- Email: [email protected]
A novel solvent system for blending of polyurethane and heparin
ARTICLE IN PRESS
Biomaterials 24 (2003) 3915–3919
A novel solvent system for blending of polyurethane and heparin Qiang Lv, Chuanbao Cao*, Hesun Zhu Research Center of Material Science, Beijing Institute of Technology, Beijing 100081, China Received 17 November 2002; accepted 8 April 2003
Abstract To improve the blood-compatibility of polyurethane, the co-solvent of tetrahydrofuran and water, a new solvent system for blending polyurethane and heparin, was proposed. After solvent casting, heparin was blended in a polyurethane film. The ATRFTIR was used to analyze the surface chemical element and the contact angle was measured to investigate the hydrophilicity of the surface of the PUs. As the amount of heparin increased, the surface hydrophilicity was increased and all the clot times exceeded the measurement limit of the clot detection instrument when the heparin loaded on the polyurethane films was 3%, 5% and 7%. After the films were immersed in the phosphate buffered saline for 30 days, the activated partial thromboplastin time and thrombin time still exceeded the measurement limit of the clot detection instrument. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Blood compatibility; Heparin; Blending; Polyurethane
1. Introduction Segmented polyurethanes have been extensively used for the construction of cardiovascular devices because of their desirable physical properties and relative thromboresistance compared to other materials used in cardiovascular application [1,2], but thrombus formation still exists in small diameter tubing. To enhance the blood compatibility of polyurethanes, many different approaches have been studied. Heparin is an important anticoagulant, used clinically to minimize thrombus formation on artificial surfaces [3]. It has the ability to interact strongly with antithrombin III to prevent the formation of fibrin clot [4,5]. There are two general methods to develop bloodcompatible polymeric materials using heparin. One method uses chemical immobilization of heparin and the other is the heparin-releasing system. Covalent immobilization of heparin onto a polymer surface can provide long lasting antithrombogenicity. Heparin can be directly bound to the surface by insertion of either functional groups [6–8] or spacer arms [9], but the activity of heparin was significantly *Corresponding author. Tel.: +86-68913469; fax: +86-68915023. E-mail address: [email protected] (C. Cao).
decreased compared to raw heparin. In a heparinreleasing system, heparin is slowly released for a short duration and the bioactivity of heparin is maintained at a high level. Therefore, a heparin-releasing system is more suitable for a short-term clinical application than the covalently coated heparin system. There are several methods studied for the formulation of a heparinreleasing system [10,11]. In those methods, most releasing systems are useless because heparin was released within several hours, so a novel formulation for controlled release of heparin is necessary. Hyum Tae Moon et al. [12] reported that the heparin-DOCA, having an amphiphilic property, was homogeneously mixed with polyurethane in the co-solvent of dioxane, propanol and water. After casting the film, heparinDOCA was homogeneously dispersed as nanoparticles in polyurethane films. However, the losing of the activity of heparin was inevitable and the system could not be used in covalent immobilization of heparin because heparin has grafted with DOCA. In this study, we utilize polyurethane and the new solvent system to prepare a heparin-releasing system by simple solvent casting. It was found that heparin homogeneously mixed with polyurethane in the cosolvent of tetrahydrofuran and water if the ratio was suitable. Then heparin was blended in the polyurethane
0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00266-7
ARTICLE IN PRESS 3916
Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
films by solvent casting. The release rate, surface property and antithrombogenicity were studied.
