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Heparin as a molecular spacer immobilized on microspheres to improve blood compatibility in hemoperfusion
Carbohydrate Polymers 205 (2019) 89–97
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Heparin as a molecular spacer immobilized on microspheres to improve blood compatibility in hemoperfusion Qi Dang, Chun-Gong Li, Xin-Xin Jin, Ya-Jin Zhao, Xiang Wang
T
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Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Heparin Spacer Blood compatibility L-Phenylalanine Endotoxin
Heparin, a highly sulfated linear polysaccharide, with anticoagulation function and blood compatibility is widely used as a biomaterials in medical application, but the most importance of heparin is its structure function as the macromolecular space arm. In this study, heparin as a spacer was covalently immobilized on the chloromethylated polystyrene microspheres (Ps) and then connected with L-phenylalanine forming the Ps-Hep-Phe structure, which was developed for endotoxin adsorption in hemoperfusion. The grafting density of heparin reach the maximum when the initial concentration of heparin solution was 5 mg/mL. The adsorbents with the heparin as a spacer showed the prolonged clotting times, low protein adsorption, and reduced the hemolysis rate, indicating that heparin-modified adsorbents have great blood compatibility. The adsorption capacity of PsHep-Phe for endotoxin was 25.15 EU/g in dynamic adsorption, higher than that of Ps. Therefore, this study imply that heparin would be promising for modification of adsorbents in hemoperfusion.
1. Introduction Heparin, a highly sulfated linear polysaccharide as a common anticoagulant, interacts with antithrombinⅢ, then accelerating the formation of thrombin-antithrombin (TAT) complexes that inhibits the activated factors XIa, IXa, Xa, Ila and the activity of thrombin, thereby inhibiting the conversion of fibrinogen to fibrin and preventing the coagulation (Verhamme, Bock, & Jackson, 2004). Meanwhile, heparin act as a catalyst is released from the complex and binds to the next antithrombin molecule again (Liu et al., 2014). Because of its excellent anticoagulation, surface heparinization has been attracted attention in various fields, such as scaffolds (Bao et al., 2015), drug delivery (Sakiyama-Elbert, 2014), especially blood purification (Duo & Stenken, 2011). Blood purification is a kind of extracorporeal circuit treatment to remove pathogenic substances, and the urgent problem is that protein adsorption, platelet adhesion and activation, and clot formation may be caused by the coagulation cascade reaction when the surface is in contact with the blood (Leszczak & Popat, 2014). So a high dose of heparin need to be administration to prevent the thrombotic events in extracorporeal therapy, but this may put the patients at high risk of bleeding or other severe side effects (Aldenhoff et al., 2004). Therefore, heparinized surface is urgent to improve the blood compatibility in blood purification. Heparin was initially in design of membranes or adsorbents by
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physical method including surface coating (Badr, Gouda, Abdel-Sattar, & Sayour, 2014) and physical blending (Wang et al., 2012) to improve the blood compatibility of biomaterials. However, heparin attached on the surface in these ways would shed quickly and soon expose the nonanticoagulant surface on the environment (You, Kang, Byun, & Lee, 2011). Stability of heparin on surface in these ways still need to be improved. Heparin covalently immobilized on surface can enhance the stability (Yang et al., 2012). Heparin has a number of chemically reactive functional groups containing carboxyl and amino groups/chain, which can be capable of either covalently attaching to a supporting matrix or accepting the introduction of a linker or spacer for attachment to a matrix (Murugesan, Xie, & Linhardt, 2008). And heparin itself carries a large number of negative charges that can interact with positively charged pathogenic, such as low-density lipoprotein (LDL) with strongly electropositive. Heparin was immobilized covalently on polysulfone sheets as a ligand to achieve selective adsorption of LDL by electrostatic interaction (Huang, Guduru, Xu, Vienken, & Groth, 2010). However, it is well known that many pathogenic toxin was not positive charge, like endotoxin in sepsis and bilirubin in liver disease. In this moment, heparin as a ligand does not work well in adsorption. Thus, it is better choice of heparin as a spacer to connect effective ligands by reaction of carboxyl groups or amino groups on heparin, which may perform its structural function and biological function thoroughly. In addition, repulsive force caused by the strong negative charge can be
Corresponding author. E-mail address: [email protected] (X. Wang).
https://doi.org/10.1016/j.carbpol.2018.08.067 Received 23 June 2018; Received in revised form 5 August 2018; Accepted 15 August 2018 Available online 01 September 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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immobilized heparin on Ps with Phe as a ligand (Ps-Hep-Phe) through an EDC/NHS coupling method to remove endotoxin. The blood compatibility and the performance of endotoxin adsorption were investigated to evaluate the application of heparin as a spacer in the blood purification field.
generated between the chains, which makes the heparin chains difficult to curl and crosslink holding a linear structure and increases the size of the connecting arm swing space for better contact with the target. Therefore, heparin as a spacer has a great potential application in blood purification. The available spacer of heparin was verified through an example with endotoxin. Endotoxin, one of primary pathogenic factors, is considered one of the principal biological substances causing sepsis or septic shock (Marshall et al., 2004). Therefore, it is eager to find an effective methods to reduce the endotoxin level in blood therapeutically. Endotoxin is an integral component of the outer membrane of Gram-negative bacteria, which is structurally related bacterial lipopolysaccharides (LPS) (Erridge, Bennett-Guerrero, & Poxton, 2002). Lipid A, the hydrophobic anchor of LPS, is a glucosamine-based phospholipid that makes up the outer monolayer of the outer membranes of most Gram-negative bacteria. Due to its phosphate groups (pK11.3, pK28.2), the endotoxin molecule is negatively charged in physiological media (Raetz & Whitfield, 2002). Polymyxin B is a polycationic antibiotic with well-known specific binding activity for endotoxin that neutralizes its toxicity (Shoji, Tani, Hanasawa, & Kodama, 1998). However, PMX-B suffered from neurotoxicity and nephrotoxicity itself with high price, which limited its wide application in clinical blood purification (Tani, Shoji, Guadagni, & Perego, 2010). In recently, the amino as the safe and effective ligand on biomaterials have been carried out to remove endotoxin. Lysine was immobilized covalently onto cellulose beads for the endotoxin removal by electrostatic interactions, having high endotoxin-binding efficiency (Fang, Wei, & Yu, 2004). Histidine as a ligand was grafted on flat-sheet nylon membranes and the adsorption capacity of endotoxin is superior to the pristine membranes (de Almeida, Almeida, Fingola, & Ferraz, 2016). However, there is no much researches about L-phenylalanine (Phe) as a ligand to adsorb endotoxin. Phe is relatively strong hydrophobic and neutral aromatic amino acid with pKa = 5.48 that has a benzene ring as a lateral group, which contributes to the hydrophobicity of biomolecules. Thus, Phe may be a potential ligand for lipid-soluble toxins removal, such as LPS, due to its hydrophobicity, low cost and lack of toxicity. However, the adsorbents used to remove endotoxins may be not only expected to high efficiency, but also good blood compatibility. To meet these challenge, heparin as a spacer was introduced into the Ps with Phe as a ligand, as shown in Fig. 1. Ps are chemical intermediates characterized by porous, suitable mechanical properties and chemical stability, and the functional groups of chloromethyl facilitate the modification, but its blood compatibility and hydrophilicity need to be improved. The introduction of heparin as a spacer can enhance the blood compatibility of microspheres, and then connected with Phe would further adsorbed endotoxin better. In this study, thus, we
