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Preparation, characterization and anticoagulation of curcumin-eluting controlled biodegradable coating stents
Journal of Controlled Release 116 (2006) 42 – 49 www.elsevier.com/locate/jconrel
Preparation, characterization and anticoagulation of curcumin-eluting controlled biodegradable coating stents Ch. J. Pan a,b,c , J.J. Tang a,b , Y.J. Weng a,b , J. Wang a,b , N. Huang a,b,⁎ a
Key Laboratory for Advanced Technologies of Materials, The Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China b Key Laboratory of Surface Engineering of Artificial Organs of Sichuan, SouthWest JiaoTong University, Chengdu 610031, China c School of Bioengineering, Chongqing University, Chongqing 400044, China Received 22 March 2006; accepted 29 August 2006 Available online 8 September 2006
Abstract Curcumin is pharmaceutically active in many ways, having properties including anticoagulation, anti-proliferation, anti-inflammatory, and may be used to fabricate drug-eluting stents to treat in-stent restenosis after stent implantation. Here we describe our investigations of curcumin-eluting PLGA coatings formed using the biodegradable polymer PLGA (polylactic acid-co-glycolic acid) as drug carrier and uniformly fabricated on the surface of 316L stainless steel stents by an ultrasonic spray method. Three doses were explored — low dose (∼ 140 μg per stent or 115 μg/cm2), moderate dose (∼ 280 μg per stent or 230 μg/cm2), and high dose (∼ 490 μg per stent or 408 μg/cm2). Pre- and post-expansion morphologies of the stent coating were examined by optical microscopy (OM) and scanning electron microscopy (SEM), indicating that the coating not only was very smooth and uniform but also had the ability to withstand the compressive and tensile strains imparted without cracking from the stent during the expansion process. Atomic force microscopy (AFM) images indicated the topography of the PLGA-only and moderate dose curcumin-eluting stent that showed an average roughness below 1 nm; no drug particles could be seen on the stent surface, indicating that curcumin can be mixed with PLGA at the molecular level using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier Transform Infrared (FTIR) spectroscopy and X-ray electron spectroscopy (XPS), with results suggesting that there was no chemical reaction between curcumin and the drug. The results of in vitro measurements of drug release from curcumin-eluting stents showed that all the curcumin-eluting stents studied exhibited a nearly linear sustained-release profile with no significant burst releases within the measurement period. The in vitro anticoagulation behavior of curcumin-eluting stents was investigated by static platelet adhesion and APTT (activated partial thromboplastin time) tests, revealing that the anticoagulation properties of curcumin-eluting stents are superior to those for stainless steel stents and PLGA-only-coated stents. The anticoagulation behavior of curcumin stents improved significantly as the drug dose was increased. © 2006 Elsevier B.V. All rights reserved. Keywords: Curcumin; Stent; Hemocompatibility; Coating; Restenosis
1. Introduction Although the introduction of coronary stents has significantly improved the treatment of patients with coronary artery disease, restenosis due to neointimal proliferation following stent im-
⁎ Corresponding author. Key Laboratory for Advanced Technologies of Materials, The Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China. Tel./fax: +86 28 87600625. E-mail address: [email protected] (N. Huang). URL: http://www.biomatchina.com (N. Huang). 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.08.023
plantation and associated with a return of ischemic symptoms, has remained a critical concern [1–3]. The mechanism of in-stent restenosis includes recoil and remodeling of the artery, thrombus formation, neointima hyperplasia due to vascular smooth muscle cell (VSMC) proliferation and inflammatory reaction [4–6]. It is generally believed that VSMC proliferation and intracellular matrix synthesis in response to stent-induced inflammatory reactions are the major mechanisms of in-stent restenosis [6]. Orally administered drugs may cause toxic reaction and may not achieve adequate local drug concentration, and thus do not resolve this problem successfully [7]. Consequently much attention has been devoted to developing treatments that target
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VSMC proliferation, the putative key event in the formation of intimal hyperplasia following stent implantation. Recently, drug-eluting stents with synthetic polymer coatings, which release anti-proliferative drugs such as rapamycin and paclitaxel over a period of several weeks or months, have opened up a new paradigm in the treatment of in-stent restenosis (ISR) [8–11]. These drug-eluting stents can provide luminal scaffolding that eventually eliminates the recoil and remodeling of coronary vessels. Additionally, the drug released from the coating can achieve high local drug concentration and thus prevent cellular proliferation, thrombus formation or inflammation. After the drug elutes from the polymer coating, however, the residual synthetic polymer coating remains in the body. Eventually, the permanent presence of the nonreabsorbable polymer may lead to complications such as an exaggerated inflammatory response and neointimal hyperplasia at the implant site [12,13]. It is, therefore, desirable to develop a drug-eluting stent with a biocompatible and biodegradable coating to prevent these unfavorable effects. Poly(lactic acid-co-glycolic acid), PLGA, a biodegradable polymer, which has a number of desirable properties such as good biocompatibility and biodegradation, is a material that is used extensively for controlled delivery of protein and peptide drugs [14], for the manufacture of medical devices [15] and wound dressings [16], as well as for fabricating scaffolds in tissue engineering [17]. Using PLGA as a drug carrier for stent coatings may be a good candidate method. Curcumin (diferuloyl methane, Fig. 1), a major chemical component of turmeric, has low intrinsic toxicity and possesses a wide rage of pharmacological activity, including antithrombus, anti-oxidation and anti-proliferation properties [18– 20], suggesting that it could be very suitable for drug-eluting stents because of these desirable characteristics. In vitro research suggests that curcumin can inhibit platelet aggregation induced by ADP, epinephrine, and collagen [21,22]. Curcumin can also prevent platelet aggregation by platelet-activated factors, and inhibits the formation of thromboxane A2 (TXA2) by platelets [23]. Curcumin inhibits not only serumstimulated- but also growth factor-stimulated-proliferation of vascular smooth cells (VSMC) [29]. In addition, cell cycle analysis of curcumin-treated VSMC has revealed a G0/G1 arrest, and a reduction in the percentage of cells in S phase. The anti-proliferative effect of curcumin may partly be mediated through inhibition of protein tyrosine kinase activity and c-myc mRNA expression [30]. Though many curcumin anticoagulation mechanisms have been studied, the anticoagulation behavior of curcumin in materials, especially biodegradable polymers such as PLGA, has not been investigated.
