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Improved blood compatibility of rapamycin-eluting stent by incorporating curcumin
Colloids and Surfaces B: Biointerfaces 59 (2007) 105–111
Improved blood compatibility of rapamycin-eluting stent by incorporating curcumin C.J. Pan a,b , J.J. Tang a , Z.Y. Shao a , J. Wang a , N. Huang a,∗ a
Key Laboratory of Advanced Materials Technology, Education Ministry, Southwest Jiaotong University, Chengdu 610031, China b School of Bioengineering, Chongqing University, Chongqing 400044, China Received 20 March 2007; received in revised form 10 April 2007; accepted 25 April 2007 Available online 29 April 2007
Abstract This paper dealt with improving the blood compatibility of the rapamycin-eluting stent by incorporating curcumin. The rapamycin- and rapamycin/curcumin-loaded PLGA (poly(d,l-lactic acid-co-glycolic acid)) coatings were fabricated onto the surface of the stainless steel stents using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier transform infrared spectroscopy (FTIR). The optical microscopy and scanning electron microscopy (SEM) images of the drug-eluting stents indicated that the surface of all drug-eluting stents was very smooth and uniform, and there were not webbings and “bridges” between struts. There were not any cracks and delaminations on stent surface after expanded by the angioplasty balloon. The in vitro platelet adhesion and activation were investigated by static platelet adhesion test and GMP140 (P-selection), respectively. The clotting time was examined by activated partially prothromplastin time (APTT) test. The fibrinogen adsorption on the drug-loaded PLGA films was evaluated by enzyme-linked immunosorbent assay (ELISA). All obtained data showed that incorporating curcumin in rapamycin-loaded PLGA coating can significantly decrease platelet adhesion and activation, prolong APTT clotting time as well as decrease the fibrinogen adsorption. All results indicated that incorporating curcumin in rapamycin-eluting coating obviously improve the blood compatibility of rapamycin-eluting stents. It was suggested that it may be possible to develop a drug-eluting stent which had the characteristics of not only good anti-proliferation but also improved anticoagulation. © 2007 Elsevier B.V. All rights reserved. Keywords: Drug-eluting stent; Rapamycin; Curcumin; Blood compatibility
1. Introduction Although the introduction of coronary stents has significantly improved the treatment of patients with coronary artery disease, in-stent restenosis remains the major drawback of stent implantation [1,2]. The mechanism of in-stent restenosis consists of a series of biological reactions such as thrombus formation, vascular smooth cell (VSMC) migration and proliferation, inflammatory response and negative remodeling and so on [3–5]. The metallic stent provides the scaffolding that virtually eliminates the negative remodeling of vessel and acute occlusion. The coating on metallic stent which contains anti-proliferative drugs can elute drug to prevent in-stent restenosis aroused by VSMC migration and proliferation. For example, the CypherTM rapamycin-eluting stent and TAXUSTM paclitaxel-eluting stent ∗
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are widely used in clinical application. However, the drug carrier of these two drug-eluting stents is nonbiodegradable polymer (the mixing polymer of PEVA and PBMA, polyethylene-covinyl acetate/poly-n-butyl methacrylate for CypherTM and SIBS, poly(styrene-b-isobutylene-b-styrene) for TAXUSTM ) [6]. After drug elutes completely from the stent, the remaining synthetic nonbiodegradable polymer may cause some clinical syndromes such as exaggerated inflammatory reaction, neointima formation and late thrombosis [35–37]. So, it is imperative to develop a novel drug-eluting stent using biodegradable polymer as drug carrier which has not only anti-proliferation but also anticoagulation. Rapamycin (also called sirolimus; Fig. 1a) is a macrocyclic lactone drug approved by FDA [7]. In vitro and in vivo studies indicate that it can significantly prevent proliferation of vascular smooth muscle cell of human and inhibit in-stent restenosis due to neointima formation [8–10]. Rapamycin also has anti-inflammatory activity [11]. So, the rapamycin-eluting
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Fig. 1. The chemical structure of rapamycin (a) and curcumin (b).
stents were extensively studied by many people and many meaningful results were obtained. However, there remains the problem of in-stent restenosis due to lack of anticoagulation. Curcumin (diferuloyl methane; Fig. 1b) has low intrinsic toxicity and possesses a wide range of pharmacological activities, including anti-thrombus, anti-oxidation and antiproliferation [12–14]. More extensive in vitro studies show that biodegrabable PLLA stent grafts loaded with curcumin could reduce the inflammatory responses of platelets and leukocytes [15]. In our previous study, we confirmed that the curcumineluting stents have good blood compatibility when compared to the control samples [16]. In this study, we hypothesized that loading rapamycin and curcummin together in coating of drugeluting stents can make the in-stent restenosis rate decreased significantly because of antiproliferation for rapamycin and good anticoagulation for curcumin. Rapamycin and rapamycincurcumin mixing drug-eluting stents were prepared and the in vitro blood compatibility of these drug-eluting stents and control stents were investigated. 2. Materials and methods 2.1. Materials, reagents and antibodies The bare stainless steel stents with outer diameter 1.65 mm and wall thickness 0.11 mm were prepared in our lab. The PLGA (d,l-LA/GA = 85:15, Mη = 95, 800) resin was provided by Sichuan Zhuxin Biomaterials Co. Ltd. Rapamycin (HPLC purity > 98.3%) was purchased from Fujian Kerui Drug Co. Ltd. Curcumin (HPLC purity > 98.6%) was bought from Shanghai Yousi Biological tTechnology Company. The following antibodies and solutions, purchased from SIGMA, were used: phosphate buffer solution (PBS) buffer, 0.02 g/ml calf serum albumin closed solution (diluted with PBS), horseradish peroxide-enzyme labeled goat anti-human fibrinogen antibody, horseradish peroxide-enzyme labeled goat anti-rat polyclonal antibody, rat anti-human CD62P antibody, TMB (3,3 ,5,5 -tetramethylbenzidine) substrate. All proteins and antibodies were used according to the manufactures recommendations. For clear antibody solutions, no further purification was performed. All other reagents used in this study were analytical grade.