2. Materials and methods 2.1. Materials Chloromethylated polystyrene Resin (HC2001-2-4, average diameter: 572.9 μm, size distribution: 365.2–820.5 μm) was provided by Tianjin Nankai Synthesis Technology co. LTD., heparin (sodium salt, ≥160U/mg) and Phe were purchased from Beijing Solarbio Science & Technology Co., Ltd. Toluidine Blue (TB), 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxy-succinimide (NHS) and FITC-LPS (from Escherichia coli 055:B5) were purchased from Sigma–Aldrich. Endotoxin standards, Chromogenic End-point Tachypleus Amebocyte Lysate(TAL) for endotoxin detection and endotoxin-free water were purchased from Xiamen Horseshoe Crab Reagent Manufactory (Xiamen, China). The Tachypleus Amebocyte Lysate for kinetic turbidity kit was purchased from Zhanjiang A&C Biological Ltd. Venous blood samples were obtained from healthy adult male volunteers, who provided written informed consents to participate in the study. The study protocols were approved by Chongqing University and Ethics Committee. 2.2. Pretreatment and amination of Ps Acid-base cleaned Ps were dispersed in N,N-Dimethylformamide (DMF) for 2 h to activate the −CH2Cl and then added into ethylenediamine (EDA) stirred for 7 h at 80 ℃ (the volume ratio of DMF:EDA = 1:2) in 500 ml three-necked round-bottomed flask. The obtained microspheres (Ps-NH2) were washed with the phosphate buffer saline (PBS) and a large amount of double distilled water alternately until the effluent solution was neutral. 2.3. Immobilization of heparin on microspheres with Phe connected Ps-NH2 was submerged into heparin and EDC/NHS solution (5 mg/ ml of heparin and EDC in citrate buffer solution: 0.2 M Na2HPO4 and 0.1 M citric acid, adjusted to pH = 4.7 with 1 M NaOH, molar ratio of EDC to NHS = 1:1) for 24 h at 37℃ to bind heparin covalently. The heparin modified Ps-NH2(Ps-Hep) was treated at 37℃ for 24 h with a solution of L-Phe (5 mg/ml) and EDC in citrate buffer solution like the above. 2.4. The density of heparin on microspheres surface The density of heparin bound to Ps-NH2 surface was assayed by the TB colorimetric method (Huang et al., 2010). The amount of immobilized heparin can be calculated by comparison with a standard curve using soluble heparin of known concentration determined by UV–vis spectrophotometry at 631 nm. 2.5. Characterization Fourier-transform infrared spectra (FTIR) were recorded on a Nicolet iN10 spectrophotometer (Thermo, US) between 4000 and 400 cm−1. Thirty-two scans were taken for each spectrum at a normal resolution of 4 cm−1. XPS spectra were carried out on the X-ray photoelectron spectrometer (Thermo Scientific™, K-Alpha+™, US), employing Al Kα excitation radiation. Zeta potential measurements were performed with a Zetasizer Nano ZS 90 analyzer (Malvern Instruments Ltd, UK) at 25.0 ± 0.1℃ using distilled water (pH = 7.4). The variety of contact angle was measured
Fig. 1. Schematic presentation of Phe and heparin immobilized adsorbent for endotoxin removal. 90
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at room temperature on a contact angle meter (GBX, France) equipped with video capture. 2.6. Blood compatibility 2.6.1. Plasma coagulation and protein adsorption The obtained fresh blood was centrifuged at 3000 rpm for 10 min to obtain platelet-poor plasma. The adsorbents (200 mg) was equilibrated with physiological saline for several hours and then added into 0.6 ml plasma. After incubating 37℃ for 30 min, the activated partial thromboplastic time (APTT), thrombin time (TT) and fibrinogen (FIB) of the plasma were evaluated by blood coagulation analyzer (CA600, Sysmex Corporation, Kobe, Japan). 200 mg of adsorbents were added into 0.6 ml fresh plasma centrifuged at 2000 rpm for 10 min and then shaken at 37℃ for 2 h. The change of albumin (ALB) and globulin (GLB) was measured by the automatic biochemical analyzer (AU680, Beckman coulter, American). 2.6.2. Platelet factor 4 (PF-4) released on different microspheres PF-4 was measured by a commercially enzyme linked immunosorbent assay kit (ELISA, RayBio) to evaluate platelet activation on different surfaces (Leszczak & Popat, 2014). Human venous blood was separated by centrifugation of the whole blood at 1200 rpm for 10 min, and the plasma was incubated with the 50 mg of microspheres for 1 h. Supernatant was diluted 1:200 times and the detections were conducted according to the protocol from manufacturer. Fig. 2. Schematic diagram presents the immobilization process of Ps-Hep-Phe.
2.6.3. Hemolysis test 100 mg of adsorbents were incubated with erythrocyte suspension (2% in normal saline solution) at 37 ℃ for 1 h. Negative and positive controls were blood samples diluted with 95% normal saline and deionized water in the absence of hydrogel respectively. The supernatant solution was obtained after centrifugation (1000 rpm for 5 min) and measured at 540 nm. The hemolysis rate was calculated as follows: Hemolysis rate (%) = (A-A0)/(A100-A0)×100%, in which A, A100, and A0 are the absorbance of the sample, positive control, and negative control, respectively. The incubated microspheres were rinsed with normal saline solution two times and fix with 2.5 wt% glutaraldehyde solution overnight. Then, the microspheres were dehydrated sequentially with 50, 60, 70, 80, 90 and 100 (v/v) ethanol and finally dried at room temperature. The morphology of red cells adhesion were observed using a scanning electron microscope (SEM, ZEISS EVO18, Germany) at 3 kV with four randomly selected SEM images.
2.7.3. Adsorption of endotoxin in human plasma Dynamic adsorption of endotoxin in human plasma was evaluated by kinetic-turbidimetric method. 1.0 g of Ps-Hep-Phe adsorbents were packed into a glass column and 5 ml human plasma was cycled through the column for 2 h with flow rate of 4 ml/min of a constant current pump at room temperature. The amount of adsorbed endotoxin at different time were investigated. 3. Results and discussions 3.1. Synthesis and characterization of microspheres Surface modification processing with well-defined polymeric structures and original structure can improve some surface properties of microsphere (Azzaroni, 2012). Fig. 2 shows that the heparin, as a spacer, was tethered covalently at one end to Ps-NH2 through the EDC/ NHS coupling chemistry and then connected with L-Phe as the same way.
2.7. Endotoxin adsorption 2.7.1. FITC-LPS adsorption in physiological saline The adsorption property of the adsorbents was evaluated qualitatively by removing FITC-LPS. 2 ml of 1ug/mL FITC-LPS solution was added and incubated darkly at 37◦C for 2 h. Then, the microspheres were rinsed with PBS and distilled water, and visualized under a fluorescence microscope (IX71 OLYMPUS, Japan).
3.1.1. The density of heparin on microspheres In this process, the grafting amount of heparin that has a number of chemically reactive functional groups determines the density of effective ligand on microspheres. Fig. 3(A) shows the initial concentration of heparin made difference to the heparin density on the surface of PsHep. It was found that the grafting density of heparin almost approached maximum when the initial concentration of heparin was more than 5 mg/ml. And it was found that the color of microspheres after stained with TB is different. Fig. 3(B) shows the photographs of Ps, Ps-NH2, heparinadsorbed Ps-NH2 and Ps-Hep after TB staining. The original Ps was white without being stained because of high hydrophobicity. And PsNH2 was purple which might be attributed to the improvement of hydrophilic resulting in adsorption of TB. After immobilization of heparin, Ps-Hep became a light blue. As a control, the heparin-adsorbed PS-NH2 that was Ps-NH2 immersed into heparin solution without EDC/NHS was purple, same as the color of Ps-NH2, which indicated that the heparin was adsorbed physically on surface and washed away easily. This
2.7.2. Endotoxin adsorption in physiological saline measured by TAL Static adsorption: Endotoxin adsorption in physiological saline was evaluated by TAL (Chromogenic). 100 mg of Ps, Ps-NH2, Ps-Hep, PsHep-Phe was added into 1 ml of physiological saline solution with the concentration of endotoxin as about 5 EU/ml and shaken at 37℃for 2 h. The adsorption capacity of endotoxin was calculated by determining the endotoxin concentration of the upper solution after adsorption. Dynamic adsorption: 0.5 g of Ps-Hep-Phe was packed in a glass column (100 mm × 10 mm) with constant current pump. 5 ml of physiological saline solution which contained the endotoxin concentration of about 5EU/ml were perfused into the column for 2 h with flow rate of 4 ml/min at room temperature. The amount of adsorbed endotoxin was measured at different time. 91
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Fig. 3. (A) Effects of the initial concentration of heparin on the heparin density on the microspheres; (B) Digital images of toluidine blue staining for Ps, Ps-NH2, Heparin-adsorbed Ps-NH2 and Heparin-modified Ps-NH2 (Ps-Hep).
