared the bleeding prevention capability of UDCA/bPEI25 nanoparticles with negatively charged PS nanoparticles. Compared with the treatment via saline, i.v. injection of PS NP could not reduce the bleeding time of injury in the femoral artery (Figure 6C). By contrast, either bPEI25 or UDCA/bPEI25 nanoparticles showed significant hemostatic activity compared to that of PS NPs. We then examined the mechanisms underlying the hemostatic efficacy of i.v. injected UDCA/bPEI25 nanoparticles. According to previous studies, i.v. injected nanoparticles functionalized with different moieties may halt bleeding by binding to activated platelets.21,23,24,26 Therefore, we examined biodistribution of i.v. administered UDCA/ bPEI25 nanoparticles based on Cy7.5-labeled bPEI25 (Figure S3B,C). After i.v. administration, detection of Cy7.5 fluorescence signals indicated that UDCA/Cy7.5-bPEI25 nanoparticles were rapidly cleared from the circulation (Figure 6D, top panel), and about 80% of them were eliminated within 3 h (Figure 6D, bottom panel). This result is in line with the previous finding that positively charged nanoparticles generally exhibit a rapid blood clearance profile and short blood circulation.56 Further examination of distribution in major organs by ex vivo fluorescence imaging revealed the majority of nanoparticles were accumulated in liver, spleen, and lung at 3 h after i.v. injection (Figure 6E), agreeing with that observed for other positively charged nanoparticles with different compositions.56−58 Interestingly, we also observed accumulation of fluorescent signals at the injured site of femoral artery in the case of UDCA/Cy7.5-bPEI25 nanoparticles (Figure 6F), while the normal control segment of the femoral artery without injury showed negligible fluorescence signals that were comparable to those treated with saline. By contrast, only weak fluorescent signals could be observed at the injured site of the femoral artery when a control negatively charged nanoparticle of Cy7.5labeled PS NP was i.v. administered at 6.0 mg/kg (Figures S7 and S8). Additional immunofluorescence analyses were performed for the sections based on the blood clots and injured vessels collected from the femoral artery of rats subjected to different treatments. We could clearly observe the

was reduced from 120, 99, to 74 s when its concentration was enhanced from 1.0, 2.0, to 4.0 mg/mL, respectively (Figure 4D). These results indicated that UDCA/bPEI25 nanoparticles could effectively staunch bleeding of liver injury in rats. Subsequently, a femoral artery bleeding model was employed,23 which was also established in rats according to the previously reported procedures (Figure 4E).23,31 After an injury was induced by puncture with a 26-gauge needle in the femoral artery, we observed blood spurting and significant bleeding in proximity of the injured site (Figure 4F). Quantitative measurement revealed the mean bleeding time was 375 s for rats treated with 300 μL of saline (Figure 4G). When the injured site was treated with 300 μL of aqueous bPEI25 solution at 2.5 mg/mL, the bleeding time was reduced to 174 s. More significant hemostatic effects were achieved by nanohemostats, with the bleeding time of 154, 94, 90, and 65 s for nanoparticles assembled by UDCA/bPEI25 at 1:1, 2:1, 3:1, and 4:1, respectively. Similar to those observed in the models of tail transection and liver bleeding, we also observed potentiated hemostatic efficacy with increased concentration of nanohemostats (Figure 4H). In this case, the bleeding time was 171, 143, and 108 s for UDCA/bPEI25 nanoparticles at 1.0, 2.0, and 4.0 mg/mL, respectively. Accordingly, assembled UDCA/ bPEI25 nanoparticles can also serve as effective nanohemostats to halt bleeding in the femoral artery. Collectively, the above results strongly suggested that assembled UDCA/bPEI25 nanoparticles can function as effective nanohemostats to promote thrombus formation and notably reduce bleeding times in different bleeding models. The hemostatic activity of these positive nanoparticles was mainly achieved by electrostatic force-induced platelet aggregation. While the cationic polymer bPEI25 itself also showed hemostatic efficacy, assembly into nanoparticles further amplified this activity by remarkably increasing the charge density on the particle surface. In addition, considerable absorption of fibrinogen on nanoparticles may enhance platelet aggregation55 and facilitate the formation of robust fibrin networks,33 thereby contributing to the development of blood clots. To further demonstrate our notion, we compared the hemostatic effect of UDCA/bPEI25 nanoparticles with a negatively charged PS nanoparticle (PS NP) in another cohort of experiments, and a clinically used absorbable gelatin sponge was used as a control hemostat. The spherical PS NP with mean size of 172 nm and ζ-potential of −31.5 mV was employed (Figure 5A,B and Figure S7). As anticipated, a PS NP at 2.5 mg/mL showed no hemostatic efficacy in three models including tail amputation in mice, liver bleeding in rats, and femoral artery bleeding in rats, when compared with the saline group (Figure 5C−E). In the case of the gelatin sponge, whereas it displayed more significant effects with respect to reducing bleeding times in the examined models than bPEI25, its hemostatic activity was comparable to or slightly lower than that of UDCA/bPEI25 nanoparticles at 2.5 mg/mL. On the basis of these promising results, we further examined the hemostatic capability of different formulations in rabbits. Similar to the findings in murine models, topical treatment with PS NP at 2.5 mg/mL could not effectively reduce bleeding times in two models of liver injury and femoral artery injury in rabbits (Figure 5F,G). Compared to the saline control, bPEI25 exhibited notable hemostatic effects in two rabbit models, while more significant efficacy was observed for UDCA/bPEI25 nanoparticles at the same concentration of 2.5 mg/mL. Of note, the hemostatic activity of UDCA/bPEI25 nanoparticles 9965