Fig. 1. Chemical structure of curcumin.
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Fig. 2. Schematic of the ultrasonic spraying system.
In the work described here, the preparation of curcumineluting stents using PLGA as the drug carrier, and its various in vitro characteristics, drug release profiles and anticoagulation are reported. 2. Materials and methods 2.1. Materials The stent material used in this study was annealed 316LVM surgical grade steel tubing (outer diameter 1.65 mm and wall thickness 0.11 mm) which was provided by Northwest Research Institute of Color Metal. The curcumin was purchased from Xi'an Zhongxin Biochemical Co. Ltd., and was of purity above P 96% (HPLC grade). The PLGA (LA:GA = 85:15, Mη¼ 95; 800 resin was endowed by Chengdu Zhuxin Biomaterials Co. Ltd., China, and was of purity above 98%. All other reagents used in this study were analytical grade. 2.2. Preparation of curcumin-eluting stents Firstly the stainless steel tube was formed into a stent by a laser cutting machine. The stents were carefully polished using an electrochemical method, and then cleaned ultrasonically with acetone, ethyl alcohol and distilled water in sequence. The cleaned stents were kept under vacuum for 48 h to evaporate the residual water. A 1 wt.% solution of PLGA containing curcumin was sprayed onto the cleaned stents. A schematic of the spray system is shown in Fig 2. The micro-liquid, which is controlled by a microsyringe, is atomized by ultrasonic atomization and then sprayed onto the stent surface. The stent is traversed and rotated during the spray process. The whole system can be controlled by a computer. We remark parenthetically that not all of the solution is sprayed onto the stent surface; the efficiency of the overall spray system is about 56%. The mass of the coating sprayed onto the stent surface can be obtained by weighing the stent before and after spraying. Three doses of curcumin-eluting stents were prepared: a low-dose stent (∼ 140 μg per stent or 115 μg/cm2), a moderate-dose stent (∼ 280 μg per stent or 230 μg/cm2), and a high-dose stent (∼ 490 μg per stent or 408 μg/cm2). Control stents were prepared by the same method, for which the coating was only the polymer. The thickness of these coatings was about 10 μm and the coating weight for each stent was 1400 μg.
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The topography of the stent coatings was also determined by atomic force microscopy (AFM, SPI3800W probe station and SPA-400 SPM unit system, Japan). Samples were imaged in the dry state before elution of curcumin. AFM images of PLGAonly stents and moderate-dose curcumin-eluting stents were obtained. The scanning range was 1 × 1 μm2. 2.6. Release profile of curcumin
Fig. 3. Optical microscope images and SEM micrographs of metallics stents spray-coated with PLGA and curcumin: a (×40) and c (×50) are pre-expansion images; b (×40) and d (×200) are post-dilation images.
2.3. Fabrication of curcumin-loaded films A 1 wt.% solution of polymer (PLGA) resin was prepared by dissolving it in ethyl acetate. The solution was used to cast thin polymer films on carefully cleaned glass petri dishes. The films were allowed to slowly dry in air to evaporate the solvent and then smooth, non-porous films were obtained. Cast films were kept under vacuum to evaporate residual solvent. Curcuminloaded films were prepared in the same manner by dissolving 10% (low-dose), 20% (moderate-dose), and 35% (high-dose) (w/w) curcumin with polymer resin in ethyl acetate, with the same evaporation and storage steps. 2.4. Structure of curcumin-loaded films The structure of PLGA, curcumin and curcumin-loaded films was examined by FTIR (Fourier Transform Infrared) spectroscopy and XPS (X-ray photo-electron spectroscopy). The scanning FTIR range was 4000 cm− 1 to 400 cm− 1. The XPS spectra were obtained using an XSAM800 (Kratos Inc, England). The instrument was equipped with a monochromatic Mg Kα (1253.6 eV) X-ray source operated at 12 kV at a pressure of 5–7 × 10− 9 mbar. The reference binding energy was set at 284.6 eV for C–C and C–H of the C1s core level spectra.
The curcumin release profile was investigated in vitro by incubating an individual curcumin-eluting stent in a 2.0 ml capped glass tube containing 1.5 ml medium at 37 °C and pH 7.4. The medium consisted of phosphate-buffered saline containing 10% ethanol (v/v) [33] because the limit of solubility of curcumin in water makes it impossible to study in buffer. At selected times, the incubation medium was completely removed for analysis and replaced with fresh medium. An aliquot of the sampled medium was measured by high performance liquid chromatography (HPLC) (Waters 2695 separations module and Waters 2487 dual λ absorbance detector). The detection wavelength was fixed at 430 nm. The flow rate was 1 ml/min and the flow phase consisted of acetonitrile and water (65:35, v/v). Results were expressed as cumulative micrograms and weight percent of curcumin released as a function of time. 2.7. In vitro anticoagulation evaluation The in vitro anticoagulation behavior of drug-loaded films and stents was investigated by platelet adhesion measurements for films and activated partial thromboplastin time (APTT) test for stents. The curcumin-eluting polymer films and the control films (PLGA-only film) were cut into 10 × 10 mm2 samples. Whole blood was centrifuged at 1500 rpm and the upper platelet-rich plasma (PRP) obtained. The film samples were immersed in platelet-rich plasma for 2 h at 37 °C. After washing with 0.9% NaCl solution, they were fixed using 0.2% glutaraldehyde solution for 1 h and then fixed using 0.5% glutaraldehyde solution for 12 h, then washed again with 0.9% NaCl three times, and subsequently immersed in 50, 50, 75, 90, 100% ethanol solutions for 5 min and dried in a dessicator. Samples were sputtered with gold before being imaged by scanning electron microscopy (SEM).
2.5. Morphology examination and balloon-expansion tests The surface morphology of the curcumin-eluting stents was examined by Motic three-dimensional optical microscopy (OM) and scanning electron microscopy (SEM, Quanta 200, Philips) before balloon expansion. The stent was mounted onto an angioplasty balloon and the balloon was dilated to 3.0 mm at a pressure of 3.5 atm. The post-expansion stents were examined by OM and SEM.