Fig. 4. Characterization of microspheres. (A) FTIR spectra and (B) XPS spectra of Ps, Ps-NH2, Ps-Hep and Ps-Hep-Phe. (C) XPS C1 s core-level spectra of Ps, Ps-Hep and Ps-Hep-Phe.
et al., 2015). These results proved that heparin had been grafted covalently onto microspheres, which was consistent with the TB staining. For the spectra of Ps-Hep-Phe, the peak of bending vibration of C–N was 1260 cm-1, and the peak for −COOe groups overlapped with − NH2 groups to form a wide peak at about 1580 cm−1 (Poon, Bin Zhu, Shen, Chan-Park, & Ng, 2007), which attributed to the a large number of –NH2 on Phe. XPS spectra were to further prove the chemical structure and reaction process on the modified microspheres. The survey scan spectra of the microspheres were shown in Fig. 4(B) and the elemental compositions from XPS spectra are listed in Table 1. For the control Ps surface has peaks at 284.7 eV, 271.1 eV and 201.1 eV, corresponding to the binding energy of C 1s and Cl 1s, Cl 2p respectively. Compared with the Ps, all modified microspheres showed a new peak at 400.1 eV, corresponding to the binding energy of N 1 s (Yang, Tian, Dai, Hu, & Xu, 2006).The nitrogen content of Ps-NH2 surface was increased significantly, and the chlorine content were decreased obviously from 5.32% for Ps to 0.26% for Ps-NH2, indicating that the –NH2 groups had been grafted successfully on microsphere surface. After binding with heparin, the oxygen content was increased significantly from 8.10% for Ps-NH2 to 23.37% for Ps-Hep, and the sulfur content, which was consistent with the peaks of S 1s, S 2p (Xiang et al., 2014), was increased
demonstrated that heparin might be mainly grafted covalently on microspheres, which was more stable. And further proof need to be measured by FTIR and XPS spectra. 3.1.2. FTIR and XPS The FTIR was employed to detect the chemical structure of microspheres. The Fig. 4(A) shows the FTIR spectra of microspheres at wavelengths from 400 to 4000 cm−1. Compared with the original Ps, the two new peaks at 3370 and 3310 cm-1 for Ps-NH2 were attributed to the antisymmetric and symmetric stretching vibrations of amino groups (Tzoneva et al., 2008), and the peaks around 1261 cm-1 and 671 cm-1 for Ps-NH2 were disappeared that was assigned to the disappearance of the in-plane bending vibration of C–H binding with chlorine groups and stretching vibrations of CeCl, which confirmed the successful immobilization of amino (eNH2) groups. For the spectra of Ps-Hep, the new peak at 3440 cm-1 was attributed to the stretching vibrations of −OH(Shi, Meng, Xu, Du, & Zhang, 2013), while the new peaks at 1660 cm-1 and 1260 cm-1 were attributed to the stretching vibrations of C]O and the bending vibration of C–N in secondary amide respectively (Wang et al., 2013) which indicated the successful synthesis of secondary amides. The wide peaks around 1060 cm-1 were ascribed to the stretching vibrations of the sulfonic (eSO3e) groups (Nie, Ma, Xia 92
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Table 1 Chemical composition of microspheres calculated from XPS survey scans. Microsphere
Ps Ps-NH2 Ps-Hep Ps-Hep-Phe
Element (Atom %) C
O
N
S
Cl
82.31 81.43 65.22 65.44
9.06 8.1 23.37 23.32
2.49 9.42 8.03 7.87
0.27 0.33 2.52 2.20
5.32 0.26 0.29 0.48
sharply to 2.52% for Ps-Hep because of the rich oxygen content and sulfonic groups of the immobilized heparin, indicating the successful immobilization of heparin on microspheres. The chemical change of microsphere surface can also be analyzed further from the high-resolution core-level spectra of C1 s through the different binding energy, as shown in Fig. 4(C1–C3). The C1s core-level spectrum of PS could be fitted by two peaks. One peak at 284.7 eV was assigned to the carbon skeleton(CeC/C]C) for the backbone of Ps microspheres. But there was the other peak at 286.5 eV for C–O, which may be the impurity on the synthetic process of Ps. With the immobilization of heparin on the microspheres surface, the binding energy at 285.3 eV was attributed to C–S bond of heparin, which was overlapped with the C–N bond and the peak at 286.5 eV was assigned the bond C–O. In addition, the signal observed at 288.2 eV (C]O) could be ascribed to attributed to the C atoms in the amide groups of grafting heparin (or phenylalanine), which proved the more formation of the amides groups in modification process. These results further confirmed that heparin was successfully immobilized on microspheres and Phe was grafted mostly on heparin of Ps-Hep.
Fig. 5. (A) Images of water contact angle at 3000 ms for Ps, Ps-NH2, Ps-Hep and Ps-Hep-Phe; (B) The dynamic water contact angles of microspheres within 3000 ms.
3.1.3. Zeta potential and water contact angle Table 2 presents the zeta potential of microspheres surface. It was found that the zeta potential of Ps-NH2 was increased significantly from -17.3 eV to 33.4 eV. This increase in surface potential can be attributed to the positive charge of grafted –NH2 on microspheres, which was consistent with the other report (Xiang et al., 2014). After binding with the heparin, the zeta potential was decreased obviously, from 33.4 eV for Ps-NH2 to 17.2 eV, which was attributed to the immobilized heparin possessing the rich negative groups. The zeta potential continued to reduce, from 17.2 eV for Ps-Hep to 10.3 eV for Ps-Hep-Phe, which was assigned to the negatively ionizable carboxylic groups of Phe in distilled water.
angle of Ps-Hep-Phe was 11.8° closed to that of Ps-Hep. Meanwhile, the water contact angle was attenuated in different degree with the prolonged time, which was attributed to the permeation of water into the microporous and capillary absorption. 3.2. Blood compatibility 3.2.1. Clotting time APTT and TT are usually used to evaluate coagulation abnormalities in the intrinsic and extrinsic pathway respectively (Popovic et al., 2012). Fig. 6(A) presents the TT and APTT of different microspheres. Compared with that of plasma control, there was no change in TT and APTT for the Ps and Ps-NH2, and the APTT for Ps-Hep, Ps-Hep-Phe were prolonged in different degrees. The increment of APTT for the Ps-Hep and Ps-Hep-Phe might be attributed to the combination between –SO3and the coagulation factors or the combination of functional groups and Ca2+ in the blood plasma (Nie, Tang et al., 2014). Surprisingly, the APTT for Ps-Hep-Phe was increased much more than that for Ps-Hep which might be assigned to the Phe affected the activation of Factor XIa (Smith et al., 2016). In order to ensure the increment whether resulted from heparin or Phe, the clotting time of Ps-NH2 immobilized with Phe and no heparin as a spacer (Ps-Phe) was measured. It was found that the APTT for Ps-Phe was no much change with Ps-Hep, but was much lower than Ps-Hep-Phe, indicating that anticoagulation of Ps-Hep-Phe was contributed by heparin and Phe together. The TT for the Ps-Hep, PsHep-Phe and Ps-Phe was prolonged slightly compared with that for plasma, which attributed to the effects of −COOe and eSO3e groups on fibrinolytic system. The results of APTT and TT confirmed the satisfactory blood compatibility of the Ps-Hep-Phe.
3.1.4. Hydrophilicity Water contact angle was a common approach to evaluate the hydrophilicity of biomaterials surface, which may endow the bio-interface with favorable anti-fouling property. Fig.5(A) presents the images of static water contact angle at 3000 ms, and Fig. 5(B) shows the water dropped within 3000 ms. When the water dropped at 3000 ms, the water contact angle of pristine Ps is around 131.6° demonstrating that the surface of Ps was high hydrophobicity. The water contact angle was decreased obviously, from 131.6° for Ps to 47.4° for Ps-NH2, which was assigned to the presence of hydrophilic amino groups. After binding with the heparin, the water contact angle was decreased to 8.4° for PsHep, which is attributed to the heparin possessing the hydrophilic groups, such as –SO3-, −COO- and −OH groups. And the water contact Table 2 Zeta potential of Ps and modified microspheres. Samples
Zeta potential(mV)
Ps Ps-NH2 Ps-Hep Ps-Hep-Phe
−21.4 ± 4.6 30.1 ± 3.0 17.3 ± 3.4 8.2 ± 2.3
3.2.2. Protein adsorption and platelet activation Protein adsorption is considered to be first event occurring on biomaterials surface contacted with blood, and the adsorbed protein will mediate the subsequent biological responses, including platelet adhesion and thrombus formation on surfaces (Ma et al., 2014). Therefore, 93
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Fig. 6. (A) TT and APTT values of normal plasma and microspheres. (B) FIB and (C) ALB and GLB adsorptionamounts on these microspheres; (D) The concentrations of PF-4 released of these microspheres with the plasma, which can act as indicator for platelet activation.