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of 6.0 mg/kg (Figure 6G). Ex vivo imaging revealed significant fluorescence signals of Cy7.5-PS NP in liver and spleen at 3 h after i.v. injection (Figure S9A), which were largely retained in these organs 1 week post-administration (Figure S9B). These results indicated that PS NPs accumulated in both liver and spleen could not be metabolized or excreted. In the case of UDCA/Cy7.5-bPEI25 nanoparticles, whereas considerable accumulation was observed in the liver, spleen, and lung at 3 h (Figure 6E), it was notably reduced 1 week after i.v. administration (Figure S9B). Accordingly, i.v. administered UDCA/bPEI25 nanoparticles could be absorbed and/or eliminated from the body. Further, acute toxicity evaluation was performed for UDCA/ bPEI25 nanoparticles assembled at a weight ratio of 2:1. After i.v. injection of UDCA/bPEI25 nanoparticles in rats at 24.0 mg/kg via tail vein, all the treated animals showed normal behaviors and gradual body weight gain (Figure S10A). During the examined time period, all the treated rats survived. At day 14, rats were euthanized. The organ index of representative major organs isolated from UDCA/bPEI25 nanoparticletreated rats was comparable to that of rats administered with saline (Figure S10B). Also, treatment with nanoparticles did not cause abnormal changes in representative hematological parameters (Figure S11). Measurement of biochemical markers relevant to hepatic and kidney functions revealed comparable levels of alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea (Figure S12). Inspection on hematoxylin and eosin (H&E)-stained histological sections of typical major organs including heart, liver, spleen, lung, kidney, and thymus indicated normal microstructure (Figure S13). No detectable injury or significant infiltration of inflammatory cells could be found in these sections. These results suggested that, at 24.0 mg/kg, UDCA/bPEI25 nanoparticles are safe for i.v. administration. Although it has been recognized that nonspecific binding or induced platelet activation has potential risk of adverse thrombotic events, such as embolism and stroke, our results indicated good safety profile for i.v. administered UDCA/bPEI25 nanoparticles at the examined dose. It should be noted that severe toxicity occurred when their dose was further increased. At 30 mg/kg, for the examined 6 rats, 5 of them died. This finding is coincident with the fact that polyplexes based on bPEI25 or its derivatives have been widely studied for in vivo gene therapy.60−62 Effect of PEI Structure on Hemostatic Efficacy of Assembled Nanohemostats. Encouraged by the promising results based on nanoparticles assembled by UDCA/bPEI25, we examined whether effective nanohemostats can be constructed using PEIs with other structures. To this end, branched PEI with Mw of 1.8 kDa (bPEI1.8) and linear PEI with Mw of 10 kDa (lPEI10) were employed. Assembly of UDCA/bPEI1.8 was conducted with procedures similar to those used for bPEI25. At a UDCA/bPEI1.8 weight ratio of 2:1, nanoparticles with an average diameter of 369 nm and ζpotential of 54.5 mV were formed (Figure 8A and Figure S14). In the case of UDCA/lPEI10 nanoparticles, they were prepared by dialysis of a solution mixture in water, which was formed by adding UDCA in DMSO dropwise into aqueous solution of lPEI10 under sonication. At a weight ratio of 2:1 for UDCA/ lPEI10, spherical particles with a mean size of 206 nm and ζpotential of 28.3 mV were afforded (Figure 8B and Figure S14). These results demonstrated that positively charged nanoparticles can also be efficiently assembled by UDCA and PEIs with different structures.

presence of UDCA/Cy5-bPEI25 nanoparticles in the blood clot and injured vessel (Figure 7). This is consistent with the ex vivo

Figure 7. Immunofluorescence images illustrating the colocalization of UDCA/Cy5-bPEI25 nanoparticles with representative plasma proteins at the injured sites in the fermoral artery of rats. Fluorescence images of sections separately stained by FITC-labeled antibodies to (A) albumin or (B) fibrinogen-β. UDCA/Cy5-bPEI25 nanoparticles were administered to rats by i.v. injection at 6.0 mg/ kg. After 5 min, a femoral artery injury was induced by a 26-gauge needle. Immediately post-hemostasis, rats were euthanized. The blood clots at the injured site and the injured vessles were collected for immunofluorescence analysis. The scale bars represent 20 μm.

