Preparation of a biocompatible stent surface by plasma polymerization followed by chemical grafting of drug compounds

Abstract

The preparation of a biocompatible stent surface was investigated by plasma polymerization of 1,2-diaminocyclohexane (DACH) followed by chemical grafting of α-lipoic acid (ALA). The plasma polymerization resulted in the deposition of a thin polymer film containing amine groups. ALA could be grafted chemically to the thin film through the formation of an amide bond in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (carbodiimide). The ALA-grafted film showed good mechanical stability and blood compatibility, and a stent coated with the film was shown to be effective at inhibiting neointimal hyperplasia.

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1. Introduction

The implantation of a stent in coronary artery is widely practised for the treatment of patients with coronary artery disease.^1,2 At first, bare metal stents were used, but these caused late complications such as restenosis and stent thrombosis.³ To reduce the complications, drug-eluting stents^4–6 were developed. These are typically made by coating a metallic stent with a polymer matrix followed by incorporation of drug compounds into the matrix. The drug compounds are then slowly released, either on their own or with polymer molecules. Drug-eluting stents are effective to some extent for the reduction of the rates of restenosis and stent thrombosis by preventing proliferation or thrombus formation, but have some problems related to characteristics of the polymer matrix. Unless the polymer matrix has excellent biocompatibility and mechanical stability with good adhesion, it can promote vessel wall responses such as platelet aggregation, inflammation, and formation of hyperplastic neointima following drug release, and can be ablated or delaminated during sterilization steps or in blood vessels with high flow pressures. Various polymers (tested without drugs) have shown such vessel responses,^7–10 and expansion or delamination of a polymer matrix after operation has also been observed.^11,12 However, it has been particularly difficult to develop a biocompatible polymer film for application with drug-eluting stents.¹³ Therefore, there is a necessity to develop a coating technology that will allow the development of a polymer matrix with excellent biocompatibility and mechanical stability for the successful operation of stent implants.

In this work, plasma polymerization of 1,2-diaminocyclohexane (DACH) was used to coat a bare metal stent with a polymer matrix. Plasma polymerization is an excellent process for the deposition of thin pinhole-free films, and, in general, the deposited films are highly cross-linked, with good chemical, thermal and mechanical stabilities, and with good adhesion to various substrates.¹⁴ In addition, drug compounds with carboxyl groups can be chemically grafted on to the polymer matrix, since the films deposited by plasma polymerization of DACH have amine groups available.¹⁵ After deposition of the films, α-lipoic acid (ALA) was chemically grafted onto the polymer matrix to improve its biocompatibility. ALA has been used in the treatment of pathology of diabetes for the scavenging of oxygen radicals.¹⁶ These species are known to play a role in atherogenesis,¹⁷ and ALA has actually been used to inhibit the development of neointimal hyerplasia.^18–20

2. Experimental

2.1 Coating of a polymer matrix by plasma polymerization of DACH

The bare metal stents used in this study were MAC^® stents (3.0 × 18 mm, AMG, Raesfeld-Erle, Germany), which were cleaned before use in 1:1 water/ethanol solution using an ultrasonicator. All reagents used in this study were analytical grade and used without further purification. The cleaned stents were placed in a home-made tubular R.F. plasma reactor, and a 50 mL flask containing liquid DACH monomer (99%, Sigma-Aldrich Co.) was connected to the reactor. The pressure was decreased to 10⁻³ torr using a vacuum pump and the plasma was generated with a radiofrequency power generator. The plasma reaction system has been described in previous reports.²¹ The stents were pretreated with Ar + H₂ (flow rate: 9.64/4.10 SCCM) plasma at 60 W for 5 minutes. Then, DACH vapour was introduced to the reactor at a flow rate of 0.89 SCCM. Plasma polymerization was carried out at 100 W for 5 minutes and then at 60 W for 15 minutes, in order to deposit a polymer matrix. Following this, all samples were left in the reactor for 15 minutes to avoid contamination by oxygen.