Fig. 4. High resolution 1-μm topography AFM images in air of the surface of PLGA-coated (left) and moderate-dose curcumin-eluting stent (right).
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struts. The coating on the stent has the ability to withstand the compressive and tensile strains imparted without cracking in the stent expansion process. Tapping mode AFM images of the surface of PLGA-coated and curcumin-eluting stents are shown in Fig. 4. It can be seen that the surface of the stent coated with PLGA-only is very smooth, with an average roughness of about 0.14 nm. After loading curcumin in the PLGA coating, the roughness of the stent is greater than for the PLGA-only-coated stent, with an average roughness of curcumin-eluting stent about 0.55 nm. However, no drug particles can be seen on the stent surfaces from the SEM and AFM images; this indicates that curcumin can be mixed with PLGA at the molecular level using an ultrasonic atomization spray method. 3.2. Surface structure of the coating films Fig. 5. FTIR spectra of curcumin-eluting coating film.
For the activated partial thromboplastin time (APTT) measurements, curcumin-eluting and control stents were put into a special testing tube. A 100 μl fresh human platelet-poorplasma (PPP), obtained by centrifuging whole blood at 3000 rpm, and 100 μl actin-activated cephthaloplastin reagent were added into the tube containing stents, followed by the addition of a 0.03 M CaCl2 solution (100 μl) after 3 min incubation at 37 °C. The clotting time of the plasma solution was measured by a coagulometer (Clot 1A, Innova Co.).
The surface structure of curcumin-eluting PLGA films was investigated by FTIR and XPS. Fig. 5 shows the FTIR spectra of PLGA, curcumin and curcumin-eluting coating films. It can be seen that the two kinds of polymer films (curcumin and curcumin-loaded film) have similar structure. The FTIR
3. Results and discussion 3.1. Stent morphology and balloon-expansion tests For drug-eluting stents, surface topography is considered to have an important influence on stent performance. If the stent has webbings and “bridges” between the struts, the coating may break off from the stent when the stent is dilated by a balloon catheter, and thrombus and VSMC proliferation can occur at the site where the coating breaks off [24]. A smooth surface coating can significantly decrease injury to blood vessels. Furthermore, a smooth stent surface is believed to reduce platelet activation and aggregation, consequently leading to less thrombus formation and neointimal proliferation [28]. PLGA coatings containing curcumin on metallic stents can be clearly visualized by optical microscopy because the curcumin is yellow. Fig. 3a–d shows OM and SEM images of curcumin-eluting stents. As can be seen in Fig. 3a and b, the PLGA coatings containing curcumin are very smooth and the curcumin can be visualized clearly before dilation by an angioplasty balloon. The coatings are very uniform and smooth. There are no cracks or webbings between struts. OM and SEM micrographs after expansion of a PLGA- and curcumin-coated stent are shown in Fig. 3b and d. It can be seen that there is no delamination or destruction of the coating on the stent after expansion with an angioplasty balloon. This indicates that the stent coating is sufficiently flexible to allow balloon expansion of the stent without cracking or peeling from the
Fig. 6. C1s XPS of (a) PLGA, and (b) circumin-eluting coating film.
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structure of curcumin and curcumin-loaded PLGA film is different from PLGA film because a peak at 1620 cm− 1 is found in the spectra of curcumin and curcumin-eluting PLGA films. The peak at 1620 cm− 1 is a stretching vibration peak of the carbon–carbon double bond in curcumin. One of the major peaks observed for the three films was around 3500 cm− 1, most probably due to –OH vibrations of intermolecularly-bonded OH groups. In particular, the strong peak at 2998 cm− 1 and 2952 cm− 1 and the rather weak peak at 1380 cm− 1 in PLGA film and curcumin-eluting film can be assigned to the stretching and deformation of methyl groups, suggesting a significant amount of branching in the polymer films. The strong peak at 1750 cm− 1 is due to C_O adsorption. The carbon–carbon double bonds at 1620 cm− 1 were found in the spectra of curcumin-eluting PLGA film and curcumin and not found in PLGA-only films, indicating that curcumin exists in the polymer film. The peak at 1620 cm− 1 in curcumin did not shift in curcumin-eluting film, suggesting that curcumin may be in a dispersed state in PLGA films. As shown in Fig. 6, the two C1s XPS spectra of PLGA and curcumin-eluting polymer films are not the same. The peaks in the two graphs at 284.6 eV, 285.7 eV, and 286.9 eV are
Fig. 8. SEM images of post-release stent (a, ×40; b, ×160; c, ×600; d, ×2000).
attributed to C–C single bonds, C–O single bonds, and C_O double bonds, respectively. The peak in Fig. 6b at 289.3 eV is attributed to C_C double bonds which only exist in curcumin, suggesting that there is some curcumin on the surface of the coating film. In the chemical structure of curcumin, C_C and –OH are active groups which may lead to chemical reaction with PLGA. However, according to the data shown in Fig. 6, C_C exists in films after curcumin loading, indicating that there is no chemical reaction between curcumin and PLGA, and thus curcumin may be dispersed in the PLGA polymer matrix. 3.3. Curcumin release profile The results of in vitro elution of curcumin from different types of curcumin-loaded stents are shown in Fig. 7. Generally speaking, drug release from a polymer-based matrix takes place with an initial burst release [25]. As shown in Fig. 7, however, all the curcumin-loaded stents exhibited a nearly linear sustained-release profile with no significant burst release over a period of 18 days. Replacing the releasing medium every day established a significant concentration gradient of curcumin between the drug-eluting stent and the release medium, and thus led to a linear constant drug diffusion [34]. The drug release profile of curcumin can be analyzed using the following linear relationship [26]. Mt ¼ kd t M0
Fig. 7. In vitro release profile of circumin from three types of coated stents: (a) shows the cumulative amount of curcumin, and (b) shows the release weight percent.
