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Terminally Alkylated Heparin. 1. Antithrombogenic Surface Modifier Takehisa Matsuda*,† and Tomoko Magoshi‡ Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, and Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565-8565, Japan Received June 5, 2001; Revised Manuscript Received September 10, 2001
Terminally alkylated heparin was prepared by reducing the terminal end of heparin and subsequent lactone formation, followed by ring-opening reaction with alkylamine. The alkyl groups used include butyl, octyl, lauryl, and stearyl. These alkylated heparins adsorbed on the poly(ethylene terephthalate) film from their respective aqueous solutions. The adsorptivity and its stability in buffer solution, complexation compatibility with antithrombin III (ATIII), were enhanced with larger alkyl-group-derivatized heparins. These were assessed using a confocal laser scanning microscope. The “heparin surfactant” developed here may be used for ensured short-term “system antithrombogenicity” of assembled extracorporeal circulatory devices or circuits. Introduction Although various blood-compatible surface designs have been explored over the years,1-17 reliable and clinically used surface process technologies applicable to medical devices have been limited. Among the surface modification techniques explored, surface derivatization and immobilization of heparin on surfaces and its sustained release have been extensively studied for more than 3 decades. Heparin, a polysaccharide composed mainly of alternating units of sulfonated glucuronic acid and glucosamine derivatives, which is clinically used as an anticoagulant, delays or inhibits the blood coagulation cascade by catalyzing the inactivation of thrombin (T) by complexing with ATIII. Therefore, heparinization has been recognized as the most proven potent and reliable thromboresistant surface with a short-term guaranteed antithrombogenicity and has been infused in clinically available blood-contacting circuit and hollow-fibertype extracorporeal devices such as artificial oxygenator (cardiopulmonary bypass) and artificial dialysis (or hemodialyzes or hemoperfusion). Without heparin, it can be said that such extracorporeal circulation has not been realized. Various research groups have continuously investigated new design concepts, to develop processing techniques and to improve the pharmacological effects and stability of surfacebound heparin in streaming blood. In particular, Kim et al.18 and Olsson et al.19 have independently pursued their own technologies over almost 2 decades. Recently, their excellent review papers dealing with these two different techniques have been independently presented. The surface heparinization technologies involved include ionic complexation of heparin with an aminated surface with or without further stabilization with glutaraldehyde, covalent * To whom correspondence may be addressed. Tel: 81-92-642-6210. Fax: 81-92-642-6212. E-mail:
[email protected]. † Kyushu University. ‡ National Cardiovascular Center Research Institute.
bonding of heparin on surfaces with or without a spacer with hydrophilic or hydrophobic arm,16 organic solution coating of heparin complexed with alkylamine,5 heparin-immobilized hydrogel layered on a surface,3,4 and heparin immobilized to aminated polyethylene.12 As for the configuration of heparin on surfaces, there appear to be three different configurations. One is heparin laid-down via multiple-point bonding of heparin with surface amino groups via electrostatic interaction or covalent bonding, the second is heparin covalently bound to a surface via an alkylene or poly(ethylene glycol) spacer arm, thus exposing it to the blood stream, and the last is an end-point configuration in which a heparin molecule stands up at its reducing terminal end by specific chemical reaction with a surface functional group. Regardless of the surface process technology employed, these methods are required for surface functional derivatization prior to heparinization. These surface heparinization techniques have been realized on some parts of devices, but not in the entire extracorporeal system which is assembled from different devices and parts at the site of the surgical operation, each of which is made of different materials. On the other hand, if the surface heparinization technique, which can be achieved after assembling an extracorporeal system composed of devices, connectors, and circuits at the site of the surgical operation, is available just prior to extracorporeal circulation, at least a short-term “system antithrombogenicity” which is free from thrombus at the entire blood-contacting surface of the extracorporeal system may be further guaranteed. The proposed and designed surface heparinization is performed using an adsorptiondriven technique using an alkylated heparin as a “heparin surfactant”, which is carried out using its buffered solution as a priming solution before blood circulation. This paper will describe the preparation of alkylated heparin, surface adsorption characteristics, the thrombin capture ability in the
10.1021/bm0100965 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/09/2001
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Figure 1. Synthetic route of terminally alkylated heparin and heparan sulfate.
