Following vascular bypass interventions, autologous saphenous vein grafts are prone to fail due to intimal hyperplasia development. An atorvastatin (ATV) eluting mesh coated with poly(lactic-co-glycolic) acid (PLGA), was designed to prevent the development of this pathology. Different parameters were investigated to optimise the drug loading efficacy. A dose of 1.6 mg of atorvastatin was sucessfully loaded on a tubular 5 cm long mesh. The most important parameter influencing ATV loading was shown to be the concentration of the drug in the coating solvent. A formulation was developed to provide in vitro an ATV release profile combining a fast (over 3 days) and a sustained release (over 40 days). The amount of PLGA polymer coating as well as the molecular weight of the polymer were optimized to achieve these kinetics. The macroporosity of the mesh was preserved after coating, while its elasticity was slightly decreased. At this development stage, a proof-of-concept of the administration on an ex vivo model is warranted.
Keywords: mesh, PLGA, atorvastatin, coating, PET, mechanical stress
Introduction
The vascular bypass is a surgical procedure to detour the stenotic areas of arteries, by implanting a vascular graft, most commonly an autologous saphenous vein graft. It is estimated that, only in Europe, more than 300.000 patients received coronary and peripheral artery bypass grafts [1].
However, within 5 years postoperatively, graft failure occurs in up to 50 % of cases [2]. Following a vascular surgery, two physiological mechanisms are typically expressed by the vascular tissue: i) the hyperproliferation of vascular smooth muscle cells and ii) the increase of the calibre of the vessel (wall remodeling) [3]. Although these two mechanisms are physiological, they can be deleterious to the vessel. On the one hand, the cellular proliferation can potentially generate intimal hyperplasia (IH), causing the obstruction of the lumen of the graft vessel (also known as restenosis) [4]. On the other hand, the excessive expansion, reduces the mechanical strength of the vessel walls and can provoque the formation of aneurysms [5].
Although various local medical devices as well as drug delivery systems have been explored for the prevention of perivascular graft failure, no product has reached the market (see CHAPTER I of the
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
114 thesis). We discussed that this might be due to the difficulty to combine the need for a sustained release of an appropriate drug to inhibit vascular smooth muscle cell proliferation and for a mechanical support to prevent aneurysm formation. We have previously evaluated in vitro the efficacy of atorvastatin (ATV) for inhibiting human vascular smooth muscle cell proliferation and migration [6, 7]. Later, we showed in vivo that only a carefully designed ATV’s release profile efficiently reduced IH by 68 % [8]. As far as mechanical support is concerned, the clinical use of a polyethylene terephthalate (PET) mesh (without drug) for supporting varicosed vein grafts for peripheric bypass led to a lower primary patency as compared to patients not receiving the mesh [9, 10]. In contrast, a sirolimus-eluting PET mesh demonstrated superior efficacy compared to non-drug loaded PET mesh in a rabbit model on autologous graft implantation for up to six weeks after surgery [11]. This demonstrated that only the mechanical support offered by the mesh might not be sufficient to reduce IH. We therefore hypothesized that by combining the feature of mechanical support with the sustained release of ATV, the efficacy of the formulation would increase.
To account for mechanical support, we chose a commercially available tubular PET mesh (Provena®). Its microfilaments knitted in a honeycomb pattern provide elasticity to the mesh, as well as the flexibility to allow facile application. Macroporosity provides the necessary space to allow for neo-adventitial vasa vasorum formation. This PET mesh was shown to reduce IH ex vivo [12], by altering the hemodynamic forces exerted on the saphenous vein in the arterial circulation [13].
Achieving sustained drug release kinetics from a mesh or a stent system has always been a challenge [14, 15]. Polymeric mesh coating techniques have been widely explored for sustaining the release of hydrophilic molecules such as antibiotics to prevent infection after hernia repair [14, 16-18]. To coat meshes or stents [19], various techniques have been used, such as dip coating [20], spray coating [18, 21] but also plating or sputtering for metal stents [22]. Dip coating consists in dipping the device in a polymer-drug solution (for hydrophobic drugs) or emulsion (for hydrophilic drugs) and then dry it by air-drying or freeze-drying [14], in order to provide a continuous smooth coating. Instead, spray coating consists in depositing micro-droplets, produced by a nozzle under pressure, on the device.
