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Blood compatibility of a ferulic acid (FA)-eluting PHBHHx system for biodegradable magnesium stent application
Materials Science and Engineering C 52 (2015) 37–45
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Blood compatibility of a ferulic acid (FA)-eluting PHBHHx system for biodegradable magnesium stent application Erlin Zhang a,⁎, Feng Shen b,c a b c
Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, Northeastern University, Shenyang 110819, China Shenzhen Salubris Biomedical Engineering Co., LTD, Shenzhen 518102, China School of Materials Sciences and Engineering, Harbin Institute of Technology, Harbin 150001, China
a r t i c l e
i n f o
Article history: Received 4 December 2014 Received in revised form 4 February 2015 Accepted 23 March 2015 Available online 25 March 2015 Keywords: Ferulic acid PHBHHx Blood compatibility Hemolysis Alkali treatment Anticoagulation
a b s t r a c t Magnesium stent has shown potential application as a new biodegradable stent. However, the fast degradation of magnesium stent limited its clinic application. Recently, a biodegradable and drug-eluting coating system was designed to prevent magnesium from fast degradation by adding ferulic acid (FA) in poly (3-hydroxybutyrateco-3-hydroxyhexanoate) (PHBHHx) by a physical method. In vitro study has demonstrated that the FA-eluting system exhibited strong promotion to the endothelialization, which might be a choice for the stent application. In this paper, the hemolysis rate, the plasma recalcification time (PRT), the plasma prothrombin time (PT) and the kinetic clotting time of the FA-eluting films were investigated and the platelet adhesion was observed in order to assess the blood compatibility of the FA-eluting PHBHHx films in comparison with PHBHHx film. The results have shown that the addition of FA had no influence on the hemolysis, but prolonged PRT, PT and the clotting time and reduced the platelet adhesion and activation, displaying that the FA-eluting PHBHHx exhibited better blood compatibility than PHBHHx. In addition, the effect of alkali treatment on the blood compatibility of FA-eluting PHBHHx was also studied. It was indicated that alkali treatment had no effect on the hemolysis and the coagulation time, but enhanced slightly the platelet adhesion. All these demonstrated that FA-eluting PHBHHx film had good blood compatibility and might be a candidate surface coating for the biodegradable magnesium stent. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Percutaneous transluminal coronary angioplasty (PTCA) has been proven to be an effective procedure performed to reduce blockages in coronary arteries [1]. Although providing intra-arterial support with bare metal stents (BMS) dramatically improves the angiographic and clinical outcome of patients to a restenosis rate of 20–30%, in-stent restenosis (ISR) still remains a major limitation for this approach with exaggerated intimal hyperplasia [1]. The advent of drug-eluting stent (DES), which release drugs such as sirolimus and paclitaxel for localized delivery, is a major advancement in the evolution of stents. However, there is a risk of late stent thrombosis (LST) associated with DES [2,3]. It is widely accepted that endothelial and smooth muscle cell damage, unavoidable in PTCA and stent placement, is a cause of restenosis. Therefore, it is always desired to prevent neointimal hyperplasia and to accelerate endothelialization. The drugs released from the surface
⁎ Corresponding author at: Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, P.O. Box 350, Northeastern University, Shenyang 110819, China. E-mail address: [email protected] (E. Zhang).
http://dx.doi.org/10.1016/j.msec.2015.03.054 0928-4931/© 2015 Elsevier B.V. All rights reserved.
polymer coating, however, not only inhibit the proliferation of vascular smooth muscle cells (VSMCs) but also inhibit the proliferation of endothelia cells (ECs), which prevents the fast endothelialization. In addition, the biostable polymer coating also causes inflammation due to its acid reaction. Recently, biodegradable stents have been attracted much attention worldwide due to their biodegradability, such as biodegradable polymer stent [4–8] and magnesium stent [4,9–11]. The first approach was the use of biodegradable polymers. Unfortunately, most of these polymers induced during the absorption process marked inflammatory changes leading to severe intimal hyperplasia or thrombotic occlusion [12]. Biodegradable magnesium stent shows much advantage over the biodegradable polymer stent due to its high strength and alkali degradation production. However, the fast degradation of magnesium alloys in a biological environment limits their clinic application. One of the effective methods to control the degradation of the magnesium alloys is surface coating, in which a biodegradable polymer coating with antiproliferative drug has been applied to the magnesium stent in many studies [10,13]. The biodegradable drug-eluting coating can adjust the degradation rate of the magnesium stent and the drug release on the other hand can effectively reduce the ISR. In the previous studies, poly-L-lactic acid
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(PLLA) and poly(dl-lactide-co-glycolide) (PLGA) are often used as the drug carrier coating, and paclitaxel and sirolimus (rapamycin) are often used as the antiproliferative drug. Recently, a new biodegradable and drug-eluting system for magnesium stent coating was developed, in which poly (3-hydroxybutyrateco-3-hydroxyhexanoate) (PHBHHx) was used as the drug delivery coating and ferulic acid (FA; 4-hydroxy-3-methoxy cinnamic acid) was used as the antiproliferative drug [14]. PHBHHx has recently been considered as potential material for tissue engineering applications such as angiogenesis and arterial repair due to the biodegradability, good blood compatibility [15] and good biocompatibility to several cell types [16–21]. FA is the main efficacious ingredient in Angelicae sinensis and is also present abundantly in various fruits and vegetables, such as bananas, citrus fruits, bamboo shoots, eggplants and cabbages [22,23]. FA exhibits many physiological functions including antioxidant, antimicrobial, anti-inflammatory, antithrombosis, antihypercholesterolemic, anticancer activities, and spermatozoa activating bioactivity [23]. The non-cytotoxicity of FA to both human umbilical vein endothelial cells (HUVECs) and VSMCs, and the promotion function on angiogenesis were also reported [24]. The promotion function of FA to HUVECs was widely reported [22,25–27]. It was found that FA at a range of concentration from 0.1 μg/mL to 10 μg/mL could markedly improve the proliferation of endothelial cell line (ECV304) and DNA synthesis in a dosedependent manner with a significant decrease in the percentage of cells in the G0/G1 phase and a significant increase in the S phase [22]. Lin et al. [27] reported that FA (10− 6–10− 4 M) induced significant angiogenesis of HUVECs in vitro without cytotoxicity via vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and hypoxic-induced factor (HIF) -1α mRNA. Further research revealed that FA upregulated cyclin D1 and VEGF mRNA [22]. It was also reported that the promotion of blood vessel regeneration by FA was observed in vitro and in vivo [28]. Meanwhile, Hou et al. [29] reported that FA significantly inhibited angiotensin II-induced VSMC proliferation in a dose-dependent manner by elevating the protein content of p21waf1/cip1, decreasing expression of cyclin D1 and inhibiting phosphorylation of retinoblastoma protein. Similar result was also reported by Wang et al. [24]. All these results indicate that FA has the biological function to promote the proliferation of ECs and inhibit the proliferation of VSMCs, which could be one of the best drug selections for the drugeluting coating system for stent application. Based on the above consideration, it is reasonable to design a new biodegradable and drug-eluting coating system for metallic stents by combining FA as the drug and PHBHHx as the drug delivery. It is proposed that after the implantation of this drug-eluting system, FA released from the coating will promote the endothelialization and prevent the neointimal hyperplasia, which provides a possibility to reduce ISR and LST. Previous study has shown that the FA-eluting PHBHHx coating had good bonding strength to magnesium substrate and the FA released from the FA-eluting PHBHHx film enhanced the adhesion, spreading and proliferation of HUVECs in a dose-dependent manner, effectively inhibited H2O2-induced injury and promoted NO production of HUVECs [14]. As a biomaterial to be used in contact with blood, the good blood compatibility is a basic requirement. PHBHHx has been proven to show good blood compatibility [15]. But it is still not known whether the addition of FA has influence on the blood compatibility, although the anticoagulant ability of FA was reported elsewhere [30,31]. In this paper, FA-eluting PHBHHx films with 5% and 10% FA were prepared by adding FA in PHBHHx by a physical method based on previous results [14,32]. At the very early stage of this research, the blood compatibility of this film was investigated to assess the possibility of FA-eluting PHBHHx system for biodegradable magnesium stents. In addition, alkali treatment has often been used as an effective surface modification method for biomedical polymers to improve the cell–matrix adhesion [33,34]. The effect of an alkali treatment on the blood compatibility of FA-eluting PHBHHx films was also reported.