imaging result (Figure 6F). Moreover, we found the colocalization of UDCA/Cy5-bPEI25 nanoparticles with both albumin and fibrinogen-β, agreeing with the result of proteomic analysis that fibrinogen and albumin were mainly absorbed proteins on UDCA/bPEI25 nanoparticles upon incubation with rat plasma (Figure S5). Consequently, preferential targeting of UDCA/bPEI25 nanoparticles at the injured site of the femoral artery can be attributed to their opsonization by fibrinogen and albumin, which was supported by previous studies that particles coated with fibrinogen or fibrinogenmimicking peptides were able to effectively initiate blood clot formation and therefore reduce bleeding in different animal models.21,22,37,55,59 Of note, the number of representative blood cells including red blood cells, white blood cells, and platelets, and the level of hemoglobin were all in the normal range after treatment with i.v. injected UDCA/bPEI25 nanoparticles at the examined dose 9966

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Figure 8. Hemostatic effects of nanoparticles based on UDCA and various PEIs. (A) Assembly of nanoparticles by UDCA and branched PEI with Mw of 1.8 kDa (bPEI1.8). (B) Nanoparticles assembled by UDCA and linear PEI with Mw of 10 kDa (lPEI10). The left panels show TEM images, and size distribution profiles are in the right panels. Bleeding times in various models including (C) tail amputation, (D) liver bleeding, and (E) femoral artery bleeding. (F) Times required for hemostasis in the injured femoral artery in rats i.v. administered with assembled nanohemostats. For all assembled nanoparticles, the weight ratio of UDCA/PEI was 2:1. All data are mean ± SE (C,D,E, n = 3; F, n = 4) of independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.

UDCA to form well-structured nanoparticles with positive surface charges. While PEIs of various structures promoted hemostasis when they were applied topically or i.v. administered, their co-assembly with UDCA afforded nanohemostats with more potent hemostatic efficacy. Notably, UDCA itself has no any hemostatic activity. The effect of enhanced hemostatic capacity by nanoassembly might be attributed to the increased surface charge density on nanoparticles compared to the corresponding cationic PEI because these cationic hemostats exerted hemostatic activity by electrostatic force-mediated bridging or cross-linking effect as well as opsonization via fibrinogen and albumin. Previously, remarkably enhanced hemostatic efficacy was realized for targeted nanoparticles by increasing surface ligand density.25 On the other hand, enhanced margination most probably contributed to the more effective bleeding prevention capability of i.v. administered nanoparticles when compared with the free polymer.63,64 Nevertheless, extensive and in-depth studies are necessary to elucidate the exact mechanisms underlying the hemostatic efficacy of assembled positive PEI nanoparticles. Effect of Cholic Acid Structure on Hemostatic Efficacy of Assembled Nanohemostats. To further expand the applications of this type of nanohemostats and interrogate the

Subsequently, we interrogated the hemostatic efficacy of nanoparticles assembled by UDCA and bPEI1.8 or lPEI10 under different bleeding conditions. In the tail amputation model in mice, treatment with either bPEI1.8 or lPEI10 alone considerably reduced bleeding times from 741 to 403 or 445 s, respectively (Figure 8C), which was similar to that observed for bPEI25. This effect was additionally augmented by assembly into nanoparticles. The bleeding time was 268 and 297 s for nanohemostats assembled with bPEI1.8 or lPEI10 at a UDCA/ PEI weight ratio of 2:1, respectively. Also, in both liver and femoral artery bleeding models in rats, we found that aqueous solution of either bPEI1.8 or lPEI10 could more effectively halt bleeding than the saline control (Figure 8D,E), while their hemostatic capacity was remarkably promoted by assembly with UDCA to form nanoparticles. Moreover, i.v. injection of bPEI1.8 or lPEI10 was able to significantly reduce bleeding time at an injury site of the femoral artery in rats when compared with those treated with saline (Figure 8F). Again, assembled UDCA/bPEI1.8 or UDCA/lPEI10 nanoparticles afforded more desirable efficacy. In combination with the results of bPEI25 and its assembled nanoparticles, these findings substantiated that, regardless of their structures, cationic polymers of PEIs can assemble with 9967

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Figure 9. Hemostatic efficacy of nanohemostats assembled by bPEI25 and various cholic acid derivatives. (A) TEM images and (B) size distribution profiles of nanoparticles assembled by bPEI25 and various cholic acids. Bleeding times in various murine models including (C) tail amputation, (D) liver injury, and (E) femoral artery injury. (F) Times required for hemostasis in the injured femoral artery in rats i.v. administered with assembled nanohemostats. For all assembled nanoparticles, the weight ratio of cholic acid to bPEI25 was 2:1. Data are mean ± SE (C,D,E, n = 3; F, n = 4) of independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.