samples was measured. When the platelets activated during aggregation, PF-4 is released from α-granules in a platelet release reaction, so PF-4 is usually used as an indicator of platelet activation (Nie, Ma, Cheng, Deng & Zhao, 2015). As shown in Fig. 6(D), there is a dramatic increase in PF-4 released in plasma for Ps-NH2that might be attributed to the high density of positive charge, which suggested that platelet activation probably occurred. After the modification with heparin, the PF-4 released in plasma was significantly decreased, indicating that platelet activation was suppressed, which was contributed to the immobilized heparin polymers on adsorbents (Nie et al., 2017). Although PF-4 released of Ps-Hep-Phe was slightly increased compared with PsHep, Ps-Hep-Phe still kept the lower level of PF-4 released compared with Ps-Phe, which demonstrated that the platelet activation on the PsHep-Phe with heparin as a spacer did not occur or was gently suppressed.
protein adsorption is one of important factors to evaluate the biocompatibility of materials. In considering surface-induced thrombosis, FIB is a central protein in the thrombus formation. Fig. 6(B) shows the FIB adsorption amounts of microspheres. We noted that heparin-modified adsorbents show the significantly decreased FIB adsorbed amount. The introduction of heparin into the Ps-Hep-Phe adsorbents still kept the lower FIB adsorbed amount than that of Ps-Phe without the heparin as a spacer, which was assigned to the improvement of hydrophilic, indicating that heparin as a spacer could reduce the FIB adsorption amount (Pan, Hou, Zhang, Dong, & Ding, 2014). To investigate further protein adsorption of PsHep-Phe, ALB and GLB were evaluated as shown in Fig. 6(C), because they are abundant protein in plasma that keep body balanced. After modifying, the adsorbed amount of ALB and GLB was decreased. And the Ps-Hep-Phe still kept the low adsorption of ALB and GLB. Meanwhile, it was found that the results of protein adsorption were highly related to the hydrophilic/hydrophobic properties of biomaterial surfaces. Some researchers reported that water contact angle played an important role in the protein adsorption (Xu, Bauer, & Siedlecki, 2014). From the view of the surface energy, surface wettability and protein adsorption both are the energy-driven process that involves the properties of both the substrate surface. So the amount of protein adsorption on the surface can be reflect from the water contact angles. Moreover, the surface of biomaterial was hydrophilic which tend to combine with the water molecules, and the ability to adsorb protein was more weaken (Nie, Qin et al., 2014). To investigate the platelet activation after the interaction with microspheres, the amount of PF-4 released in surface-exposed plasma
3.2.3. Hemolysis test Hemolysis is a reliable method to estimate the hemocompatibility of biomaterials in contact with blood (Wu et al., 2017; Zhao, Ma, Zeng, Tu & Zhao, 2016). Fig. 7F shows the degree of hemolysis obtained with Ps, Ps-NH2, Ps-Hep, Ps-Hep-Phe and Ps-Phe. All the microspheres showed a low degree of hemolysis (< 2%); but the adsorbents with the heparin as a spacer further reduced the degree of hemolysis. As same, the hemolysis rate of Ps-Hep-Phe was lower than that of Ps-Phe, showing good blood compatibility. Fig. 7G also shown the visual observation of the hemolytic phenomenon, consistent with the previous quantitative spectrophotometric measurement. And the morphologies and adhesion of erythrocytes adhered on Ps, Ps-NH2, Ps-Hep, Ps-Hep-Phe and Ps-Phe 94
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Fig. 7. The morphologies of erythrocytes treated with (A)Ps, (B) Ps-NH2, (C)Ps-Hep, (D) Ps-Hep-Phe, (E) Ps-Phe, respectively; (F) Hemolysis rate of microspheres; (G) Visual observations of hemolysis.
3.3. Endotoxin adsorption
microspheres Fig. 7(A–E) were observed by SEM. It was observed that Ps had little adhesion of erthrocytes, which may be due to the superhydrophobic surface of Ps. After the treatment of EDA, erythrocytes adhered on the Ps-NH2 surface became more and some of them showed ridges. Then the introduction of heparin into surface, Ps-Hep and PsHep-Phe showed extremely little adhesion of erythrocytes and the morphologies of erythrocytes with biconcave shape and smooth surfaces similar to normal red cells, demonstrating the good blood compatibility and nontoxicity. More erythrocytes adhered on surface of PsPhe without heparin also confirmed this point, indicating that the incorporation of heparin into microspheres could reduce the risk to erythrocytes in hemoperfusion. Therefore, heparin as a spacer to connected Phe and Ps was a better choice to improve the great blood compatibility of adsorbents.
3.3.1. FITC-LPS adsorption The adsorption of fluorescent LPS was used for qualitative evaluation. The fluorescence intensity was observed by fluorescence microscopy to evaluate the adsorption capacity of FITC-LPS as shown in Fig. 8(A), and in order to avoid visual error, the fluorescence intensity was calculated by image-pro plus, as shown in Fig. 8(B). The adsorption capacity of Ps was weak with the lowest fluorescence intensity. And the fluorescence intensity was increased with the modification. Ps-Hep-Phe had a larger fluorescence intensity than Ps. This indicated indirectly that Ps-Hep-Phe had an improvement in adsorption capacity of endotoxin.
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Fig. 8. (A) Images (10 × 4) of FITC-LPS adsorption for 2 h on microspheres; (B) The fluorescence intensity of microspheres after FITC-LPS adsorption calculated by image-pro plus. (C) The endotoxin adsorption capacity of the Ps and the modified microspheres; (D) Effects of adsorption time on endotoxin adsorption capacity; (E) Effects of adsorption time on endotoxin adsorption capacity in human plasma.
equilibrium time of Ps-Hep-Phe was 60 min, which could be meets the requirements in clinic.
3.3.2. Adsorption of endotoxin in physiological saline solution measured by TAL Fig. 8(C) shows the static adsorption capacity for the all microspheres. The adsorption capacity of pristine Ps was 18.03EU/g. The adsorption capacity of Ps-NH2 was increased obviously due to the positive group of –NH2. Then the introduction of heparin into microspheres, there was no obvious drop in the adsorption cpacity of Ps-Hep compared with Ps-NH2 which might be mainly attributed to the positive charge of Ps-Hep as revealed by the zeta potential test (Chandy & Rao, 2000). After connected with Phe, the adsorption capacity of Ps-Hep-Phe was more than that of other microspheres. This increase might be attributed to the hydrophobic amino acid of Phe that could interacted with the Lipid A of endotoxin by the hydrophobic interaction (Anspach, 2001; Jirawutthiwongchai et al., 2016). Moreover, the extension of the spacer with heaprin could reduce the steric hindrance between the ligand and the endotoxin molecule which might also contribute to the adsorption capacity of Ps-Hep-Phe (Zhi, Mei, Li, Hou & Wang, 2005). These indicated that the heparin as a spacer connected matrix with Phe still performed well in adsorption capacity for endotoxin removal. To study further the kinetic adsorption process of Ps-Hep-Phe, the Fig. 8(D) shows the relationship between adsorption capacity of endotoxin and adsorption time in the physiological saline solution. There was a rapid increased before initial 30 min because of the frequent collision between the exposed active sites and a large amount of endotoxin monomers. With the increment of time, the rate of adsorption efficiency was gradually slower. When the time reached 90 min, the adsorption capacity of endotoxin was 25.15 EU/g.
4. Conclusion In this study, heparin as a spacer was covalently immobilized onto Ps successfully and connected with ligand of Phe, which involved condensation reaction between −COOH and –NH2 through the EDC/ NHS coupling chemistry for the endotoxin removal. And the heparinmodified microspheres displayed the great hydrophilic property. The heparin as a spacer immobilized onto the microspheres still plays an important role in the protein adsorption and anticoagulant of adsorbents. Moreover, Phe connected with biological macromolecular chains can swing in a wider range to capture endotoxin molecules and the Ps-Hep-Phe still keeps the pretty adsorption capacity of endotoxin compared with the Ps. Therefore, heparin as a molecular spacer will be an effective way to improving the performance of adsorbents in blood purification. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant No. 11572065), Development, clinical trial and registration of hemoperfusion (No. cstc2018jszx-cyzd0582) and Chongqing University Postgraduates’ Innovation Project (No. CYB15028).