2.2. Fabrication of drug-eluting stent The spray solutions were prepared by dissolving drug and polymer in the dichloromethane. The whole spraying system is illustrated in Fig. 2. The liquid ejected from microsyringe was atomized and focused by untrasonic atomization equipment, and then the focused liquid beam was sprayed onto the surface of stainless steel stents. The stent kept rotating and moving during spraying process to get a homogeneous coating. The rapamycineluting stent (called RES) with 100 g drug and the rapamycincurcumin-eluting stent (called RCES) with 100 g rapamycin and 100 g curcumin were prepared. The controlled stent (called PLGA) with PLGA only was prepared using the same method. The stainless steel stent was labeled SS. 2.3. Fabrication of drug-eluting polymer films The PLGA films and drug-loaded PLGA films were prepared by casting method. The casting solution was obtained by dissolving drug and PLGA in chloroform according to the drug prescription of drug-eluting stents. The solution was slowly poured into cleaned glass Petri dishs to obtain coating films. The films were allowed to slowly evaporate the solvent for 48 h and then kept in vacuum to evaporate the residual solvent for 72 h. The labels of these films were the same as the drug-eluting stents. 2.4. FTIR analysis The surface structure of RES and RCES films was investigated by Fourier transform infrared spectroscopy (FTIR,
Fig. 2. The schematic diagram of ultrasonic atomization spraying system.
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ST-IR20SX, NICOLET Co. Ltd., American). The scanning wavenumber scope was 4000–400 cm−1 . 2.5. Morphology of drug-eluting stents and balloon-expansion After sterilized by epoxy ethane in Sichuan New Century Polymer Co. Ltd. in China, the surface morphology of the drug-eluting stent was observed by optical microscopy (OM) and scanning electron microscopy (SEM, Quanta200, Philipis). Then, the same drug-eluting stent was mounted onto the angioplasty balloon and then dilated at the pressure of 4.0 atm. The stent morphology after deployment was also examined by SEM. 2.6. Platelet adhesion The platelet-rich plasma (PRP) was obtained by centrifuging the fresh whole blood of healthy volunteer at 1500 rpm (revolutions per minute). The control samples (stainless steel and PLGA film) and drug-loading PLGA films were incubated in PRP for 2 h. These samples were rinsed by 0.9% NaCl solution and then fixed by 0.2% glutaraldehyde solution for 1 h and 0.5% glutaraldehyde solution for 12 h, respectively. The samples were washed again for three times using 0.9% NaCl solution and subsequently immersed into 50, 50, 75, 90, 100% ethanol solutions in sequence for 5 min. After dried in a dessicator, the samples were sputtered with gold before being imaged by scanning electron microscopy (SEM). 2.7. APTT For the activated partial thromboplastin time (APTT) measurements, the drug-eluting and control stents were put into a special test tube. The 100 l fresh human platelet-poor-plasma (PPP) which was obtained by centrifuging the whole blood at 3000 rpm, and 100 l actin-activated cephthaloplastin reagent were added into the above tube containing stents, followed by the addition of a 0.03 M CaCl2 solution (100 l) after 3 min incubation at 37 ◦ C. The clotting time of the plasma solution was measured by a coagulometer (Clot 1A, Innova Co.). 2.8. Fibrinogen adsorption test Fibrinogen adsorption was evaluated using enzyme-linked immunosorbent assay which is based on the interaction antigen–antibody reaction [17]. For in vitro fibrinogen adsorption detection, the fresh whole blood of a volunteer was centrifuged at 3000 rpm to obtain platelet-poor plasma (PPP). Each sample (1 cm × 1 cm) was immersed in 500 l PPP for 120 min at 37 ◦ C after incubated in PBS for 3 h at 37 ◦ C to equilibrate the surface. The samples were rinsed with PBS closed solution containing calf serum albumin and then moved into a new 24-well culture plate. Thereafter, the films were incubated in 200 l horseradish peroxide-enzyme goat anti-human fibrinogen antibody for 60 min at 37 ◦ C then rinsed with PBS closed solution containing calf serum albumin for three times. The films were moved into a new 24-well culture plate and then
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200 l TMB substrate was added into each well. The absorbance of the solution was measured at 492 nm after 10 min of the enzyme reaction when 50 l stop solution (1 mol/l H2 SO4 ) of the reaction was injected into the 24-well culture plate. These measurements were carried out five times for each sample and averaged. 2.9. Platelet activation evaluation (GMP140, P-selection) Firstly, 10 ml fresh blood sample from a healthy volunteer was centrifuged for 15 min at 1500 rpm to obtain platelet-rich plasma (PRP) for GMP140 determination. GMP140 expression in plasma was determined using a common enzyme-linked immunosorbent assay [18]. The samples with the same size (10 mm × 10 mm) in 24-well culture plate were cultured in PRP for 2 h at 37 ◦ C and then rinsed with calf serum albumin PBS solution for three times. Thereafter, these samples were shifted into a new 24-well culture plate and 200 l rat anti-human CD62P antibody solution was injected into each well, respectively, for culturing 1 h at 37 ◦ C. Followed by rinsing three times, 200 l horseradish peroxide-enzyme goat anti-rat polyclonal antibody was added into each well containing sample. After cultured 1 h at 37 ◦ C, these samples were rinsed three times using PBS stop solution then transferred into a new 24-well culture plate and then 140 l TMB solution was injected into each well. The absorbance of the solution was measured at 450 nm after 10 min of the enzyme reaction when the stop solution (1 mol/l H2 SO4 ) of the reaction was injected into the 24-well culture plate. These measurements were carried out five times for each sample and averaged. 3. Results and discussion 3.1. The morphology and balloon expansion For drug-eluting stents, the smooth surface can decrease the friction between stent and vessel wall and thus inhibit vessel injury significantly. Smooth stent surface can significantly inhibit platelet deposition and thus improve the blood compatibility of stents [19,20]. The OM and SEM surface morphology images of the drug-eluting stents in this work after sterilized by epoxy ethane sterilizer are shown in Fig. 3. It can be seen that the coating on the stent surface was very smooth and uniform. After dilation by angioplasty, the coating was still smooth and uniform (Fig. 3c, e and f). There were no webbings and “bridges” between struts (Fig. 3). No delamination and destruction were observed (Fig. 3c, e and f), indicating that the coating can adhere tightly to the stent surface. Furthermore, the coating can withstand the compressive and expansion strains imparted during mounting and deployment process. 3.2. The FTIR analysis In the present paper, the surface structure of drug-eluting coating films was examined by FTIR. The results are shown in Fig. 4. There were some differences among three films. The major peak at 3490 cm−1 in three films was most probably due
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Fig. 3. The OM (a–c) and SEM (d–f) images of drug-eluting stents (a, d, RES; b, c, e, f, RCES; c, e, f, after depolyment). The surface of all stents was very smooth and uniform. No webbings were observed between struts.