extended to a large number of carboxyl-bearing hydrophobic compounds (Figure 1A). We then explored the hemostatic capability of nanoparticles assembled by bPEI25 and various cholic acids in different bleeding models. Compared with aqueous solution of bPEI25, all the assembled nanoparticles more significantly reduced bleeding times in a tail transection model in mice as well as in liver and femoral artery bleeding models in rats (Figure 9C−E). Whereas comparable hemostatic effects were achieved for nanohemostats assembled by UDCA, CDCA, HDCA, DOCA, and CA, nanoparticles based on DHCA or LCA showed relative less efficacy in these three bleeding models. This is especially true in the case of LCA-based nanoparticles. Because DHCAor LCA-derived nanohemostats had relatively larger size, these results implied that an increase in particle size may impair the

spectrum of compounds that can be used for assembly of nanohemostats, different cholic acids were studied to prepare nanoparticles by assembling them with bPEI25. For this purpose, cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), deoxycholic acid (DOCA), dehydrocholic acid (DHCA), and lithocholic acid (LCA) were utilized. After dialysis of bPEI25 with various cholic acids at a weight ratio of 1:2, TEM visualization showed the formation of nanoparticles regardless of different cholic acids (Figure 9A). Measurement by DLS revealed relatively narrow size distribution (Figure 9B and Figure S15), with mean size varying from 90 to 600 nm (Figure S15A). Despite their different sizes, all of these nanoparticles exhibited comparable positive surface charges (Figure S15B). These results suggested that the formation of nanoparticles by assembly of PEIs can be 9968

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positive nanoparticles based on PEIs and cholic acids may serve as easily scalable, cost-effective, safe, topically applicable or injectable nanohemostats for prehospital care or bleeding control in the hospital settings and on the battlefield. They may also be utilized for the treatment of coagulation dysfunction resulting from thrombocytopenia and/or platelet function disorders.

hemostatic capacity of nanoparticles. This should be related to the reduced specific surface area, in consideration of the fact that nanoparticles function as supramolecular cross-linkers to mediate platelet aggregation, activation, and hemostasis, and therefore, smaller particles can more efficiently interact with blood components due to their high surface area to volume ratio. Also, preadministration of nanohemostats based on various cholic acids by i.v. injection notably reduced bleeding times of femoral artery injury in rats, compared to the controls of saline and aqueous bPEI25 solution (Figure 9F). In this case, DHCAor LCA-based nanohemostats displayed more potent hemostatic activity compared to nanoparticles assembled by other cholic acids. This was different from the hemostatic effect after topical applications in other three bleeding models, as illustrated in Figure 9C−E. These results indicated that different mechanisms might be involved in the hemostatic processes for locally applied or i.v. injected nanoparticles. It has been demonstrated that the size of particles has a very important effect on their margination that refers to the distribution propensity of circulating particles toward the vessel walls.64 While margination of particles is enhanced by increased size, smaller particles display reduced margination propensity since they tend to flow along with blood.65 Additionally, in the bloodstream, platelets circulate in close proximity to the vascular wall because they are physically excluded from bulk flow by red blood cells, largely resulting from their size, shape, and flexibility.65 Consequently, i.v. administered nanoparticles with larger sizes may have more chance to contact platelets, thereby leading to more potent bleeding prevention capability when compared with the smaller particles. Nevertheless, we cannot exclude the possibility that i.v. injected nanohemostats may target injury sites and prevent bleeding through other different mechanisms since these nanoparticles can interact with diverse blood cells and the full spectrum of biomolecules in plasma. Indeed, augmented vascular targeting and enhanced thrombosis were achieved by different nanoplatforms via interactions with varied blood elements.66−68

