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Alleviation of Polycation-Induced Blood Coagulation by the Formation of Polypseudorotaxanes with Macrocyclic Cucurbit[7]uril Qiaoxian Huang,† Qian Cheng,† Xiangjun Zhang,† Hang Yin,†,‡ Lian-Hui Wang,‡ and Ruibing Wang*,† †
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau, China ‡ Key Laboratory for Organic Electronics and Information Displays, Jiangsu Key Laboratory of Biosensors, Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, 9 Wenyuan Road, Nanjing 210023, China ACS Appl. Bio Mater. Downloaded from pubs.acs.org by DURHAM UNIV on 09/07/18. For personal use only.
S Supporting Information *
ABSTRACT: Hexadimethrine bromide (HB), a polycation to neutralize heparin and control internal bleeding, may also cause serious blood coagulation that may be life-threatening. Reversal of HB by heparin and vice versa might potentially lead to a vicious circle of alternative bleeding and blood coagulation. In this Letter, a biocompatible synthetic nanoreceptor, cucurbit[7]uril (CB[7]), was demonstrated to dramatically alleviate HB-induced blood coagulation in vitro as well as in vivo, through the formation of HB@CB[7] polypseudorotaxanes. This discovery, for the first time, suggests the significant clinical potential of a synthetic receptor in alleviating the side effects and toxicity of a polycationic drug. KEYWORDS: cucurbit[7]uril, hexadimethrine bromide, polypseudorotaxanes, blood coagulation
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the red blood cells (RBCs), these polycations (including protamine and HB) could induce RBCs’ aggregation and subsequent blood coagulation by interacting with RBCs via electrostatic interactions.6 Consequently, intravenous injection of overdose polycations (such as HB) often induces pulmonary hypertension due to the physical embolization of the pulmonary vessels by RBCs aggregation/coagulation.7 Further reversing HB with heparin would lead to a vicious cycle of risks of coagulation and bleeding in the circulatory system. Thus, an ideal solution is to use electrostatically neutral species that are inert in the circulatory system, to neutralize HB in vivo. Cucurbit[n]urils (CB[n], typically n is 5, 6, 7, 8, or 10) are a family of rigid, pumpkin-shaped, synthetic cyclic oligomers with n representing the number of glycoluril units that are interconnected by methylene bridges. Each CB[n] possesses one nanosized hydrophobic cavity and two openings laced with carbonyl groups.8 The neutral yet dipole-negative carbonyl portals of CB[n] have enabled these macrocyclic nanoreceptors to complex various cationic molecules with relatively strong binding affinities.8−10 Within this family of host molecules, CB[7] (Figure 1) seems to be the most popular nanoreceptor in pharmaceutical and biomedical sciences,
recisely balancing between bleeding and blood clotting is always a challenging task.1 For instance, systemic heparinization is commonly employed to prevent deep vein and arterial thrombosis, pulmonary embolism, and arterial thromboembolism during or after a surgery; subsequently, heparin neutralizers are often administered to play against the anticoagulation effect of the extra heparin, particularly for those patients with risks of internal hemorrhage and stroke.2 As a FDA-approved heparin neutralizer, protamine sulfate is generally used for the treatment of heparin overdose.3 For a large number of patients exhibiting an allergic response to protamine,4 a protamine-like, synthetic polycationic species, hexadimethrine bromide (HB, also known as polybrene) (Figure 1) replaces protamine to neutralize heparin in clinics.5 However, due to the relatively high negative-charge density on
Figure 1. Molecule structures of cucurbit[7]uril (left) and hexadimethrine bromide (right). The protons of hexadimethrine bromide are letter labeled. © XXXX American Chemical Society
Received: August 6, 2018 Accepted: September 4, 2018 Published: September 4, 2018 A
DOI: 10.1021/acsabm.8b00401 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
Figure 2. (a) 1H NMR spectra of HB (1.0 mM with regard to the hexyl group) in deuterated water, without and with 0.5 and 1.5 equiv of CB[7]. CB[7] and HDO’s proton resonances were, respectively, labeled with ● and ○. (b) Scheme of HB@CB[7] host−guest complexes. (c) Top: Thermogram of titrating 0.04 mL of CB[7] (1.0 mM) into 0.2 mL of HB solution (0.1 mM with regard to its repeating unit) (pH 7.4) at 25 °C. Bottom: The plot of enthalpic changes during the above-described titration, against the molar ratios of host/guest (“one set of binding sites” isothermal model).