2. Experiment 2.1. Materials The polyurethane was presented from the polymer laboratory of our university. The synthesis method employed here was a two-step method, in which the first step was to use poly(ethylene oxide-tetramethylene oxide) provided by LiMing chemical research institute and isophorone diisocyanate (IPDI) to synthesize a prepolymer, and the second step was to use 1,4butanediol as an extender to react with the prepolymer to produce the final product. The average molecular weight of poly(ethylene oxide-tetramethylene oxide) was 4850 and the ratio of ethylene oxide/tetramethylene oxide was 50/50. As a result, the average molecular weight of PU determined by gel permeation chromatography (Wasters 150-C, 1 ml/min, 30 C) was about 64,000. The mechanical properties were measured on the Instron-6022 Test machine at 20 C and 65% RH and the head speed was 10 mm/min. The tensile strength of PU was about 8.34 MPa and ultimate elongation was 636%. 2.2. Preparation of heparin blended polyurethane film Polyurethane (1.3 g) was dissolved in 15 ml tetrahydrofuran, and then a 2 ml different concentration heparin water solution was mixed with it. The weight ratio of heparin was 3%, 5% and 7% compared with polyurethane, respectively. The mixing solution was spread on a glass plate (6 6 cm2) and the solvent was evaporated at room temperature. The films were dried in a drying oven at 60 C for 24 h for testing.
2.4. Release test of heparin from polyurethane film The amount of heparin released from the film was determined using the toluidine blue method as reported in the literature [8,13,14]. For keeping the thickness of the films identical to each other, all films tested were made at the same experimental conditions. A known amount of heparin aqueous solution (2 ml) was added to the toluidine blue solution (3 ml) and the mixed solution was adequately vibrated. Hexane (3 ml) was then added, and the mixture was well shaken so that the toluidine blue–heparin complex was extracted into the organic layer. The toluidine blue that remained in the aqueous phase was determined by measuring the absorbance at 631 nm and the concentration of heparin in the aqueous solution was obtained and used as a calibration curve to determine the amount of released heparin. To study the release rate of heparin, the polyurethane film (3 cm 4 cm) containing heparin was immersed in PBS solution (12 ml, pH 7.4) for 24 h. Periodically, the supernatant (2 ml) of the PBS solution was replaced with a fresh one in order to maintain a sink condition. And then the absorbance at 631 nm of the supernatant was measured and the released amount of heparin was calculated according to the standard curve. The standard curve (Fig. 3) was obtained according to the method of Smith et al. The standard curve was linear in the range of 0–40 mg heparin/ml and the heparin content was obtained directly from the standard curve. 2.5. In vitro coagulation time tests for films The nephelometry measurements, including PT, activated partial thromboplastin time (APTT) and thrombin time (TT), were performed with the coagulation instrument Coag-A-Mate XM (Organon Teknika, USA) which measures the change of luminosity when
a
2.3. Characterizations of heparin blended polyurethane film The surfaces of PUs were investigated using attenuated total reflectance infrared spectroscope (ATRFTIR, SYSTEM-2000, PE, USA). After the film was immersed in phosphate buffered saline (PBS) for 1 h, the surface morphologies were studied to compare with the initial morphologies by scanning electron microscope (SEM, JSM-35C, JEOL, Japan). The water contact angles of the film surfaces were measured by putting a droplet of deionized water on the surface of the polymer films using the JY-82 contact angle apparatus (ChengDe, China). With each specimen, the measurement was repeated at different sites, and average values were obtained for the contact angles.
b c
d
e 3500
3000
2500
2000
1500
1000
Wavenumber cm-1
Fig. 1. ATR-FTIR spectra of: (a) PU, (b) PU-3 wt%He, (c) PU5 wt%He, (d) PU-7 wt%He and (e) FTIR spectra of heparin.
ARTICLE IN PRESS Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
light traverses the plasma sample. Briefly, the tested films were incubated with healthy human blood plasma in a transparent plastic tube, and the reagents for each coagulation time test were added to the tube immediately.