EXPERIMENTAL SECTION Materials. Cholic acid (CA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), deoxycholic acid (DOCA), dehydrocholic acid (DHCA), lithocholic acid (LCA), N-hydroxysuccinimide (NHS), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl), and branched polyethylenimine with molecular weight (Mw) of 1.8 kDa (bPEI1.8) or 25 kDa (bPEI25) were purchased from Sigma-Aldrich (USA). Linear polyethylenimine with Mw of 10 kDa (lPEI10) and a negatively charged polystyrene (PS) nanoparticle (NP) (polybead carboxylate microspheres 0.2 μm) were obtained from Polysciences, Inc. (USA). Antiserum albumin and antifibrinogen-β antibodies were received from Santa Cruz Biotechnology, Inc. FITC-labeled hamster anti-mouse CD61 antibody (FITC-CD61) was supplied by BD Biosciences (USA). Cyanine 5 NHS ester (Cy5), cyanine 7.5 NHS ester (Cy7.5), and cyanine 7.5 amine were provided by Lumiprobe (USA). FITC-labeled goat anti-rabbit IgG (H+L) and FITC-labeled goat anti-mouse IgG (H+L) were purchased from Beyotime Biotechnology (China). Adenosine 5′-diphosphate (ADP) was supplied by Aladdin Reagent Co., Ltd. (Shanghai, China). All the other reagents are commercially available and used as received. Preparation of Positively Charged Nanoparticles by SelfAssembly. Self-assembly of nanoparticles was carried out by a dialysis procedure.42 In the case of branched PEIs, the pairs of various cholic acids and bPEIs at defined weight ratios were dissolved in a common solvent of DMSO. The polymer concentration was maintained at 10 mg/mL in all cases for the preparation of assembled positive nanoparticles. Thus, obtained solution was dialyzed against deionized water at 25 °C. The outer aqueous solution was exchanged every 2 h. After 24 h of dialysis, samples were collected for further analysis without any other treatments. For the preparation of nanoparticles based on a linear PEI of lPEI10, lPEI10 was dissolved in deionized water at 10 mg/mL, into which DMSO solution containing UDCA was added dropwise under sonication. Thus, obtained solution mixture was subjected to dialysis procedures similar to those employed for bPEIs. Cholic acid and its derivatives including CA, UDCA, CDCA, HDCA, DOCA, DHCA, and LCA were utilized to prepare nanohemostats. Fabrication of Fluorescence-Labeled Nanoparticles. Either Cy5-labeled bPEI25 (Cy5-bPEI25) or Cy7.5-labeled bPEI25 (Cy7.5bPEI25) was synthesized by reaction of 2.4 mg of Cy5 or Cy7.5 NHS ester with 10 mg of bPEI25 in 1 mL of DMSO at 40 °C under dark conditions. After 12 h, Cy5-bPEI25 or Cy7.5-bPEI25 was collected, which was employed to prepare fluorescent nanoparticles in the presence of UDCA, with a UDCA/Cy5-bPEI25 or UDCA/Cy7.5bPEI25 weight ratio of 2:1, according to the above-mentioned procedures. To prepare the Cy7.5-labeled negatively charged PS nanoparticle (Cy7.5-PS NP), 4 mg of NHS and 12 mg of EDC·HCl were added into 16 mL of PBS (at pH 7.4) containing 50 mg of PS NP, and the obtained suspension was magnetically stirred at room temperature for 12 h. Then 0.8 mg of Cy7.5 amine was added, and the reaction was performed at 4 °C for 24 h. The labeled nanoparticle Cy7.5-PS NP was subsequently collected by centrifugation at 21 750g for 10 min, followed by thorough washing. Characterization of Nanoparticles. FTIR spectra were recorded on a PerkineElmer spectrometer (100S, USA). 1H NMR spectra were acquired on an Agilent NMR system (600 MHz DD2, USA). Particle size and ζ-potential measurements were conducted on a Malvern Zetasizer Nano ZS instrument at 25 °C. Transmission electron microscopy observation was performed on a TECNAI-10 microscope

CONCLUSIONS In summary, positively charged PEI nanoparticles can be efficiently prepared by cholic-acid-mediated self-assembly of PEIs with either branched or linear architecture. Regardless of different structures of cholic acids and PEIs, well-shaped nanoparticles with relatively narrow size distribution could be formed. Both molecular simulation and experimental characterizations demonstrated that assembly of these nanoparticles was driven by multiple noncovalent forces between cholic acid and PEI, including electrostatic, H-bonding, and hydrophobic interactions. These assembled nanoparticles were able to induce platelet aggregation and platelet activation in both whole blood and platelet-rich plasma. Whereas PEI itself could staunch bleeding in different animal models including mouse tail amputation as well as liver injury and femoral artery injury in rats or rabbits, assembled nanoparticles showed more potent hemostatic effects independent of different PEIs and cholic acids. Moreover, i.v. injection of different nanoparticles could effectively reduce bleeding times of femoral artery injury in rats by preferential accumulation at the injured vascular site. Furthermore, preliminary in vivo experiments in rats revealed that i.v. administration of relatively low doses of nanoparticles assembled by UDCA and branched PEI with Mw of 25 kDa showed good safety profile. These findings suggested that 9969