Figure 2b). This hypothesis was further supported by the observation of downfield shifts of Hd (Δδ = 0.062), He (Δδ = 0.243), and Hf (Δδ = 0.460), implying that these protons were located near the carbonyl laces, but outside the pocket of CB[7], thus experiencing deshielding effects. Furthermore, the complexation behaviors (including the binding stoichiometry, binding affinity Ka, and thermodynamic information such as ΔH and TΔS) between CB[7] and HB were investigated by isothermal titration calorimetry (ITC) in an aqueous solution. As shown in Figure 2c, the thermogram evolved during the complexations of HB and CB[7], as measured by ITC, afforded a complexation constant of 1.04 (±0.19) × 107 M−1 and defined the binding ratio of 1:1 between the repeating unit of HB and the host under neutral conditions. Additionally, the respective enthalpic (ΔH) and entropic changes (TΔS) of −31.3 and 8.74 kJ/mol, derived from the same set of ITC titration curve (Figure 2c) suggest that the complexation process was mainly enthalpy-driven, likely attributed to both Coulombic forces and H-bonding between the HB and the synthetic receptor. Such a strong complexation affinity of HB@ CB[7] may ensure a competitive complexation of HB by CB[7] even under in vivo conditions. It was previously reported that HB can induce RBCs aggregation by diminishing intercellular distances.19 To assess whether CB[7] is able to alleviate HB-induced RBCs agglutination in vitro, as a result of threading of HB by CB[7], a hemagglutination assay was performed and the cells were examined by optical microscopy. As shown in Figure 3, RBCs treated with HB (0.5 mM and 1.0 mM) exhibited obvious agglutination in a dose-dependent manner, consistent with a previous report.20 Conversely, very minor agglutination was observed when the RBCs were incubated with HB accompanied by CB[7] (1.0 equiv to the repeating unit of HB), implying that the threading of HB by CB[7] dramatically alleviated its RBC-aggregation-inducing activity, likely due to the supramolecular complexation of HB by CB[7] and formation of polypseudorotaxane. Meanwhile, CB[7] alone did not exhibit any observable side effects in vitro, similar to the control group, implying its bioinert nature in this system. Inspired by the promising results with the RBC cell line, we further examined if CB[7] may alleviate HB-induced RBC
attributed to its biocompatibility and reasonable watersolubility.11,12 For instance, Zhang et al. reported that encapsulation of a small cationic molecule, viologen, by CB[7], significantly attenuated its cytotoxicity in vitro.13 We have recently demonstrated that CB[7] not only inhibited a small toxic molecule-induced seizure in both zebrafish and mice models14 but also concealed the taste of the bitterest substance in the world.15 In addition to complexing small cationic, bioactive molecules and modulating their toxicity and bioactivity, CB[7] also exhibited complexation properties with polycations. For instance, Zhang et al. demonstrated that CB[7] formed polypseudorotaxane with polylysine and turned off its antibacterial activity in vitro.16 Similarly, CB[7] was found to complex with polyethylenimine and a cationic dendrimer, respectively, by our research group and Kim’s research group and subsequently to reduce these polycations’ cytotoxicity without compromising their gene transfection efficiencies when employed as gene carriers in vitro.17,18 With the preliminary success of polycationic toxicity modulation in vitro, we hypothesized that directly threading CB[7] onto a HB polymeric chain may form relatively stable polypseudorotaxanes, and the positive charges of HB would be effectively shielded by the carbonyl portals of CB[7] via cation−dipole interactions. As a consequence, HB-induced RBCs aggregation and blood coagulation would be significantly alleviated in vitro and in vivo without the risk of causing further hemorrhage that would be otherwise caused by heparin as a neutralizing agent, as CB[7] is likely bioinert in the circulatory system. This would be the first in vivo study of the protective effects of CB[7] on the side effects of a polycationic species. We first examined whether the repeating hexyl unit (with an ammonium ion on each side) of HB is encapsulated by the cavity of CB[7] by 1H NMR spectroscopy accompanied by 2D COSY NMR spectroscopy. As shown in Figure 2a and Figure S1, with CB[7] (concentration varied from 0 to 1.5 equiv of the hexyl unit) present under neutral pH conditions, all of the protons of the hexyl unit (Ha,b,c) of HB experienced a significant shift to the upper field, with Δδ values (complexation-induced shifts) of −0.857, −0.717, and −0.560 ppm for Ha, Hb, and Hc, respectively, suggesting that the hexyl chain was entirely included inside the CB[7]’s cavity (as shown in B
DOI: 10.1021/acsabm.8b00401 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