3. Results and discussion 3.1. Surface infrared analysis Fig. 1 showed ATR-FTIR spectra of PU-0%He(a), PU-3%He(b), PU-5%He(c), PU-7%He(d) and the Table 1 Water contact angles of different polyurethane films Heparin rate
Contact angle ( )
3% Heparin 5% Heparin 7% Heparin
7272 6871 5473
3917
FTIR spectra of heparin as typical examples. In the spectrum of PU(b)–(d), the peak in 2944 cm 1 increased and the peak in 1460 cm 1 moved to low wave number which may be due to the effect of heparin. On the other hand, the new peak in 891 cm 1 which exists in the spectra of the heparin was also found in the spectra of (b)–(d) and the peak in 2358 cm 1, though it had no absorption in heparin, represented the stretching vibration of sulfonamide (–NHSO3) reported by some researchers [15,16] and the result may mean there are some interaction between polyurethane and heparin such as the hydrogen bond. At the same time, the point that the area of peaks in 2358 cm 1 augment when the ratio of heparin increases partly suggests the surface content of heparin increases. 3.2. Water contact angle To investigate the hydrophilicity of the PUs, water contact angle measurements were carried out (Table 1). The PUs blended with heparin were relatively
(a)
10 µm
10 µm
10 10 m µm
µm 1010 m
10 µm
10 µm
10 µm
10 µm
(b)
(c)
(d)
Fig. 2. SEM of surface morphologies of heparin loaded polyurethane films before and after immersing in PBS for 1 h: (a) 0 wt%heparin, (b) 3 wt%heparin, (c) 5 wt%heparin, (d) 7 wt%heparin.
ARTICLE IN PRESS Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
3918
hydrophilic; PUs 1–3 exhibited contact angles of 7272, 6871 and 5473, respectively. These results agree with the increase of heparin content in the polyurethane.
3.3. Release of heparin from polyurethane films As shown in Fig. 2, when the heparin-loaded film was soaked in PBS for 1 h, there were many ridges at the surface when heparin was released out of the films, but the size and density of the ridges were not increased with the increasing amount of loaded heparin. The density of the ridges was most crowded for the 5 wt% heparin loading films and most sparse for the 7 wt% heparin loading one. Furthermore, the SEM images have the change accordant with the percentage of released amount of heparin shown in Fig. 4. For 3 wt%, 5 wt% and 7 wt% heparin loading films (Fig. 4), the percentages of released amounts of heparin for 24 h were 6.13%, 7.19% and 3.61%, respectively. This may be due to the difference in the interlaced structure of the different content of heparin and polyurethane. For the heparin-loaded films, heparin was quickly released within the first one hour, followed
Absorbance at 631nm
2.5
2.0
1.5
1.0
0.5
0.00
0.05
0.10
0.15
by the relative sustained release rate for one day. This might be because of the fact that heparin located near the surface would have had a short distance to travel to the film surface. Although the relation of heparin content and release rate needed further research, the blended amount of heparin was an important parameter in controlling the release rate.
3.4. In vitro blood compatibility The PT, APTT and TT were widely used for the clinical detection of the abnormality of blood plasma. In recent times, they were applied in the evaluation of in vitro antithrombogenicity of biomaterials. The normal ranges of PT, APTT and TT for a healthy blood plasma were regarded as 1173, 2874 and 1675 s, respectively. The results are given in Table 2. The three times all exceeded the measurement limit of the clot-detection instrument indicated that heparin blended with polyurethane kept excellent blood-compatibility and the plasma was not coagulated with polyurethane since the released heparin was enough to prevent the formation of fibrin clot. Table 3 shows the APTT, PT and TT of the films containing 5% heparin, which was immersed in PBS for one month. After 30 days, although PT had decreased to 12 s, the APTT and TT still exceeded the measurement limit of the clot-detection instrument. Since we have studied that polyurethane containing heparin reduced the PT time to 15 s when it was located in the desiccator for five days at 20 C, the reduction of PT may be partly due to the loss of heparin bioactivity as the films still preserved enough heparin to keep the films’ bloodcompatibility within about 30 days.
0.20 Table 2 Activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) of Pu films containing heparin
Heparin amount(mg)
Fig. 3. Grades curve of heparin amount.
Heparin relative release percent (%)
Heparin rate
Clot time (s)
8 7
b
6
a
0% 3% 5% 7%
5
Heparin Heparin Heparin Heparin
TT
APTT
PT
15.1 >150 >150 >150
46.8 >200 >200 >200
25.0 >150 >150 >150
4
c
3
Table 3 Clot times of Pu films containing 5 wt% heparin immersed in PBS for different number of days
2 1
Immerse time
Clot time (min)
0 0
5
10
15
20
25
Time (h)
Fig. 4. Release profiles of heparin from polyurethane films: (a) 3 wt%heparin, (b) 5 wt%heparin, (c) 7 wt%heparin.
