Sustained VEGF delivery via PLGA nanoparticles promotes vascular growth

Abstract

Technologies to increase tissue vascularity are critically important to the fields of tissue engineering and cardiovascular medicine. Currently, limited technologies exist to encourage angiogenesis and arteriogenesis in a controlled manner. In the present study, we describe an injectable controlled release system consisting of VEGF encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The majority of VEGF was released gradually over 2-4 days from the NPs as determined by an ELISA release kinetics experiment. An in vitro aortic ring bioassay was used to verify the bioactivity of VEGF-NPs compared with empty NPs and no treatment. A mouse femoral artery ischemia model was then used to measure revascularization in VEGF-NP-treated limbs compared with limbs treated with naked VEGF and saline. 129/Sv mice were anesthetized with isoflurane, and a region of the common femoral artery and vein was ligated and excised. Mice were then injected with VEGF-NPs, naked VEGF, or saline. After 4 days, three-dimensional microcomputed tomography angiography was used to quantify vessel growth and morphology. Mice that received VEGF-NP treatment showed a significant increase in total vessel volume and vessel connectivity compared with 5 microg VEGF, 2.5 microg VEGF, and saline treatment (all P < 0.001). When the yield of the fabrication process was taken into account, VEGF-NPs were over an order of magnitude more potent than naked VEGF in increasing blood vessel volume. Differences between the VEGF-NP group and all other groups were even greater when only small-sized vessels under 300 mum diameter were analyzed. In conclusion, sustained VEGF delivery via PLGA NPs shows promise for encouraging blood vessel growth in tissue engineering and cardiovascular medicine applications.

Fig. 1.

A: transmission electron micrograph of lyophilized 50:50 poly(lactic-co-glycolic acid) (PLGA) VEGF encapsulated nanoparticles (VEGF-NPs). Inset: higher-magnification image. Scale bar = 1 μm. B: release kinetics of 50:50 PLGA VEGF-NPs. By 4 days, 89% was released. Values are means ± SE; n = 4.

Fig. 2.

Aortic ring angiogenesis assay. Top: representative images of aortic rings in the presence of saline, empty NPs, and VEGF-NPs. Discrete sprouts are indicative of a proangiogenic environment. Bottom: nean number of sprouts per ring over time in the various conditions. Nine replicates were performed. The rate of formation followed a gradual exponential curve and was greatest for the VEGF-NP group. Values are means ± SE; n = 9 for all groups. *Significance between the VEGF-NP group and either of the two control groups at P < 0.05.

Fig. 3.

Limb images and microcomputerized tomography (microCT)-measured parameters. A: representative three-dimensional microCT angiograms of hindlimbs from the different treatment groups [VEGF-NP (V-NP), 5 μg VEGF (V 5ug), 2.5 μg VEGF (V 2.5ug), saline, day 0 (d0), and nonsurgery (Non surg)]. B: total vessel volume in the surgery limb compared with the nonsurgery limb. Day 0 vessel volume was subtracted from each group. C: <300 μm vessel volume in the surgery limb compared with the nonsurgery limb. Day 0 vessel volume was subtracted from each group. D: vessel connectivity. Day 0 connectivity was subtracted from each group. For B–D, n = 16 for the saline group, n = 11 for the 2.5 μg VEGF group, n = 13 for the 5 μg VEGF group, n = 12 for the VEGF-NP group, and n = 69 for the nonsurgery group. Values are means ± SE. ★Significance to all other groups at P < 0.05; *significance between two groups at bracket ends at P < 0.05.

Fig. 4.

A: distribution of mean vessel diameter of the VEGF-NP group compared with the other experimental and control groups. The x-axis represents vessel diameter, and the y-axis represents the frequency. B: mean vessel diameter for each experimental and control group. n = 16 for the saline group, n = 11 for the 2.5 μg VEGF group, n = 13 for the 5 μg VEGF group, n = 12 for the VEGF-NP group, and n = 7 for the day 0 group. Values are means ± SE. ★Significance to all other groups at P < 0.05; *significance between two groups at bracket ends at P < 0.05.

Fig. 5.

Vessel presence in the ischemic hindlimb musculature (endothelial cells were stained with lectin, and nuclei were counterstained with Hoechst stain). Magnification: ×400. Endothelial cells are denoted with an arrow. A: VEGF-NP group. B: saline group. C: nonsurgery/nonischemic control limb.