2.2 Chemical grafting of ALA onto the DACH-coated polymer matrix

10.0 mL (1.21 mmol) of ALA solution (25 mg/mL, thioctic acid^®600T, VIATRIS GmbH & Co. KG) and 0.36 g (1.21 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (carbodiimide, Sigma-Aldrich Co.) were dissolved in 4.00 mL of 0.075 M trisodium citrate dehydrate (Yakuri Pure Chemicals Co.) at pH 5. The solution was kept for 1 hour at ambient temperature to activate the carboxyl groups of ALA. Then, DACH-coated stents were immersed in the activated solution to promote covalent bonding through the formation of amide bonds between the amine groups in the polymer matrix and the carboxylic groups of ALA (Scheme 1). After 1 hour, the ALA-grafted stents were removed from the solution, washed with distilled water (5 mL × 3), and dried for 24 hours at 20 °C in a desiccator.

Scheme 1 Preparation of ALA-grafted stents.

2.3 Analyses and measurements

The surface morphology of the ALA-grafted stent was observed by SEM (S-4700, Hitachi), while the chemical structure of the coated polymer matrix was analyzed by FT-IR (FT/IR-430, Miracle, Jasco) and electron spectroscopy for chemical analysis (ESCA, VG Multilab 2000, ThermoVG Scientific). FT-IR measurements were performed in the attenuated total reflection mode. The ESCA instrument was equipped with a monochromatic MgKα X-ray source at a pressure of 10⁻¹⁰ torr. The reference binding energy was set at 285.0 eV for aliphatic carbons (C–H, C–C) of the C1s core-level spectra.

The thickness of the DACH film was determined indirectly by measuring the thickness of a film deposited on a quartz crystal of a thickness monitor (XTC/2, Inficon) located inside a plasma reactor.

The adhesion strength of the polymer matrix to a bare metal stent surface was evaluated by simple tape test. Commercial adhesive tape was attached to the matrix and quickly detached. The surfaces of the stent and the adhesive tape were examined by eye and using FT-IR.

The blood compatibility of ALA-grafted film was evaluated by an in vitroplatelet adhesion test. Round stainless steel plates with a diameter of 1 cm were used, with platelet concentrate (PC) and fresh frozen plasma (FFP) from human blood supplied by the Korean Red Cross (Gwangju branch). PC was diluted with FFP to prepare optimal platelet-rich plasma (PRP, platelet number: 25.2 × 10⁴/µL). Control plates (stainless steel plates) and prepared ALA-grafted plates were put into a 24-well tissue culture testplate (SPL Life Science), respectively, and then 1.0 mL of phosphate-buffered saline (PBS) was poured into each well, and allowed to equilibriate at 37 °C. After 1 hour, the PBS in each well was replaced with 1.0 mL of PRP. After incubation for 1 hour at 37 °C, the PRP was collected, and the plates rinsed with PBS. Platelets adhered to the plates were fixed with 2% glutaraldehyde (Sigma-Aldrich, 1.0 mL) at 4 °C for 24 hours and then dried with 50, 60, 70, 80, 90 and 100% ethanol solutions in sequence for 5 minutes each. After drying in a desiccator, the surfaces of all plates were examined by SEM.

The amount of released ALA from the drug-coated stents was determined using UV spectroscopy (Safire²™, Tecan Co.). ALA-grafted stents were incubated at 37 °C in 1 mL PBS containing 10% ethanol (v/v). The solution was completely taken out after 24 hours and replaced with fresh PBS solution. The amount of ALA from the stent surface contained in the solution was determined by its UV absorption spectrum at 330 nm. For comparison, the same test was carried out on films of PLGA (poly(dl-lactide-co-glycolide)), which is used for the controlled delivery of drugs for the manufacture of medical devices.²²DACH-coated stents were spray-coated with (a) PGLA as a control, and (b) PLGA containing 10 wt% ALA. The ALA-containing PLGA films were prepared by dissolving 0.1 g of ALA in CH₂Cl₂ containing 1.0 wt% PLGA, 50:50, MW 40,000–75,000). All PLGA-coated stents were dried in a vacuum oven for 4 days to remove residual CH₂Cl₂solvent before the test.