ð1Þ
Mt Where M is the release fraction of the drug, t is the time, and k a 0 constant parameter of the release system. In theory, a sustained release of antirestenotic drugs for at least three weeks is required to prevent smooth muscle cell
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biodegradation; in fact, the mass loss rate and molecular weight of PLGA used here reach about 60% after 86 days in PBS (data not shown). SEM images of curcumin-eluting stents which released for 18 days in PBS medium are shown in Fig. 8. Many micropores can be seen in the stent surface after immersion of the stent in release medium for 18 days, perhaps hastening the subsequent biodegradation process. 3.4. In vitro anticoagulation Platelet adhesion measurements and activated partial thromplastin time (APTT) tests were used to evaluate the in vitro anticoagulation of drug-eluting films with different drug doses and control films. Thrombotic occlusion of stent angioplasty resulting from activated platelets is a complication of stent implantation. Curcumin can improve the hemocompatibility of biomaterials. Research results of Nguyen et al. indicate that curcumin significantly decreases platelet aggregation and activation and improves the blood compatibility of PLLA fibers [27]. More extensive in vitro studies have shown that biodegradable PLLA stents loaded with curcumin reduce the inflammatory response of platelets and leukocytes. Platelet aggregation occurs when fibrinogen molecules bind to the activated GPllb/llla receptor and connect platelets to one another [36]. The results of platelet adhesion experiments are indicated in Fig. 9. Many platelets adhere onto the stainless steel plate; furthermore, many adhered platelets are aggregated and activated, suggesting that the blood compatibility of stainless steel is very poor. In the case of PLGA-only film, platelets adhered on the film surface were generally singular with a rounded shape typical of platelets in an inactivated state, a few pseudopodia and deformation were observed. Drug-eluting films with different drug doses show significantly less platelet adhesion and aggregation than the PLGA-only film and stainless steel plate. Platelets adhered on the surface of curcumin-loaded PLGA films are not activated and the extension of pseudopodia is not observed. Adhered platelets on the surface of curcumin-loaded film decreased
Fig. 9. SEM images of platelet adhesion experiment in vitro (a, e — stainless steel; b, f — PLGA-only film; c, g — low-dose film; d, h — high-dose film).
migration and proliferation [35]. Based on the release profile shown in Fig. 7, the average sustained-release rates of curcumin for low-dose stents, moderate-dose stents, and high-dose stents were 5.26 μg per day (3.76% by weight), 7.37 μg per day (2.63% by weight), and 8.26 μg per day (1.68% by weight), respectively. Thus, based on these release rates, it can be concluded that the sustained-release duration of curcumin was ∼ 27 days for low-dose stents, ∼ 38 days for medium-dose stents, and ∼ 59 days for high-dose stents — all entirely satisfactory for a releasing requirement of three weeks. We note that the calculated release period does not consider the effect of
Fig. 10. APTT results for drug-eluting stents and control stent. (S0 — stainless steel stent; S1 — PLGA-only stent; S2 — low-dose stent; S3 — moderate-dose stent; S4 — high-dose stent).
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significantly with increasing drug dose. The curcumin released from drug-loaded films might inhibit the cyclooxygenase pathway by blocking the GPllb/llla receptor, inhibiting platelet aggregation leading to the formation of blood clots [37]. The cascade of reactions involved in the coagulation of blood is an autocatalytic and self-limiting process converting zymogene to an active form in the presence of proteolytic enzyme thrombin [31]. Research on anticoagulation for curcumin shows that curcumin prolongs the clotting time for both human and rat plasma by approximately 1.6-, 2.1- and 1.1-fold, as shown by thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, respectively, when compared to a control [32]. To examine the in vitro anticoagulation of drug-eluting stents and control stents, the APTT (activated partial thromboplastin time), which shows the bioactivity of intrinsic blood coagulation factors, was examined, and the results are shown in Fig. 10. The APTT time of bare stents (S0) is shorter than for plasma, indicating that bare stents may activate the coagulation process. The APTTs of all drug-eluting stents are prolonged compared to S0 and S1. For instance, the APTT of S4 increased from 40.6 s to 47.35 s. This indicates that activation of the intrinsic blood coagulation system is suppressed by curcumin released from the drug-eluting stent, and the extent of suppression is related to the curcumin content of the drugeluting stents. With the increase of curcumin drug dose, the APTT is prolonged, indicating that the blood compatibility of curcumin-eluting stents is improved. We speculate that the curcumin-prolonged APTT time could be due to the presence of hydrophobic groups in curcumin moiety [32]. Additionally, the hydroxyl groups in curcumin may prevent protein adsorption and thus improve the blood compatibility of the samples. These results all indicate that curcumin-eluting stents have good anticoagulation characteristics, and may prevent thrombus formation after stent implantation. 4. Conclusions In conclusion, the results obtained in this study suggest that curcumin-eluting stents, as developed in our work, have the ability to withstand compressive and tensile strains imparted without cracking from the stent during the expansion process. Curcumin can be mixed with PLGA at the molecular level using an ultrasonic atomization spray method, as shown by AFM images. Curcumin may be dispersed in the PLGA matrix according, as shown by FTIR and XPS. The stents have a sustained-release profile without initial burst release. Compared to bare stents and a control with PLGA-only coating, our drugcoated stents exhibit excellent anticoagulation. References [1] Ron Waksman, Drug-eluting stents, from bench to bed, Cardiovascular Radiation Medicine 3 (2002) 226–241. [2] E. Ragar, G. Sianos, P.W. Serruys, Stent development and local drug delivery, British Medical Bulletin 59 (2001) 227–248. [3] J. Gunn, D. Cumberland, Stent coatings and local drug delivery: state of the art, European Heart Journal 20 (1999) 1693–1700.