2. Materials and methods 2.1. Preparation of material and sample PHBHHx (with 12 mol% HHx and a molecular weight of 200,000 Da) was kindly provided by Dr. Qiong Wu (Tsinghua University, Beijing, China). Ferulic acid (FA) with a purity of ≥ 98% was purchased from Nanjing Zelang Medical Technology Co., Ltd. PHBHHx, FA and ethyl acetate (analytical grade, Sinopharm Chemical Reagent Co., Ltd, Shenyang, China) were used to prepare FA-eluting samples. Briefly, 0.25 g of PHBHHx and 5 wt.% FA or 10 wt.% FA were dissolved in 5 mL ethyl acetate to prepare 5% drug-eluting or 10% drug-eluting PHBHHx material (named PHBHHx + 5%FA and PHBHHx + 10%FA), respectively. Then, the solutions were poured into a Petri dish and kept at 30 °C under vacuum for 24 h and then at 35 °C for 48 h to allow the complete evaporation of the ethyl acetate. The prepared film was about 80–100 μm in thickness. Samples with a dimension of 10 mm × 15 mm for different measurements were cut from the films. Before the tests, the samples were sterilized by ultraviolet radiation for 4 h. 2.2. Blood and plasma Blood obtained from a healthy volunteer was mixed with acid citrate dextrose, and centrifuged at 3000 rpm and 1500 rpm for 15 min to get platelet-poor plasma (PPP) and platelet rich plasma (PRP), respectively. 2.3. Alkali treatment FA-eluting films were immersed in NaOH solution (2 mol L−1) for 120 min. Then, the films were taken out and rinsed extensively with deionized water until the pH of the rinsing water became neutral. Samples after alkali treatment were denoted as PHBHHx + 5%FA + Alkali and PHBHHx + 10%FA + Alkali, respectively. 2.4. Scanning electron microscopy The samples were posted on an aluminum stump and coated with gold under vacuum. The morphology observation of the films was conducted on a scanning electron microscopy (SEM) with a model of SSX-550 (SHIMADZU, Japan) with an acceleration voltage of 5 kV. 2.5. Contact angle measurement Water contact angle (θ) of the films was determined on a contact angle goniometer (POWEREACH, Shanghai, China). Deionized water was used as a probe liquid. Water contact angle was measured by five independent measurements and was presented as mean ± standard deviation. 2.6. Drug release measurement For FA release measurement, the FA-load films were immersed in a simulated plasma solution with a liquid volume/surface area ratio of 2.5 mL/cm2 at 37 °C for preset duration. The cumulative FA release was measured on a high performance liquid chromatography (HPLC, SHIMADZU LC2010) with Diamonsil C18 column and acetonitrile-0.1% phosphate as mobile phase at a flow rate of 1 mL/min at 20 °C. Triple tests were conducted for each sample. 2.7. Hemolysis assay Hemolysis test was conducted with reference to ISO 10993-4: Selection of test for interaction with blood and ISO 10993-12: Sample preparation and reference materials. The blood sample was prepared by mixing 8 mL anticoagulated whole blood with 10 mL normal saline. Extract liquids were prepared by immersing PHBHHx sample and the
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drug-eluting samples in normal saline for 24 h at 37 °C (surface area/ volume = 6 cm2/mL). Then, 10 mL extract liquid and 10 mL normal saline were put into a test tube and then 0.2 mL blood sample was added. The test tube was incubated at 37 °C for 1 h and then centrifuged at 2500 rpm for 5 min. Optical density (O.D.) of the supernate was measured at a wavelength of 545 nm by a spectrophotometer (S53/54 UV–VIS Spectrophotometer, Shanghai LingGuang, China). 10 mL distilled water with 0.2 mL blood sample was used as the positive sample and 10 mL normal saline with 0.2 mL blood sample as the negative control. Three parallel experiments were performed for each sample. The hemolysis rate (HR) was calculated as follow
HR ¼
Dt −Dnc 100% Dpc −Dnc
ð1Þ
where HR is the hemolysis rate, and Dt, Dpc and Dnc are the absorbency of the sample, the positive control and the negative control, respectively.
2.8. Plasma recalcification time (PRT) and plasma prothrombin time (PT) measurements The coagulation times were determined with a semiautomatic blood coagulation analyzer using mechanical end point determination (GF2000 II, CaiHong Analytical, China). In all tests, glass silicide and glass were used as a negative control and a positive control, respectively. For PRT measurement, 150 μL of PRP was carefully added on the film samples' surface and incubated at 37 ± 1 °C for 1 min, then, 100 μL residual PRP was taken to a test cup followed by the addition of 100 μL of calcium chloride solution (0.025 mol L− 1) immediately at 37 ± 1 °C. A mechanical end point determination was used to measure the formation of fibrin clot. For PT measurement, 150 μL of PPP was carefully added on the test samples' surface and incubated at 37 ± 1 °C for 1 min. After this, 100 μL of PPP was taken out to the test cup, simultaneously with the addition of 100 μL of thromboplastin reagent (Zhong Tai, Wuhan, China) at 37 ± 1 °C. The tests were repeated three times for each sample.
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2.11. Statistical analysis Data were presented as means ± standard deviation. Statistical comparisons were performed using Student's t-test. p b 0.05 was considered statistically significant. 3. Results 3.1. Surface morphology Fig. 1 shows a smooth and dense surface microstructure of PHBHHx film. Fig. 2 presents the surface morphologies of the FAeluting PHBHHx films before and after alkali treatment. It was observed that PHBHHx + 5%FA film (Fig. 2a) had a smooth and dense surface while many pores with less than 100 μm size were observed on the surface of PHBHHx + 10%FA film (Fig. 2c). PHBHHx + 10%FA film was much rougher than PHBHHx + 5%FA film. After the alkali treatment, as shown in Fig. 2b and d, there were more holes on the surface due to the etching function of NaOH. Compared with the surface microstructure before the treatment, it can be concluded that alkali treatment led to a much rougher surface with more pores. 3.2. Water contact angle Fig. 3 shows the water contact angles of PHBHHx and the FA-eluting PHBHHx films before and after alkali treatment. The contact angle of PHBHHx film was about 90.2° ± 1.3°. The addition of 5% FA reduced the contact angle significantly to 87.4° ± 1.1° (p b 0.05) and further increase in FA content (10%FA) decreased the contact angle significantly to 83.3° ± 0.9° (p b 0.05). It can be deduced that the addition of FA reduced the water contact angle significantly in a dose-dependent manner, in turn, improved the hydrophilicity of the film significantly. After the alkali treatment, the contact angles of the PHBHHx + 5%FA and PHBHHx + 10%FA were reduced significantly to 52.4° ± 0.9° and 50.6° ± 1.4° (p b 0.05), respectively. Before the alkali treatment, the contact angles of these films were in a range of 83–90°, indicating that the films were still hydrophobic films. After the alkali treatment, the contact angle was dramatically reduced to 50–52°, indicating that the alkali treatment changed the film from hydrophobic to hydrophilic film.
2.9. Kinetic clotting time
3.3. Drug release
100 μL of anticoagulant blood was carefully added on the test samples and 10 μL of calcium chloride solution (0.2 mol L−1) was added simultaneously, and time was recorded immediately. After 10, 30, 50, 70 and 90 min, respectively, the surface of the test sample was flowed slowly by the distilled water separately and the flowed liquid was collected in a 96-well plate. O.D. value of the surplus extricate hemoglobin in the flowed liquid was measured in a spectrophotometer (S53/54 UV– VIS Spectrophotometer, Shanghai, China) at a wavelength of 545 nm. Glass silicide and glass were used as a negative control and a positive control, respectively.