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extracted, and platelets were reconstituted in PPP to a final concentration of 5 × 108 platelets/mL. In Vitro Examination of Aggregation and Activation of Platelets. In 96-well plates, 100 μL of reconstituted platelets in PPP at 5 × 108 platelets/mL was added to each well, in which 10 μL of UDCA/bPEI25 (at a weight ratio of 2:1) assembled nanoparticles at 2.5 mg/mL was added. In the control group, the same volume of saline was added. Immediately after this procedure, samples were fixed for 1 h by adding an equal volume of 2% glutaraldehyde, and the 96-well plate was agitated for 1 min on an orbital shaker at 180 rpm. After incubation for 10 min at room temperature, samples were observed by phase contrast microscopy (Olympus CKX41, Japan). To prepare specimens for scanning electron microscopy, samples were fixed by 2% glutaraldehyde and dehydrated with an ethanol gradient. Subsequently, specimens were mounted on freshly cleaved mica and coated with gold−palladium. SEM images were taken on a S-3400N II electron microscope (Hitachi, Japan). To determine the platelet aggregation curve by a resistance method, 1.0 mL of blood anticoagulated with heparin was treated with 50 μL of aqueous solution containing either UDCA/bPEI25 nanoparticles (UDCA/bPEI25 = 2:1) or a platelet agonist ADP at 37 °C under magnetic stirring at 1200 rpm. The final concentration of UDCA/ bPEI25 nanoparticles was 0.25 or 0.5 mg/mL, and the dose of ADP was 10 or 100 μM. After 5 min, platelet aggregation curves were measured for 8 min by a resistance aggregometer (Chrono-Log Corp, Haventown, PA). The aggregation rate and the slope were then calculated. In vitro aggregation and activation of platelets were also examined by immunofluorescence analysis. To this end, 500 μL of freshly prepared PRP was suspended in 3 mL of HEPES buffer, into which 50 μL of FITC-CD61 (0.5 mg/mL) was added, followed by incubation at room temperature for 40 min. Then the samples were centrifuged at 1600g for 5 min. After the buffer was removed, platelets were reconstituted in HEPES buffer to a final concentration of 5 × 108 platelets/mL. Subsequently, 150 μL of this HEPES suspension containing 5 × 108 platelets/mL was added to each well. This was followed by the addition of 15 μL of UDCA/Cy5-bPEI25 nanoparticles (the weight ratio was 2:1, 2.5 mg/mL), while HEPES buffer was added as a control. Immediately following this procedure, samples were fixed for 1 h using 2% glutaraldehyde by incubation for 10 min at room temperature. Samples were then imaged by confocal laser scanning microscopy on a fluorescence microscope (Leica, Heidelberg, Germany). In Vivo Hemostasis in a Mouse Tail Transection Model. C57BL/6 mice (18−23 g) were employed in this experiment. Typically, the tail was cleaned and disinfected. Then the tail was amputated at a site 5 mm from the end of tail, and the tail was immersed in saline, bPEI25 solution (2.5 mg/mL), or aqueous solution containing UDCA/bPEI25 nanoparticles (2.5 mg/mL) or PS nanoparticles (2.5 mg/mL) in tubes that were maintained at 37 °C. The clinically used absorbable gelatin sponge (Jinling Pharmaceutical Co., Ltd., China) was used as the positive control, which was directly applied to the injured site. The tail bleeding time, that is, the time required for bleeding to cease (no blood flow within 1 min), was determined as described previously.69 The bleeding time was taken as the average value of three independent experiments. Based on the similar procedures, the hemostatic capability of nanoparticles assembled by UDCA and different PEIs or assembled with bPEI25 and various cholic acids was examined. All the procedures were performed by the same operator blinded to the treatment groups. In Vivo Hemostasis in a Liver Injury Model in Rats and Rabbits. Sprague−Dawley rats (230−250 g) were anesthetized, and the abdomen was opened in the rostral-to-caudal direction to expose the liver. Injury of 2 mm deep into the left lobe of the liver, with a length of 0.6−0.8 cm, was cut using a scalpel, separating the two halves of the lobe transversely.31 Subsequently, 300 μL of aqueous solution containing various assembled nanoparticles was applied to the site of injury by a 27-gauge needle. In the control groups, the same volume of saline or bPEI solution at 2.5 mg/mL was applied to the injured site. All the procedures were performed by the same operator blinded to