presence of ATIII, and its alkyl chain length dependency. The latter two subjects were assessed using a confocal laser scanning microscope (CLSM). Experimental Section Materials. All solvents and reagents including heparin were obtained from Wako Pure Chemicals Inc. (Osaka, Japan). Heparan sulfate sodium salt was purchased from Seikagaku Co., Ltd. (Tokyo, Japan). Antithrombin III (ATIII), thrombin (T), and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). The medium DMEM was obtained from GIBCO (Grand Island, NY), and fetal bovine serum (FBS) was obtained from Life Technologies (New York, USA). The poly(ethylene terephthalate) (PET) film was obtained from Bellco Glass Inc. (New Jersey, USA). The polystyrene, poly(vinyl chloride), and poly(vinyl alcohol) films were obtained from Minamide Corp. (Osaka, Japan). General Methods. Purification of heparin, oxidized heparin, and alkylated heparin was achieved using a dialysis membrane (molecular weight cutoff ) 12000-14000, Wako). The ion-exchange resin used was Dowex 50X8 (H+) (Dow Chemicals, Midland, MI). 1H NMR spectra were recorded on a JEOL JNM-GX270 FT-NMR spectrometer (270 MHz, Tokyo, Japan). X-ray photoelectron spectra recording for the determination of surface chemical composition was carried out using an ESCA-750 instrument (Shimadzu Corporation, Kyoto, Japan) at a 15° takeoff angle. Wettability of treated surfaces was evaluated using the sessile drop technique with a contact angle meter (CA-D, Kyowa Kaimenkagaku Co., Ltd., Tokyo, Japan). The surfaces of photocured films were observed by scanning electron microscopy (SEM) using a JEOL JSM-6301F (Tokyo, Japan) microscope. Fluorescence on the surface was observed using a confocal laser scanning microscope (CLSM, 543 nm excitation, Bio-Rad Lab., Hercules, CA). Gel permeation chromatographic (GPC) analysis in water was carried out with HPLC-8020 instrument
(Tosoh, Tokyo) (column, Tosoh TSK gel R-5000) using poly(ethylene glycol) standards. Synthesis of Alkylated Heparin. Alkylated heparin was synthesized using a similar procedure reported for preparation of alkylated condroitin sulfate, alkylated hyaluronan, and alkylated heparin with few modifications.20 Figure 1 shows the sequential procedures for preparation of alkylated heparin with different sized hydrophobic tails. Heparin sodium salt (4.0 g, molecular weight ) 12000; 198.6 IU/mg) was dissolved in water and passed through a Dowex 50X8 (H+) column. The eluate was dialyzed followed by lyophilization to obtain heparin (3.8 g, 3.2 × 10-4 mol). The reducing end of heparin was oxidized with iodide (0.8 g, 6.3 × 10-3 mol) in 20% aqueous methanol solution (100 mL) for 6 h at room temperature. The completion of the oxidation was assessed by the 3, 5-dinitrosalicylic acid method.20 The reaction solution was added to ethanol containing 4% (w/v) potassium hydroxide (200 mL). The white precipitate thus obtained was filtered, dissolved in water and subjected to dialysis. Upon freeze-drying of the dialyzed solution, oxidized heparin was obtained. There was little significant difference in GPC elution pattern before and after oxidation. The product dissolved in water was passed through a Dowex 50X8 (H+) column. Upon freeze-drying of the eluate, the lactoneheparin was obtained (3.0 g, 2.5 × 10-4 mol). n-Butylamine (0.1 mol, 1.8 × 10-3 mol) or n-octylamine (0.1 mL, 6.0 × 10-4 mol) was added to lactone-heparin (100 mg, 8.3 × 10-5 mol) dissolved in N,N-dimethylformamide (5.0 mL). This mixture was stirred for 8 h at 80 °C. The reaction mixture was concentrated and then dissolved in water. The dilute reaction mixture was passed through a Dowex 50X8 (H+) column. The eluate was extensively dialyzed followed by freeze-drying to obtain the heparin alkylated with butyl- (108.8 mg, 9.1 × 10-5 mol) or octylamine (88.8 mg, 7.4 × 10-5 mol). Laurylamine (22.9 mg, 1.3 × 10-4 mol) or stearylamine (33.7 mg, 1.3 × 10-4 mol) was dissolved in chloroform and added to a lactone-heparin (150 mg, 1.3 × 10-5 mol) solu-
Terminally Alkylated Heparin