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[32] Panchatcharam Manikandan, Miriyala Sumitra, Srinivasan Aishwarya, Bhakthavatsalam Murali Manohar, Beema Lokanadamc, Rengarajulu Puvanakrishnan, Curcumin modulates free radical quenching in myocardial ischaemia in rats, The International Journal of Biochemistry and Cell Biology 36 (2004) 1977–1990. [33] Frank Alexis, Subbu S. Venkatraman, Santosh Kumar Rath, Freddy Boey, In vitro study of release mechanisms of paclitaxel and rapamycin from drug-incorporated biodegradable stent matrices, Journal of controlled release 98 (2004) 67–74. [34] Mei-Chin Chen, Hsiang-Fa Liang, Ya-Ling Chiu, et al., A novel drugeluting stent spray-coated with multi-layers of collagen and sirolimus, Journal of Controlled Release 108 (2005) 178–189. [35] J. Eduardo Sousa, Patrick W. Serruys, Marco A. Costa, New frontiers in cardiology, drug-eluting stents: part l, Circulation 107 (2003) 2274–2279. [36] J. Lefkovits, E.F Plow, E.J. Topol, Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine, New England Journal of Medicine 332 (1995) 1553–1559. [37] M.T. Huang, T. Lysz, T. Ferraro, et al., Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis, Cancer Research 51 (1991) 813–819.
Preparation, characterization and anticoagulation of curcumin-eluting controlled biodegradable coating stents Ch. J. Pan a,b,c , J.J. Tang a,b , Y.J. Weng a,b , J. Wang a,b , N. Huang a,b,⁎ a
Key Laboratory for Advanced Technologies of Materials, The Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China b Key Laboratory of Surface Engineering of Artificial Organs of Sichuan, SouthWest JiaoTong University, Chengdu 610031, China c School of Bioengineering, Chongqing University, Chongqing 400044, China Received 22 March 2006; accepted 29 August 2006 Available online 8 September 2006
Abstract Curcumin is pharmaceutically active in many ways, having properties including anticoagulation, anti-proliferation, anti-inflammatory, and may be used to fabricate drug-eluting stents to treat in-stent restenosis after stent implantation. Here we describe our investigations of curcumin-eluting PLGA coatings formed using the biodegradable polymer PLGA (polylactic acid-co-glycolic acid) as drug carrier and uniformly fabricated on the surface of 316L stainless steel stents by an ultrasonic spray method. Three doses were explored — low dose (∼ 140 μg per stent or 115 μg/cm2), moderate dose (∼ 280 μg per stent or 230 μg/cm2), and high dose (∼ 490 μg per stent or 408 μg/cm2). Pre- and post-expansion morphologies of the stent coating were examined by optical microscopy (OM) and scanning electron microscopy (SEM), indicating that the coating not only was very smooth and uniform but also had the ability to withstand the compressive and tensile strains imparted without cracking from the stent during the expansion process. Atomic force microscopy (AFM) images indicated the topography of the PLGA-only and moderate dose curcumin-eluting stent that showed an average roughness below 1 nm; no drug particles could be seen on the stent surface, indicating that curcumin can be mixed with PLGA at the molecular level using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier Transform Infrared (FTIR) spectroscopy and X-ray electron spectroscopy (XPS), with results suggesting that there was no chemical reaction between curcumin and the drug. The results of in vitro measurements of drug release from curcumin-eluting stents showed that all the curcumin-eluting stents studied exhibited a nearly linear sustained-release profile with no significant burst releases within the measurement period. The in vitro anticoagulation behavior of curcumin-eluting stents was investigated by static platelet adhesion and APTT (activated partial thromboplastin time) tests, revealing that the anticoagulation properties of curcumin-eluting stents are superior to those for stainless steel stents and PLGA-only-coated stents. The anticoagulation behavior of curcumin stents improved significantly as the drug dose was increased. © 2006 Elsevier B.V. All rights reserved. Keywords: Curcumin; Stent; Hemocompatibility; Coating; Restenosis
1. Introduction Although the introduction of coronary stents has significantly improved the treatment of patients with coronary artery disease, restenosis due to neointimal proliferation following stent im-
⁎ Corresponding author. Key Laboratory for Advanced Technologies of Materials, The Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China. Tel./fax: +86 28 87600625. E-mail address: [email protected] (N. Huang). URL: http://www.biomatchina.com (N. Huang). 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.08.023
plantation and associated with a return of ischemic symptoms, has remained a critical concern [1–3]. The mechanism of in-stent restenosis includes recoil and remodeling of the artery, thrombus formation, neointima hyperplasia due to vascular smooth muscle cell (VSMC) proliferation and inflammatory reaction [4–6]. It is generally believed that VSMC proliferation and intracellular matrix synthesis in response to stent-induced inflammatory reactions are the major mechanisms of in-stent restenosis [6]. Orally administered drugs may cause toxic reaction and may not achieve adequate local drug concentration, and thus do not resolve this problem successfully [7]. Consequently much attention has been devoted to developing treatments that target
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VSMC proliferation, the putative key event in the formation of intimal hyperplasia following stent implantation. Recently, drug-eluting stents with synthetic polymer coatings, which release anti-proliferative drugs such as rapamycin and paclitaxel over a period of several weeks or months, have opened up a new paradigm in the treatment of in-stent restenosis (ISR) [8–11]. These drug-eluting stents can provide luminal scaffolding that eventually eliminates the recoil and remodeling of coronary vessels. Additionally, the drug released from the coating can achieve high local drug concentration and thus prevent cellular proliferation, thrombus formation or inflammation. After the drug elutes from the polymer coating, however, the residual synthetic polymer coating remains in the body. Eventually, the permanent presence of the nonreabsorbable polymer may lead to complications such as an exaggerated inflammatory response and neointimal hyperplasia at the implant site [12,13]. It is, therefore, desirable to develop a drug-eluting stent with a biocompatible and biodegradable coating to prevent these unfavorable effects. Poly(lactic acid-co-glycolic acid), PLGA, a biodegradable polymer, which has a number of desirable properties such as good biocompatibility and biodegradation, is a material that is used extensively for controlled delivery of protein and peptide drugs [14], for the manufacture of medical devices [15] and wound dressings [16], as well as for fabricating scaffolds in tissue engineering [17]. Using PLGA as a drug carrier for stent coatings may be a good candidate method. Curcumin (diferuloyl methane, Fig. 1), a major chemical component of turmeric, has low intrinsic toxicity and possesses a wide rage of pharmacological activity, including antithrombus, anti-oxidation and anti-proliferation properties [18– 20], suggesting that it could be very suitable for drug-eluting stents because of these desirable characteristics. In vitro research suggests that curcumin can inhibit platelet aggregation induced by ADP, epinephrine, and collagen [21,22]. Curcumin can also prevent platelet aggregation by platelet-activated factors, and inhibits the formation of thromboxane A2 (TXA2) by platelets [23]. Curcumin inhibits not only serumstimulated- but also growth factor-stimulated-proliferation of vascular smooth cells (VSMC) [29]. In addition, cell cycle analysis of curcumin-treated VSMC has revealed a G0/G1 arrest, and a reduction in the percentage of cells in S phase. The anti-proliferative effect of curcumin may partly be mediated through inhibition of protein tyrosine kinase activity and c-myc mRNA expression [30]. Though many curcumin anticoagulation mechanisms have been studied, the anticoagulation behavior of curcumin in materials, especially biodegradable polymers such as PLGA, has not been investigated.