(Philips, The Netherlands) operating at an acceleration voltage of 80 kV. Formvar-coated copper grids were used to prepare specimens by dipping the grid into aqueous solution of various nanoparticles, and extra solution was blotted with filter paper. After water was evaporated at room temperature, samples were observed directly without staining. Molecular Modeling of Interactions between UDCA and PEI. Interactions between a branched PEI (bPEI) and a typical cholic acid derivative UDCA were theoretically interrogated by molecular modeling via molecular docking and molecular dynamic (MD) simulation according to the literature.41 Specifically, the repeat unit of bPEI was built in 3D coordinates using the MOE’s (molecular operating environment software package, Chemical Computing Group, Canada) builder tool. The polymer chain was built by a head-to-tail connection with 40 structural units, and five repeat units were employed to build a short-chain oligopolymer to simulate polymer−polymer interactions in the docking process. The 3D structures of UDCA, bPEI, short-chain bPEI, and water molecules were preoptimized before running simulation using the all atom MMFF94x force field with no constraints. Subsequently, UDCA and preoptimized water molecules were docked into the minimized, hydrated bPEI structures using the AutoDock4.2 software package to estimate the binding energy and intermolecular energies. UDCA was docked into the minimized UDCA molecule to estimate UDCA− UDCA interactions. To find the effect of bPEI conformation on UDCA−bPEI interactions, the UDCA/bPEI complex with the lowest energy, obtained from docking, was employed to perform MD simulations in water environment using MOE software. Finally, the conformation of bPEI obtained from MD stimulations was used in docking studies to predict changes in intermolecular interactions and putative binding sites between bPEI25 and UDCA. Animals. Male Sprague−Dawley rats (230−250 g), male C57BL/6 (18−23 g), and male rabbits (2.0−2.5 kg) were obtained from the Animal Center at the Third Military Medical University. Animals were housed in standard rat/mouse/rabbit cages under conditions of optimum light and temperature at 22 ± 1 °C, with ad libitum access to water and food. All the animals were acclimatized to the laboratory for at least 3 days before experiments. Animal care and experiments were carried out in accordance with procedures approved by Animal Care and Use Committees of Third Military Medical University. Examination of In Vitro Plasma Protein Absorption on UDCA/bPEI25 Nanoparticles. First, proteins adsorbed on nanoparticles were quantified by proteomic analysis. Specifically, 500 μL of aqueous solution of UDCA/bPEI25 nanoparticles (UDCA/bPEI25 = 2:1, 2.5 mg/mL) was incubated with 5 mL of rat plasma freshly collected from male Sprague−Dawley rats. After 5 min of incubation, nanoparticles were collected by centrifugation at 21 750g. Proteins were eluted from the recovered nanoparticles by adding an equal volume of lysis buffer containing 8 M urea and 2% CHAPS and incubation at 95 °C for 5 min and precipitated using the ProteoExtract kit (Merck KGaA, Darmstadt, Germany).51 The precipitated proteins were then solubilized in 25 mM ammonium bicarbonate containing 0.1% RapiGest (Waters, USA) at 80 °C for 15 min. Proteins were reduced by adding 5 mM dithiothreitol and incubation at 56 °C for 45 min. Free cysteines were alkylated with 15 mM of iodoacetamide (Sigma, USA) at 25 °C for 1 h in the dark. Subsequently, 0.2 μg sequencing grade modified trypsin (Promega, USA) was added, and the samples were incubated overnight at 37 °C. After digestion, RapiGest SF was hydrolyzed by adding 10 mM HCl, followed by incubation at 37 °C for 10 min. The resulting precipitate was removed by centrifugation at 13 000g for 15 min at 4 °C, and the supernatant was transferred into autosampler vials for peptide analysis via a LTQ Orbitrap Velos Pro mass spectrometer. Collection of Platelets. After rats were anesthetized, blood was collected and stored in anticoagulant tubes. In addition, platelet-rich plasma (PRP) was prepared by centrifugation of blood at 85g for 10 min at room temperature. After the resulting PRP supernatant was harvested, the residue was centrifuged at 765g for 10 min to obtain platelet-poor plasma (PPP). Then PRP was resuspended in HEPES buffer and centrifuged at 800g for 10 min. Subsequently, buffer was 9970

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ACS Nano the treatment groups. The time for complete arrest of bleeding (no blood flow within 10 s) was recorded in three independent experiments by the same researcher. In a separate study, the hemostatic activity of UDCA/bPEI25 nanoparticles (2.5 mg/mL) was compared with PS NP (2.5 mg/mL) and a gelatin sponge. Also, a liver injury model was established in rabbits (2.0−2.5 kg). In this case, injury with a depth of 2 mm and a length of 1.0−1.2 cm was cut in the left lobe of the liver. Subsequently, 1.0 mL of aqueous solution containing bPEI25, UDCA/bPEI25 nanoparticles, or PS NP at the same concentration of 2.5 mg/mL was applied to the site of injury by a 27-gauge needle. In the control group, the same volume of saline was used. In addition, a gelatin sponge was used as the positive control, which was directly applied to the injured site. The time for complete arrest of bleeding was recorded according to the aforementioned method. Hemostasis in a Femoral Bleeding Model in Rats. Sprague− Dawley rats (230−250 g) were utilized in this cohort of study. After anesthesia of rats, the skin was removed, and the overlying muscles were cut to expose the femoral artery and sciatic nerve.31 Injury at the middle segment of the femoral artery was induced by puncture with a 26-gauge needle. Then 300 μL of saline, bPEI solution at 2.5 mg/mL, or aqueous solutions containing different nanoparticles was separately applied over the injured site by a 27-gauge needle. In a separate study, the hemostatic capability of UDCA/bPEI25 nanoparticles was compared with that of PS NP and a gelatin gel. The time for complete arrest of bleeding (no blood flow within 10 s) was determined. Three independent experiments were performed. All the procedures were performed by the same operator blinded to the treatment groups. The same bleeding model was established in rabbits (2.0−2.5 kg). In this case, injury at the middle segment of the femoral artery was induced by a 23-gauge needle. Then the similar treatment regimens as those used in the rat model were adopted to interrogate the hemostatic activity of various agents. In Vivo Prevention of Bleeding by Intravenously Injected Nanoparticles. After Sprague−Dawley rats (230−250 g) were anesthetized, saline, bPEI solution (2.5 mg/mL), or aqueous solution containing different nanoparticles (concentrations varying from 1.0, 2.0, to 4.0 mg/mL) was i.v. injected. The total dose of bPEI and different nanoparticles was 6.0 mg/kg of body weight. After 5 min of administration, injury at the middle segment of the femoral artery was induced by puncture with a 26-gauge needle. Then the time required to cease bleeding for at least 10 s was recorded as the bleeding time. Four independent experiments were carried out. In the same bleeding model in rats and through the similar procedures, we compared the hemostatic capability of UDCA/bPEI25 nanoparticles and PS NP. Of note, all the procedures were performed by the same operator blinded to the treatment groups. Ex Vivo Imaging of Accumulation of Nanoparticles at the Injured Femoral Artery Site after Intravenous Injection in Rats. After anesthesia of Sprague−Dawley rats (230−250 g), UDCA/Cy7.5bPEI25 nanoparticles (the weight ratio of UDCA/Cy7.5-bPEI25 was 2:1) in saline was i.v. administered via tail vein at 6.0 mg/kg. The control rats received saline injection. After 5 min, an injury was induced by puncture with a 26-gauge needle in the femoral artery. Then the injured segment of the femoral artery was isolated. In another control group, rats without injury in the femoral artery were treated i.v. injection with the same dose of UDCA/Cy7.5-bPEI25 nanoparticles. After 5 min, a segment of the femoral artery at the same location as that in the injured group was collected. Ex vivo fluorescence imaging was performed on a living imaging system (IVIS Spectrum, PerkinElmer, USA). Following the similar procedures, the accumulation of positively charged UDCA/bPEI25 nanoparticles at the injured site was compared with that of PS NP. Both of them were labeled with Cy7.5 and administered at the same dose of 6.0 mg/kg via i.v. injection. Immunofluorescence Analysis of Serum Albumin and Fibrinogen Absorbed on UDCA/bPEI25 Nanoparticles Accumulated at the Injured Site after Intravenous Injection. Absorption of typical proteins including serum albumin and fibrinogen