3.3.3. Adsorption of endotoxin in human plasma The microspheres as adsorbents used in hemoperfusion, need to the high removal efficiency in clinical treatment. Thus, adsorption equilibrium time should be confirmed to ensure patients against harms in the therapy process. Fig. 8(E) shows the adsorption efficiency of the PsHep-Phe. The adsorption capacity reached a plateau when the adsorption time exceeded 60 min which was similar with other reports (Cao, Zhu, Zhang, & Dong, 2016; Fang et al., 2004), and the adsorption capacity was up to 15.00 EU/g at 60 min. In clinical treatment, the contact time between materials and plasma should be avoided too long which should be better not more than 2 h. So the adsorption
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Heparin as a molecular spacer immobilized on microspheres to improve blood compatibility in hemoperfusion Qi Dang, Chun-Gong Li, Xin-Xin Jin, Ya-Jin Zhao, Xiang Wang
T
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Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Heparin Spacer Blood compatibility L-Phenylalanine Endotoxin
Heparin, a highly sulfated linear polysaccharide, with anticoagulation function and blood compatibility is widely used as a biomaterials in medical application, but the most importance of heparin is its structure function as the macromolecular space arm. In this study, heparin as a spacer was covalently immobilized on the chloromethylated polystyrene microspheres (Ps) and then connected with L-phenylalanine forming the Ps-Hep-Phe structure, which was developed for endotoxin adsorption in hemoperfusion. The grafting density of heparin reach the maximum when the initial concentration of heparin solution was 5 mg/mL. The adsorbents with the heparin as a spacer showed the prolonged clotting times, low protein adsorption, and reduced the hemolysis rate, indicating that heparin-modified adsorbents have great blood compatibility. The adsorption capacity of PsHep-Phe for endotoxin was 25.15 EU/g in dynamic adsorption, higher than that of Ps. Therefore, this study imply that heparin would be promising for modification of adsorbents in hemoperfusion.
1. Introduction Heparin, a highly sulfated linear polysaccharide as a common anticoagulant, interacts with antithrombinⅢ, then accelerating the formation of thrombin-antithrombin (TAT) complexes that inhibits the activated factors XIa, IXa, Xa, Ila and the activity of thrombin, thereby inhibiting the conversion of fibrinogen to fibrin and preventing the coagulation (Verhamme, Bock, & Jackson, 2004). Meanwhile, heparin act as a catalyst is released from the complex and binds to the next antithrombin molecule again (Liu et al., 2014). Because of its excellent anticoagulation, surface heparinization has been attracted attention in various fields, such as scaffolds (Bao et al., 2015), drug delivery (Sakiyama-Elbert, 2014), especially blood purification (Duo & Stenken, 2011). Blood purification is a kind of extracorporeal circuit treatment to remove pathogenic substances, and the urgent problem is that protein adsorption, platelet adhesion and activation, and clot formation may be caused by the coagulation cascade reaction when the surface is in contact with the blood (Leszczak & Popat, 2014). So a high dose of heparin need to be administration to prevent the thrombotic events in extracorporeal therapy, but this may put the patients at high risk of bleeding or other severe side effects (Aldenhoff et al., 2004). Therefore, heparinized surface is urgent to improve the blood compatibility in blood purification. Heparin was initially in design of membranes or adsorbents by
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physical method including surface coating (Badr, Gouda, Abdel-Sattar, & Sayour, 2014) and physical blending (Wang et al., 2012) to improve the blood compatibility of biomaterials. However, heparin attached on the surface in these ways would shed quickly and soon expose the nonanticoagulant surface on the environment (You, Kang, Byun, & Lee, 2011). Stability of heparin on surface in these ways still need to be improved. Heparin covalently immobilized on surface can enhance the stability (Yang et al., 2012). Heparin has a number of chemically reactive functional groups containing carboxyl and amino groups/chain, which can be capable of either covalently attaching to a supporting matrix or accepting the introduction of a linker or spacer for attachment to a matrix (Murugesan, Xie, & Linhardt, 2008). And heparin itself carries a large number of negative charges that can interact with positively charged pathogenic, such as low-density lipoprotein (LDL) with strongly electropositive. Heparin was immobilized covalently on polysulfone sheets as a ligand to achieve selective adsorption of LDL by electrostatic interaction (Huang, Guduru, Xu, Vienken, & Groth, 2010). However, it is well known that many pathogenic toxin was not positive charge, like endotoxin in sepsis and bilirubin in liver disease. In this moment, heparin as a ligand does not work well in adsorption. Thus, it is better choice of heparin as a spacer to connect effective ligands by reaction of carboxyl groups or amino groups on heparin, which may perform its structural function and biological function thoroughly. In addition, repulsive force caused by the strong negative charge can be
Corresponding author. E-mail address: [email protected] (X. Wang).
https://doi.org/10.1016/j.carbpol.2018.08.067 Received 23 June 2018; Received in revised form 5 August 2018; Accepted 15 August 2018 Available online 01 September 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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immobilized heparin on Ps with Phe as a ligand (Ps-Hep-Phe) through an EDC/NHS coupling method to remove endotoxin. The blood compatibility and the performance of endotoxin adsorption were investigated to evaluate the application of heparin as a spacer in the blood purification field.
generated between the chains, which makes the heparin chains difficult to curl and crosslink holding a linear structure and increases the size of the connecting arm swing space for better contact with the target. Therefore, heparin as a spacer has a great potential application in blood purification. The available spacer of heparin was verified through an example with endotoxin. Endotoxin, one of primary pathogenic factors, is considered one of the principal biological substances causing sepsis or septic shock (Marshall et al., 2004). Therefore, it is eager to find an effective methods to reduce the endotoxin level in blood therapeutically. Endotoxin is an integral component of the outer membrane of Gram-negative bacteria, which is structurally related bacterial lipopolysaccharides (LPS) (Erridge, Bennett-Guerrero, & Poxton, 2002). Lipid A, the hydrophobic anchor of LPS, is a glucosamine-based phospholipid that makes up the outer monolayer of the outer membranes of most Gram-negative bacteria. Due to its phosphate groups (pK11.3, pK28.2), the endotoxin molecule is negatively charged in physiological media (Raetz & Whitfield, 2002). Polymyxin B is a polycationic antibiotic with well-known specific binding activity for endotoxin that neutralizes its toxicity (Shoji, Tani, Hanasawa, & Kodama, 1998). However, PMX-B suffered from neurotoxicity and nephrotoxicity itself with high price, which limited its wide application in clinical blood purification (Tani, Shoji, Guadagni, & Perego, 2010). In recently, the amino as the safe and effective ligand on biomaterials have been carried out to remove endotoxin. Lysine was immobilized covalently onto cellulose beads for the endotoxin removal by electrostatic interactions, having high endotoxin-binding efficiency (Fang, Wei, & Yu, 2004). Histidine as a ligand was grafted on flat-sheet nylon membranes and the adsorption capacity of endotoxin is superior to the pristine membranes (de Almeida, Almeida, Fingola, & Ferraz, 2016). However, there is no much researches about L-phenylalanine (Phe) as a ligand to adsorb endotoxin. Phe is relatively strong hydrophobic and neutral aromatic amino acid with pKa = 5.48 that has a benzene ring as a lateral group, which contributes to the hydrophobicity of biomolecules. Thus, Phe may be a potential ligand for lipid-soluble toxins removal, such as LPS, due to its hydrophobicity, low cost and lack of toxicity. However, the adsorbents used to remove endotoxins may be not only expected to high efficiency, but also good blood compatibility. To meet these challenge, heparin as a spacer was introduced into the Ps with Phe as a ligand, as shown in Fig. 1. Ps are chemical intermediates characterized by porous, suitable mechanical properties and chemical stability, and the functional groups of chloromethyl facilitate the modification, but its blood compatibility and hydrophilicity need to be improved. The introduction of heparin as a spacer can enhance the blood compatibility of microspheres, and then connected with Phe would further adsorbed endotoxin better. In this study, thus, we