Fig. 4 shows the drug release from the drug-eluting films with immersion time. The effect of the alkali treatment on the drug release was also shown in the figure. For both PHBHHx + 5%FA film and PHBHHx + 10%FA film, there was a tendency that the cumulative
2.10. In vitro platelet adhesion Before the test, the test samples were immersed in phosphatebuffered saline (PBS, pH = 7.3) for 20 min. Then the film samples were laid on the bottoms of the wells a 24-well plate, submerged with PRP and incubated at 37 ± 1 °C for 0.5 h and 3 h, respectively. After this, the film samples were washed gently with PBS three times to remove non-adhered platelets, and then fixed with 2.5% buffered glutaraldehyde solution overnight in a refrigerator at 4 °C. After fixation, the test samples were washed and dehydrated in a graded ethanol series (50%, 60%, 70%, 80%, 90% and 100% for 15 min each). Surface gold coating was conducted on all samples for SEM observation.
Fig. 1. Surface microstructure of PHBHHx film.
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Fig. 2. Surface micrographs of the drug-eluting PHBHHx films before and after NaOH treatment. a) PHBHHx + 5%FA, b) PHBHHx + 5%FA + Alkali, c) PHBHHx + 10%FA, d) PHBHHx + 10%FA + Alkali.
drug release increased rapidly at the first 24 h and then slowed down during the following immersion. Due to the high FA content in PHBHHx + 10%FA film, the released drug was much higher than that from PHBHHx + 5%FA film at all intervals. After alkali treatment, the cumulative drug release dropped sharply for both PHBHHx + 5%FA and PHBHHx + 10%FA films. For examples, the cumulative drug releases were 28.2 μg and 37.7 μg for PHBHHx + 5%FA and PHBHHx + 10%FA films, corresponding to only 14.9% and 13.5% the values before the treatment, respectively. NaOH has a very strong etching ability. After 120 min immersion in NaOH solution, the films have been etched seriously as indicated by the holes on the surface in Fig. 2. The drug on the surface and
subsurface would dissolve in the solution, which might be the main reason for the sharp reduction in the cumulative drug release.
Fig. 3. Water contact angle of PHBHHx and the different drug-eluting PHBHHx films. (*p b 0.05).
Fig. 4. In vitro drug release curves of the drug-eluting PHBHHx films before and after the NaOH treatment.
3.4. Hemolysis HR values of the samples are listed in Table 1. HR of PHBHHx was 1.429, less than the recommended value of 5%, suggesting that PHBHHx had a good hemolysis property. HR vales of PHBHHx + 5%FA and PHBHHx + 10%FA were 0.831 and 0.498, respectively, indicating that the addition of FA reduced HR value slightly. Also, HR values of PHBHHx + 5%FA + Alkali and PHBHHx + 10%FA + Alkali were much lower than the recommended value, showing that the alkali treatment also reduced HR slightly. It has to be pointed that HR values of all test
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Table 1 Optical density and hemolysis rates of the test samples. Sample no.
PHBHHx
PHBHHx + 5%FA
PHBHHx + 5% + Alkali
PHBHHx +10%FA
PHBHHx +10%FA + Alkali
Negative control
Positive control
1 2 3 HR (%)
0.013 0.009 0.015 1.429
0.01 0.01 0.006 0.831
0.01 0.004 0.007 0.548
0.008 0.005 0.007 0.498
0.011 0.006 0.01 0.880
0.003 0.003 0.005 –
0.581 0.631 0.605 –
samples were much less than the recommended value of 5%, displaying the addition of FA and the alkali treatment have no influence on the hemolysis properties of the film. 3.5. PRT and PT Fig. 5 shows PRT values of PHBHHx film and the FA-eluting PHBHHx films before and after the alkali treatment. PHBHHx had a PRT of 280 min. It was shown that the PHBHHx + 5%FA had a significant longer PRT value than PHBHHx (p b 0.05) while PHBHHx + 10%FA had a significant longer PRT value than PHBHHx + 5%FA (p b 0.05), indicating that FA significantly prolonged the PRT properties of the film in a dosedependent manner. Although difference was found in PRT values of the FA-eluting PHBHHx films before and after alkali treatment, the difference was not significant (p N 0.05), indicating that the alkali treatment had limited influence on PRT of the FA-eluting PHBHHx films. Fig. 6 shows PT values of PHBHHx film and the FA-eluting PHBHHx films before and after the alkali treatment. It was evident that both PHBHHx + 5%FA film and PHBHHx + 10%FA film had significant longer PT values than PHBHHx (p b 0.05) but no significant difference was found between PHBHHx + 10%FA and PHBHHx + 5%FA (p N 0.05). Difference was also found in PT values of the FA-eluting PHBHHx films before and after alkali treatment, but the difference was not significant (p N 0.05), confirming that the alkali treatment has limited influence on PT of the FA-eluting PHBHHx films. 3.6. Kinetic clotting time Fig. 7 shows the kinetic clotting time of the test films. Generally, the time when O.D. value is equal to 0.1 is defined as initial clotting time while the time when O.D. value is equal to 0.01 is defined as whole clotting time. Table 2 summarizes the initial clotting time and the whole clotting time obtained from Fig. 7. The initial clotting time and the whole clotting time of the PHBHHx + 5%FA were both longer than those of PHBHHx, indicating that the addition of FA prolonged the clotting time. It can also be found that the clotting time increased with
Fig. 5. PRT of PHBHHx and the FA-eluting PHBHHx before and after alkali treatment (*p b 0.05).
the increase in FA content in the film. These results confirm that FA had a potential function to improve the anticoagulant properties. Although difference was observed in the initial clotting of the FAeluting films before and after the alkali treatment, the increase in the clotting time was limited, the difference was not significant (p N 0.05), indicating that the alkali treatment had limited influence on PRT of the FA-eluting PHBHHx films. 3.7. Platelet adhesion Fig. 8 shows the morphology of the adhered platelets on PHBHHx and the FA-eluting PHBHHx films after 0.5 h incubation. Several platelets with round shape were observed on the surface of PHBHHx film while only a few platelets with round shape were observed on the FAeluting films, indicating that the platelets were not activated at that moment in both cases. No difference in platelet number and shape was found before and after the alkali treatment, displaying that the alkali treatment has no influence on the platelet adhesion and activation at the early stage. After incubation for 3 h, as shown in Fig. 9, most platelets spread on the surface of PHBHHx with pseudopodia while the platelet number increased, indicating that the platelets were activated at this moment. Meanwhile, more platelets were also observed on the FA-eluting PHBHHx films and the platelets spread on the surface. However, the platelet number and the change in the morphology of the platelets on the FA-eluting films were much less than that on PHBHHx at all intervals. After the alkali treatment, more platelets were found on the films after 3 h incubation, but no difference in the platelet morphology was found, indicating that the alkali treatment enhanced the adhesion of platelet but did not promote the activation. 4. Discussion A biomaterial, to be used as a drug-eluting coating on metallic stent, should meet the following requirements: (1) good cell biocompatibility; (2) good blood compatibility, including good hemolysis and
Fig. 6. PT of PHBHHx and FA-eluting PHBHHx before and after alkali treatment (*p b 0.05).