tion in dimethylformamide (5 mL) neutralized with tri-nbutylamine (0.1 mL). The reaction mixture was there stirred for 12 h at 80 °C. The reaction mixture was concentrated and washed with chloroform to remove unreacted laurylamine (or stearylamine). The residue obtained was dissolved in water and passed through a Dowex 50X8 (H+) column. The eluate on dialysis and freeze-drying gave the respective alkylated heparin with lauryl- (126.3 mg, 1.1 × 10-5 mol) or stearylamine (134.8 mg, 1.1 × 10-5 mol). These alkylated heparins, regardless of size of alkyl group, were completely soluble in water 30% and 70% aqueous ethanolic solutions. Fluorescence-labeled stearyl group-bearing heparin was prepared by condensation reaction with 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF, Sigma) according to our method previously reported9 (the number of DTAF incorporated was around 2.7 per molecule). Surface Characterization. The surface chemical composition and wettability of surfaces either coated with a solution of alkylated heparin or adsorbed from an aqueous solution were determined by electron spectroscopy for chemical analysis (ESCA) analysis and water contact angle measurements, respectively. PET films (f15 mm), which were thoroughly washed sequentially with ethanol, acetone, and distilled water prior to treatment, was immersed in 1 wt % solution of the alkylated heparin for 3 h, after which different washing methods were employed: (1) simple washing with water, (2) washing with water and subsequently with 30% ethanol solution, and (3) washing with water, followed by sequential washing with 30% and 70% ethanol solutions. One film was retained without any rinsing. Untreated PET film was used as the control. Platelet Adhesion. PET films treated with a solution of alkylated heparin and an untreated PET film were used as samples and control, respectively, for the platelet adhesion study. Platelets were isolated from the venous blood of healthy volunteers. Platelet-rich plasma (PRP) was obtained through centrifugation (900 rpm, 15 min, 20 °C) of whole blood anticoagulated with 3.8% sodium citrate. The substrates were placed on the bottom of a 24-well tissue culture dish (Iwaki Glass Co. Ltd., Chiba, Japan) and were incubated in PRP (5.0 × 105 cells/well) for 30 min at 37 °C. After incubation, substrates were gently washed with physiological saline solution (PBS). Platelets adhering to the substrates were fixed with 3% glutaraldehyde with 0.1 M cacodylate buffer solution at room temperature for 30 min followed by fixation with 1% osmium tetraoxide. Samples were dehydrated using an ethanol-graded series. Critical-point-dried SEM samples of platelets adhered on the film surfaces were obtained using a critical point dryer (HCP-2, Hitachi Co. Ltd., Tokyo, Japan) and sputter-coated platinum and examined using a scanning electron microscope. The relative numbers of adhered platelets were determined from 10 different SEM microphotographs. Biological Activity of Alkylated Heparin. PET films treated with 1 wt % solutions of alkylated heparin were immersed in a buffer solution of antithrombin III (ATIII, 1 unit/mL), followed by immersion in a thrombin solution (T; 10 unit/mL). Then, these films were washed thoroughly with a buffer solution for fluorescent staining. All steps were
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performed for 30 min at 4 °C. The heparin-treated surfaces were treated with 1% bovine serum albumin (BSA) used as a blocking agent in PBS for 30 min, which will block the nonspecific adsorption of (ATIII and T antibodies proteins and avidins), onto the nonheparinized surface. The heparin-ATIII-T trimolecular complex formed surfaces on using an enzyme-labeled antibody technique (ABC kit; Vector Laboratories, Inc., Burlingame, USA) were visualized under a CLSM. Staining was performed in accordance with the manufacturer’s instruction. Briefly, the first step involved treatment with a buffer solution containing sheep monoclonal antibody. Then, a biotinylate secondary antibody containing buffer solution was applied, and avidin and biotinylated alkaline phosphatase mixed solution were subsequently added to the surface. Alkaline phosphatase substrate kit 1 was then used. The colorimetric enzymesubstrate reaction allows for visualization of only fixed proteins. Images of the surfaces were obtained by CLSM. Results Preparation of Alkylated Heparin and Heparan Sulfate. Figure 1 shows schematics of preparation of heparins with different chain lengths of the alkyl group at the terminal end according to the method developed previously for lipidderivatized polysaccharides, such as heparin, heparan sulfate, chondroitin sulfate, and hyaluronan.20 Three reactions were sequentially conducted: iodide cleavage of termini (step 1), its lactone ring formation (step 2), followed by ring-opening reaction with alkylamine leading to the formation of an amide group with alkyl end (step 3). This resulted in the formation of alkylated heparin. The completion of oxidation was assessed by the 