Preparation and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(D,L-lactic-co-glycolic acid) microspheres using a double emulsion/solvent evaporation technique

Biodegradable Poly(lactic-co-glycolic acid; PLGA), microspheres encapsulating the angiogenic protein recombinant human vascular endothelial growth factor (rhVEGF) were formed to achieve VEGF release in a sustained manner. These microspheres are a promising delivery system which can be used for therapeutic angiogenesis. The PLGA microspheres incorporating two different initial loading amounts of rhVEGF have been prepared by a modified water-in-oil-in-water (w/o/w) double emulsion/solvent evaporation technique. The microspheres have been characterized by particle size distribution, environmental scanning electron microscopy (ESEM), light microscopy, encapsulation efficiency and their degradation was studied in vitro. The rhVEGF released from microspheres was quantified by the competitive enzyme-linked immunosorbent assay (ELISA) and human umbilical vein endothelial cell (HUVEC) proliferation assay was used to assess biological activity of the released VEGF. The microspheres were spherical with diameters of 10-60 mu m and the encapsulation efficiency was between 46% and 60%. The release kinetics of rhVEGF was studied for two different amounts: 5 mu g VEGF (V5) and 50 mu g VEGF (V50) per 500 mg starting polymer. The total protein (VEGF:BSA) release increased up to 4 weeks for two rhVEGF concentrations. The ELISA results showed that the burst release for V5 and V50 microspheres were 4 and 27 ng/mL, respectively. For V5, the microspheres showed an initial burst release, followed by a higher steady-state release until 14 days. VEGF release increased up to 2 weeks for V50 microsphere. HUVEC proliferation assay showed that endothelial cells responded to bioactive VEGF by proliferating and migrating.</.