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Fig. 7. Kinetic clotting time of the test films.
anticoagulation properties; (3) biodegradability, for biodegradable coating application; and (4) ability to inhibit neointimal hyperplasia or/and to promote endothelialization. Good cell biocompatibility includes non-cytotoxicity and cell adhesion property. Both PHBHHx and FA have no cell toxicity [35]. PHBHHx can be degraded in vitro. However, after 4 week immersion in a simulated plasma solution, only 30 wt.% was degraded [14]. The addition of FA accelerated the degradation in a dose-dependent way. But even for the PHBHHx + 10%FA, only 55 wt.% was degraded after 4 week immersion [14], which indicates that this coating system could protect the magnesium stent from the fast degradation and guarantee the drug release at least within 4 weeks. In addition, it was also indicated that FA could enhance the HUVEC adhesion and proliferation, reduce the H2O2-induced injury and promote NO production [14]. However, the cell adhesion property on the surface of PHBHHx is not satisfied due to the poor hydrophilic property. Several surface modification techniques have been used to improve the surface hydrophilicity of polymer materials including surface hydrolysis [36], grafting technique [37], ultraviolet treatment [38], alkali treatment [33,34] and plasma treatment [39]. Also it was shown that NaOH treatment improved the cell adhesion, spreading and proliferation and had no influence on the H2O2 induced injury and NO production [14]. Good blood biocompatibility is another basic requirement. PHBHHx has shown very good blood compatibility [15]. The addition of FA in PHBHHx enhanced the proliferation of HUVECs, on the other hand, might bring about influence on the blood compatibility of PHBHHx. Surface morphology shown in Figs. 1 and 2 indicated that the addition of FA in PHBHHx produced some small holes with a size of less than 100 μm in the film, which contributes to the significant reduction in the contact angle in comparison with PHBHHx film. Alkali treatment created many large holes on the surface of the FA-eluting films, as shown in Fig. 2b and d, which dramatically reduces the contact angle from 83–87° to 50–52°, as shown in Fig. 3. The sharp improvement in the hydrophilicity will benefit cell adhesion and proliferation, including platelet. It is always desired that the drug in a drug-eluting system can be released gradually with the degradation of drug carrier. In this study, FA was mixed with PHBHHx by a physical method and no reaction happened between FA and PHBHHx to protect the physical and biological functions of FA. The drug release curves of the FA-eluting films in Fig. 4 clearly showed that FA was released in a gradual manner rather than a burst manner, displaying that the FA release can be successfully controlled. However, the alkali treatment dramatically reduced the FA cumulative release due to the etching function although it significantly improved the hydrophilicity of the FA-eluting PHBHHx film. Therefore, it is very necessary to optimize the alkali treatment in the next step to obtain a balance between the good hydrophilicity and the drug release behavior.
It can be found from Table 1 that the HR values of PHBHHx and the FA-eluting films were both much less than the recommended value of 5% by ISO standard, indicating that both PHBHHx and FA have no destruction to erythrocyte. Plasma coagulation properties of a biomaterial indicate the possibility of this material to react with blood component and cause thrombosis [40–42]. Long PRT, PT and the initial clotting are always desired. However, no recommended values are given in ISO standard. As for the blood coagulation mechanism, it is widely accepted that there exist three pathways which can lead to thrombosis: an intrinsic pathway, an extrinsic pathway, and a common pathway. PRT is normally used to assess the intrinsic pathway and PT is used to evaluate the extrinsic pathway and common coagulation. Significant increase was observed in PRT and PT of the FA-eluting PHBHHx films in comparison with the value of PHBHHx, which indicates that the FA-eluting film has less activation to both intrinsic pathway and extrinsic pathway and common coagulation than PHBHHx. In addition, the longer initial clotting time and whole clotting time of the FA-eluting PHBHHx than those of PHBHHx also indicate that the FA-eluting films exhibited a weaker activation degree to the hemagglutination factor XII than PHBHHx. All these results clearly display that FA inhibited the coagulation of the blood system, especially in a dose-dependent manner in PRT. It can be also found that no significant difference in PRT, PT and the initial clotting time was found before and after the alkali treatment, indicating that the alkali treatment has limited influence on the anticoagulation time. However, the alkali treatment dramatically reduced the cumulative FA release, as shown in Fig. 4, it can be deduced that small amount of FA can significantly extend the coagulation time. On the other hand, this result suggests that the anticoagulation function of FA would not be dose-dependent when the FA content is more than a critical value. Serious platelet adhesion and activation on the surface of a biomaterial are the other main factors which cause thrombosis. After 0.5 h incubation, a few platelets were observed on the surface of both PHBHHx and the FA-eluting films. Even after 3 h incubation, only several platelets were observed on the surface of PHBHHx film. Although some of them spread on the surface and were activated, the situation was still not serious. On the contrary, the platelet number on the FA-eluting films was less than the number on PHBHHx and no difference in the shape change of the adhered platelet can be found between PHBHHx and the FAeluting film, indicating that FA reduced the platelet number and had no influence on the activation of platelet. When platelets are contacted with the film, two factors will affect the platelet adhesion. One is the hydrophilicity of the film. Good hydrophilicity promotes the adhesion of platelet. Another factor is FA released from the film. The addition of FA in PHBHHx improved the hydrophilicity of PHBHHx, which should promote the platelet adhesion. But only a few platelets were found on the FA-eluting film, which strongly indicates that FA has a function to resist the platelet adhesion. Alkali treatment improved the hydrophilicity of the FA-eluting film but reduced FA release, as shown in Figs. 3 and 4, which might be the main reason why alkali treatment enhanced the platelet adhesion. Polymers normally have poor cell adhesion properties due to their smooth surface and low hydrophilicity. Although the addition of FA reduced the contact angle as stated in the above section, the contact angle was still as high as 80°–82° and hydrophilicity still needs to be improved for good cell adhesion. Alkali treatment as an effective method to improve the hydrophilicity reduced FA release, which led to enhancement in the platelet adhesion. So it is necessary to find out other surface modifications in the next step, which could improve the hydrophilicity without deteriorative effect of the FA release. Although the alkali treatment affects the FA release, the alkali environment resulted from the degradation of magnesium stent will not influence the FA release due to the fact the FA-eluting system will be degraded first and then the degradation of magnesium stent will happen. All these results strongly demonstrated that FA-eluting PHBHHx had very good blood biocompatibility. With the consideration of the good
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Table 2 The initial clotting time and the whole clotting time of the test samples (min). Samples
PHBHHx
PHBHHx + 5%FA
PHBHHx + 5% + Alkali
PHBHHx + 10%FA
PHBHHx + 10%FA + Alkali
Initial clotting time Whole clotting time
50 82
65 N90
70 N90
72 N90
73 N90
bonding strength to magnesium substrate and the promotion function to endothelialization [14], the FA-eluting PHBHHx system has shown potential as a candidate for the drug-eluting surface coating system of magnesium stent. It has to be pointed that the FA-eluting system is not degraded uniformly in physiological environment and also physiological solution can be penetrated into defects of coating, which might cause the localized and filiform corrosion of magnesium stent and then might lead to a failure of implantation. Previous study [14,32] has shown that the degradation rate of PHBHHx coating can be adjusted by the selection of molecular weight of PHBHHx, the coating thickness and the FA content. Therefore, it is necessary in the next step to optimize the
whole stent system parameters in order to get a good drug-eluting stent system. 5. Conclusion From the above research results, the following conclusions can be drawn: 1) FA-eluting PHBHHx showed good hemolysis and long PRT, PT and the initial clotting time. 2) FA had a function to inhibit the coagulation of the blood system and resist the platelet adhesion.
Fig. 8. Platelet adhesion on PHBHHx film and the FA-eluting PHBHHx films contacted with plasma for 0.5 h. a) PHBHHx, b) PHBHHx + 5%FA, c) PHBHHx + 5%FA + Alkali, d) PHBHHx + 10%FA, e) PHBHHx + 10%FA + Alkali.
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Fig. 9. Platelet adhesion on PHBHHx film and the FA-eluting PHBHHx films contacted with plasma for 3 h. a) PHBHHx, b) PHBHHx + 5%FA, c) PHBHHx + 5%FA + Alkali, d) PHBHHx + 10%FA, e) PHBHHx + 10%FA + Alkali.
