No perivascular system is available on the market for the prevention of IH, despite the abundant literature on this route of administration (see CHAPTER I of the thesis). We explored the formulation of a perivascular system delivering ATV, dedicated to the treatment of IH, combining two key properties: appropriate drug release kinetics and constriction of the vessel. Parameters affecting the ATV release from a PLGA coating on a macroporous PET mesh were explored. This study aimed at optimizing the coating of the mesh in order to: i) maximize the ATV loading, ii) improve the unidirectional release of ATV, iii) ensure an in vitro drug release profile combining a fast initial release over the first 3 days, followed by a sustained release over 28 days and iv) adjust the mechanical properties of the mesh.
Drug loading on the mesh
To optimize loading efficacy, we investigated the following parameters: concentration of polymer and drug in the coating solution, number of coating layers, type of polymer (molecular weight). The mass of polymer adhering by the dipping method on the mesh was explored. When consecutive coating layers were applied, the amount of polymer adhering on the mesh did not significantly increase: the first dipping increased the mesh mass by 20 %, while the consecutive 2nd and 3rd coatings only added 10 % to the pre-existent polymer mass (Fig. 2). However, when considering the ATV loading on the mesh, it was independent of the polymer concentration in the coating solution.
(Fig. 3). This limited loading arises from the equilibrium between the steady ATV concentration in the coating and the amount of deposited coating. This could be explained by an almost constant solution volume drawn by the mesh before drying step, irrespectively of the amount of polymer adhering on the mesh. The lack of influence of solution’s viscosity on the amount of solution drawn might be related to the highly open porous mesh structure. In this case, solvent-PET interfacial tension might play a more critical role.
Interestingly, the increase of ATV loading is not linearly proportional to the number of coatings.
Indeed, the amount of drug or polymer adhering on the mesh doubled only after three consecutive coatings. This is probably due to the fact that a certain amount of polymer is re-dissolved once in the solution. In all cases, the most efficient approach to improve drug loading was to increase ATV concentration in the coating solution (Fig. 3). Indeed, a proportional increase of the loading was observed. Finally, the molecular weight of the polymer did not affect the drug-loading efficacy, confirming a viscosity-independent deposition of the coating solution. We thus concluded that to improve drug loading, ATV concentration in the coating solvent must be maximized. Adding coating layers can also be beneficial.
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
119 Under these conditions, a loading of 40 µg per mg of mesh or 320 µg per cm of mesh was achieved with a single coating containing 0.6 % (w/v) of ATV and 10 % (w/w) of PLGA. Thus for a 5-cm mesh, 1.6 mg of ATV will be deposited. In a mouse model we have demonstrated that a dose of 50 mg/kg or 1 mg/mouse, was necessary for the inhibition of IH administered as gel and microparticles [8]. This perivascular system might satisfy an ex vivo proof of concept, even at low dosage. However, for a clinical application, higher dose should be envisioned.
1 3 510 20 1 3 5 1020 1 3 510 20
Figure 2. Effect of polymer’s molecular weight, concentration in the coating solution and number of coating layers on the mass of PLGA adhering on the mesh. PLGA mass adhering on the mesh is expressed as the percentage ratio of the mass of polymer adhering on the mesh to the initial mass of the mesh. nm: not measured. Each point is a mean ± SD (n = 3).
Figure 3. Effect of polymer’s molecular weight and concentration in the coating solvent, number of coating layer and ATV concentration in the coating solvent on the ATV loading of coated meshes.
Each point is a mean ± SD (n = 3).
3.2. PLCL external coating
To improve directional release of the drug from the meshes, following dip-coating in an ATV/PLGA solution they were externally spray-coated with PLCL. In Fig. 4, meshes cross-sections are presented as two distinct layers of coating on SEM. According to these micrographs, the thickness of the PLGA layer is of 5 to 15 µm, while the thickness of PLCL is of 10 µm. The thickness of the matrix
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
120 corresponds to the amount of polymer deposited on the mesh. The coating’s surface and matrix appeared smooth and uninterrupted (Fig. 4). This is important, as a discontinuous porous surface would allow for fast water penetration and accelerate the drug release process. The efficacy of the unidirectional coating needs further validation (e.g.. ex vivo or in vivo).
Figure 4. A.: Cross-section of the mesh dip-coated with PLGA and externally spray-coated with PLCL, as observed on SEM. B.: Schematic illustration of the mesh cross-section, denoting the different layers of the coatings.
3.3. In vitro drug release profile
We have previously shown that an optimal ATV release profile is necessary to prevent IH. This involves a two-phase release kinetic with a combination of a fast (over 3 days), followed by a sustained (over 4 weeks) release [8]. We attempted to reproduce this drug release profile on a polymer coated perivascular mesh system.
The impact of the polymer’s and the drug’s concentration in the coating solution, the polymer’s molecular weight, as well as the effect of the number of coating layers on the drug release profile were investigated (Fig. 5). For RG502 all the release curves were monophasic, typical for diffusion kinetics. Similar patterns were obtained for RG503 and RG504 at 1 % and 3 % w/v concentrations for single coatings. Triphasic release curves were observed for RG503 and RG504 for 10 % w/v placed in a single coating. A reduction of the burst release (20-40 % instead of 50-60%) was observed for 3 % w/v with a triple coating for RG503 and RG504. As shown previously, increasing PLGA’s concentration in the coating solution would increase polymer deposition (Fig. 2) and consequently the matrix thickness. Thus, it is inferred that the increasing thickness of the matrix resulted in a shifted release curve. It is widely acknowledged that the steady state release rate of a drug from a PLGA slab is inversely proportional to matrix thicknesss [26]. Similar observations relating the thickness of the matrix with the release curve shape were made for spherical PLGA systems [27].