3. Results and discussion

3.1 Deposition of polymer matrix by plasma polymerization of DACH and grafting reaction of ALA

Plasma polymerization of DACH resulted in the deposition of a thin film with thickness of 13.0 nm onto a bare metal stent surface, which was determined by multiplying the deposition rate (measured with a thickness monitor) by the deposition time.

Fig. 1 shows the FT-IR spectra of a DACH film and an ALA-grafted film. In the spectrum of the DACH film, aliphatic C–H bands are observed at 2920, 2857, 1467 and 1376 cm⁻¹. A C–N stretching band and an N–H bending vibration are observed at 1100 cm⁻¹ and 1635 cm⁻¹, respectively. A broad band due to N–H stretching is observed in the region 3200–3500 cm⁻¹, and is assigned to a polymeric amine. This indicates that the DACH films contained amine groups, allowing ALA to be grafted chemically to the film through the formation of amide bonds. Following grafting, absorption bands due to the C=O stretching and N–H bending vibrations of the amide bond are observed at 1644 cm⁻¹ and 1569 cm⁻¹. The C=O stretching of amide occurs at lower frequency than normal carbonyl band due to a resonance effect, indicating that most of the amine groups of the DACH film in the matrix were involved in this grafting reaction.

Fig. 1 FT-IR spectra of a film deposited with (a) DACH and (b) after ALA grafting onto the DACH surface.

Fig. 2 shows an ESCA wide-scan spectra and atomic percentages of elements for the DACH film and ALA-grafted film. The DACH film shows not only C1s and N1s peaks, but also an O1s peak, which seems to be due to oxidation of the film following exposure to air.

Fig. 2 ESCA wide-scan spectra and atomic percent of a DACH film (at 60 W) and an ALA-grafted surface.

It is well known that the films deposited by plasma polymerization are rich in free radicals, and can easily be oxidized in air. The ALA-grafted film shows additional S2s and S2p peaks, indicating that ALA molecules are in the surface layer. Also, the atomic percentage of oxygen in the film is higher than that in the DACH film, which is possibly due to oxygen molecules in the amide groups.

Fig. 3 shows the C1s peaks for the DACH film and the ALA-grafted film. The spectra consist of several peak signals. Two peak signals appear at almost the same binding energies in both spectra; a C–C peak at 285.0 eV and a C–O or C–N peak at 286.2 or 286.0 eV. The other peak signals at 287.7 eV (DACH film) and 288.7 eV (ALA-grafted film) are assigned to the carbonyl group (C=O). However, the binding energy of the C=O carbon in the ALA-grafted film (288.7 eV) is higher than that in the DACH film (287.7 eV). The reason for this shift to higher binding energy upon grafting seems to be due to the transformation of C=O to amide groups, indicating that ALA has been chemically grafted onto the surface of DACH film.

Fig. 3 High-resolution ESCA C1s spectra of a DACH film (at 60 W) and an ALA-grafted film.

3.2 Mechanical stability of the polymer matrix

The polymer matrix coated at 60 W showed poor adhesion to a bare metal stent surface – it failed in the adhesion test with adhesive tape, and sometimes peeled off after the grafting reaction. However, the films coated at higher discharge power, 100 W, passed the adhesion test, although they contained a lower concentration of amine groups, which may be explained by a characteristic of plasma polymerization. In plasma polymerization, monomer molecules fragment to form small reactive species, and a film is deposited on a substrate surface by interaction of the reactive species with the substrate surface and with each other. The degree of fragmentation also increases, and the interactions become more energetic as discharge power increases. As the degree of fragmentation increases, a smaller proportion of monomer molecules retain their original chemical structure, but the adhesion of the deposited film becomes stronger. Therefore, a two-step plasma reaction was used in the coating of the polymer matrix to achieve good adhesion and a high concentration of amine groups – coating at 100 W for 5 min followed by coating at 60 W for 15 min. The polymer matrix prepared in this way was very stable even after the grafting of ALA.