Five days Thirty days
TT
APTT
PT
>150 >150
>200 >200
11.8 12.4
ARTICLE IN PRESS Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
4. Conclusion In this study, the appropriate solvent was used to dissolve heparin and polyurethane. The simple solvent casting method allows heparin to be released slowly from the polymeric film and the rate of drug release can be controlled by the amount of drug being loaded. In the films, heparin can keep excellent bioactivity and in a relatively long time the films shows favorable blood compatibility. So we think that it could be especially useful when applied to devices designed for mediumtime use.
Acknowledgements This study is supported by 973 project (G1999064705) of China and 863 project (2002AA326030) of China.
References [1] Kolff WJ, Akutsu T, Dreyer B, Norton H. Artificial heart in the chest and use of polyurethanes for making hearts, valves and aortas. Trans Am Soc Artif Intern Organs 1959;5:298. [2] Boretos JW, Pierce WS, Baier RE, Leroy AF, Donachy HJ. Surface bulk characteristics of a polyether urethane for artificial hearts. J Biomed Mater Res 1975;9:327. [3] Adil D. Heparin-immobolized poly(2-hydroxyethylmethacrylate)based microspheres. J Appl Sci 1999;74:655–62. [4] Damus PS, Hicks M, Rosenberg RB. Anticoagulant action of heparin. Nature 1997;246:355. [5] Casu B. Structure and biological activity of heparin. Adv Carbohydr Chem Biochem 1985;43:51–134.
3919
[6] Park KD, Okano T, Nojiri C, Kim SW. Heparin immobilization onto segmented polyurethane urea surfaces—effect of hydrophilic spacers. J Biomed Mater Res 1988;22:977. [7] Jung-Sook Bae, Eun-Jin Seo, Inn-Kyu Kang. Synthesis and charaterization of heparinized polyurethanes using plasma glow discharge. Biomaterials 1999;20:529–37. [8] Inn-Kyu Kang, Oh Hyeong Kwon, Yonng Moo Lee, Yong Kiel Sung. Preparation and surface characterization of functional group-grafted and heparin-immobilized polyurethanes by plasma glow discharge. Biomaterials 1996;17:841–7. [9] Zun Chen, Ruifeng Zhang, Makoto Kodama, Tadao Nakaya. Anticoagulant surface prepared by the heparinization of ionic polyurethane film. J Appl Polym Sci 2000;75:382–90. [10] Garner B, Georgevich A, Hodgson AJ, Liu L, Wallace GG. Polyurethane–heparin composites as stimulus-responsive substrates for endothelial cell growth. J Biomed Mater Res 1999;44:121–9. [11] Kjeld Christensen, Rolf Larsson, Hakan Emanuelsson, Graciela Elgue, Anders Larsson. Heparin coating of the stent graft: effects on platelets, coagulation and complement activation. Biomaterials 2001;22:349–35. [12] Hyun Tae Moon, Yong-Kyu Lee, Joon Koo Han, Youngro Byun. A novel formulation for controlled release of heparin-DOCA conjugate dispersed as nanoparticles in polyurethane film. Biomaterials 2001;22:281–9. [13] Young Jin Kim, Inn-Kyu Kang, Man Woo Huh, Sung-Chul Yoon. Surface characterization and in vitro blood compatibility of poly(ethylene terephthalate) immobilized with insulin and/or heparin using plasma glow discharge. Biomaterials 2000;21: 121–30. [14] Ito Y, Sisido M, Imanishi Y. Synthesis and antithrombogenicity of anionic polyurethanes. J Biomed Mater Res 1986;20:1157–77. [15] Kang K, Kwon OH, Byun KH. Surface modification of polyetherurethaneureas and their antithrombogenicity. J Mater Sci: Mater Med 1996;7:135–40. [16] Chandy T, Gladwin SD, et al. Use of plasma glow for surface engineering biomolecules to enhande blood compatibility of Dacron and PTFE vascular prosthesis. Biomaterials 2000; 21:699–712.