Fig. 1. Chemical structure of curcumin.
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Fig. 2. Schematic of the ultrasonic spraying system.
In the work described here, the preparation of curcumineluting stents using PLGA as the drug carrier, and its various in vitro characteristics, drug release profiles and anticoagulation are reported. 2. Materials and methods 2.1. Materials The stent material used in this study was annealed 316LVM surgical grade steel tubing (outer diameter 1.65 mm and wall thickness 0.11 mm) which was provided by Northwest Research Institute of Color Metal. The curcumin was purchased from Xi'an Zhongxin Biochemical Co. Ltd., and was of purity above P 96% (HPLC grade). The PLGA (LA:GA = 85:15, Mη¼ 95; 800 resin was endowed by Chengdu Zhuxin Biomaterials Co. Ltd., China, and was of purity above 98%. All other reagents used in this study were analytical grade. 2.2. Preparation of curcumin-eluting stents Firstly the stainless steel tube was formed into a stent by a laser cutting machine. The stents were carefully polished using an electrochemical method, and then cleaned ultrasonically with acetone, ethyl alcohol and distilled water in sequence. The cleaned stents were kept under vacuum for 48 h to evaporate the residual water. A 1 wt.% solution of PLGA containing curcumin was sprayed onto the cleaned stents. A schematic of the spray system is shown in Fig 2. The micro-liquid, which is controlled by a microsyringe, is atomized by ultrasonic atomization and then sprayed onto the stent surface. The stent is traversed and rotated during the spray process. The whole system can be controlled by a computer. We remark parenthetically that not all of the solution is sprayed onto the stent surface; the efficiency of the overall spray system is about 56%. The mass of the coating sprayed onto the stent surface can be obtained by weighing the stent before and after spraying. Three doses of curcumin-eluting stents were prepared: a low-dose stent (∼ 140 μg per stent or 115 μg/cm2), a moderate-dose stent (∼ 280 μg per stent or 230 μg/cm2), and a high-dose stent (∼ 490 μg per stent or 408 μg/cm2). Control stents were prepared by the same method, for which the coating was only the polymer. The thickness of these coatings was about 10 μm and the coating weight for each stent was 1400 μg.
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The topography of the stent coatings was also determined by atomic force microscopy (AFM, SPI3800W probe station and SPA-400 SPM unit system, Japan). Samples were imaged in the dry state before elution of curcumin. AFM images of PLGAonly stents and moderate-dose curcumin-eluting stents were obtained. The scanning range was 1 × 1 μm2. 2.6. Release profile of curcumin
Fig. 3. Optical microscope images and SEM micrographs of metallics stents spray-coated with PLGA and curcumin: a (×40) and c (×50) are pre-expansion images; b (×40) and d (×200) are post-dilation images.
2.3. Fabrication of curcumin-loaded films A 1 wt.% solution of polymer (PLGA) resin was prepared by dissolving it in ethyl acetate. The solution was used to cast thin polymer films on carefully cleaned glass petri dishes. The films were allowed to slowly dry in air to evaporate the solvent and then smooth, non-porous films were obtained. Cast films were kept under vacuum to evaporate residual solvent. Curcuminloaded films were prepared in the same manner by dissolving 10% (low-dose), 20% (moderate-dose), and 35% (high-dose) (w/w) curcumin with polymer resin in ethyl acetate, with the same evaporation and storage steps. 2.4. Structure of curcumin-loaded films The structure of PLGA, curcumin and curcumin-loaded films was examined by FTIR (Fourier Transform Infrared) spectroscopy and XPS (X-ray photo-electron spectroscopy). The scanning FTIR range was 4000 cm− 1 to 400 cm− 1. The XPS spectra were obtained using an XSAM800 (Kratos Inc, England). The instrument was equipped with a monochromatic Mg Kα (1253.6 eV) X-ray source operated at 12 kV at a pressure of 5–7 × 10− 9 mbar. The reference binding energy was set at 284.6 eV for C–C and C–H of the C1s core level spectra.
The curcumin release profile was investigated in vitro by incubating an individual curcumin-eluting stent in a 2.0 ml capped glass tube containing 1.5 ml medium at 37 °C and pH 7.4. The medium consisted of phosphate-buffered saline containing 10% ethanol (v/v) [33] because the limit of solubility of curcumin in water makes it impossible to study in buffer. At selected times, the incubation medium was completely removed for analysis and replaced with fresh medium. An aliquot of the sampled medium was measured by high performance liquid chromatography (HPLC) (Waters 2695 separations module and Waters 2487 dual λ absorbance detector). The detection wavelength was fixed at 430 nm. The flow rate was 1 ml/min and the flow phase consisted of acetonitrile and water (65:35, v/v). Results were expressed as cumulative micrograms and weight percent of curcumin released as a function of time. 2.7. In vitro anticoagulation evaluation The in vitro anticoagulation behavior of drug-loaded films and stents was investigated by platelet adhesion measurements for films and activated partial thromboplastin time (APTT) test for stents. The curcumin-eluting polymer films and the control films (PLGA-only film) were cut into 10 × 10 mm2 samples. Whole blood was centrifuged at 1500 rpm and the upper platelet-rich plasma (PRP) obtained. The film samples were immersed in platelet-rich plasma for 2 h at 37 °C. After washing with 0.9% NaCl solution, they were fixed using 0.2% glutaraldehyde solution for 1 h and then fixed using 0.5% glutaraldehyde solution for 12 h, then washed again with 0.9% NaCl three times, and subsequently immersed in 50, 50, 75, 90, 100% ethanol solutions for 5 min and dried in a dessicator. Samples were sputtered with gold before being imaged by scanning electron microscopy (SEM).