This technique allows to produce multiple layers of coatings. However, it generates a rough non-continuous coating, requiring a subsequent heat curing to smoothen the surface.
One of the main adverse effects of ATV administered orally, is rhabdomyolysis. In the case of perivascular systems, the toxicity on muscular tissues proximal to the site of application -notably the heart in the case of coronary bypasses- should be absolutely avoided. To reduce the amount of drug diffusing in neighbouring tissues, unidirectional drug release systems were developed [23-25]. They require casting or electrospining the outer layer of sheaths or wraps, with a coating containing drug-free non permeable polymer. However, these techniques cannot be applied in the case of macroporous structures, as in our case, since the pores would be eventually be obstructed. To explore
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
115 this aspect, spray-coating was employed to develop a method enabling the production of a drug-free poly(L-lactide-co-caprolactone) (PLCL) layer. The choice of PLCL was based on the fact that it has excellent elasticity and is unlikely to stiffen the mesh. PLCL also has a degradation rate substantially lower compared to PLGA and and therefore would not resorb before the end of ATV release.
The aim of this study was to investigate the parameters affecting the loading and release profile of a statin, from a PLGA-coated PET multifilament mesh. Ultimately a perivascular atorvastatin releasing mesh was designed with fine-tuned drug-release kinetics, adapted to the development of the pathology. A layer of PLCL covering externally the mesh was added to enable unidirectional release of the drug. Mechanical properties were also investigated as they are of importance in the handling of the mesh during the surgical procedure and contribute to the supportive activity of the mesh.
Materials and methods
Materials
Poly(D,L-lactic-co-glycolic acid ) (PLGA) ester-terminated at 50:50 molar ratio Resomer® was used, either RG502 (i.v. 0.16-0.24 dL/g), RG504 (i.v. 0.45-0.60 dL/g) (Evonik Industries AG, Darmstadt, Germany) or RG503 (i.v. 0.32-0.44 dL/g) (Boehringer Ingelheim GmbH, Germany).
Poly(D,L-lactide-co-caprolactone) (PLCL) at D,L-lactide 40 mol %, (Sigma-Aldrich, Saint-Louis, USA), atorvastatin calcium (Chemos GmbH; Regenstauf; Germany), chloroform (Chromasolv®
plus for HPLC, ≥ 99.9 %, 0.5-1.0 % ethanol as stabilizer, Sigma-Aldrich Chemie GmbH; Steinheim;
Germany), Tributyl-O-acetylcitrate 98 % (TBAC) (Aldrich, Saint-Louis, USA), Tissue-Tek®
O.C.T.™ Compound (Sakura®; Alphen aan den Rijn; Netherlands) were used as received.
Polyethylene terephthalate (PET) Provena® 5 mm of diameter tubular mesh at 76 meshes/cm2, 8 mg/cm was a generous gift from B. Braun Medical AG, Aesculap, Sempach, Switzerland. All other chemicals were of analytical grade.
Methods
Coating
For dip-coating, solutions were prepared by mixing 1 or 6 mg of ATV and different amounts of PLGA (ranging from 10 mg to 200 mg) in 1 mL acetone and placing in an ultrasound bath for 20 min. Addition of 30 mg of TBAC was also tested in some cases to improve the mechanical properties of the coating. When 10 mg of ATV were added in the mixture, filtration (PVDF 0.22 µm) was necessary to remove the excess of non-dissolved ATV. A 5 mm-long tubular mesh was immersed in the coating solution and then submitted to a gentle flux of air. When successive immersions were
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
116 planned, the meshes were allowed to dry for 10 sec between the coating intervals. The mesh was then left to dry overnight at 37 ⁰C.