3) Alkali treatment had no influence on hemolysis, PRT, PT and the initial clotting time of the FA-eluting PHBHHx films, but enhanced the platelet adhesion slightly. Acknowledgment The authors would like to acknowledge the financial support from National Natural Science Foundation (no. 31271024/C1002, no. 31470930/C1002), Foundation for Key Program of Ministry of Education, China(no. 313014), Heilongjiang Universities' Science and Technology innovation team program (no. 2012TD010), Nature Science Foundation of Heilongjiang Province for Distinguished Young Scholars (no. JC201205) and funding from Northeastern University (985 program) and Program LZ2014018, China. References [1] G. Mani, M.D. Feldman, D. Patel, M. Agrawal, Coronary stents: a materials perspective, Biomaterials 28 (2007) 1689–1710.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Blood compatibility of a ferulic acid (FA)-eluting PHBHHx system for biodegradable magnesium stent application Erlin Zhang a,⁎, Feng Shen b,c a b c
Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, Northeastern University, Shenyang 110819, China Shenzhen Salubris Biomedical Engineering Co., LTD, Shenzhen 518102, China School of Materials Sciences and Engineering, Harbin Institute of Technology, Harbin 150001, China
a r t i c l e
i n f o
Article history: Received 4 December 2014 Received in revised form 4 February 2015 Accepted 23 March 2015 Available online 25 March 2015 Keywords: Ferulic acid PHBHHx Blood compatibility Hemolysis Alkali treatment Anticoagulation
a b s t r a c t Magnesium stent has shown potential application as a new biodegradable stent. However, the fast degradation of magnesium stent limited its clinic application. Recently, a biodegradable and drug-eluting coating system was designed to prevent magnesium from fast degradation by adding ferulic acid (FA) in poly (3-hydroxybutyrateco-3-hydroxyhexanoate) (PHBHHx) by a physical method. In vitro study has demonstrated that the FA-eluting system exhibited strong promotion to the endothelialization, which might be a choice for the stent application. In this paper, the hemolysis rate, the plasma recalcification time (PRT), the plasma prothrombin time (PT) and the kinetic clotting time of the FA-eluting films were investigated and the platelet adhesion was observed in order to assess the blood compatibility of the FA-eluting PHBHHx films in comparison with PHBHHx film. The results have shown that the addition of FA had no influence on the hemolysis, but prolonged PRT, PT and the clotting time and reduced the platelet adhesion and activation, displaying that the FA-eluting PHBHHx exhibited better blood compatibility than PHBHHx. In addition, the effect of alkali treatment on the blood compatibility of FA-eluting PHBHHx was also studied. It was indicated that alkali treatment had no effect on the hemolysis and the coagulation time, but enhanced slightly the platelet adhesion. All these demonstrated that FA-eluting PHBHHx film had good blood compatibility and might be a candidate surface coating for the biodegradable magnesium stent. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Percutaneous transluminal coronary angioplasty (PTCA) has been proven to be an effective procedure performed to reduce blockages in coronary arteries [1]. Although providing intra-arterial support with bare metal stents (BMS) dramatically improves the angiographic and clinical outcome of patients to a restenosis rate of 20–30%, in-stent restenosis (ISR) still remains a major limitation for this approach with exaggerated intimal hyperplasia [1]. The advent of drug-eluting stent (DES), which release drugs such as sirolimus and paclitaxel for localized delivery, is a major advancement in the evolution of stents. However, there is a risk of late stent thrombosis (LST) associated with DES [2,3]. It is widely accepted that endothelial and smooth muscle cell damage, unavoidable in PTCA and stent placement, is a cause of restenosis. Therefore, it is always desired to prevent neointimal hyperplasia and to accelerate endothelialization. The drugs released from the surface
⁎ Corresponding author at: Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, P.O. Box 350, Northeastern University, Shenyang 110819, China. E-mail address: [email protected] (E. Zhang).
http://dx.doi.org/10.1016/j.msec.2015.03.054 0928-4931/© 2015 Elsevier B.V. All rights reserved.
polymer coating, however, not only inhibit the proliferation of vascular smooth muscle cells (VSMCs) but also inhibit the proliferation of endothelia cells (ECs), which prevents the fast endothelialization. In addition, the biostable polymer coating also causes inflammation due to its acid reaction. Recently, biodegradable stents have been attracted much attention worldwide due to their biodegradability, such as biodegradable polymer stent [4–8] and magnesium stent [4,9–11]. The first approach was the use of biodegradable polymers. Unfortunately, most of these polymers induced during the absorption process marked inflammatory changes leading to severe intimal hyperplasia or thrombotic occlusion [12]. Biodegradable magnesium stent shows much advantage over the biodegradable polymer stent due to its high strength and alkali degradation production. However, the fast degradation of magnesium alloys in a biological environment limits their clinic application. One of the effective methods to control the degradation of the magnesium alloys is surface coating, in which a biodegradable polymer coating with antiproliferative drug has been applied to the magnesium stent in many studies [10,13]. The biodegradable drug-eluting coating can adjust the degradation rate of the magnesium stent and the drug release on the other hand can effectively reduce the ISR. In the previous studies, poly-L-lactic acid
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(PLLA) and poly(dl-lactide-co-glycolide) (PLGA) are often used as the drug carrier coating, and paclitaxel and sirolimus (rapamycin) are often used as the antiproliferative drug. Recently, a new biodegradable and drug-eluting system for magnesium stent coating was developed, in which poly (3-hydroxybutyrateco-3-hydroxyhexanoate) (PHBHHx) was used as the drug delivery coating and ferulic acid (FA; 4-hydroxy-3-methoxy cinnamic acid) was used as the antiproliferative drug [14]. PHBHHx has recently been considered as potential material for tissue engineering applications such as angiogenesis and arterial repair due to the biodegradability, good blood compatibility [15] and good biocompatibility to several cell types [16–21]. FA is the main efficacious ingredient in Angelicae sinensis and is also present abundantly in various fruits and vegetables, such as bananas, citrus fruits, bamboo shoots, eggplants and cabbages [22,23]. FA exhibits many physiological functions including antioxidant, antimicrobial, anti-inflammatory, antithrombosis, antihypercholesterolemic, anticancer activities, and spermatozoa activating bioactivity [23]. The non-cytotoxicity of FA to both human umbilical vein endothelial cells (HUVECs) and VSMCs, and the promotion function on angiogenesis were also reported [24]. The promotion function of FA to HUVECs was widely reported [22,25–27]. It was found that FA at a range of concentration from 0.1 μg/mL to 10 μg/mL could markedly improve the proliferation of endothelial cell line (ECV304) and DNA synthesis in a dosedependent manner with a significant decrease in the percentage of cells in the G0/G1 phase and a significant increase in the S phase [22]. Lin et al. [27] reported that FA (10− 6–10− 4 M) induced significant angiogenesis of HUVECs in vitro without cytotoxicity via vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and hypoxic-induced factor (HIF) -1α mRNA. Further research revealed that FA upregulated cyclin D1 and VEGF mRNA [22]. It was also reported that the promotion of blood vessel regeneration by FA was observed in vitro and in vivo [28]. Meanwhile, Hou et al. [29] reported that FA significantly inhibited angiotensin II-induced VSMC proliferation in a dose-dependent manner by elevating the protein content of p21waf1/cip1, decreasing expression of cyclin D1 and inhibiting phosphorylation of retinoblastoma protein. Similar result was also reported by Wang et al. [24]. All these results indicate that FA has the biological function to promote the proliferation of ECs and inhibit the proliferation of VSMCs, which could be one of the best drug selections for the drugeluting coating system for stent application. Based on the above consideration, it is reasonable to design a new biodegradable and drug-eluting coating system for metallic stents by combining FA as the drug and PHBHHx as the drug delivery. It is proposed that after the implantation of this drug-eluting system, FA released from the coating will promote the endothelialization and prevent the neointimal hyperplasia, which provides a possibility to reduce ISR and LST. Previous study has shown that the FA-eluting PHBHHx coating had good bonding strength to magnesium substrate and the FA released from the FA-eluting PHBHHx film enhanced the adhesion, spreading and proliferation of HUVECs in a dose-dependent manner, effectively inhibited H2O2-induced injury and promoted NO production of HUVECs [14]. As a biomaterial to be used in contact with blood, the good blood compatibility is a basic requirement. PHBHHx has been proven to show good blood compatibility [15]. But it is still not known whether the addition of FA has influence on the blood compatibility, although the anticoagulant ability of FA was reported elsewhere [30,31]. In this paper, FA-eluting PHBHHx films with 5% and 10% FA were prepared by adding FA in PHBHHx by a physical method based on previous results [14,32]. At the very early stage of this research, the blood compatibility of this film was investigated to assess the possibility of FA-eluting PHBHHx system for biodegradable magnesium stents. In addition, alkali treatment has often been used as an effective surface modification method for biomedical polymers to improve the cell–matrix adhesion [33,34]. The effect of an alkali treatment on the blood compatibility of FA-eluting PHBHHx films was also reported.