2.5. Morphology examination and balloon-expansion tests The surface morphology of the curcumin-eluting stents was examined by Motic three-dimensional optical microscopy (OM) and scanning electron microscopy (SEM, Quanta 200, Philips) before balloon expansion. The stent was mounted onto an angioplasty balloon and the balloon was dilated to 3.0 mm at a pressure of 3.5 atm. The post-expansion stents were examined by OM and SEM.
Fig. 4. High resolution 1-μm topography AFM images in air of the surface of PLGA-coated (left) and moderate-dose curcumin-eluting stent (right).
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struts. The coating on the stent has the ability to withstand the compressive and tensile strains imparted without cracking in the stent expansion process. Tapping mode AFM images of the surface of PLGA-coated and curcumin-eluting stents are shown in Fig. 4. It can be seen that the surface of the stent coated with PLGA-only is very smooth, with an average roughness of about 0.14 nm. After loading curcumin in the PLGA coating, the roughness of the stent is greater than for the PLGA-only-coated stent, with an average roughness of curcumin-eluting stent about 0.55 nm. However, no drug particles can be seen on the stent surfaces from the SEM and AFM images; this indicates that curcumin can be mixed with PLGA at the molecular level using an ultrasonic atomization spray method. 3.2. Surface structure of the coating films Fig. 5. FTIR spectra of curcumin-eluting coating film.
For the activated partial thromboplastin time (APTT) measurements, curcumin-eluting and control stents were put into a special testing tube. A 100 μl fresh human platelet-poorplasma (PPP), obtained by centrifuging whole blood at 3000 rpm, and 100 μl actin-activated cephthaloplastin reagent were added into the tube containing stents, followed by the addition of a 0.03 M CaCl2 solution (100 μl) after 3 min incubation at 37 °C. The clotting time of the plasma solution was measured by a coagulometer (Clot 1A, Innova Co.).
The surface structure of curcumin-eluting PLGA films was investigated by FTIR and XPS. Fig. 5 shows the FTIR spectra of PLGA, curcumin and curcumin-eluting coating films. It can be seen that the two kinds of polymer films (curcumin and curcumin-loaded film) have similar structure. The FTIR
3. Results and discussion 3.1. Stent morphology and balloon-expansion tests For drug-eluting stents, surface topography is considered to have an important influence on stent performance. If the stent has webbings and “bridges” between the struts, the coating may break off from the stent when the stent is dilated by a balloon catheter, and thrombus and VSMC proliferation can occur at the site where the coating breaks off [24]. A smooth surface coating can significantly decrease injury to blood vessels. Furthermore, a smooth stent surface is believed to reduce platelet activation and aggregation, consequently leading to less thrombus formation and neointimal proliferation [28]. PLGA coatings containing curcumin on metallic stents can be clearly visualized by optical microscopy because the curcumin is yellow. Fig. 3a–d shows OM and SEM images of curcumin-eluting stents. As can be seen in Fig. 3a and b, the PLGA coatings containing curcumin are very smooth and the curcumin can be visualized clearly before dilation by an angioplasty balloon. The coatings are very uniform and smooth. There are no cracks or webbings between struts. OM and SEM micrographs after expansion of a PLGA- and curcumin-coated stent are shown in Fig. 3b and d. It can be seen that there is no delamination or destruction of the coating on the stent after expansion with an angioplasty balloon. This indicates that the stent coating is sufficiently flexible to allow balloon expansion of the stent without cracking or peeling from the
Fig. 6. C1s XPS of (a) PLGA, and (b) circumin-eluting coating film.
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structure of curcumin and curcumin-loaded PLGA film is different from PLGA film because a peak at 1620 cm− 1 is found in the spectra of curcumin and curcumin-eluting PLGA films. The peak at 1620 cm− 1 is a stretching vibration peak of the carbon–carbon double bond in curcumin. One of the major peaks observed for the three films was around 3500 cm− 1, most probably due to –OH vibrations of intermolecularly-bonded OH groups. In particular, the strong peak at 2998 cm− 1 and 2952 cm− 1 and the rather weak peak at 1380 cm− 1 in PLGA film and curcumin-eluting film can be assigned to the stretching and deformation of methyl groups, suggesting a significant amount of branching in the polymer films. The strong peak at 1750 cm− 1 is due to C_O adsorption. The carbon–carbon double bonds at 1620 cm− 1 were found in the spectra of curcumin-eluting PLGA film and curcumin and not found in PLGA-only films, indicating that curcumin exists in the polymer film. The peak at 1620 cm− 1 in curcumin did not shift in curcumin-eluting film, suggesting that curcumin may be in a dispersed state in PLGA films. As shown in Fig. 6, the two C1s XPS spectra of PLGA and curcumin-eluting polymer films are not the same. The peaks in the two graphs at 284.6 eV, 285.7 eV, and 286.9 eV are
Fig. 8. SEM images of post-release stent (a, ×40; b, ×160; c, ×600; d, ×2000).
attributed to C–C single bonds, C–O single bonds, and C_O double bonds, respectively. The peak in Fig. 6b at 289.3 eV is attributed to C_C double bonds which only exist in curcumin, suggesting that there is some curcumin on the surface of the coating film. In the chemical structure of curcumin, C_C and –OH are active groups which may lead to chemical reaction with PLGA. However, according to the data shown in Fig. 6, C_C exists in films after curcumin loading, indicating that there is no chemical reaction between curcumin and PLGA, and thus curcumin may be dispersed in the PLGA polymer matrix. 3.3. Curcumin release profile The results of in vitro elution of curcumin from different types of curcumin-loaded stents are shown in Fig. 7. Generally speaking, drug release from a polymer-based matrix takes place with an initial burst release [25]. As shown in Fig. 7, however, all the curcumin-loaded stents exhibited a nearly linear sustained-release profile with no significant burst release over a period of 18 days. Replacing the releasing medium every day established a significant concentration gradient of curcumin between the drug-eluting stent and the release medium, and thus led to a linear constant drug diffusion [34]. The drug release profile of curcumin can be analyzed using the following linear relationship [26]. Mt ¼ kd t M0
Fig. 7. In vitro release profile of circumin from three types of coated stents: (a) shows the cumulative amount of curcumin, and (b) shows the release weight percent.