3,5-dinitrosalicylic method.20 The reaction was monitored by 1H NMR spectroscopy to determine the completion of the reaction performing carefully controlled oxidative cleavage. GPC analyses before and after modification showed that there was little significant change in GPC pattern, indicating that oxidative cleavage of main backbone did not occur. Figure 2 shows the ring-opening reaction time vs the integrated ratio of methylene protons ascribed to stearylamine against methyl protons ascribed to the acetyl group of heparin. Chloroform extraction and extensive dialysis, both of which removed unreacted alkylamine, followed by freeze-drying, produced white powder. The ratio ascribed to stearylamine increased with reaction time and remained unchanged beyond 8 h. Even at a high concentration and longer reaction times of step 1 and step 2, the maximal ratio achieved was around 2.0. This indicates that the reaction appeared to be completed within 8 h under these particular reaction conditions. Stearyl-group-end-capped heparin was designated as Hep-C18. Heparins having alkyl groups such as butyl, octyl, and lauryl were also prepared after ratios similar to that of Hep-C18, as mentioned above, were maximum, respectively. These alkylated heparins were designated as Hep-C4, Hep-C8, and Hep-C12, respectively. Heparan sulfate having a stearyl group (designated as HS-C18) was also prepared according to the reactions described above. All the alkylated heparins prepared were soluble in water and 70% aqueous ethanolic solution.
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Figure 2. Relationship between the reaction time of ring-opening amidation and the integral ratio of peaks in 1H NMR spectroscopy.
Figure 3. Procedures of adsorption of alkylated heparin in aqueous solution and adsorption in washing solutions. ESCA and water contact angle measurements were carried out at each step.
Adsorption. A poly(ethylene terephthalate) (PET) disk film was immersed in an aqueous solution containing 1 wt % alkylated heparin for 3 h and was subsequently subjected to sequential washing with water and 30% and 70% aqueous ethanol solutions. At each washing step, air-dried surfaces were subjected to ESCA measurement and contact angle measurement as shown in Figure 3. Figure 4 shows dependencies of elemental ratios of N/C and S/C, determined by ESCA measurement, on the washing steps. Irrespective of the type of alkylated heparin, extensive washing resulted in both reduced N/C and S/C ratios. Higher N/C and higher S/C ratios mean a larger amount of alkylated heparin on the PET surface since N and S were derived only from heparin. Hep-C12 appeared to have the highest adsorptive character, followed by Hep-C18. The lowest one was shown by HepC4. Nonmodified heparin was not adsorbed on PET surface, judging at least from the relative elemental ratios, N/C and S/C. These values became negligible after washing with water as was Hep-C4. Therefore, we did not examine biological activity of the nonmodified heparin-treated surface. The adsorption character of HS-C18 was almost identical to that of Hep-C4.
Figure 4. ESCA elemental changes of adsorbed surfaces with alkylated heparin with different chain lengths as a function of washing steps (1) nonwashing, (2) water, (3) 30% ethanol aqueous solution, and (4) 70% ethanol aqueous solution: Hep-C4 (9); Hep-C8 ([); Hep-C12 (b); Hep-C18 (2); HS-C18 (4).
On the other hand, as shown in Figure 5, water contact angle measurements indicated that, by washing at each step, both advancing and receding contact angles were increased, irrespective of the type of alkyl groups. In terms of receding contact angles, HS-C18 was minimal followed by those of Hep-C12 and Hep-C18. The least wettable one was HepC4. Table 1 shows the polymeric substrate dependency of
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Terminally Alkylated Heparin
Figure 5. Changes in water contact angle of adsorbed surfaces with alkylated heparin with different chain lengths as a function of washing steps (1) nonwashing, (2) water, (3) 30% ethanol aqueous solution, and (4) 70% ethanol aqueous solution: Hep-C4 (9); Hep-C8 ([); Hep-C12 (b); Hep-C18 (2); HS-C18 (4). Table 1. Water Wettability of Hep-C18 Treated Polymeric Surfaces and Its Dependency on Washing Solutionsa polymer PET
PST
PVC
PVA
contact angle (deg)
treatment/ washing step
advancing
receding
nontreated I II III nontreated I II III nontreated I II III nontreated I II III
72 38 62 68 85 20 75 78 92 25 82 80 74 25 52 62
56 5 24 38 80 5 31 52 90