2. Materials and methods 2.1. Preparation of material and sample PHBHHx (with 12 mol% HHx and a molecular weight of 200,000 Da) was kindly provided by Dr. Qiong Wu (Tsinghua University, Beijing, China). Ferulic acid (FA) with a purity of ≥ 98% was purchased from Nanjing Zelang Medical Technology Co., Ltd. PHBHHx, FA and ethyl acetate (analytical grade, Sinopharm Chemical Reagent Co., Ltd, Shenyang, China) were used to prepare FA-eluting samples. Briefly, 0.25 g of PHBHHx and 5 wt.% FA or 10 wt.% FA were dissolved in 5 mL ethyl acetate to prepare 5% drug-eluting or 10% drug-eluting PHBHHx material (named PHBHHx + 5%FA and PHBHHx + 10%FA), respectively. Then, the solutions were poured into a Petri dish and kept at 30 °C under vacuum for 24 h and then at 35 °C for 48 h to allow the complete evaporation of the ethyl acetate. The prepared film was about 80–100 μm in thickness. Samples with a dimension of 10 mm × 15 mm for different measurements were cut from the films. Before the tests, the samples were sterilized by ultraviolet radiation for 4 h. 2.2. Blood and plasma Blood obtained from a healthy volunteer was mixed with acid citrate dextrose, and centrifuged at 3000 rpm and 1500 rpm for 15 min to get platelet-poor plasma (PPP) and platelet rich plasma (PRP), respectively. 2.3. Alkali treatment FA-eluting films were immersed in NaOH solution (2 mol L−1) for 120 min. Then, the films were taken out and rinsed extensively with deionized water until the pH of the rinsing water became neutral. Samples after alkali treatment were denoted as PHBHHx + 5%FA + Alkali and PHBHHx + 10%FA + Alkali, respectively. 2.4. Scanning electron microscopy The samples were posted on an aluminum stump and coated with gold under vacuum. The morphology observation of the films was conducted on a scanning electron microscopy (SEM) with a model of SSX-550 (SHIMADZU, Japan) with an acceleration voltage of 5 kV. 2.5. Contact angle measurement Water contact angle (θ) of the films was determined on a contact angle goniometer (POWEREACH, Shanghai, China). Deionized water was used as a probe liquid. Water contact angle was measured by five independent measurements and was presented as mean ± standard deviation. 2.6. Drug release measurement For FA release measurement, the FA-load films were immersed in a simulated plasma solution with a liquid volume/surface area ratio of 2.5 mL/cm2 at 37 °C for preset duration. The cumulative FA release was measured on a high performance liquid chromatography (HPLC, SHIMADZU LC2010) with Diamonsil C18 column and acetonitrile-0.1% phosphate as mobile phase at a flow rate of 1 mL/min at 20 °C. Triple tests were conducted for each sample. 2.7. Hemolysis assay Hemolysis test was conducted with reference to ISO 10993-4: Selection of test for interaction with blood and ISO 10993-12: Sample preparation and reference materials. The blood sample was prepared by mixing 8 mL anticoagulated whole blood with 10 mL normal saline. Extract liquids were prepared by immersing PHBHHx sample and the
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drug-eluting samples in normal saline for 24 h at 37 °C (surface area/ volume = 6 cm2/mL). Then, 10 mL extract liquid and 10 mL normal saline were put into a test tube and then 0.2 mL blood sample was added. The test tube was incubated at 37 °C for 1 h and then centrifuged at 2500 rpm for 5 min. Optical density (O.D.) of the supernate was measured at a wavelength of 545 nm by a spectrophotometer (S53/54 UV–VIS Spectrophotometer, Shanghai LingGuang, China). 10 mL distilled water with 0.2 mL blood sample was used as the positive sample and 10 mL normal saline with 0.2 mL blood sample as the negative control. Three parallel experiments were performed for each sample. The hemolysis rate (HR) was calculated as follow
HR ¼
Dt −Dnc 100% Dpc −Dnc
ð1Þ
where HR is the hemolysis rate, and Dt, Dpc and Dnc are the absorbency of the sample, the positive control and the negative control, respectively.
2.8. Plasma recalcification time (PRT) and plasma prothrombin time (PT) measurements The coagulation times were determined with a semiautomatic blood coagulation analyzer using mechanical end point determination (GF2000 II, CaiHong Analytical, China). In all tests, glass silicide and glass were used as a negative control and a positive control, respectively. For PRT measurement, 150 μL of PRP was carefully added on the film samples' surface and incubated at 37 ± 1 °C for 1 min, then, 100 μL residual PRP was taken to a test cup followed by the addition of 100 μL of calcium chloride solution (0.025 mol L− 1) immediately at 37 ± 1 °C. A mechanical end point determination was used to measure the formation of fibrin clot. For PT measurement, 150 μL of PPP was carefully added on the test samples' surface and incubated at 37 ± 1 °C for 1 min. After this, 100 μL of PPP was taken out to the test cup, simultaneously with the addition of 100 μL of thromboplastin reagent (Zhong Tai, Wuhan, China) at 37 ± 1 °C. The tests were repeated three times for each sample.
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2.11. Statistical analysis Data were presented as means ± standard deviation. Statistical comparisons were performed using Student's t-test. p b 0.05 was considered statistically significant. 3. Results 3.1. Surface morphology Fig. 1 shows a smooth and dense surface microstructure of PHBHHx film. Fig. 2 presents the surface morphologies of the FAeluting PHBHHx films before and after alkali treatment. It was observed that PHBHHx + 5%FA film (Fig. 2a) had a smooth and dense surface while many pores with less than 100 μm size were observed on the surface of PHBHHx + 10%FA film (Fig. 2c). PHBHHx + 10%FA film was much rougher than PHBHHx + 5%FA film. After the alkali treatment, as shown in Fig. 2b and d, there were more holes on the surface due to the etching function of NaOH. Compared with the surface microstructure before the treatment, it can be concluded that alkali treatment led to a much rougher surface with more pores. 3.2. Water contact angle Fig. 3 shows the water contact angles of PHBHHx and the FA-eluting PHBHHx films before and after alkali treatment. The contact angle of PHBHHx film was about 90.2° ± 1.3°. The addition of 5% FA reduced the contact angle significantly to 87.4° ± 1.1° (p b 0.05) and further increase in FA content (10%FA) decreased the contact angle significantly to 83.3° ± 0.9° (p b 0.05). It can be deduced that the addition of FA reduced the water contact angle significantly in a dose-dependent manner, in turn, improved the hydrophilicity of the film significantly. After the alkali treatment, the contact angles of the PHBHHx + 5%FA and PHBHHx + 10%FA were reduced significantly to 52.4° ± 0.9° and 50.6° ± 1.4° (p b 0.05), respectively. Before the alkali treatment, the contact angles of these films were in a range of 83–90°, indicating that the films were still hydrophobic films. After the alkali treatment, the contact angle was dramatically reduced to 50–52°, indicating that the alkali treatment changed the film from hydrophobic to hydrophilic film.
2.9. Kinetic clotting time
3.3. Drug release
100 μL of anticoagulant blood was carefully added on the test samples and 10 μL of calcium chloride solution (0.2 mol L−1) was added simultaneously, and time was recorded immediately. After 10, 30, 50, 70 and 90 min, respectively, the surface of the test sample was flowed slowly by the distilled water separately and the flowed liquid was collected in a 96-well plate. O.D. value of the surplus extricate hemoglobin in the flowed liquid was measured in a spectrophotometer (S53/54 UV– VIS Spectrophotometer, Shanghai, China) at a wavelength of 545 nm. Glass silicide and glass were used as a negative control and a positive control, respectively.