ð1Þ
Mt Where M is the release fraction of the drug, t is the time, and k a 0 constant parameter of the release system. In theory, a sustained release of antirestenotic drugs for at least three weeks is required to prevent smooth muscle cell
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biodegradation; in fact, the mass loss rate and molecular weight of PLGA used here reach about 60% after 86 days in PBS (data not shown). SEM images of curcumin-eluting stents which released for 18 days in PBS medium are shown in Fig. 8. Many micropores can be seen in the stent surface after immersion of the stent in release medium for 18 days, perhaps hastening the subsequent biodegradation process. 3.4. In vitro anticoagulation Platelet adhesion measurements and activated partial thromplastin time (APTT) tests were used to evaluate the in vitro anticoagulation of drug-eluting films with different drug doses and control films. Thrombotic occlusion of stent angioplasty resulting from activated platelets is a complication of stent implantation. Curcumin can improve the hemocompatibility of biomaterials. Research results of Nguyen et al. indicate that curcumin significantly decreases platelet aggregation and activation and improves the blood compatibility of PLLA fibers [27]. More extensive in vitro studies have shown that biodegradable PLLA stents loaded with curcumin reduce the inflammatory response of platelets and leukocytes. Platelet aggregation occurs when fibrinogen molecules bind to the activated GPllb/llla receptor and connect platelets to one another [36]. The results of platelet adhesion experiments are indicated in Fig. 9. Many platelets adhere onto the stainless steel plate; furthermore, many adhered platelets are aggregated and activated, suggesting that the blood compatibility of stainless steel is very poor. In the case of PLGA-only film, platelets adhered on the film surface were generally singular with a rounded shape typical of platelets in an inactivated state, a few pseudopodia and deformation were observed. Drug-eluting films with different drug doses show significantly less platelet adhesion and aggregation than the PLGA-only film and stainless steel plate. Platelets adhered on the surface of curcumin-loaded PLGA films are not activated and the extension of pseudopodia is not observed. Adhered platelets on the surface of curcumin-loaded film decreased
Fig. 9. SEM images of platelet adhesion experiment in vitro (a, e — stainless steel; b, f — PLGA-only film; c, g — low-dose film; d, h — high-dose film).
migration and proliferation [35]. Based on the release profile shown in Fig. 7, the average sustained-release rates of curcumin for low-dose stents, moderate-dose stents, and high-dose stents were 5.26 μg per day (3.76% by weight), 7.37 μg per day (2.63% by weight), and 8.26 μg per day (1.68% by weight), respectively. Thus, based on these release rates, it can be concluded that the sustained-release duration of curcumin was ∼ 27 days for low-dose stents, ∼ 38 days for medium-dose stents, and ∼ 59 days for high-dose stents — all entirely satisfactory for a releasing requirement of three weeks. We note that the calculated release period does not consider the effect of
Fig. 10. APTT results for drug-eluting stents and control stent. (S0 — stainless steel stent; S1 — PLGA-only stent; S2 — low-dose stent; S3 — moderate-dose stent; S4 — high-dose stent).
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significantly with increasing drug dose. The curcumin released from drug-loaded films might inhibit the cyclooxygenase pathway by blocking the GPllb/llla receptor, inhibiting platelet aggregation leading to the formation of blood clots [37]. The cascade of reactions involved in the coagulation of blood is an autocatalytic and self-limiting process converting zymogene to an active form in the presence of proteolytic enzyme thrombin [31]. Research on anticoagulation for curcumin shows that curcumin prolongs the clotting time for both human and rat plasma by approximately 1.6-, 2.1- and 1.1-fold, as shown by thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, respectively, when compared to a control [32]. To examine the in vitro anticoagulation of drug-eluting stents and control stents, the APTT (activated partial thromboplastin time), which shows the bioactivity of intrinsic blood coagulation factors, was examined, and the results are shown in Fig. 10. The APTT time of bare stents (S0) is shorter than for plasma, indicating that bare stents may activate the coagulation process. The APTTs of all drug-eluting stents are prolonged compared to S0 and S1. For instance, the APTT of S4 increased from 40.6 s to 47.35 s. This indicates that activation of the intrinsic blood coagulation system is suppressed by curcumin released from the drug-eluting stent, and the extent of suppression is related to the curcumin content of the drugeluting stents. With the increase of curcumin drug dose, the APTT is prolonged, indicating that the blood compatibility of curcumin-eluting stents is improved. We speculate that the curcumin-prolonged APTT time could be due to the presence of hydrophobic groups in curcumin moiety [32]. Additionally, the hydroxyl groups in curcumin may prevent protein adsorption and thus improve the blood compatibility of the samples. These results all indicate that curcumin-eluting stents have good anticoagulation characteristics, and may prevent thrombus formation after stent implantation. 4. Conclusions In conclusion, the results obtained in this study suggest that curcumin-eluting stents, as developed in our work, have the ability to withstand compressive and tensile strains imparted without cracking from the stent during the expansion process. Curcumin can be mixed with PLGA at the molecular level using an ultrasonic atomization spray method, as shown by AFM images. Curcumin may be dispersed in the PLGA matrix according, as shown by FTIR and XPS. The stents have a sustained-release profile without initial burst release. Compared to bare stents and a control with PLGA-only coating, our drugcoated stents exhibit excellent anticoagulation. References [1] Ron Waksman, Drug-eluting stents, from bench to bed, Cardiovascular Radiation Medicine 3 (2002) 226–241. [2] E. Ragar, G. Sianos, P.W. Serruys, Stent development and local drug delivery, British Medical Bulletin 59 (2001) 227–248. [3] J. Gunn, D. Cumberland, Stent coatings and local drug delivery: state of the art, European Heart Journal 20 (1999) 1693–1700.
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