to –OH vibrations of intermolecularly bonded OH groups. The strong peak at 2998 and 2952 cm−1 and the rather weak peak at 1380 cm−1 in three films can be assigned to the stretching and deformation of methyl groups. The strong peak at 1750 cm−1 in curcumin and RCES is due to C O adsorption. However, this peak shifted to about 1700 cm−1 in RES film because of the presence of rapamycin. The carbon–carbon double bonds at 1620 cm−1 were found in spectra of curcumin film and RCES film. All results indicate that the curcumin and rapamycin exist in PLGA films. 3.3. In vitro platelet adhesion Fig. 5 shows the average platelets number adhered on the different films from ten SEM images (×500). It can be seen that
Fig. 4. The FTIR spectra of RES, curcumin and RCES films.
many platelets deposited onto stainless steel plate surface. In the case of PLGA and RES film, the platelets decreased significantly when compared to stainless steel. After incorporated curcumin in RES, the adhered platelets further reduced. For aggregation platelets, there was no distinct difference among stainless steel, PLGA and RES films. However, the aggregated platelets adhered on RCES film surface decreased distinctly. All results indicated that incorporated curcumin in rapamycin-loaded films can significantly inhibit platelets adhering and aggregating and thus may obviously improve the blood compatibility of the rapamycin-eluting stent. The morphology of platelets adhered on different material surface is shown in Fig. 6. For stainless steel, the adhered platelets almost aggregated and were activated. Many pseudopodia were observed on SS (Fig. 6a). In the case of PLGA (Fig. 6b), the aggregated platelets had decreased when compared to stainless steel; some pseudopodia were observed, indicating
Fig. 5. Platelet adhesion on different material surfaces (culture time: 2 h). Data are expressed as means ± standard deviations for n = 10.
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Fig. 6. The morphology of platelets adhered on stainless steel (a), PLGA (b), RES (c) and RCES (d). Culture time: 2 h.
that its blood compatibility was better than SS. The morphology of platelets adhered on rapamycin-loaded PLGA film was similar with PLGA. However, after incorporating curcumin in rapamycin-eluting film, the adhered platelets on RCES were not activated and the extension of pseudopodia and deformation was not observed (Fig. 6d). The adhered platelets were generally singular with the rounded shape typical of platelets in the inactivated state. It was indicated that incorporating curcumin could improve the blood compatibility of rapamycin-eluting film. Platelet activation and adhesion are known to occur during cardiopulmonary bypass, hemodialysis, as well as cardiovascular implant such as blood vessel stent [21]. In the present paper, the platelet adhesion behavior on different materials was examined and the results were given in Figs. 5 and 6. It can be seen from the figures that curcumin can significantly decrease the platelets adhesion, aggregation and activation of rapamycin-loaded films and thus improve its blood compatibility. It was reported that curcumin can inhibit platelet aggregation induced by ADP, epinephrine, and collagen in vitro and slightly increase prostacylin synthesis [22]. Moreover, curcumin additionally blocks platelet aggregation by platelet-activating factor, collagen and arachidonic acid, and inhibits as well the formation of thromboxane A2 (TXA2) by platelets [23]. Curcumin is also a potential inhibitor of platelet-activating factor, and of reactive oxygen-generating enzymes, including cyclooxygenase and lipoxygenase [24,25]. All these properties of curcumin can contribute to the blood compatibility of rapamycin-loaded PLGA films.
ing curcumin in rapamycin-eluting stent might play an important role in inhibiting the activities of some clotting factors of blood plasma involved in APTT. Blood coagulation involves a series of proteolytic reactions resulting in the formation of fibrin clot [26]. Thrombin is formed following a cascade reactions where an inactive factor becomes enzymatically active following surface contact or after proteolytic cleavage by other enzymes; the newly activated enzyme then activates another inactive precursor factor [26]. The research on anticoagulation for curcumin shows that curcumin prolongs the clotting time of both human as well as rat plasma to approximately 1.1-fold as shown by activated partial thromboplastin time (APTT) assays when compared to control sample [27]. The results of the present paper indicate that curcumin can prolong the clotting time of rapamycin-loaded PLGA film as showed by APTT. This provided a probable pathway for curcumin to inhibit thrombus formation. The APTT shows the bioactivity of intrinsic blood coagulation factors, so, curcumin improves the blood compatibility of rapamycin-loaded film by preventing activation of intrinsic blood coagulation factors. The time prolonged by curcumin could be due to the presence of hydrophobic groups in curcumin moiety [27]. 3.5. Fibrinogen adsorption evaluation The amount of adsorbed fibrinogen on the surfaces of different materials is shown in Fig. 8. It was showed that many
3.4. APTT The APTT test is widely used for the clinical detection of abnormality of blood plasma and for the primary screening of the anticoagulative chemicals. The results of our present work are shown in Fig. 7. The normal APTT time for a healthy blood plasma was regarded to be about 37.6 s (Fig. 7). When the blood plasma was incubated with the stainless steel stent and PLGAcoated stent, the corresponding data of APTT were 33.6 and 36.9 s, indicating that neither stainless steel nor PLGA influenced the thrombogenicity of the blood plasma, they could cause occurrence of intrinsic coagulation. However, when the blood plasma was incubated with rapamycin-eluting stent, the APTT became a litter larger than plasma. After incorporating curcumin in rapamycin-eluting stent, the APTT was prolonged from 38 s for RES to 43.9 s for RCES, indicating RCES can prolong the occurrence of intrinsic pathway system. Therefore, incorporat-
Fig. 7. The APTT of different stents. Columns and error bars represent means ± S.D. for n = 6.
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Fig. 8. Fibrinogen adsorption on different material surfaces. Columns and error bars represent means ± S.D. for n = 5.
