Poly(N-isopropylacrylamide)/N-tert-butylacrylamide (NiPAAm/NtBAAm) co-polymers are a novel class of thermoresponsive co-polymers (TP) under investigation as drug delivery systems (Doorty et al., 2003). These co-polymers have the potential to be used as intra-vascular stent coatings, for delivery of vasculoprotective agents to injured coronary vasculature. One candidate agent is vascular endothelial growth factor (VEGF), which could in principle stimulate re-endothelialisation (i) of vessels denuded following angioplasty and (ii) of the stent itself (Zachary et al., 2000). This study aimed to assess the ability of these co-polymers to incorporate and release VEGF and to determine the ability of VEGF to modulate endothelial cell growth on such surfaces.

Co-polymer films were prepared using a 50:50 (w/w) mixture of non cross-linkable and cross-linkable co-polymers of the following monomer compositions (w/w): 85:15 NiPAAm:NtBAAm) and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (cross-linking agent) respectively. Following cross-linking by UV irradiation, Incorporation into films was achieved by incubating them at 4 °C with solutions containing VEGF₁₆₅ (200ng/ml) for 7 days. VEGF₁₆₅ subsequently released at 37 °C was quantified by ELISA. Human aortic endothelial cells (HAEC) were grown on 65/35 and 50/50 co-polymer films at
37 °C for up to 6 days in the absence/presence of VEGF₁₆₅ (20ng/ml). Cell proliferation was measured at
24 h intervals in terms of AlamarBlue reduction. Cell viability in the absence/presence of VEGF₁₆₅ was assessed at 24 h and 10 days post seeding by flow cytometry, using annexin-V and propidium iodide staining. Significant differences were identified following analysis by one-way ANOVA, using the software package SAS version 6.12.

Co-polymer films loaded with 200 ng VEGF₁₆₅ released the growth factor for up to 6 days. (0.4 ng by 24 h, total release 1.35 ng, or 0.67% of amount loaded). At 6 days, cell numbers established on 50/50 co-polymer and control tissue culture polystyrene (TCP) surfaces were similar. Cell morphology was unaltered by growth on 50/50 or 65/35 films, and neither surface induced apoptosis or necrosis of adherent cells. HAEC growth on 50/50 and 65/35 films was significantly improved by VEGF₁₆₅: at day 7, cells grown on 50/50 and 65/35 films in the presence of VEGF₁₆₅ reduced AlamarBlue to a significantly greater extent (p<0.005 and p<0.05 respectively) than those grown in the absence of the growth factor, indicating an increase in cell number in the presence of VEGF₁₆₅ . There was no significant difference in viability between cells grown for 24 h on 50/50, 65/35 and TCP ± VEGF₁₆₅ .

This study has established that NiPAAm-derived co-polymers can be used to delivery VEGF for an extended time period. It has also shown that such co-polymers can support the growth of an endothelial monolayer, with maintenance of viability. We conclude that this system has therapeutic potential for local delivery of pro-angiogenic molecules to injured blood vessels.

Doorty KB, et al. (2003) Cardiovasc Pathol 12, 105-110
Zachary I, et al., (2000) Arterioscler Thromb Vasc Biol 20, 1512-1520

Supported by the Irish Heart Foundation and Enterprise Ireland