Biomaterials 24 (2003) 3915–3919
A novel solvent system for blending of polyurethane and heparin Qiang Lv, Chuanbao Cao*, Hesun Zhu Research Center of Material Science, Beijing Institute of Technology, Beijing 100081, China Received 17 November 2002; accepted 8 April 2003
Abstract To improve the blood-compatibility of polyurethane, the co-solvent of tetrahydrofuran and water, a new solvent system for blending polyurethane and heparin, was proposed. After solvent casting, heparin was blended in a polyurethane film. The ATRFTIR was used to analyze the surface chemical element and the contact angle was measured to investigate the hydrophilicity of the surface of the PUs. As the amount of heparin increased, the surface hydrophilicity was increased and all the clot times exceeded the measurement limit of the clot detection instrument when the heparin loaded on the polyurethane films was 3%, 5% and 7%. After the films were immersed in the phosphate buffered saline for 30 days, the activated partial thromboplastin time and thrombin time still exceeded the measurement limit of the clot detection instrument. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Blood compatibility; Heparin; Blending; Polyurethane
1. Introduction Segmented polyurethanes have been extensively used for the construction of cardiovascular devices because of their desirable physical properties and relative thromboresistance compared to other materials used in cardiovascular application [1,2], but thrombus formation still exists in small diameter tubing. To enhance the blood compatibility of polyurethanes, many different approaches have been studied. Heparin is an important anticoagulant, used clinically to minimize thrombus formation on artificial surfaces [3]. It has the ability to interact strongly with antithrombin III to prevent the formation of fibrin clot [4,5]. There are two general methods to develop bloodcompatible polymeric materials using heparin. One method uses chemical immobilization of heparin and the other is the heparin-releasing system. Covalent immobilization of heparin onto a polymer surface can provide long lasting antithrombogenicity. Heparin can be directly bound to the surface by insertion of either functional groups [6–8] or spacer arms [9], but the activity of heparin was significantly *Corresponding author. Tel.: +86-68913469; fax: +86-68915023. E-mail address: [email protected] (C. Cao).
decreased compared to raw heparin. In a heparinreleasing system, heparin is slowly released for a short duration and the bioactivity of heparin is maintained at a high level. Therefore, a heparin-releasing system is more suitable for a short-term clinical application than the covalently coated heparin system. There are several methods studied for the formulation of a heparinreleasing system [10,11]. In those methods, most releasing systems are useless because heparin was released within several hours, so a novel formulation for controlled release of heparin is necessary. Hyum Tae Moon et al. [12] reported that the heparin-DOCA, having an amphiphilic property, was homogeneously mixed with polyurethane in the co-solvent of dioxane, propanol and water. After casting the film, heparinDOCA was homogeneously dispersed as nanoparticles in polyurethane films. However, the losing of the activity of heparin was inevitable and the system could not be used in covalent immobilization of heparin because heparin has grafted with DOCA. In this study, we utilize polyurethane and the new solvent system to prepare a heparin-releasing system by simple solvent casting. It was found that heparin homogeneously mixed with polyurethane in the cosolvent of tetrahydrofuran and water if the ratio was suitable. Then heparin was blended in the polyurethane
0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00266-7
ARTICLE IN PRESS 3916
Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
films by solvent casting. The release rate, surface property and antithrombogenicity were studied.
2. Experiment 2.1. Materials The polyurethane was presented from the polymer laboratory of our university. The synthesis method employed here was a two-step method, in which the first step was to use poly(ethylene oxide-tetramethylene oxide) provided by LiMing chemical research institute and isophorone diisocyanate (IPDI) to synthesize a prepolymer, and the second step was to use 1,4butanediol as an extender to react with the prepolymer to produce the final product. The average molecular weight of poly(ethylene oxide-tetramethylene oxide) was 4850 and the ratio of ethylene oxide/tetramethylene oxide was 50/50. As a result, the average molecular weight of PU determined by gel permeation chromatography (Wasters 150-C, 1 ml/min, 30 C) was about 64,000. The mechanical properties were measured on the Instron-6022 Test machine at 20 C and 65% RH and the head speed was 10 mm/min. The tensile strength of PU was about 8.34 MPa and ultimate elongation was 636%. 2.2. Preparation of heparin blended polyurethane film Polyurethane (1.3 g) was dissolved in 15 ml tetrahydrofuran, and then a 2 ml different concentration heparin water solution was mixed with it. The weight ratio of heparin was 3%, 5% and 7% compared with polyurethane, respectively. The mixing solution was spread on a glass plate (6 6 cm2) and the solvent was evaporated at room temperature. The films were dried in a drying oven at 60 C for 24 h for testing.