fibrinogens adhered on the surface of stainless steel plate. The fibrinogen adsorption amount of SS was about 1.26-fold when compared to that of PLGA. When the rapamycin-eluting PLGA film was incubated in human plasma, the adsorbed fibrinogen amount significantly decreases. After incorporating curcumin in rapamycin-eluting film, the fibrinogen adsorption further reduced when compared to that of RES. The fibrinogen adsorption of RCES was 54.2% of RES, 15.3% of PLGA and 12.2% of stainless steel, respectively. It clearly indicated that incorporating curcumin decrease fibrinogen adsorption of RES. Fibrinogen adsorption has been considered as the major role for blood compatibility of biomaterials [28]. Pre-coated fibrinogen has been demonstrated to enhance surface induced thrombosis [29]. In addition, fibrinogen has been shown to be a key factor in thrombosis because a specific receptor for the fibrinogen molecule, the glycoprotein complex IIb-IIIA (GP IIb-IIIA), is found on the platelet membrane [30]. Formation of these fibrinogen–GP IIb-IIIA complexes greatly enhances platelet activation [31]. The results showed in Fig. 8 indicated that fibrinogen adsorption on SS and PLGA was much heavier than RES and RCES. The adsorbed fibrinogen tended to cover the surface of SS in a thick multilayer pattern and probably undergoes a serious change of its conformation, which enhances its affinity for platelet and effect on platelet activation. The contrary result obtained from RES and RCES indicated the less fibrinogen adsorption may hardly change its conformation and effect on platelet activation. The least fibrinogen adsorption on RCES indicated its blood compatibility may be the best. Therefore, these RCES film on stainless steel stent surface may inhibit fibrinogen and thus improve the blood compatibility of implanting stent. 3.6. P-selection assay (GMP140) P-selection, also called GMP140, is only present on the activated platelets surface, and thus may be used as a marker of platelet activation on biomaterials [32]. The activated platelets
Fig. 9. The GMP140 on different material surfaces (expressed as absorbency value). Columns and error bars represent means ± S.D. for n = 5.
on different materials as indicated by absorbency values are shown in Fig. 9. It was indicated that adhered platelets on stainless steel (SS) surface were activated remarkably when contacted with human plasma. The absorbency value of PLGA decreases, indicating that the activated platelets were less than SS. The activated platelets on RES were only about 64.1% of PLGA and 39.5% of SS. However, after incorporating curcumin in RES, the amount of activated platelets on RCES were reduced to 24.0% of RES, 15.4% of PLGA and 9.5% of PLGA, suggesting that few platelets were activated by RCES when incubated in human plasma. The results were consistent with platelet adhesion result and fibrinogen adsorption results. It is well known that platelet activation is an important aspect in the interaction of biomaterials with blood [33]. The activation of platelets leads to a rapid change of platelet morphology with formation of pseudopodia, spreading and the onset of release reaction [34]. It is a valuable characterization of biomaterials haemocompatibility to understand the mechanism including in the adhesion, activation and release action of platelets. The present research showed that incorporating curcumin in rapamycin-eluting PLGA film can significantly prevent platelet activation and thus improve the blood compatibility of the stent. Forenamed fibrinogen adsorption results indicated that curcumin in RES film can reduce fibrinogen adsorption. The less adsorbed fibrinogen on RCES can hardly undergo conformation change and thus fewer integrated locations of platelet would expose, so, little platelets would be activated when incubated in human plasma. 4. Conclusions The rapamycin-eluting and curcumin/rapamycin-eluting stents using PLGA as drug carrier were prepared using an ultrasonic atomization spray system. The surface of all drugeluting stents was uniform and smooth. No webbings and “bridges” were observed between struts. There were not cracks on stent surface during mounting onto angioplasty and deployment process, indicating that the coating can withstand the
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compressive and deployment strains imparted during this process. Incorporating curcumin in rapamycin-loading PLGA film can reduce fibrinogen adsorption significantly, thus decrease platelet adhesion and activation. All results of haemocompatibility tests demonstrated that incorporating curcumin in rapamycin-loaded PLGA coating can improve the blood compatibility of rapamycin-eluting stent. This provide a new way for developing drug-eluting stents which have not only good anticoagulation but also anti-proliferation. Acknowledgements This work is financially supported by the key basic research program no. 2005CB623904, NSFC-RGC 30318006# of Natural Science Fund of China and the innovation fund of Southwest Jiaotong University, China. References ¨ [1] R.S. JUrgen, V. Vitali, W.H. John, et al., Biological aspects of radiation and drug-eluting stents for the prevention of restenosis, Cardiovasc. Res. 63 (2004) 22–30. [2] R. Virmani, F.D. Kolodgie, A. Farb, A. Lafont, Drug-eluting stents: are human and animal studies comparable? Heart 89 (2) (2003) 133–138. [3] C.L. Harry, N.O. Stephen, M.K. Levon, Coronary in-stent restenosis: current status and future strategies, J. Am. College Cardiol. 39 (2) (2002) 183–193. [4] M.R. Bennett, M. O’Sullivan, Mechanisms of angioplasty and stent restenosis: implications for design of rational therapy, Pharmacol. Ther. 91 (2001) 149–166. [5] A.A.F. Gordon, Y.A. Tony, The mechanism of coronary restenosis: insights from experimental models, Int. J. Exp. Pathol. 81 (2000) 63–88. [6] E. Regar, G. Sianos, P.W. Serruys, Stent development and local drug delivery, Br. Med. Bull. 59 (2001) 227–248. [7] C.G. Groth, L. Backman, J.M. Morales, et al., Sirolimus (rapamycin)-based therapy in human renal transplantation: similar efficacy and different toxicity compared with cyclosporine: Sirolimus European Renal Transplant Study Group, Transplantation 67 (1999) 1036–1042. [8] S.O. Marx, T. Jayaraman, L.O. Go, A.R. Marks, Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells, Circ. Res. 76 (1995) 412–417. [9] M. Poon, S.O. Marx, R. Gallo, et al., Rapamycin inhibits vascular smooth muscle cell migration, J. Clin. Invest. 98 (1996) 2277–2283. [10] C.R. Gregory, X. Huang, R.E. Pratt, et al., Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement, Transplantation 59 (1995) 655–661. [11] R. Gallo, A. Padurean, T. Jayaraman, et al., Inhibition of thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle, Circulation 99 (1999) 2164–2170. [12] K.T. Nguyen, S.H. Su, A. Sheng, et al., In vitro hemocompatibility studies of drug-loaded poly-(l-lactic acid) fibers, Biomaterials 24 (2003) 5191–5201. [13] T.N. Kytai, S. Nishat, P.S. Kajal, et al., Molecular responses of vascular smooth muscle cells and phagocytes to curcumin-eluting bioresorbable stent materials, Biomaterials 25 (2004) 5333–5346. [14] M.W. Waylon, A.H. Lucy, F.A. Steve, et al., Anti-oxidant activities of curcumin and related enones, Bioorg. Med. Chem. 13 (2005) 3811–3820. [15] R. Srivastava, M. Dikshit, R.C. Srimal, B.N. Dhawan, Antithrombotic effect of curcumin, Thromb. Res. 40 (3) (1985) 413–417. [16] C.J. Pan, J.J. Tang, Y.J. Weng, J. Wang, N. Huang, Preparation characterization and anticoagulation of curcumin-eluting controlled biodegradable coating stents, J. Controlled Release 116 (2006) 42–49.