2. Materials and methods 2.1. Materials Chloromethylated polystyrene Resin (HC2001-2-4, average diameter: 572.9 μm, size distribution: 365.2–820.5 μm) was provided by Tianjin Nankai Synthesis Technology co. LTD., heparin (sodium salt, ≥160U/mg) and Phe were purchased from Beijing Solarbio Science & Technology Co., Ltd. Toluidine Blue (TB), 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxy-succinimide (NHS) and FITC-LPS (from Escherichia coli 055:B5) were purchased from Sigma–Aldrich. Endotoxin standards, Chromogenic End-point Tachypleus Amebocyte Lysate(TAL) for endotoxin detection and endotoxin-free water were purchased from Xiamen Horseshoe Crab Reagent Manufactory (Xiamen, China). The Tachypleus Amebocyte Lysate for kinetic turbidity kit was purchased from Zhanjiang A&C Biological Ltd. Venous blood samples were obtained from healthy adult male volunteers, who provided written informed consents to participate in the study. The study protocols were approved by Chongqing University and Ethics Committee. 2.2. Pretreatment and amination of Ps Acid-base cleaned Ps were dispersed in N,N-Dimethylformamide (DMF) for 2 h to activate the −CH2Cl and then added into ethylenediamine (EDA) stirred for 7 h at 80 ℃ (the volume ratio of DMF:EDA = 1:2) in 500 ml three-necked round-bottomed flask. The obtained microspheres (Ps-NH2) were washed with the phosphate buffer saline (PBS) and a large amount of double distilled water alternately until the effluent solution was neutral. 2.3. Immobilization of heparin on microspheres with Phe connected Ps-NH2 was submerged into heparin and EDC/NHS solution (5 mg/ ml of heparin and EDC in citrate buffer solution: 0.2 M Na2HPO4 and 0.1 M citric acid, adjusted to pH = 4.7 with 1 M NaOH, molar ratio of EDC to NHS = 1:1) for 24 h at 37℃ to bind heparin covalently. The heparin modified Ps-NH2(Ps-Hep) was treated at 37℃ for 24 h with a solution of L-Phe (5 mg/ml) and EDC in citrate buffer solution like the above. 2.4. The density of heparin on microspheres surface The density of heparin bound to Ps-NH2 surface was assayed by the TB colorimetric method (Huang et al., 2010). The amount of immobilized heparin can be calculated by comparison with a standard curve using soluble heparin of known concentration determined by UV–vis spectrophotometry at 631 nm. 2.5. Characterization Fourier-transform infrared spectra (FTIR) were recorded on a Nicolet iN10 spectrophotometer (Thermo, US) between 4000 and 400 cm−1. Thirty-two scans were taken for each spectrum at a normal resolution of 4 cm−1. XPS spectra were carried out on the X-ray photoelectron spectrometer (Thermo Scientific™, K-Alpha+™, US), employing Al Kα excitation radiation. Zeta potential measurements were performed with a Zetasizer Nano ZS 90 analyzer (Malvern Instruments Ltd, UK) at 25.0 ± 0.1℃ using distilled water (pH = 7.4). The variety of contact angle was measured
Fig. 1. Schematic presentation of Phe and heparin immobilized adsorbent for endotoxin removal. 90
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at room temperature on a contact angle meter (GBX, France) equipped with video capture. 2.6. Blood compatibility 2.6.1. Plasma coagulation and protein adsorption The obtained fresh blood was centrifuged at 3000 rpm for 10 min to obtain platelet-poor plasma. The adsorbents (200 mg) was equilibrated with physiological saline for several hours and then added into 0.6 ml plasma. After incubating 37℃ for 30 min, the activated partial thromboplastic time (APTT), thrombin time (TT) and fibrinogen (FIB) of the plasma were evaluated by blood coagulation analyzer (CA600, Sysmex Corporation, Kobe, Japan). 200 mg of adsorbents were added into 0.6 ml fresh plasma centrifuged at 2000 rpm for 10 min and then shaken at 37℃ for 2 h. The change of albumin (ALB) and globulin (GLB) was measured by the automatic biochemical analyzer (AU680, Beckman coulter, American). 2.6.2. Platelet factor 4 (PF-4) released on different microspheres PF-4 was measured by a commercially enzyme linked immunosorbent assay kit (ELISA, RayBio) to evaluate platelet activation on different surfaces (Leszczak & Popat, 2014). Human venous blood was separated by centrifugation of the whole blood at 1200 rpm for 10 min, and the plasma was incubated with the 50 mg of microspheres for 1 h. Supernatant was diluted 1:200 times and the detections were conducted according to the protocol from manufacturer. Fig. 2. Schematic diagram presents the immobilization process of Ps-Hep-Phe.
2.6.3. Hemolysis test 100 mg of adsorbents were incubated with erythrocyte suspension (2% in normal saline solution) at 37 ℃ for 1 h. Negative and positive controls were blood samples diluted with 95% normal saline and deionized water in the absence of hydrogel respectively. The supernatant solution was obtained after centrifugation (1000 rpm for 5 min) and measured at 540 nm. The hemolysis rate was calculated as follows: Hemolysis rate (%) = (A-A0)/(A100-A0)×100%, in which A, A100, and A0 are the absorbance of the sample, positive control, and negative control, respectively. The incubated microspheres were rinsed with normal saline solution two times and fix with 2.5 wt% glutaraldehyde solution overnight. Then, the microspheres were dehydrated sequentially with 50, 60, 70, 80, 90 and 100 (v/v) ethanol and finally dried at room temperature. The morphology of red cells adhesion were observed using a scanning electron microscope (SEM, ZEISS EVO18, Germany) at 3 kV with four randomly selected SEM images.
2.7.3. Adsorption of endotoxin in human plasma Dynamic adsorption of endotoxin in human plasma was evaluated by kinetic-turbidimetric method. 1.0 g of Ps-Hep-Phe adsorbents were packed into a glass column and 5 ml human plasma was cycled through the column for 2 h with flow rate of 4 ml/min of a constant current pump at room temperature. The amount of adsorbed endotoxin at different time were investigated. 3. Results and discussions 3.1. Synthesis and characterization of microspheres Surface modification processing with well-defined polymeric structures and original structure can improve some surface properties of microsphere (Azzaroni, 2012). Fig. 2 shows that the heparin, as a spacer, was tethered covalently at one end to Ps-NH2 through the EDC/ NHS coupling chemistry and then connected with L-Phe as the same way.
2.7. Endotoxin adsorption 2.7.1. FITC-LPS adsorption in physiological saline The adsorption property of the adsorbents was evaluated qualitatively by removing FITC-LPS. 2 ml of 1ug/mL FITC-LPS solution was added and incubated darkly at 37◦C for 2 h. Then, the microspheres were rinsed with PBS and distilled water, and visualized under a fluorescence microscope (IX71 OLYMPUS, Japan).
3.1.1. The density of heparin on microspheres In this process, the grafting amount of heparin that has a number of chemically reactive functional groups determines the density of effective ligand on microspheres. Fig. 3(A) shows the initial concentration of heparin made difference to the heparin density on the surface of PsHep. It was found that the grafting density of heparin almost approached maximum when the initial concentration of heparin was more than 5 mg/ml. And it was found that the color of microspheres after stained with TB is different. Fig. 3(B) shows the photographs of Ps, Ps-NH2, heparinadsorbed Ps-NH2 and Ps-Hep after TB staining. The original Ps was white without being stained because of high hydrophobicity. And PsNH2 was purple which might be attributed to the improvement of hydrophilic resulting in adsorption of TB. After immobilization of heparin, Ps-Hep became a light blue. As a control, the heparin-adsorbed PS-NH2 that was Ps-NH2 immersed into heparin solution without EDC/NHS was purple, same as the color of Ps-NH2, which indicated that the heparin was adsorbed physically on surface and washed away easily. This
2.7.2. Endotoxin adsorption in physiological saline measured by TAL Static adsorption: Endotoxin adsorption in physiological saline was evaluated by TAL (Chromogenic). 100 mg of Ps, Ps-NH2, Ps-Hep, PsHep-Phe was added into 1 ml of physiological saline solution with the concentration of endotoxin as about 5 EU/ml and shaken at 37℃for 2 h. The adsorption capacity of endotoxin was calculated by determining the endotoxin concentration of the upper solution after adsorption. Dynamic adsorption: 0.5 g of Ps-Hep-Phe was packed in a glass column (100 mm × 10 mm) with constant current pump. 5 ml of physiological saline solution which contained the endotoxin concentration of about 5EU/ml were perfused into the column for 2 h with flow rate of 4 ml/min at room temperature. The amount of adsorbed endotoxin was measured at different time. 91
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Fig. 3. (A) Effects of the initial concentration of heparin on the heparin density on the microspheres; (B) Digital images of toluidine blue staining for Ps, Ps-NH2, Heparin-adsorbed Ps-NH2 and Heparin-modified Ps-NH2 (Ps-Hep).