2.4. Release test of heparin from polyurethane film The amount of heparin released from the film was determined using the toluidine blue method as reported in the literature [8,13,14]. For keeping the thickness of the films identical to each other, all films tested were made at the same experimental conditions. A known amount of heparin aqueous solution (2 ml) was added to the toluidine blue solution (3 ml) and the mixed solution was adequately vibrated. Hexane (3 ml) was then added, and the mixture was well shaken so that the toluidine blue–heparin complex was extracted into the organic layer. The toluidine blue that remained in the aqueous phase was determined by measuring the absorbance at 631 nm and the concentration of heparin in the aqueous solution was obtained and used as a calibration curve to determine the amount of released heparin. To study the release rate of heparin, the polyurethane film (3 cm 4 cm) containing heparin was immersed in PBS solution (12 ml, pH 7.4) for 24 h. Periodically, the supernatant (2 ml) of the PBS solution was replaced with a fresh one in order to maintain a sink condition. And then the absorbance at 631 nm of the supernatant was measured and the released amount of heparin was calculated according to the standard curve. The standard curve (Fig. 3) was obtained according to the method of Smith et al. The standard curve was linear in the range of 0–40 mg heparin/ml and the heparin content was obtained directly from the standard curve. 2.5. In vitro coagulation time tests for films The nephelometry measurements, including PT, activated partial thromboplastin time (APTT) and thrombin time (TT), were performed with the coagulation instrument Coag-A-Mate XM (Organon Teknika, USA) which measures the change of luminosity when
a
2.3. Characterizations of heparin blended polyurethane film The surfaces of PUs were investigated using attenuated total reflectance infrared spectroscope (ATRFTIR, SYSTEM-2000, PE, USA). After the film was immersed in phosphate buffered saline (PBS) for 1 h, the surface morphologies were studied to compare with the initial morphologies by scanning electron microscope (SEM, JSM-35C, JEOL, Japan). The water contact angles of the film surfaces were measured by putting a droplet of deionized water on the surface of the polymer films using the JY-82 contact angle apparatus (ChengDe, China). With each specimen, the measurement was repeated at different sites, and average values were obtained for the contact angles.
b c
d
e 3500
3000
2500
2000
1500
1000
Wavenumber cm-1
Fig. 1. ATR-FTIR spectra of: (a) PU, (b) PU-3 wt%He, (c) PU5 wt%He, (d) PU-7 wt%He and (e) FTIR spectra of heparin.
ARTICLE IN PRESS Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
light traverses the plasma sample. Briefly, the tested films were incubated with healthy human blood plasma in a transparent plastic tube, and the reagents for each coagulation time test were added to the tube immediately.