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Improved blood compatibility of rapamycin-eluting stent by incorporating curcumin C.J. Pan a,b , J.J. Tang a , Z.Y. Shao a , J. Wang a , N. Huang a,∗ a
Key Laboratory of Advanced Materials Technology, Education Ministry, Southwest Jiaotong University, Chengdu 610031, China b School of Bioengineering, Chongqing University, Chongqing 400044, China Received 20 March 2007; received in revised form 10 April 2007; accepted 25 April 2007 Available online 29 April 2007
Abstract This paper dealt with improving the blood compatibility of the rapamycin-eluting stent by incorporating curcumin. The rapamycin- and rapamycin/curcumin-loaded PLGA (poly(d,l-lactic acid-co-glycolic acid)) coatings were fabricated onto the surface of the stainless steel stents using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier transform infrared spectroscopy (FTIR). The optical microscopy and scanning electron microscopy (SEM) images of the drug-eluting stents indicated that the surface of all drug-eluting stents was very smooth and uniform, and there were not webbings and “bridges” between struts. There were not any cracks and delaminations on stent surface after expanded by the angioplasty balloon. The in vitro platelet adhesion and activation were investigated by static platelet adhesion test and GMP140 (P-selection), respectively. The clotting time was examined by activated partially prothromplastin time (APTT) test. The fibrinogen adsorption on the drug-loaded PLGA films was evaluated by enzyme-linked immunosorbent assay (ELISA). All obtained data showed that incorporating curcumin in rapamycin-loaded PLGA coating can significantly decrease platelet adhesion and activation, prolong APTT clotting time as well as decrease the fibrinogen adsorption. All results indicated that incorporating curcumin in rapamycin-eluting coating obviously improve the blood compatibility of rapamycin-eluting stents. It was suggested that it may be possible to develop a drug-eluting stent which had the characteristics of not only good anti-proliferation but also improved anticoagulation. © 2007 Elsevier B.V. All rights reserved. Keywords: Drug-eluting stent; Rapamycin; Curcumin; Blood compatibility
1. Introduction Although the introduction of coronary stents has significantly improved the treatment of patients with coronary artery disease, in-stent restenosis remains the major drawback of stent implantation [1,2]. The mechanism of in-stent restenosis consists of a series of biological reactions such as thrombus formation, vascular smooth cell (VSMC) migration and proliferation, inflammatory response and negative remodeling and so on [3–5]. The metallic stent provides the scaffolding that virtually eliminates the negative remodeling of vessel and acute occlusion. The coating on metallic stent which contains anti-proliferative drugs can elute drug to prevent in-stent restenosis aroused by VSMC migration and proliferation. For example, the CypherTM rapamycin-eluting stent and TAXUSTM paclitaxel-eluting stent ∗
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are widely used in clinical application. However, the drug carrier of these two drug-eluting stents is nonbiodegradable polymer (the mixing polymer of PEVA and PBMA, polyethylene-covinyl acetate/poly-n-butyl methacrylate for CypherTM and SIBS, poly(styrene-b-isobutylene-b-styrene) for TAXUSTM ) [6]. After drug elutes completely from the stent, the remaining synthetic nonbiodegradable polymer may cause some clinical syndromes such as exaggerated inflammatory reaction, neointima formation and late thrombosis [35–37]. So, it is imperative to develop a novel drug-eluting stent using biodegradable polymer as drug carrier which has not only anti-proliferation but also anticoagulation. Rapamycin (also called sirolimus; Fig. 1a) is a macrocyclic lactone drug approved by FDA [7]. In vitro and in vivo studies indicate that it can significantly prevent proliferation of vascular smooth muscle cell of human and inhibit in-stent restenosis due to neointima formation [8–10]. Rapamycin also has anti-inflammatory activity [11]. So, the rapamycin-eluting
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Fig. 1. The chemical structure of rapamycin (a) and curcumin (b).
stents were extensively studied by many people and many meaningful results were obtained. However, there remains the problem of in-stent restenosis due to lack of anticoagulation. Curcumin (diferuloyl methane; Fig. 1b) has low intrinsic toxicity and possesses a wide range of pharmacological activities, including anti-thrombus, anti-oxidation and antiproliferation [12–14]. More extensive in vitro studies show that biodegrabable PLLA stent grafts loaded with curcumin could reduce the inflammatory responses of platelets and leukocytes [15]. In our previous study, we confirmed that the curcumineluting stents have good blood compatibility when compared to the control samples [16]. In this study, we hypothesized that loading rapamycin and curcummin together in coating of drugeluting stents can make the in-stent restenosis rate decreased significantly because of antiproliferation for rapamycin and good anticoagulation for curcumin. Rapamycin and rapamycincurcumin mixing drug-eluting stents were prepared and the in vitro blood compatibility of these drug-eluting stents and control stents were investigated. 2. Materials and methods 2.1. Materials, reagents and antibodies The bare stainless steel stents with outer diameter 1.65 mm and wall thickness 0.11 mm were prepared in our lab. The PLGA (d,l-LA/GA = 85:15, Mη = 95, 800) resin was provided by Sichuan Zhuxin Biomaterials Co. Ltd. Rapamycin (HPLC purity > 98.3%) was purchased from Fujian Kerui Drug Co. Ltd. Curcumin (HPLC purity > 98.6%) was bought from Shanghai Yousi Biological tTechnology Company. The following antibodies and solutions, purchased from SIGMA, were used: phosphate buffer solution (PBS) buffer, 0.02 g/ml calf serum albumin closed solution (diluted with PBS), horseradish peroxide-enzyme labeled goat anti-human fibrinogen antibody, horseradish peroxide-enzyme labeled goat anti-rat polyclonal antibody, rat anti-human CD62P antibody, TMB (3,3 ,5,5 -tetramethylbenzidine) substrate. All proteins and antibodies were used according to the manufactures recommendations. For clear antibody solutions, no further purification was performed. All other reagents used in this study were analytical grade.