The surface morphology of a stent is known to be very important because rough surfaces and cracks can induce rapid restenosis and thrombosis.²³ The surface morphology of the polymer matrix was therefore examined by SEM. SEM images of a stent coated with an ALA-grafted polymer matrix show a smooth and uniform surface, without any cracking (Fig. 4a,b). No trace of abrasion or delamination was seen even after immersion in PBS for 1 month at 37 °C with shaking, indicating that the ALA-grafted polymer matrix has high mechanical stability (Fig. 4c,d).

Fig. 4 SEM images of a ALA-grafted stent (a, b) before and (c, d) after washing in PBS for 1 month.

3.3 Blood compatibility test

The ALA-grafted polymer matrix showed much better blood compatibility compared with a bare metal surface when evaluated by an in vitroplatelet adhesion test with human blood PRP. Fig. 5 shows SEM images of a bare metal surface and the surface of an ALA-grafted polymer matrix after the platelet adhesion test. The total adhered platelet number is significantly lower on the ALA-grafted matrix surface than on a bare metal surface. In addition, the platelets on the matrix show no aggregation, while those on the bare metal surface have a highly aggregated pseudopodium shape. Platelets are known to have a highly aggregated pseudopodium morphology when they are active.²⁴ This result indicates that grafted ALA has played a key role in the inhibition of platelet aggregation, which leads to the formation of blood clots. Moreover, a stent coated with an ALA-grafted polymer matrix was found to be effective for the inhibition of neointimal hyperplasia when evaluated in a porcine artery stent restenosis model,²⁰ which can be attributed to immobilized ALA on the stent surface. In a 2-week in vitrodrug release test, there was no trace of ALA release from the ALA-grafted stent surface, while a certain amount of released ALA was continuously detected from a physically loaded stent (ALA contained on a PLGA-coated stent) as shown in Fig. 6. This indicates that ALA was chemically grafted on the surface of the DACH-coated stent. Vascular inflammation is one of the main mechanisms in the development of neointimal hyperplasia after angioplasty or stenting, and subsequent abnormal overgrowth of vascular smooth muscle cells (VSMCs) is an aggravating factor in this process. ALA has been reported to block the proliferation and migration of VSMCs by decreasing the level of reactive oxygen species, thereby alleviating the risk of neointimal hyperplasia.¹⁹ALA immobilized on a stent could exert an anti-proliferative effect on abnormal VSMC growth, specifically in the infarct area, and reduce the side effect on normal VSMCs.²⁵

Fig. 5 SEM images of adhered platelets onto a bare metal stent with (a) 250× and (b) 3000× magnification, and ALA-grafted stent with (c) 250× and (d) 3000× magnification.

Fig. 6 UV absorbance at 330 nm by ALA molecules released from a PLGA film (control), a physically loaded stent, and a ALA-grafted stent as a function of time in an in vitrodrug release test.

4. Conclusions

Neointimal hyperplasia, which is triggered by an inflammatory response to foreign materials, is a main cause of in-stent restenosis, but can be inhibited if the stent is modified to have good blood compatibility by coating with drug compounds. We have shown that plasma polymerization of DACH is a suitable process for coating a stent with polymer, and that drugs with carboxylic groups able to inhibit neointimal hyperplasia – in this case α-lipoic acid – can be chemically grafted on to the DACH surface by the formation of amide bonds, resulting in a stent with good blood compatibility.

Acknowledgements

This work was supported by KRF-2007-412-J02002 and a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R01-2007-000-20584-0 and No. R13-2008-010-01002-0).

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