3. Results and discussion 3.1. Surface infrared analysis Fig. 1 showed ATR-FTIR spectra of PU-0%He(a), PU-3%He(b), PU-5%He(c), PU-7%He(d) and the Table 1 Water contact angles of different polyurethane films Heparin rate
Contact angle ( )
3% Heparin 5% Heparin 7% Heparin
7272 6871 5473
3917
FTIR spectra of heparin as typical examples. In the spectrum of PU(b)–(d), the peak in 2944 cm 1 increased and the peak in 1460 cm 1 moved to low wave number which may be due to the effect of heparin. On the other hand, the new peak in 891 cm 1 which exists in the spectra of the heparin was also found in the spectra of (b)–(d) and the peak in 2358 cm 1, though it had no absorption in heparin, represented the stretching vibration of sulfonamide (–NHSO3) reported by some researchers [15,16] and the result may mean there are some interaction between polyurethane and heparin such as the hydrogen bond. At the same time, the point that the area of peaks in 2358 cm 1 augment when the ratio of heparin increases partly suggests the surface content of heparin increases. 3.2. Water contact angle To investigate the hydrophilicity of the PUs, water contact angle measurements were carried out (Table 1). The PUs blended with heparin were relatively
(a)
10 µm
10 µm
10 10 m µm
µm 1010 m
10 µm
10 µm
10 µm
10 µm
(b)
(c)
(d)
Fig. 2. SEM of surface morphologies of heparin loaded polyurethane films before and after immersing in PBS for 1 h: (a) 0 wt%heparin, (b) 3 wt%heparin, (c) 5 wt%heparin, (d) 7 wt%heparin.
ARTICLE IN PRESS Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
3918
hydrophilic; PUs 1–3 exhibited contact angles of 7272, 6871 and 5473, respectively. These results agree with the increase of heparin content in the polyurethane.
3.3. Release of heparin from polyurethane films As shown in Fig. 2, when the heparin-loaded film was soaked in PBS for 1 h, there were many ridges at the surface when heparin was released out of the films, but the size and density of the ridges were not increased with the increasing amount of loaded heparin. The density of the ridges was most crowded for the 5 wt% heparin loading films and most sparse for the 7 wt% heparin loading one. Furthermore, the SEM images have the change accordant with the percentage of released amount of heparin shown in Fig. 4. For 3 wt%, 5 wt% and 7 wt% heparin loading films (Fig. 4), the percentages of released amounts of heparin for 24 h were 6.13%, 7.19% and 3.61%, respectively. This may be due to the difference in the interlaced structure of the different content of heparin and polyurethane. For the heparin-loaded films, heparin was quickly released within the first one hour, followed
Absorbance at 631nm
2.5
2.0
1.5
1.0
0.5
0.00
0.05
0.10
0.15
by the relative sustained release rate for one day. This might be because of the fact that heparin located near the surface would have had a short distance to travel to the film surface. Although the relation of heparin content and release rate needed further research, the blended amount of heparin was an important parameter in controlling the release rate.
3.4. In vitro blood compatibility The PT, APTT and TT were widely used for the clinical detection of the abnormality of blood plasma. In recent times, they were applied in the evaluation of in vitro antithrombogenicity of biomaterials. The normal ranges of PT, APTT and TT for a healthy blood plasma were regarded as 1173, 2874 and 1675 s, respectively. The results are given in Table 2. The three times all exceeded the measurement limit of the clot-detection instrument indicated that heparin blended with polyurethane kept excellent blood-compatibility and the plasma was not coagulated with polyurethane since the released heparin was enough to prevent the formation of fibrin clot. Table 3 shows the APTT, PT and TT of the films containing 5% heparin, which was immersed in PBS for one month. After 30 days, although PT had decreased to 12 s, the APTT and TT still exceeded the measurement limit of the clot-detection instrument. Since we have studied that polyurethane containing heparin reduced the PT time to 15 s when it was located in the desiccator for five days at 20 C, the reduction of PT may be partly due to the loss of heparin bioactivity as the films still preserved enough heparin to keep the films’ bloodcompatibility within about 30 days.
0.20 Table 2 Activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) of Pu films containing heparin
Heparin amount(mg)
Fig. 3. Grades curve of heparin amount.
Heparin relative release percent (%)
Heparin rate
Clot time (s)
8 7
b
6
a
0% 3% 5% 7%
5
Heparin Heparin Heparin Heparin
TT
APTT
PT
15.1 >150 >150 >150
46.8 >200 >200 >200
25.0 >150 >150 >150
4
c
3
Table 3 Clot times of Pu films containing 5 wt% heparin immersed in PBS for different number of days
2 1
Immerse time
Clot time (min)
0 0
5
10
15
20
25
Time (h)
Fig. 4. Release profiles of heparin from polyurethane films: (a) 3 wt%heparin, (b) 5 wt%heparin, (c) 7 wt%heparin.