2.2. Fabrication of drug-eluting stent The spray solutions were prepared by dissolving drug and polymer in the dichloromethane. The whole spraying system is illustrated in Fig. 2. The liquid ejected from microsyringe was atomized and focused by untrasonic atomization equipment, and then the focused liquid beam was sprayed onto the surface of stainless steel stents. The stent kept rotating and moving during spraying process to get a homogeneous coating. The rapamycineluting stent (called RES) with 100 g drug and the rapamycincurcumin-eluting stent (called RCES) with 100 g rapamycin and 100 g curcumin were prepared. The controlled stent (called PLGA) with PLGA only was prepared using the same method. The stainless steel stent was labeled SS. 2.3. Fabrication of drug-eluting polymer films The PLGA films and drug-loaded PLGA films were prepared by casting method. The casting solution was obtained by dissolving drug and PLGA in chloroform according to the drug prescription of drug-eluting stents. The solution was slowly poured into cleaned glass Petri dishs to obtain coating films. The films were allowed to slowly evaporate the solvent for 48 h and then kept in vacuum to evaporate the residual solvent for 72 h. The labels of these films were the same as the drug-eluting stents. 2.4. FTIR analysis The surface structure of RES and RCES films was investigated by Fourier transform infrared spectroscopy (FTIR,
Fig. 2. The schematic diagram of ultrasonic atomization spraying system.
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ST-IR20SX, NICOLET Co. Ltd., American). The scanning wavenumber scope was 4000–400 cm−1 . 2.5. Morphology of drug-eluting stents and balloon-expansion After sterilized by epoxy ethane in Sichuan New Century Polymer Co. Ltd. in China, the surface morphology of the drug-eluting stent was observed by optical microscopy (OM) and scanning electron microscopy (SEM, Quanta200, Philipis). Then, the same drug-eluting stent was mounted onto the angioplasty balloon and then dilated at the pressure of 4.0 atm. The stent morphology after deployment was also examined by SEM. 2.6. Platelet adhesion The platelet-rich plasma (PRP) was obtained by centrifuging the fresh whole blood of healthy volunteer at 1500 rpm (revolutions per minute). The control samples (stainless steel and PLGA film) and drug-loading PLGA films were incubated in PRP for 2 h. These samples were rinsed by 0.9% NaCl solution and then fixed by 0.2% glutaraldehyde solution for 1 h and 0.5% glutaraldehyde solution for 12 h, respectively. The samples were washed again for three times using 0.9% NaCl solution and subsequently immersed into 50, 50, 75, 90, 100% ethanol solutions in sequence for 5 min. After dried in a dessicator, the samples were sputtered with gold before being imaged by scanning electron microscopy (SEM). 2.7. APTT For the activated partial thromboplastin time (APTT) measurements, the drug-eluting and control stents were put into a special test tube. The 100 l fresh human platelet-poor-plasma (PPP) which was obtained by centrifuging the whole blood at 3000 rpm, and 100 l actin-activated cephthaloplastin reagent were added into the above tube containing stents, followed by the addition of a 0.03 M CaCl2 solution (100 l) after 3 min incubation at 37 ◦ C. The clotting time of the plasma solution was measured by a coagulometer (Clot 1A, Innova Co.). 2.8. Fibrinogen adsorption test Fibrinogen adsorption was evaluated using enzyme-linked immunosorbent assay which is based on the interaction antigen–antibody reaction [17]. For in vitro fibrinogen adsorption detection, the fresh whole blood of a volunteer was centrifuged at 3000 rpm to obtain platelet-poor plasma (PPP). Each sample (1 cm × 1 cm) was immersed in 500 l PPP for 120 min at 37 ◦ C after incubated in PBS for 3 h at 37 ◦ C to equilibrate the surface. The samples were rinsed with PBS closed solution containing calf serum albumin and then moved into a new 24-well culture plate. Thereafter, the films were incubated in 200 l horseradish peroxide-enzyme goat anti-human fibrinogen antibody for 60 min at 37 ◦ C then rinsed with PBS closed solution containing calf serum albumin for three times. The films were moved into a new 24-well culture plate and then
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200 l TMB substrate was added into each well. The absorbance of the solution was measured at 492 nm after 10 min of the enzyme reaction when 50 l stop solution (1 mol/l H2 SO4 ) of the reaction was injected into the 24-well culture plate. These measurements were carried out five times for each sample and averaged. 2.9. Platelet activation evaluation (GMP140, P-selection) Firstly, 10 ml fresh blood sample from a healthy volunteer was centrifuged for 15 min at 1500 rpm to obtain platelet-rich plasma (PRP) for GMP140 determination. GMP140 expression in plasma was determined using a common enzyme-linked immunosorbent assay [18]. The samples with the same size (10 mm × 10 mm) in 24-well culture plate were cultured in PRP for 2 h at 37 ◦ C and then rinsed with calf serum albumin PBS solution for three times. Thereafter, these samples were shifted into a new 24-well culture plate and 200 l rat anti-human CD62P antibody solution was injected into each well, respectively, for culturing 1 h at 37 ◦ C. Followed by rinsing three times, 200 l horseradish peroxide-enzyme goat anti-rat polyclonal antibody was added into each well containing sample. After cultured 1 h at 37 ◦ C, these samples were rinsed three times using PBS stop solution then transferred into a new 24-well culture plate and then 140 l TMB solution was injected into each well. The absorbance of the solution was measured at 450 nm after 10 min of the enzyme reaction when the stop solution (1 mol/l H2 SO4 ) of the reaction was injected into the 24-well culture plate. These measurements were carried out five times for each sample and averaged. 3. Results and discussion 3.1. The morphology and balloon expansion For drug-eluting stents, the smooth surface can decrease the friction between stent and vessel wall and thus inhibit vessel injury significantly. Smooth stent surface can significantly inhibit platelet deposition and thus improve the blood compatibility of stents [19,20]. The OM and SEM surface morphology images of the drug-eluting stents in this work after sterilized by epoxy ethane sterilizer are shown in Fig. 3. It can be seen that the coating on the stent surface was very smooth and uniform. After dilation by angioplasty, the coating was still smooth and uniform (Fig. 3c, e and f). There were no webbings and “bridges” between struts (Fig. 3). No delamination and destruction were observed (Fig. 3c, e and f), indicating that the coating can adhere tightly to the stent surface. Furthermore, the coating can withstand the compressive and expansion strains imparted during mounting and deployment process. 3.2. The FTIR analysis In the present paper, the surface structure of drug-eluting coating films was examined by FTIR. The results are shown in Fig. 4. There were some differences among three films. The major peak at 3490 cm−1 in three films was most probably due
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Fig. 3. The OM (a–c) and SEM (d–f) images of drug-eluting stents (a, d, RES; b, c, e, f, RCES; c, e, f, after depolyment). The surface of all stents was very smooth and uniform. No webbings were observed between struts.