HB were dramatically alleviated by CB[7], likely due to supramolecular complexation and formulation of polypseudorotaxanes. These observations, in line with the results in vitro, further supported our hypothesis. To further examine if CB[7] may inhibit HB-caused blood coagulations directly in the circulatory system in vivo, mice were systemically evaluated upon i.v. administration with a single dose of normal saline (control group), CB[7] (240 mg/ kg), and HB (20 mg/kg) with and without CB[7] (120 and 240 mg/kg, in a molar ratio of 1:1 and 1:2 HB@CB[7]), respectively. The survival rate and body weight of the mice (n = 8 per group) were monitored for 15 days post administration. As shown in Figure 5a, the mice administered with free HB at a dose of 20 mg/kg died within an hour after administration, implying its significant cytotoxicity when overdosed. In contrast, the survival rates of the mice treated with HB (20 mg/kg) in the presence of 120 mg/kg and 240 mg/kg CB[7] after a 15-day follow-up were 80% and 100%, respectively. These results strongly suggest that CB[7] dramatically alleviated the toxicity of HB in vivo in a dosedependent manner and that an excess amount of CB[7] may completely inhibit HB’s toxicity from a survival rate perspective. The requirement of an excess amount of CB[7] to completely inhibit the toxicity of HB in vivo is likely attributed to the highly complexed environment in the circulatory system due to the presence of a variety of endogenous and exogenous species that might competitively bind with CB[7]. In addition, CB[7] alone at the dose up to 240 mg/kg did not cause any casualty, confirming its iv biosafety at this level. Indeed, very recently, we have demonstrated the superior biocompatibility of CB[7] in vivo, and the maximum tolerable dose (MTD) CB[7] in mice via fast i.v. administration was defined as below 200 mg/kg.22 In the current study, a slightly higher dose (240 mg/kg) did not induce observable toxicity in this mouse model, as we used a slow infusion technique, different from the fast infusion technique in the previous study. Accordingly, the 15-day bodyweight growth curve of the mice treated with CB[7] at 240 mg/kg is similar to that of the control group (Figure 5b). As expected, CB[7] alleviated HB-induced body weight variation in a dose-dependent manner (Figure 5b). The body weight of the mice administered with HB (20 mg/kg) in the presence of CB[7] (120 mg/kg) decreased during the first 2 days after the single-dose administration and subsequently recovered gradually from the third day onward. The average
Figure 3. Microscopic images of RBCs treated with a variety of concentrations of HB (0, 0.5, and 1.0 mM) without and with 1.0 equiv of CB[7], respectively, during the hemagglutination assay.
aggregation and blood coagulation in a mouse model. A mouse tail transection bleeding model (Figure 4a) previously reported as a suitable in vivo model to study hemorrhage control was employed in this investigation.21 Male C57BL/6 mice were randomly selected and placed in a prone position after general anesthesia by chloral hydrate aqueous solution (10% w/v, 0.005 mL/g). A distal 0.5 cm segment of each mouse tail was amputated with a scalpel. Each of the tails was immediately immersed in a test tube that had 2 mL of normal saline in the absence (control) or in the presence of HB (0.4 mM), CB[7] (0.4 mM), and HB@CB[7] (0.4 mM/0.4 mM), respectively, prewarmed in a water bath to 37 °C. Both the total time of bleeding and total volume of lost blood were subsequently evaluated for each mouse (with the experimental details described in the Supporting Information). As shown in Figure 4b,c, the bleeding time and total blood-loss volume for the mouse tails treated with CB[7] alone (0.4 mM) were 129.57 ± 19.62 min and 103.56 ± 24.42 μL, respectively, without significant differences from those measured for the control group (104.12 ± 13.00 min, 99.84 ± 24.59 μL), suggesting the biocompatibility and bioinert nature of CB[7] in this system. As expected, the mouse tails treated with HB (0.4 mM) exhibited a significantly reduced bleeding time and total bloodloss volume (35.09 ± 5.80 min, 11.90 ± 4.48 μL) in the mouse tail transection model, likely due to the RBC aggregation and blood coagulation effects of this polycation, as previously discussed. In contrast, HB@CB[7]-treated mouse tails considerably normalized the bleeding time and blood-loss volume to 105.45 ± 18.00 min and 91.58 ± 19.97 μL, respectively, suggesting that the blood coagulation effects of
Figure 4. Schematic diagram of the mouse tail transection model (a). Quantification of the total time of bleeding (b) and volume of lost blood (c) of mouse tails treated in saline with and without CB[7] (0.4 mM), HB (0.4 mM), and HB@CB[7] (0.4 mM/0.4 mM), respectively. Data presented are the mean ± SEM (n = 6). *P < 0.05; ns represents “no significant difference” between the experimental group and the control group. C
DOI: 10.1021/acsabm.8b00401 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
Figure 5. Top: Survival rates (a) and body weight changes (b) of C57BL/6 mice after a single-dose i.v. injection of saline, CB[7] (240 mg/kg) and HB (20 mg/kg) without and with CB[7] (120 and 240 mg/kg, respectively). Data are presented as mean value ± SEM (n = 8). The control group, CB[7] 240 mg/kg, and HB + CB[7] 240 mg/kg group are overlapped on the survival curve (100% survival) in panel a. (c) Schematic diagram showing pulmonary embolism (i) and H&E stained histological sections of the lung issues isolated from the mice administered with a single-dose i.v. injection of saline (ii), CB[7] (240 mg/kg) (iii), and HB (20 mg/kg) without (iv) and with CB[7] (120 and 240 mg/kg, respectively) (v and vi), respectively. The green arrow shows a mixed thrombus, the black arrow shows the congestion of the alveoli, and the red arrow indicates the red thrombus.