For spray-coating, solutions were prepared by dissolving PLCL in acetone at a concentration of 2 mg/mL. A 1-cm long mesh was adapted on an alveolar foam polyurethane cylinder and put in rotation at 1300 rpm (Eurostar digital, IKA-Werke, Staufen, Germany). The meshes were spray-coated with 170 µl of PLCL solution, using an airbrush system (HP-CS Eclipse, Iwata®) under 0.25 bar of pressure (Fig. 1). After each second of spraying, the mesh was allowed to dry for four seconds under pressurized air stream.
Figure 1. Schematic illustration of the spray-coating technique.
Meshes nomenclature is presented as
50A. B%. xC, atvD, E
where:A: Type of polymer (PLGA or PLCL), with regards to molecular weight B: Concentration of PLGA in the coating solution (% w/v)
C: Coating layers number
D: Concentration of ATV in the coating solution (mg/mL) E: PLCL external coating
System characterization
For drug loading quantification, ATV was extracted from the PLGA coating of meshes by immersion in 1 mL of DMSO and diluted ten-fold in acetone. The samples were analysed by reversed phase HPLC (LC module I plus, Waters corporation, Milford, USA), and ATV concentration was cleosil CC 125 / 4 120-5
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
117 C18 (Macherey-Nagel GmbH & Co. KG, Oensingen, Switzerland), maintained at 25°C. The mobile phase was 55 % acetate buffer (10 mM, pH 3) and 45 % acetonitrile, at a flow rate 1mL/min. Injection volume was 10.0 µL/min. The calibration curve was constructed by consecutive dilutions of ATV in acetonitrile/ethanol containing PLGA (0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 µg/mL). The retention time was of 7.8 min. The method has been fully validated. A limit of quantification of 500 ng/mL and limit of detection of 50 ng/mL were obtained. A trueness of 98 to 102 % was determined, and the intermediate precision was 2 %; moreover, the three replicates injected on three different days demonstrated the repeatability of the method. The quantification was conducted in triplicate.
Millenium® software was used for data analysis. The results presented as drug loading, correspond to the mass of ATV in the coating per mg of mesh. Each experiment was conducted in triplicate and the values are expressed as mean ± standard deviation.
For scanning electron microscopy imaging, longitudinally cut meshes were placed on double sided adherent conductive carbon PELCO Tabs™ and then onto metal stubs. They were dried under vacuum, sputtered with a 15–20 nm layer of gold and examined by scanning electron microscopy (SEM), using a JSM-7001FA (JEOL, Tokyo, Japan) at 5.0 kV. To obtain mesh cross-sections, meshes were immersed in Tissue-Tek and cut in a cryotome (CryostarTM NX70 Cryostat, Thermo scientific, Walldorf, Germany).
For the in vitro release kinetics, 1 cm of mesh was placed in 5 mL of PBS (0.1 M, pH 7.4) / SDS 0.1
% under sink conditions. Incubation was performed under 80 rpm constant stirring, at 37 °C (EG 110 IR, Jouan, Saint-Herblain, France). At predetermined time intervals, 500 µL of supernatant were sampled and replaced with fresh medium. The samples were analyzed by reversed phase HPLC with the method described above. Each experiment was conducted in triplicate and the values are expressed as mean ± standard error of the mean.
The meshes were also mechanically characterized. Resistance to elongation was measured using a texture analyser (TA.XTplus®, Tracomme AG, Bonstetten, Switzerland). A mesh was cut longitudinally and installed horizontally on a grip fixation (grip to grip distance 6 mm). A ‘return to start’ method was employed at ‘tension’ mode, at pre-test, test and post-test speeds of 1.0, 2.0, 10.0 mm/sec respectively. Data were analysed with Exponent® Stable Micro System software. The measures were performed in triplicate. The Young’s elasticity modulus was evaluated as the slope of the stress-strain curve up to 20 % strain. Triplicates were performed on different samples and results are presented as mean ± standard deviation.
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
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