to –OH vibrations of intermolecularly bonded OH groups. The strong peak at 2998 and 2952 cm−1 and the rather weak peak at 1380 cm−1 in three films can be assigned to the stretching and deformation of methyl groups. The strong peak at 1750 cm−1 in curcumin and RCES is due to C O adsorption. However, this peak shifted to about 1700 cm−1 in RES film because of the presence of rapamycin. The carbon–carbon double bonds at 1620 cm−1 were found in spectra of curcumin film and RCES film. All results indicate that the curcumin and rapamycin exist in PLGA films. 3.3. In vitro platelet adhesion Fig. 5 shows the average platelets number adhered on the different films from ten SEM images (×500). It can be seen that
Fig. 4. The FTIR spectra of RES, curcumin and RCES films.
many platelets deposited onto stainless steel plate surface. In the case of PLGA and RES film, the platelets decreased significantly when compared to stainless steel. After incorporated curcumin in RES, the adhered platelets further reduced. For aggregation platelets, there was no distinct difference among stainless steel, PLGA and RES films. However, the aggregated platelets adhered on RCES film surface decreased distinctly. All results indicated that incorporated curcumin in rapamycin-loaded films can significantly inhibit platelets adhering and aggregating and thus may obviously improve the blood compatibility of the rapamycin-eluting stent. The morphology of platelets adhered on different material surface is shown in Fig. 6. For stainless steel, the adhered platelets almost aggregated and were activated. Many pseudopodia were observed on SS (Fig. 6a). In the case of PLGA (Fig. 6b), the aggregated platelets had decreased when compared to stainless steel; some pseudopodia were observed, indicating
Fig. 5. Platelet adhesion on different material surfaces (culture time: 2 h). Data are expressed as means ± standard deviations for n = 10.
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Fig. 6. The morphology of platelets adhered on stainless steel (a), PLGA (b), RES (c) and RCES (d). Culture time: 2 h.
that its blood compatibility was better than SS. The morphology of platelets adhered on rapamycin-loaded PLGA film was similar with PLGA. However, after incorporating curcumin in rapamycin-eluting film, the adhered platelets on RCES were not activated and the extension of pseudopodia and deformation was not observed (Fig. 6d). The adhered platelets were generally singular with the rounded shape typical of platelets in the inactivated state. It was indicated that incorporating curcumin could improve the blood compatibility of rapamycin-eluting film. Platelet activation and adhesion are known to occur during cardiopulmonary bypass, hemodialysis, as well as cardiovascular implant such as blood vessel stent [21]. In the present paper, the platelet adhesion behavior on different materials was examined and the results were given in Figs. 5 and 6. It can be seen from the figures that curcumin can significantly decrease the platelets adhesion, aggregation and activation of rapamycin-loaded films and thus improve its blood compatibility. It was reported that curcumin can inhibit platelet aggregation induced by ADP, epinephrine, and collagen in vitro and slightly increase prostacylin synthesis [22]. Moreover, curcumin additionally blocks platelet aggregation by platelet-activating factor, collagen and arachidonic acid, and inhibits as well the formation of thromboxane A2 (TXA2) by platelets [23]. Curcumin is also a potential inhibitor of platelet-activating factor, and of reactive oxygen-generating enzymes, including cyclooxygenase and lipoxygenase [24,25]. All these properties of curcumin can contribute to the blood compatibility of rapamycin-loaded PLGA films.
ing curcumin in rapamycin-eluting stent might play an important role in inhibiting the activities of some clotting factors of blood plasma involved in APTT. Blood coagulation involves a series of proteolytic reactions resulting in the formation of fibrin clot [26]. Thrombin is formed following a cascade reactions where an inactive factor becomes enzymatically active following surface contact or after proteolytic cleavage by other enzymes; the newly activated enzyme then activates another inactive precursor factor [26]. The research on anticoagulation for curcumin shows that curcumin prolongs the clotting time of both human as well as rat plasma to approximately 1.1-fold as shown by activated partial thromboplastin time (APTT) assays when compared to control sample [27]. The results of the present paper indicate that curcumin can prolong the clotting time of rapamycin-loaded PLGA film as showed by APTT. This provided a probable pathway for curcumin to inhibit thrombus formation. The APTT shows the bioactivity of intrinsic blood coagulation factors, so, curcumin improves the blood compatibility of rapamycin-loaded film by preventing activation of intrinsic blood coagulation factors. The time prolonged by curcumin could be due to the presence of hydrophobic groups in curcumin moiety [27]. 3.5. Fibrinogen adsorption evaluation The amount of adsorbed fibrinogen on the surfaces of different materials is shown in Fig. 8. It was showed that many
3.4. APTT The APTT test is widely used for the clinical detection of abnormality of blood plasma and for the primary screening of the anticoagulative chemicals. The results of our present work are shown in Fig. 7. The normal APTT time for a healthy blood plasma was regarded to be about 37.6 s (Fig. 7). When the blood plasma was incubated with the stainless steel stent and PLGAcoated stent, the corresponding data of APTT were 33.6 and 36.9 s, indicating that neither stainless steel nor PLGA influenced the thrombogenicity of the blood plasma, they could cause occurrence of intrinsic coagulation. However, when the blood plasma was incubated with rapamycin-eluting stent, the APTT became a litter larger than plasma. After incorporating curcumin in rapamycin-eluting stent, the APTT was prolonged from 38 s for RES to 43.9 s for RCES, indicating RCES can prolong the occurrence of intrinsic pathway system. Therefore, incorporat-
Fig. 7. The APTT of different stents. Columns and error bars represent means ± S.D. for n = 6.
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Fig. 8. Fibrinogen adsorption on different material surfaces. Columns and error bars represent means ± S.D. for n = 5.