on nanoparticles was also analyzed by immunofluorescence. In brief, 5 min after i.v. injection of UDCA/Cy5-bPEI25 nanoparticles (UDCA/ Cy5-bPEI25 = 2:1) in Sprague−Dawley rats (230−250 g) at 6.0 mg/ kg, an injury in the femoral artery was induced by puncture with a 26gauge needle. Immediately after hemostasis, the blood clots and injured vessels were collected and embedded in OCT. Sections were then prepared and washed with PBS (0.01 M, pH 7.4) six times to remove the remaining fixative, followed by separate incubation with the primary antibody of serum albumin or fibrinogen-β overnight. After being thoroughly washed with PBS to remove the unbound primary antibody, tissue sections were then kept in the dark and incubated with a FITC-labeled secondary antibody for 1 h at 37 °C. Subsequently, the unbound secondary antibodies were removed by washing in PBS. Observation was carried out by confocal laser scanning microscopy. In Vivo Circulation of UDCA/bPEI25 Nanoparticles. To interrogate in vivo pharmacokinetic profiles of UDCA/bPEI25 nanoparticles after i.v. injection, 0.5 mL of aqueous solution containing UDCA/Cy7.5-bPEI25 nanoparticles (the UDCA/Cy7.5-bPEI25 weight ratio was 2:1) was i.v. injected via tail vein in Sprague−Dawley rats (230−250 g) at 6.0 mg/kg. Immediately after administration, blood was collected at predetermined time points. Fluorescence imaging of blood samples in 96-well plates was performed via a living imaging system (IVIS Spectrum, PerkinElmer, USA). The fluorescence intensity was quantified and normalized to that at 0 h. Tissue Distribution of Nanoparticles after Intravenous Injection. To evaluate tissue distribution and clearance of UDCA/ bPEI25 nanoparticles and PS NP, 0.5 mL of aqueous solution containing Cy7.5-labeled nanoparticles was administered to rats via i.v. injection at 6.0 mg/kg. At 3 h or 7 days after administration, rats were euthanized and main organs including heart, liver, spleen, lung, and kidney were isolated for fluorescence observation by a living imaging system. Preliminary Acute Toxicity Evaluation on Nanoparticles after Intravenous Injection. As a preliminary study on the safety profile of assembled nanohemostats, UDCA/bPEI25 nanoparticles were examined after i.v. injection in rats. Specifically, 12 male Sprague−Dawley rats (180−200 g) were randomly assigned into two groups (n = 6 in each group). UDCA/bPEI25 nanoparticles in saline was i.v. administered at a total dose of 24 mg/kg. In the control group, rats were i.v. injected with saline. Post-administration, body weight of rats was monitored at defined time points and their general behaviors were observed. After 2 weeks, animals were euthanized. Blood samples were collected for hematological analysis (Sysmex KX-21, Sysmex Co., Japan). Major organs including heart, liver, spleen, lung, kidney, and thymus were isolated and weighed. The organ index was calculated as the ratio of organ weight to the body weight of each rat. In addition, histopathological sections of collected organs were prepared and stained with H&E, followed by imaging with optical microscopy. Statistical Analysis. Statistical analysis was performed by SPSS 13.0 using the one-way ANOVA test. The p < 0.05 is considered to be statistically significant.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b04124. Figures S1−S15 (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9971

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