Fig. 4 shows the drug release from the drug-eluting films with immersion time. The effect of the alkali treatment on the drug release was also shown in the figure. For both PHBHHx + 5%FA film and PHBHHx + 10%FA film, there was a tendency that the cumulative
2.10. In vitro platelet adhesion Before the test, the test samples were immersed in phosphatebuffered saline (PBS, pH = 7.3) for 20 min. Then the film samples were laid on the bottoms of the wells a 24-well plate, submerged with PRP and incubated at 37 ± 1 °C for 0.5 h and 3 h, respectively. After this, the film samples were washed gently with PBS three times to remove non-adhered platelets, and then fixed with 2.5% buffered glutaraldehyde solution overnight in a refrigerator at 4 °C. After fixation, the test samples were washed and dehydrated in a graded ethanol series (50%, 60%, 70%, 80%, 90% and 100% for 15 min each). Surface gold coating was conducted on all samples for SEM observation.
Fig. 1. Surface microstructure of PHBHHx film.
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Fig. 2. Surface micrographs of the drug-eluting PHBHHx films before and after NaOH treatment. a) PHBHHx + 5%FA, b) PHBHHx + 5%FA + Alkali, c) PHBHHx + 10%FA, d) PHBHHx + 10%FA + Alkali.
drug release increased rapidly at the first 24 h and then slowed down during the following immersion. Due to the high FA content in PHBHHx + 10%FA film, the released drug was much higher than that from PHBHHx + 5%FA film at all intervals. After alkali treatment, the cumulative drug release dropped sharply for both PHBHHx + 5%FA and PHBHHx + 10%FA films. For examples, the cumulative drug releases were 28.2 μg and 37.7 μg for PHBHHx + 5%FA and PHBHHx + 10%FA films, corresponding to only 14.9% and 13.5% the values before the treatment, respectively. NaOH has a very strong etching ability. After 120 min immersion in NaOH solution, the films have been etched seriously as indicated by the holes on the surface in Fig. 2. The drug on the surface and
subsurface would dissolve in the solution, which might be the main reason for the sharp reduction in the cumulative drug release.
Fig. 3. Water contact angle of PHBHHx and the different drug-eluting PHBHHx films. (*p b 0.05).
Fig. 4. In vitro drug release curves of the drug-eluting PHBHHx films before and after the NaOH treatment.
3.4. Hemolysis HR values of the samples are listed in Table 1. HR of PHBHHx was 1.429, less than the recommended value of 5%, suggesting that PHBHHx had a good hemolysis property. HR vales of PHBHHx + 5%FA and PHBHHx + 10%FA were 0.831 and 0.498, respectively, indicating that the addition of FA reduced HR value slightly. Also, HR values of PHBHHx + 5%FA + Alkali and PHBHHx + 10%FA + Alkali were much lower than the recommended value, showing that the alkali treatment also reduced HR slightly. It has to be pointed that HR values of all test
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Table 1 Optical density and hemolysis rates of the test samples. Sample no.
PHBHHx
PHBHHx + 5%FA
PHBHHx + 5% + Alkali
PHBHHx +10%FA
PHBHHx +10%FA + Alkali
Negative control
Positive control
1 2 3 HR (%)
0.013 0.009 0.015 1.429
0.01 0.01 0.006 0.831
0.01 0.004 0.007 0.548
0.008 0.005 0.007 0.498
0.011 0.006 0.01 0.880
0.003 0.003 0.005 –
0.581 0.631 0.605 –
samples were much less than the recommended value of 5%, displaying the addition of FA and the alkali treatment have no influence on the hemolysis properties of the film. 3.5. PRT and PT Fig. 5 shows PRT values of PHBHHx film and the FA-eluting PHBHHx films before and after the alkali treatment. PHBHHx had a PRT of 280 min. It was shown that the PHBHHx + 5%FA had a significant longer PRT value than PHBHHx (p b 0.05) while PHBHHx + 10%FA had a significant longer PRT value than PHBHHx + 5%FA (p b 0.05), indicating that FA significantly prolonged the PRT properties of the film in a dosedependent manner. Although difference was found in PRT values of the FA-eluting PHBHHx films before and after alkali treatment, the difference was not significant (p N 0.05), indicating that the alkali treatment had limited influence on PRT of the FA-eluting PHBHHx films. Fig. 6 shows PT values of PHBHHx film and the FA-eluting PHBHHx films before and after the alkali treatment. It was evident that both PHBHHx + 5%FA film and PHBHHx + 10%FA film had significant longer PT values than PHBHHx (p b 0.05) but no significant difference was found between PHBHHx + 10%FA and PHBHHx + 5%FA (p N 0.05). Difference was also found in PT values of the FA-eluting PHBHHx films before and after alkali treatment, but the difference was not significant (p N 0.05), confirming that the alkali treatment has limited influence on PT of the FA-eluting PHBHHx films. 3.6. Kinetic clotting time Fig. 7 shows the kinetic clotting time of the test films. Generally, the time when O.D. value is equal to 0.1 is defined as initial clotting time while the time when O.D. value is equal to 0.01 is defined as whole clotting time. Table 2 summarizes the initial clotting time and the whole clotting time obtained from Fig. 7. The initial clotting time and the whole clotting time of the PHBHHx + 5%FA were both longer than those of PHBHHx, indicating that the addition of FA prolonged the clotting time. It can also be found that the clotting time increased with
Fig. 5. PRT of PHBHHx and the FA-eluting PHBHHx before and after alkali treatment (*p b 0.05).
the increase in FA content in the film. These results confirm that FA had a potential function to improve the anticoagulant properties. Although difference was observed in the initial clotting of the FAeluting films before and after the alkali treatment, the increase in the clotting time was limited, the difference was not significant (p N 0.05), indicating that the alkali treatment had limited influence on PRT of the FA-eluting PHBHHx films. 3.7. Platelet adhesion Fig. 8 shows the morphology of the adhered platelets on PHBHHx and the FA-eluting PHBHHx films after 0.5 h incubation. Several platelets with round shape were observed on the surface of PHBHHx film while only a few platelets with round shape were observed on the FAeluting films, indicating that the platelets were not activated at that moment in both cases. No difference in platelet number and shape was found before and after the alkali treatment, displaying that the alkali treatment has no influence on the platelet adhesion and activation at the early stage. After incubation for 3 h, as shown in Fig. 9, most platelets spread on the surface of PHBHHx with pseudopodia while the platelet number increased, indicating that the platelets were activated at this moment. Meanwhile, more platelets were also observed on the FA-eluting PHBHHx films and the platelets spread on the surface. However, the platelet number and the change in the morphology of the platelets on the FA-eluting films were much less than that on PHBHHx at all intervals. After the alkali treatment, more platelets were found on the films after 3 h incubation, but no difference in the platelet morphology was found, indicating that the alkali treatment enhanced the adhesion of platelet but did not promote the activation. 4. Discussion A biomaterial, to be used as a drug-eluting coating on metallic stent, should meet the following requirements: (1) good cell biocompatibility; (2) good blood compatibility, including good hemolysis and
Fig. 6. PT of PHBHHx and FA-eluting PHBHHx before and after alkali treatment (*p b 0.05).
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E. Zhang, F. Shen / Materials Science and Engineering C 52 (2015) 37–45
Fig. 7. Kinetic clotting time of the test films.