Fig. 4. Characterization of microspheres. (A) FTIR spectra and (B) XPS spectra of Ps, Ps-NH2, Ps-Hep and Ps-Hep-Phe. (C) XPS C1 s core-level spectra of Ps, Ps-Hep and Ps-Hep-Phe.
et al., 2015). These results proved that heparin had been grafted covalently onto microspheres, which was consistent with the TB staining. For the spectra of Ps-Hep-Phe, the peak of bending vibration of C–N was 1260 cm-1, and the peak for −COOe groups overlapped with − NH2 groups to form a wide peak at about 1580 cm−1 (Poon, Bin Zhu, Shen, Chan-Park, & Ng, 2007), which attributed to the a large number of –NH2 on Phe. XPS spectra were to further prove the chemical structure and reaction process on the modified microspheres. The survey scan spectra of the microspheres were shown in Fig. 4(B) and the elemental compositions from XPS spectra are listed in Table 1. For the control Ps surface has peaks at 284.7 eV, 271.1 eV and 201.1 eV, corresponding to the binding energy of C 1s and Cl 1s, Cl 2p respectively. Compared with the Ps, all modified microspheres showed a new peak at 400.1 eV, corresponding to the binding energy of N 1 s (Yang, Tian, Dai, Hu, & Xu, 2006).The nitrogen content of Ps-NH2 surface was increased significantly, and the chlorine content were decreased obviously from 5.32% for Ps to 0.26% for Ps-NH2, indicating that the –NH2 groups had been grafted successfully on microsphere surface. After binding with heparin, the oxygen content was increased significantly from 8.10% for Ps-NH2 to 23.37% for Ps-Hep, and the sulfur content, which was consistent with the peaks of S 1s, S 2p (Xiang et al., 2014), was increased
demonstrated that heparin might be mainly grafted covalently on microspheres, which was more stable. And further proof need to be measured by FTIR and XPS spectra. 3.1.2. FTIR and XPS The FTIR was employed to detect the chemical structure of microspheres. The Fig. 4(A) shows the FTIR spectra of microspheres at wavelengths from 400 to 4000 cm−1. Compared with the original Ps, the two new peaks at 3370 and 3310 cm-1 for Ps-NH2 were attributed to the antisymmetric and symmetric stretching vibrations of amino groups (Tzoneva et al., 2008), and the peaks around 1261 cm-1 and 671 cm-1 for Ps-NH2 were disappeared that was assigned to the disappearance of the in-plane bending vibration of C–H binding with chlorine groups and stretching vibrations of CeCl, which confirmed the successful immobilization of amino (eNH2) groups. For the spectra of Ps-Hep, the new peak at 3440 cm-1 was attributed to the stretching vibrations of −OH(Shi, Meng, Xu, Du, & Zhang, 2013), while the new peaks at 1660 cm-1 and 1260 cm-1 were attributed to the stretching vibrations of C]O and the bending vibration of C–N in secondary amide respectively (Wang et al., 2013) which indicated the successful synthesis of secondary amides. The wide peaks around 1060 cm-1 were ascribed to the stretching vibrations of the sulfonic (eSO3e) groups (Nie, Ma, Xia 92
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Table 1 Chemical composition of microspheres calculated from XPS survey scans. Microsphere
Ps Ps-NH2 Ps-Hep Ps-Hep-Phe
Element (Atom %) C
O
N
S
Cl
82.31 81.43 65.22 65.44
9.06 8.1 23.37 23.32
2.49 9.42 8.03 7.87
0.27 0.33 2.52 2.20
5.32 0.26 0.29 0.48
sharply to 2.52% for Ps-Hep because of the rich oxygen content and sulfonic groups of the immobilized heparin, indicating the successful immobilization of heparin on microspheres. The chemical change of microsphere surface can also be analyzed further from the high-resolution core-level spectra of C1 s through the different binding energy, as shown in Fig. 4(C1–C3). The C1s core-level spectrum of PS could be fitted by two peaks. One peak at 284.7 eV was assigned to the carbon skeleton(CeC/C]C) for the backbone of Ps microspheres. But there was the other peak at 286.5 eV for C–O, which may be the impurity on the synthetic process of Ps. With the immobilization of heparin on the microspheres surface, the binding energy at 285.3 eV was attributed to C–S bond of heparin, which was overlapped with the C–N bond and the peak at 286.5 eV was assigned the bond C–O. In addition, the signal observed at 288.2 eV (C]O) could be ascribed to attributed to the C atoms in the amide groups of grafting heparin (or phenylalanine), which proved the more formation of the amides groups in modification process. These results further confirmed that heparin was successfully immobilized on microspheres and Phe was grafted mostly on heparin of Ps-Hep.
Fig. 5. (A) Images of water contact angle at 3000 ms for Ps, Ps-NH2, Ps-Hep and Ps-Hep-Phe; (B) The dynamic water contact angles of microspheres within 3000 ms.
3.1.3. Zeta potential and water contact angle Table 2 presents the zeta potential of microspheres surface. It was found that the zeta potential of Ps-NH2 was increased significantly from -17.3 eV to 33.4 eV. This increase in surface potential can be attributed to the positive charge of grafted –NH2 on microspheres, which was consistent with the other report (Xiang et al., 2014). After binding with the heparin, the zeta potential was decreased obviously, from 33.4 eV for Ps-NH2 to 17.2 eV, which was attributed to the immobilized heparin possessing the rich negative groups. The zeta potential continued to reduce, from 17.2 eV for Ps-Hep to 10.3 eV for Ps-Hep-Phe, which was assigned to the negatively ionizable carboxylic groups of Phe in distilled water.
angle of Ps-Hep-Phe was 11.8° closed to that of Ps-Hep. Meanwhile, the water contact angle was attenuated in different degree with the prolonged time, which was attributed to the permeation of water into the microporous and capillary absorption. 3.2. Blood compatibility 3.2.1. Clotting time APTT and TT are usually used to evaluate coagulation abnormalities in the intrinsic and extrinsic pathway respectively (Popovic et al., 2012). Fig. 6(A) presents the TT and APTT of different microspheres. Compared with that of plasma control, there was no change in TT and APTT for the Ps and Ps-NH2, and the APTT for Ps-Hep, Ps-Hep-Phe were prolonged in different degrees. The increment of APTT for the Ps-Hep and Ps-Hep-Phe might be attributed to the combination between –SO3and the coagulation factors or the combination of functional groups and Ca2+ in the blood plasma (Nie, Tang et al., 2014). Surprisingly, the APTT for Ps-Hep-Phe was increased much more than that for Ps-Hep which might be assigned to the Phe affected the activation of Factor XIa (Smith et al., 2016). In order to ensure the increment whether resulted from heparin or Phe, the clotting time of Ps-NH2 immobilized with Phe and no heparin as a spacer (Ps-Phe) was measured. It was found that the APTT for Ps-Phe was no much change with Ps-Hep, but was much lower than Ps-Hep-Phe, indicating that anticoagulation of Ps-Hep-Phe was contributed by heparin and Phe together. The TT for the Ps-Hep, PsHep-Phe and Ps-Phe was prolonged slightly compared with that for plasma, which attributed to the effects of −COOe and eSO3e groups on fibrinolytic system. The results of APTT and TT confirmed the satisfactory blood compatibility of the Ps-Hep-Phe.
3.1.4. Hydrophilicity Water contact angle was a common approach to evaluate the hydrophilicity of biomaterials surface, which may endow the bio-interface with favorable anti-fouling property. Fig.5(A) presents the images of static water contact angle at 3000 ms, and Fig. 5(B) shows the water dropped within 3000 ms. When the water dropped at 3000 ms, the water contact angle of pristine Ps is around 131.6° demonstrating that the surface of Ps was high hydrophobicity. The water contact angle was decreased obviously, from 131.6° for Ps to 47.4° for Ps-NH2, which was assigned to the presence of hydrophilic amino groups. After binding with the heparin, the water contact angle was decreased to 8.4° for PsHep, which is attributed to the heparin possessing the hydrophilic groups, such as –SO3-, −COO- and −OH groups. And the water contact Table 2 Zeta potential of Ps and modified microspheres. Samples
Zeta potential(mV)
Ps Ps-NH2 Ps-Hep Ps-Hep-Phe
−21.4 ± 4.6 30.1 ± 3.0 17.3 ± 3.4 8.2 ± 2.3
3.2.2. Protein adsorption and platelet activation Protein adsorption is considered to be first event occurring on biomaterials surface contacted with blood, and the adsorbed protein will mediate the subsequent biological responses, including platelet adhesion and thrombus formation on surfaces (Ma et al., 2014). Therefore, 93
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Fig. 6. (A) TT and APTT values of normal plasma and microspheres. (B) FIB and (C) ALB and GLB adsorptionamounts on these microspheres; (D) The concentrations of PF-4 released of these microspheres with the plasma, which can act as indicator for platelet activation.
