In vitro and in vivo evaluation of FGF2 and FGF9 dual-loaded poly(ester amide) fibers for therapeutic angiogenesis

摘要

Introduction: In therapeutic angiogenesis, the delivery of angiogenic factors either by bolus injection or infusion results in their rapid clearance from the site of interest due to their short biological half-life. The angiogenesis process is complex, therefore therapeutic angiogenesis regimens should include the administration of multiple growth factors and recapitulate temporal presentation of these growth factors. In this study, we are reporting controlled delivery of a different 'cocktail' of growth factors; an angiogenic factor - fibroblast growth factor-2 (FGF2) and an arteriogenic factor - fibroblast growth factor-9 (FGF9). We are aiming to achieve sustained and differential release of these two growth factors from biodegradable poly(ester amide) (PEA) electrospun fibers towards targeting neovascular formation and stabilization. Materials and Methods: FGF2 and FGF9 were dual loaded into PEA fibers using a mixed blend and emulsion electrospinning technique. In vitro release kinetics of FGF2/FGF9 dual-loaded PEA fibers was studied in PBS (pH 7.4) at 37 °C for 28 days using ELISA as previously described. In vitro angiogenesis assays including Matrigel tube formation assay and Boyden chamber transwell assay were used to evaluate endothelial cell (EC) tube formation, directed smooth muscle cell (SMC) migration, and EC-SMC interaction. Ex ovo chick chorioallantoic membrane (CAM) model coupled with power Doppler ultrasound imaging was employed to assess the in vivo angiogenic capacity of our delivery system. Results and Discussion: In vitro release studies showed controlled and differential release of both factors in a bioactive form. Co-released FGF2 and FGF9 from dual loaded PEA fibers enhanced EC tube formation, directed-migration of SMCs towards PDGF-BB, and EC/SMC tube stabilization. The CAM assay showed a higher percentage increase in vascular density of the full CAM surface treated with FGF2/FGF9 dual-loaded fibers with time (Figure 1). Power Doppler 3D volumes showed the formation of smaller vessels near the CAM surface in the case of dual-loaded PEA fibers and soluble growth factors treated CAMs that were not observed in the case of unloaded PEA libers treated CAMs and the negative control, which may indicate a localized angiogenic effect (Figure 2). Conclusion: Localized angiogenesis was observed at the interface between the dual-loaded fibrous mat and the CAM surface. The CAM model had some limitations in detecting a statistically significant difference in the full CAM vasculature due to its rapid vascular growth that did not emulate the controlled and relatively slow release of FGF2 and FGF9 from the PEA fibers. Despite the aforementioned limitations, the in vitro angiogenesis assays data together with the preliminary CAM assay data suggests that FGF2/FGF9 dual loaded PEA fibers can have a potential therapeutic angiogenesis application for treatment of ischemic vascular diseases. Moving forward, a more clinically relevant model, the ischemic hind limb mouse model will be employed to better investigate the angiogenic effects of the FGF2/FGF9 dual-loaded PEA fibers.