anticoagulation properties; (3) biodegradability, for biodegradable coating application; and (4) ability to inhibit neointimal hyperplasia or/and to promote endothelialization. Good cell biocompatibility includes non-cytotoxicity and cell adhesion property. Both PHBHHx and FA have no cell toxicity [35]. PHBHHx can be degraded in vitro. However, after 4 week immersion in a simulated plasma solution, only 30 wt.% was degraded [14]. The addition of FA accelerated the degradation in a dose-dependent way. But even for the PHBHHx + 10%FA, only 55 wt.% was degraded after 4 week immersion [14], which indicates that this coating system could protect the magnesium stent from the fast degradation and guarantee the drug release at least within 4 weeks. In addition, it was also indicated that FA could enhance the HUVEC adhesion and proliferation, reduce the H2O2-induced injury and promote NO production [14]. However, the cell adhesion property on the surface of PHBHHx is not satisfied due to the poor hydrophilic property. Several surface modification techniques have been used to improve the surface hydrophilicity of polymer materials including surface hydrolysis [36], grafting technique [37], ultraviolet treatment [38], alkali treatment [33,34] and plasma treatment [39]. Also it was shown that NaOH treatment improved the cell adhesion, spreading and proliferation and had no influence on the H2O2 induced injury and NO production [14]. Good blood biocompatibility is another basic requirement. PHBHHx has shown very good blood compatibility [15]. The addition of FA in PHBHHx enhanced the proliferation of HUVECs, on the other hand, might bring about influence on the blood compatibility of PHBHHx. Surface morphology shown in Figs. 1 and 2 indicated that the addition of FA in PHBHHx produced some small holes with a size of less than 100 μm in the film, which contributes to the significant reduction in the contact angle in comparison with PHBHHx film. Alkali treatment created many large holes on the surface of the FA-eluting films, as shown in Fig. 2b and d, which dramatically reduces the contact angle from 83–87° to 50–52°, as shown in Fig. 3. The sharp improvement in the hydrophilicity will benefit cell adhesion and proliferation, including platelet. It is always desired that the drug in a drug-eluting system can be released gradually with the degradation of drug carrier. In this study, FA was mixed with PHBHHx by a physical method and no reaction happened between FA and PHBHHx to protect the physical and biological functions of FA. The drug release curves of the FA-eluting films in Fig. 4 clearly showed that FA was released in a gradual manner rather than a burst manner, displaying that the FA release can be successfully controlled. However, the alkali treatment dramatically reduced the FA cumulative release due to the etching function although it significantly improved the hydrophilicity of the FA-eluting PHBHHx film. Therefore, it is very necessary to optimize the alkali treatment in the next step to obtain a balance between the good hydrophilicity and the drug release behavior.
It can be found from Table 1 that the HR values of PHBHHx and the FA-eluting films were both much less than the recommended value of 5% by ISO standard, indicating that both PHBHHx and FA have no destruction to erythrocyte. Plasma coagulation properties of a biomaterial indicate the possibility of this material to react with blood component and cause thrombosis [40–42]. Long PRT, PT and the initial clotting are always desired. However, no recommended values are given in ISO standard. As for the blood coagulation mechanism, it is widely accepted that there exist three pathways which can lead to thrombosis: an intrinsic pathway, an extrinsic pathway, and a common pathway. PRT is normally used to assess the intrinsic pathway and PT is used to evaluate the extrinsic pathway and common coagulation. Significant increase was observed in PRT and PT of the FA-eluting PHBHHx films in comparison with the value of PHBHHx, which indicates that the FA-eluting film has less activation to both intrinsic pathway and extrinsic pathway and common coagulation than PHBHHx. In addition, the longer initial clotting time and whole clotting time of the FA-eluting PHBHHx than those of PHBHHx also indicate that the FA-eluting films exhibited a weaker activation degree to the hemagglutination factor XII than PHBHHx. All these results clearly display that FA inhibited the coagulation of the blood system, especially in a dose-dependent manner in PRT. It can be also found that no significant difference in PRT, PT and the initial clotting time was found before and after the alkali treatment, indicating that the alkali treatment has limited influence on the anticoagulation time. However, the alkali treatment dramatically reduced the cumulative FA release, as shown in Fig. 4, it can be deduced that small amount of FA can significantly extend the coagulation time. On the other hand, this result suggests that the anticoagulation function of FA would not be dose-dependent when the FA content is more than a critical value. Serious platelet adhesion and activation on the surface of a biomaterial are the other main factors which cause thrombosis. After 0.5 h incubation, a few platelets were observed on the surface of both PHBHHx and the FA-eluting films. Even after 3 h incubation, only several platelets were observed on the surface of PHBHHx film. Although some of them spread on the surface and were activated, the situation was still not serious. On the contrary, the platelet number on the FA-eluting films was less than the number on PHBHHx and no difference in the shape change of the adhered platelet can be found between PHBHHx and the FAeluting film, indicating that FA reduced the platelet number and had no influence on the activation of platelet. When platelets are contacted with the film, two factors will affect the platelet adhesion. One is the hydrophilicity of the film. Good hydrophilicity promotes the adhesion of platelet. Another factor is FA released from the film. The addition of FA in PHBHHx improved the hydrophilicity of PHBHHx, which should promote the platelet adhesion. But only a few platelets were found on the FA-eluting film, which strongly indicates that FA has a function to resist the platelet adhesion. Alkali treatment improved the hydrophilicity of the FA-eluting film but reduced FA release, as shown in Figs. 3 and 4, which might be the main reason why alkali treatment enhanced the platelet adhesion. Polymers normally have poor cell adhesion properties due to their smooth surface and low hydrophilicity. Although the addition of FA reduced the contact angle as stated in the above section, the contact angle was still as high as 80°–82° and hydrophilicity still needs to be improved for good cell adhesion. Alkali treatment as an effective method to improve the hydrophilicity reduced FA release, which led to enhancement in the platelet adhesion. So it is necessary to find out other surface modifications in the next step, which could improve the hydrophilicity without deteriorative effect of the FA release. Although the alkali treatment affects the FA release, the alkali environment resulted from the degradation of magnesium stent will not influence the FA release due to the fact the FA-eluting system will be degraded first and then the degradation of magnesium stent will happen. All these results strongly demonstrated that FA-eluting PHBHHx had very good blood biocompatibility. With the consideration of the good
E. Zhang, F. Shen / Materials Science and Engineering C 52 (2015) 37–45
43
Table 2 The initial clotting time and the whole clotting time of the test samples (min). Samples
PHBHHx
PHBHHx + 5%FA
PHBHHx + 5% + Alkali
PHBHHx + 10%FA
PHBHHx + 10%FA + Alkali
Initial clotting time Whole clotting time
50 82
65 N90
70 N90
72 N90
73 N90
bonding strength to magnesium substrate and the promotion function to endothelialization [14], the FA-eluting PHBHHx system has shown potential as a candidate for the drug-eluting surface coating system of magnesium stent. It has to be pointed that the FA-eluting system is not degraded uniformly in physiological environment and also physiological solution can be penetrated into defects of coating, which might cause the localized and filiform corrosion of magnesium stent and then might lead to a failure of implantation. Previous study [14,32] has shown that the degradation rate of PHBHHx coating can be adjusted by the selection of molecular weight of PHBHHx, the coating thickness and the FA content. Therefore, it is necessary in the next step to optimize the
whole stent system parameters in order to get a good drug-eluting stent system. 5. Conclusion From the above research results, the following conclusions can be drawn: 1) FA-eluting PHBHHx showed good hemolysis and long PRT, PT and the initial clotting time. 2) FA had a function to inhibit the coagulation of the blood system and resist the platelet adhesion.
Fig. 8. Platelet adhesion on PHBHHx film and the FA-eluting PHBHHx films contacted with plasma for 0.5 h. a) PHBHHx, b) PHBHHx + 5%FA, c) PHBHHx + 5%FA + Alkali, d) PHBHHx + 10%FA, e) PHBHHx + 10%FA + Alkali.
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E. Zhang, F. Shen / Materials Science and Engineering C 52 (2015) 37–45
Fig. 9. Platelet adhesion on PHBHHx film and the FA-eluting PHBHHx films contacted with plasma for 3 h. a) PHBHHx, b) PHBHHx + 5%FA, c) PHBHHx + 5%FA + Alkali, d) PHBHHx + 10%FA, e) PHBHHx + 10%FA + Alkali.
3) Alkali treatment had no influence on hemolysis, PRT, PT and the initial clotting time of the FA-eluting PHBHHx films, but enhanced the platelet adhesion slightly. Acknowledgment The authors would like to acknowledge the financial support from National Natural Science Foundation (no. 31271024/C1002, no. 31470930/C1002), Foundation for Key Program of Ministry of Education, China(no. 313014), Heilongjiang Universities' Science and Technology innovation team program (no. 2012TD010), Nature Science Foundation of Heilongjiang Province for Distinguished Young Scholars (no. JC201205) and funding from Northeastern University (985 program) and Program LZ2014018, China. References [1] G. Mani, M.D. Feldman, D. Patel, M. Agrawal, Coronary stents: a materials perspective, Biomaterials 28 (2007) 1689–1710.
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