samples was measured. When the platelets activated during aggregation, PF-4 is released from α-granules in a platelet release reaction, so PF-4 is usually used as an indicator of platelet activation (Nie, Ma, Cheng, Deng & Zhao, 2015). As shown in Fig. 6(D), there is a dramatic increase in PF-4 released in plasma for Ps-NH2that might be attributed to the high density of positive charge, which suggested that platelet activation probably occurred. After the modification with heparin, the PF-4 released in plasma was significantly decreased, indicating that platelet activation was suppressed, which was contributed to the immobilized heparin polymers on adsorbents (Nie et al., 2017). Although PF-4 released of Ps-Hep-Phe was slightly increased compared with PsHep, Ps-Hep-Phe still kept the lower level of PF-4 released compared with Ps-Phe, which demonstrated that the platelet activation on the PsHep-Phe with heparin as a spacer did not occur or was gently suppressed.
protein adsorption is one of important factors to evaluate the biocompatibility of materials. In considering surface-induced thrombosis, FIB is a central protein in the thrombus formation. Fig. 6(B) shows the FIB adsorption amounts of microspheres. We noted that heparin-modified adsorbents show the significantly decreased FIB adsorbed amount. The introduction of heparin into the Ps-Hep-Phe adsorbents still kept the lower FIB adsorbed amount than that of Ps-Phe without the heparin as a spacer, which was assigned to the improvement of hydrophilic, indicating that heparin as a spacer could reduce the FIB adsorption amount (Pan, Hou, Zhang, Dong, & Ding, 2014). To investigate further protein adsorption of PsHep-Phe, ALB and GLB were evaluated as shown in Fig. 6(C), because they are abundant protein in plasma that keep body balanced. After modifying, the adsorbed amount of ALB and GLB was decreased. And the Ps-Hep-Phe still kept the low adsorption of ALB and GLB. Meanwhile, it was found that the results of protein adsorption were highly related to the hydrophilic/hydrophobic properties of biomaterial surfaces. Some researchers reported that water contact angle played an important role in the protein adsorption (Xu, Bauer, & Siedlecki, 2014). From the view of the surface energy, surface wettability and protein adsorption both are the energy-driven process that involves the properties of both the substrate surface. So the amount of protein adsorption on the surface can be reflect from the water contact angles. Moreover, the surface of biomaterial was hydrophilic which tend to combine with the water molecules, and the ability to adsorb protein was more weaken (Nie, Qin et al., 2014). To investigate the platelet activation after the interaction with microspheres, the amount of PF-4 released in surface-exposed plasma
3.2.3. Hemolysis test Hemolysis is a reliable method to estimate the hemocompatibility of biomaterials in contact with blood (Wu et al., 2017; Zhao, Ma, Zeng, Tu & Zhao, 2016). Fig. 7F shows the degree of hemolysis obtained with Ps, Ps-NH2, Ps-Hep, Ps-Hep-Phe and Ps-Phe. All the microspheres showed a low degree of hemolysis (< 2%); but the adsorbents with the heparin as a spacer further reduced the degree of hemolysis. As same, the hemolysis rate of Ps-Hep-Phe was lower than that of Ps-Phe, showing good blood compatibility. Fig. 7G also shown the visual observation of the hemolytic phenomenon, consistent with the previous quantitative spectrophotometric measurement. And the morphologies and adhesion of erythrocytes adhered on Ps, Ps-NH2, Ps-Hep, Ps-Hep-Phe and Ps-Phe 94
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Fig. 7. The morphologies of erythrocytes treated with (A)Ps, (B) Ps-NH2, (C)Ps-Hep, (D) Ps-Hep-Phe, (E) Ps-Phe, respectively; (F) Hemolysis rate of microspheres; (G) Visual observations of hemolysis.
3.3. Endotoxin adsorption
microspheres Fig. 7(A–E) were observed by SEM. It was observed that Ps had little adhesion of erthrocytes, which may be due to the superhydrophobic surface of Ps. After the treatment of EDA, erythrocytes adhered on the Ps-NH2 surface became more and some of them showed ridges. Then the introduction of heparin into surface, Ps-Hep and PsHep-Phe showed extremely little adhesion of erythrocytes and the morphologies of erythrocytes with biconcave shape and smooth surfaces similar to normal red cells, demonstrating the good blood compatibility and nontoxicity. More erythrocytes adhered on surface of PsPhe without heparin also confirmed this point, indicating that the incorporation of heparin into microspheres could reduce the risk to erythrocytes in hemoperfusion. Therefore, heparin as a spacer to connected Phe and Ps was a better choice to improve the great blood compatibility of adsorbents.
3.3.1. FITC-LPS adsorption The adsorption of fluorescent LPS was used for qualitative evaluation. The fluorescence intensity was observed by fluorescence microscopy to evaluate the adsorption capacity of FITC-LPS as shown in Fig. 8(A), and in order to avoid visual error, the fluorescence intensity was calculated by image-pro plus, as shown in Fig. 8(B). The adsorption capacity of Ps was weak with the lowest fluorescence intensity. And the fluorescence intensity was increased with the modification. Ps-Hep-Phe had a larger fluorescence intensity than Ps. This indicated indirectly that Ps-Hep-Phe had an improvement in adsorption capacity of endotoxin.
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Fig. 8. (A) Images (10 × 4) of FITC-LPS adsorption for 2 h on microspheres; (B) The fluorescence intensity of microspheres after FITC-LPS adsorption calculated by image-pro plus. (C) The endotoxin adsorption capacity of the Ps and the modified microspheres; (D) Effects of adsorption time on endotoxin adsorption capacity; (E) Effects of adsorption time on endotoxin adsorption capacity in human plasma.
equilibrium time of Ps-Hep-Phe was 60 min, which could be meets the requirements in clinic.
3.3.2. Adsorption of endotoxin in physiological saline solution measured by TAL Fig. 8(C) shows the static adsorption capacity for the all microspheres. The adsorption capacity of pristine Ps was 18.03EU/g. The adsorption capacity of Ps-NH2 was increased obviously due to the positive group of –NH2. Then the introduction of heparin into microspheres, there was no obvious drop in the adsorption cpacity of Ps-Hep compared with Ps-NH2 which might be mainly attributed to the positive charge of Ps-Hep as revealed by the zeta potential test (Chandy & Rao, 2000). After connected with Phe, the adsorption capacity of Ps-Hep-Phe was more than that of other microspheres. This increase might be attributed to the hydrophobic amino acid of Phe that could interacted with the Lipid A of endotoxin by the hydrophobic interaction (Anspach, 2001; Jirawutthiwongchai et al., 2016). Moreover, the extension of the spacer with heaprin could reduce the steric hindrance between the ligand and the endotoxin molecule which might also contribute to the adsorption capacity of Ps-Hep-Phe (Zhi, Mei, Li, Hou & Wang, 2005). These indicated that the heparin as a spacer connected matrix with Phe still performed well in adsorption capacity for endotoxin removal. To study further the kinetic adsorption process of Ps-Hep-Phe, the Fig. 8(D) shows the relationship between adsorption capacity of endotoxin and adsorption time in the physiological saline solution. There was a rapid increased before initial 30 min because of the frequent collision between the exposed active sites and a large amount of endotoxin monomers. With the increment of time, the rate of adsorption efficiency was gradually slower. When the time reached 90 min, the adsorption capacity of endotoxin was 25.15 EU/g.
4. Conclusion In this study, heparin as a spacer was covalently immobilized onto Ps successfully and connected with ligand of Phe, which involved condensation reaction between −COOH and –NH2 through the EDC/ NHS coupling chemistry for the endotoxin removal. And the heparinmodified microspheres displayed the great hydrophilic property. The heparin as a spacer immobilized onto the microspheres still plays an important role in the protein adsorption and anticoagulant of adsorbents. Moreover, Phe connected with biological macromolecular chains can swing in a wider range to capture endotoxin molecules and the Ps-Hep-Phe still keeps the pretty adsorption capacity of endotoxin compared with the Ps. Therefore, heparin as a molecular spacer will be an effective way to improving the performance of adsorbents in blood purification. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant No. 11572065), Development, clinical trial and registration of hemoperfusion (No. cstc2018jszx-cyzd0582) and Chongqing University Postgraduates’ Innovation Project (No. CYB15028).
3.3.3. Adsorption of endotoxin in human plasma The microspheres as adsorbents used in hemoperfusion, need to the high removal efficiency in clinical treatment. Thus, adsorption equilibrium time should be confirmed to ensure patients against harms in the therapy process. Fig. 8(E) shows the adsorption efficiency of the PsHep-Phe. The adsorption capacity reached a plateau when the adsorption time exceeded 60 min which was similar with other reports (Cao, Zhu, Zhang, & Dong, 2016; Fang et al., 2004), and the adsorption capacity was up to 15.00 EU/g at 60 min. In clinical treatment, the contact time between materials and plasma should be avoided too long which should be better not more than 2 h. So the adsorption
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