fibrinogens adhered on the surface of stainless steel plate. The fibrinogen adsorption amount of SS was about 1.26-fold when compared to that of PLGA. When the rapamycin-eluting PLGA film was incubated in human plasma, the adsorbed fibrinogen amount significantly decreases. After incorporating curcumin in rapamycin-eluting film, the fibrinogen adsorption further reduced when compared to that of RES. The fibrinogen adsorption of RCES was 54.2% of RES, 15.3% of PLGA and 12.2% of stainless steel, respectively. It clearly indicated that incorporating curcumin decrease fibrinogen adsorption of RES. Fibrinogen adsorption has been considered as the major role for blood compatibility of biomaterials [28]. Pre-coated fibrinogen has been demonstrated to enhance surface induced thrombosis [29]. In addition, fibrinogen has been shown to be a key factor in thrombosis because a specific receptor for the fibrinogen molecule, the glycoprotein complex IIb-IIIA (GP IIb-IIIA), is found on the platelet membrane [30]. Formation of these fibrinogen–GP IIb-IIIA complexes greatly enhances platelet activation [31]. The results showed in Fig. 8 indicated that fibrinogen adsorption on SS and PLGA was much heavier than RES and RCES. The adsorbed fibrinogen tended to cover the surface of SS in a thick multilayer pattern and probably undergoes a serious change of its conformation, which enhances its affinity for platelet and effect on platelet activation. The contrary result obtained from RES and RCES indicated the less fibrinogen adsorption may hardly change its conformation and effect on platelet activation. The least fibrinogen adsorption on RCES indicated its blood compatibility may be the best. Therefore, these RCES film on stainless steel stent surface may inhibit fibrinogen and thus improve the blood compatibility of implanting stent. 3.6. P-selection assay (GMP140) P-selection, also called GMP140, is only present on the activated platelets surface, and thus may be used as a marker of platelet activation on biomaterials [32]. The activated platelets
Fig. 9. The GMP140 on different material surfaces (expressed as absorbency value). Columns and error bars represent means ± S.D. for n = 5.
on different materials as indicated by absorbency values are shown in Fig. 9. It was indicated that adhered platelets on stainless steel (SS) surface were activated remarkably when contacted with human plasma. The absorbency value of PLGA decreases, indicating that the activated platelets were less than SS. The activated platelets on RES were only about 64.1% of PLGA and 39.5% of SS. However, after incorporating curcumin in RES, the amount of activated platelets on RCES were reduced to 24.0% of RES, 15.4% of PLGA and 9.5% of PLGA, suggesting that few platelets were activated by RCES when incubated in human plasma. The results were consistent with platelet adhesion result and fibrinogen adsorption results. It is well known that platelet activation is an important aspect in the interaction of biomaterials with blood [33]. The activation of platelets leads to a rapid change of platelet morphology with formation of pseudopodia, spreading and the onset of release reaction [34]. It is a valuable characterization of biomaterials haemocompatibility to understand the mechanism including in the adhesion, activation and release action of platelets. The present research showed that incorporating curcumin in rapamycin-eluting PLGA film can significantly prevent platelet activation and thus improve the blood compatibility of the stent. Forenamed fibrinogen adsorption results indicated that curcumin in RES film can reduce fibrinogen adsorption. The less adsorbed fibrinogen on RCES can hardly undergo conformation change and thus fewer integrated locations of platelet would expose, so, little platelets would be activated when incubated in human plasma. 4. Conclusions The rapamycin-eluting and curcumin/rapamycin-eluting stents using PLGA as drug carrier were prepared using an ultrasonic atomization spray system. The surface of all drugeluting stents was uniform and smooth. No webbings and “bridges” were observed between struts. There were not cracks on stent surface during mounting onto angioplasty and deployment process, indicating that the coating can withstand the
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compressive and deployment strains imparted during this process. Incorporating curcumin in rapamycin-loading PLGA film can reduce fibrinogen adsorption significantly, thus decrease platelet adhesion and activation. All results of haemocompatibility tests demonstrated that incorporating curcumin in rapamycin-loaded PLGA coating can improve the blood compatibility of rapamycin-eluting stent. This provide a new way for developing drug-eluting stents which have not only good anticoagulation but also anti-proliferation. Acknowledgements This work is financially supported by the key basic research program no. 2005CB623904, NSFC-RGC 30318006# of Natural Science Fund of China and the innovation fund of Southwest Jiaotong University, China. References ¨ [1] R.S. JUrgen, V. Vitali, W.H. John, et al., Biological aspects of radiation and drug-eluting stents for the prevention of restenosis, Cardiovasc. Res. 63 (2004) 22–30. [2] R. Virmani, F.D. Kolodgie, A. Farb, A. Lafont, Drug-eluting stents: are human and animal studies comparable? Heart 89 (2) (2003) 133–138. [3] C.L. Harry, N.O. Stephen, M.K. Levon, Coronary in-stent restenosis: current status and future strategies, J. Am. College Cardiol. 39 (2) (2002) 183–193. [4] M.R. Bennett, M. O’Sullivan, Mechanisms of angioplasty and stent restenosis: implications for design of rational therapy, Pharmacol. Ther. 91 (2001) 149–166. [5] A.A.F. Gordon, Y.A. Tony, The mechanism of coronary restenosis: insights from experimental models, Int. J. Exp. Pathol. 81 (2000) 63–88. [6] E. Regar, G. Sianos, P.W. Serruys, Stent development and local drug delivery, Br. Med. Bull. 59 (2001) 227–248. [7] C.G. Groth, L. Backman, J.M. Morales, et al., Sirolimus (rapamycin)-based therapy in human renal transplantation: similar efficacy and different toxicity compared with cyclosporine: Sirolimus European Renal Transplant Study Group, Transplantation 67 (1999) 1036–1042. [8] S.O. Marx, T. Jayaraman, L.O. Go, A.R. Marks, Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells, Circ. Res. 76 (1995) 412–417. [9] M. Poon, S.O. Marx, R. Gallo, et al., Rapamycin inhibits vascular smooth muscle cell migration, J. Clin. Invest. 98 (1996) 2277–2283. [10] C.R. Gregory, X. Huang, R.E. Pratt, et al., Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement, Transplantation 59 (1995) 655–661. [11] R. Gallo, A. Padurean, T. Jayaraman, et al., Inhibition of thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle, Circulation 99 (1999) 2164–2170. [12] K.T. Nguyen, S.H. Su, A. Sheng, et al., In vitro hemocompatibility studies of drug-loaded poly-(l-lactic acid) fibers, Biomaterials 24 (2003) 5191–5201. [13] T.N. Kytai, S. Nishat, P.S. Kajal, et al., Molecular responses of vascular smooth muscle cells and phagocytes to curcumin-eluting bioresorbable stent materials, Biomaterials 25 (2004) 5333–5346. [14] M.W. Waylon, A.H. Lucy, F.A. Steve, et al., Anti-oxidant activities of curcumin and related enones, Bioorg. Med. Chem. 13 (2005) 3811–3820. [15] R. Srivastava, M. Dikshit, R.C. Srimal, B.N. Dhawan, Antithrombotic effect of curcumin, Thromb. Res. 40 (3) (1985) 413–417. [16] C.J. Pan, J.J. Tang, Y.J. Weng, J. Wang, N. Huang, Preparation characterization and anticoagulation of curcumin-eluting controlled biodegradable coating stents, J. Controlled Release 116 (2006) 42–49.
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