Additionally, the effect of molecular weight on the shape of the release curve, was also observed on microparticulate PLGA systems of approximately 10 µm (see CHAPTER II of the thesis).
In the case of higher ATV loading without altering the number of coating layers or polymer concentration in the coating solution (Fig. 6), the burst increases and the triphasic shape is attenuated.
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
121 Similar observations were obtained for spherical systems [28]. Additionally, the increase of drug content in the PLGA matrix, can make the encounter of water with drug molecules more likely. All in all, the obtained curves from PLGA films deposited on a mesh are very similar with the ones obtained by microparticulate PLGA of the same thickness and drug loading (see CHAPTER II of the thesis). This leads us to the conclusion that the system geometry (f.ex. film, sphere) is not so critical for drug release kinetics [26], making possible to extrapolate the results observed on microspheres to film layers of the same thickness and drug loading.
Figure 5. Left panel: Effect of PLGA’s molecular weight and concentration in the coating solution on the cumulative release. Right panel: Effect of PLGA’s molecular weight and number of coating layers on the cumulative release of ATV loaded meshes in vitro (PBS (0.1 M, pH 7.4)/SDS 0.1%) Each point is a mean ± SoEM (n = 3).
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
122 For ATV perivascular application the most adequate release at a maximum drug loading would be 504.10%.x1.atv6 (Fig. 6), ensuring a 40 % burst along with a sustained release over 40 days, [8]. On this preparation, the external PLCL coating almost did not affect the in vitro release kinetics of the drug (Fig. 6). In this experimental set-up, since the mesh is fully immersed in the release medium, it is not possible to distinguish and characterize the inward and outward fluxes of the drug. Therefore, conclusions cannot be drawn with regard to the efficacy of the PLCL coating to guide ATV release inwards. An ex vivo or in vivo system would be necessary to validate the concept.
0 1 0 2 0 3 0 4 0
0 2 0 4 0 6 0 8 0
T im e ( d a y s )
Atorvastatin (%)
5 0 4 .1 0 % .x 1 .a tv 6 5 0 4 .1 0 % . x 1 .a tv 6 .P L C L 5 0 4 .1 0 % .x 1 .a tv 1
Figure 6. Effect of ATV loading on the release profile of single coated meshes with RG504 at 10 % w/v of coating solution. The effect of the PLCL external coating is investigated.
3.4. Mechanical properties
Mechanical properties of perivascular systems are important for the efficacy of the system. For instance, stiffness of the applied system might impede its application around the vessel. For this reason, we wanted to ensure that the macroporosity of the mesh - which would impact the elasticity - would be conserved. Multiple coating layers preserve better the macroporosity of the mesh compared with highly concentrated coating solutions, while the amount of polymer adhering would remain the same. Dip coating with a solution of 20 % w/v of PLGA demonstrated a tendency to obstruct the macropores of the mesh (Fig. 7), probably due to high surface tension. Accordingly, using coating solutions at PLGA concentrations above 10 % was excluded, despite their ability to deposit a high polymer mass on the mesh, which in turn could be beneficial to obtain a thick matrix releasing the drug over an extended period of time. Mesh macroporosity was regarded as critical for its efficacy, allowing the vessel to maintain an exchange of nutrients with the surrounding tissue by developing the vital vasa vasorum and increasing the chances of graft survival [29].
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
123 Figure 7. SEM images of RG504 coated meshes. Upper panel: Eight micrographs: Effect of the variation of the polymer concentration in the coating solution. Lower panel: Six micrographs: Effect of the number of coating layers. Macropore occlusion is partial at 10% of PLGA and complete at 20% of PLGA.
While elasticity is important for application of the system around the vasculature, vessel constriction was shown to be beneficial for reducing intimal hyperplasia (see CHAPTER I of the thesis). We have thus investigated the mechanical properties of meshes before or after coating under different conditions. Interestingly, the elastic modulus of a human carotid artery (0.1 to 1 MPa) was measured to be of the same order of magnitude as the uncoated mesh [30]. A six-fold increase in ATV loading increased the Young’s modulus of elasticity proportionally. Application of three coating layers, demonstrated a three-fold increase in the rigidity of the mesh possibly also due to the increased atorvastatin loading. The Young’s modulus measured for PLA or PCL coated meshes by others was four to five times higher compared to our results with PLGA [18]. Unexpectedly, the use of TBAC, a hydrophobic plasticizer [31], did not improve the elasticity measured. Due to PLCL excellent
CHAPTER V - Design and characterization of a perivascular PLGA coated perivascular mesh sustaining the release of atorvastatin for the prevention of restenosis.
124 elastic properties, the additional coating further increased the elasticity of the mesh by 30 % , preserving a mesh elasticity close to that of vascular tissue values reported in literature [30].
Figure 8. Elongation testing of coated compared to non-coated meshes. A: Typical tensile stress–
strain curve obtained for a coated mesh. B: Effect of ATV loading. C: Effect of the number of coating layers. D: Effect of plasticizers. Each bar is the mean ± SD. Ordinary one-way ANOVA with Tukey’s multiple comparisons: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) compared to non-coated mesh.