Five days Thirty days
TT
APTT
PT
>150 >150
>200 >200
11.8 12.4
ARTICLE IN PRESS Q. Lv et al. / Biomaterials 24 (2003) 3915–3919
4. Conclusion In this study, the appropriate solvent was used to dissolve heparin and polyurethane. The simple solvent casting method allows heparin to be released slowly from the polymeric film and the rate of drug release can be controlled by the amount of drug being loaded. In the films, heparin can keep excellent bioactivity and in a relatively long time the films shows favorable blood compatibility. So we think that it could be especially useful when applied to devices designed for mediumtime use.
Acknowledgements This study is supported by 973 project (G1999064705) of China and 863 project (2002AA326030) of China.
References [1] Kolff WJ, Akutsu T, Dreyer B, Norton H. Artificial heart in the chest and use of polyurethanes for making hearts, valves and aortas. Trans Am Soc Artif Intern Organs 1959;5:298. [2] Boretos JW, Pierce WS, Baier RE, Leroy AF, Donachy HJ. Surface bulk characteristics of a polyether urethane for artificial hearts. J Biomed Mater Res 1975;9:327. [3] Adil D. Heparin-immobolized poly(2-hydroxyethylmethacrylate)based microspheres. J Appl Sci 1999;74:655–62. [4] Damus PS, Hicks M, Rosenberg RB. Anticoagulant action of heparin. Nature 1997;246:355. [5] Casu B. Structure and biological activity of heparin. Adv Carbohydr Chem Biochem 1985;43:51–134.
3919
[6] Park KD, Okano T, Nojiri C, Kim SW. Heparin immobilization onto segmented polyurethane urea surfaces—effect of hydrophilic spacers. J Biomed Mater Res 1988;22:977. [7] Jung-Sook Bae, Eun-Jin Seo, Inn-Kyu Kang. Synthesis and charaterization of heparinized polyurethanes using plasma glow discharge. Biomaterials 1999;20:529–37. [8] Inn-Kyu Kang, Oh Hyeong Kwon, Yonng Moo Lee, Yong Kiel Sung. Preparation and surface characterization of functional group-grafted and heparin-immobilized polyurethanes by plasma glow discharge. Biomaterials 1996;17:841–7. [9] Zun Chen, Ruifeng Zhang, Makoto Kodama, Tadao Nakaya. Anticoagulant surface prepared by the heparinization of ionic polyurethane film. J Appl Polym Sci 2000;75:382–90. [10] Garner B, Georgevich A, Hodgson AJ, Liu L, Wallace GG. Polyurethane–heparin composites as stimulus-responsive substrates for endothelial cell growth. J Biomed Mater Res 1999;44:121–9. [11] Kjeld Christensen, Rolf Larsson, Hakan Emanuelsson, Graciela Elgue, Anders Larsson. Heparin coating of the stent graft: effects on platelets, coagulation and complement activation. Biomaterials 2001;22:349–35. [12] Hyun Tae Moon, Yong-Kyu Lee, Joon Koo Han, Youngro Byun. A novel formulation for controlled release of heparin-DOCA conjugate dispersed as nanoparticles in polyurethane film. Biomaterials 2001;22:281–9. [13] Young Jin Kim, Inn-Kyu Kang, Man Woo Huh, Sung-Chul Yoon. Surface characterization and in vitro blood compatibility of poly(ethylene terephthalate) immobilized with insulin and/or heparin using plasma glow discharge. Biomaterials 2000;21: 121–30. [14] Ito Y, Sisido M, Imanishi Y. Synthesis and antithrombogenicity of anionic polyurethanes. J Biomed Mater Res 1986;20:1157–77. [15] Kang K, Kwon OH, Byun KH. Surface modification of polyetherurethaneureas and their antithrombogenicity. J Mater Sci: Mater Med 1996;7:135–40. [16] Chandy T, Gladwin SD, et al. Use of plasma glow for surface engineering biomolecules to enhande blood compatibility of Dacron and PTFE vascular prosthesis. Biomaterials 2000; 21:699–712.