that CB[7] significantly alleviated the HB-induced thrombus and hypoxic damage of the lungs via strong host−guest interactions. In summary, we demonstrated that the synthetic nanoreceptor, CB[7], significantly alleviated polycation-induced blood coagulation effects both in vitro and in vivo, likely attributed to threading of the polycation by CB[7] and formulation of polypseudotoxanes. This discovery exhibits the significant potential of CB[7] in clinical detoxification or reversal of polycationic agents.
body weight was, however, consistently lower than that of the control group during the 15-day period. On the other hand, the mice administered with HB (20 mg/ kg) in the presence of a higher dose of CB[7] (240 mg/kg) experienced a moderately decreased body weight during the first day and subsequently recovered quickly from the second day onward. Since the seventh day, the average body weight of this group of mice was almost identical to that of the control group, implying that excess amount of CB[7] might be necessary to completely inhibit the toxic effects of HB in vivo. This is particularly important clinically that the excess agent has to be biocompatible and that a large excess would not cause any hematological effects that heparin would otherwise induce, and CB[7] seems to fulfill these requirements. It was previously reported that an overdose of HB or polycation may cause blood coagulation in the circulatory system, which would cause pulmonary hypertension and life-threatening lung embolization.7 Thus, the lung tissues of the mice in the control, CB[7]-treated, and HB (without and with CB[7])treated groups were, respectively, collected for pathohistological analysis. As shown in Figure 5c, mixed thrombosis, alveoli congestions, and inflammatory cells infiltration were clearly visible in the lung tissue from the mice treated with free HB (at a dose of 20 mg/kg). The HB@CB[7]-treated groups exhibited normal lung parenchyma, similar to those observed in the control group and CB[7]-treated group, demonstrating
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00401. Experimental details, materials and instrumentation, 1H NMR titration, ITC experiment, hemagglutination assay, animal assay, mouse tail transection bleeding model, in vivo safety toxicity evaluation of HB in mice, statistical analysis, and 2D COSY NMR spectra of HB in D2O in the absence and presence of 1.5 equiv of CB[7] (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. D
DOI: 10.1021/acsabm.8b00401 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials ORCID
(17) Lim, Y. B.; Kim, T.; Lee, J. W.; Kim, S. M.; Kim, H. J.; Kim, K.; Park, J. S. Self-Assembled Ternary Complex of Cationic Dendrimer, Cucurbituril, and DNA: Noncovalent Strategy in Developing a Gene Delivery Carrier. Bioconjugate Chem. 2002, 13 (6), 1181−1185. (18) Huang, Q.; Li, S.; Ding, Y.-F.; Yin, H.; Wang, L.-H.; Wang, R. Macrocycle-Wrapped Polyethylenimine for Gene Delivery with Reduced Cytotoxicity. Biomater. Sci. 2018, 6 (5), 1031−1039. (19) Liu, Z.; Jiao, Y.; Wang, T.; Zhang, Y.; Xue, W. Interactions between Solubilized Polymer Molecules and Blood Components. J. Controlled Release 2012, 160 (1), 14−24. (20) Li, C.; Zhong, D.; Zhang, Y.; Tuo, W.; Li, N.; Wang, Q.; Liu, Z.; Xue, W. The Effect of the Gene Carrier Material Polyethyleneimine on the Structure and Function of Human Red Blood Cells in Vitro. J. Mater. Chem. B 2013, 1 (14), 1885−1893. (21) Cheng, J.; Feng, S.; Han, S.; Zhang, X.; Chen, Y.; Zhou, X.; Wang, R.; Li, X.; Hu, H.; Zhang, J. Facile Assembly of Cost-Effective and Locally Applicable or Injectable Nanohemostats for Hemorrhage Control. ACS Nano 2016, 10 (11), 9957−9973. (22) Zhang, X.; Xu, X.; Li, S.; Wang, L.-H.; Zhang, J.; Wang, R. A Systematic Evaluation of the Biocompatibility of Cucurbit[7]uril in Mice. Sci. Rep. 2018, 8 (1), 8819.
Hang Yin: 0000-0002-2898-844X Lian-Hui Wang: 0000-0001-9030-9172 Ruibing Wang: 0000-0001-9489-4241 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the Macau Science and Technology Development Fund, Macao SAR (FDCT/020/2015/A1), and Research Committee of University of Macau (MYRG201600165-ICMS-QRCM, MYRG2016-00008-ICMS-QRCM, and MYRG2017-00010-ICMS) for providing the financial support for this research.
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DOI: 10.1021/acsabm.8b00401 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
