Sustained release of multiple growth factors from injectable polymeric system as a novel therapeutic approach towards angiogenesis

Abstract

Purpose: The aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion.

Methods: Three groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF(165) (VEGF), or alginate containing VEGF(165) combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points.

Results: In the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles.

Conclusions: Sequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.

Fig. 1

In vitro release kinetics of pre-encapsulated PDGF and VEGF from alginate fabricated from PLG.

Fig. 2

Representative image of CD 31 staining of thigh muscle treated by intramuscular injection of alginate containing VEGF at week 3, ×200. A, alginate; B, new tissue; C, new vessel; D, inflammatory cells.

Fig. 3

Computer-assisted quantitative analyses of hindlimb blood flow in apoE^−/− mice demonstrated significantly enhanced ratio of ischemic to untreated limb blood perfusion in mice ischemic limb receiving intramuscular injections of alginate (Blank), alginate containing VEGF₁₆₅ (VEGF), or alginate containing VEGF₁₆₅ combined with PLG microspheres containing PDGF-BB (VEGF/PDGF).

Fig. 4

Representative images of micro-CT after 5 weeks intramuscular injections of alginate (Blank), alginate containing VEGF₁₆₅ (VEGF), or alginate containing VEGF₁₆₅ combined with PLG microspheres containing PDGF-BB (VEGF/PDGF).

Fig. 5

Representative images of CD31 staining from thigh muscle of apoE^−/− mice treated by intramuscular injections of alginate (Blank), alginate containing VEGF₁₆₅ (VEGF), or alginate containing VEGF₁₆₅ combined with PLG microspheres containing PDGF-BB (VEGF/PDGF) at 2, 4, and 6 weeks, ×200.

Fig. 6

Representative images of α-SMA staining from thigh muscle of apoE^–/– mice treated by intramuscular injections of alginate (Blank), alginate containing VEGF₁₆₅ (VEGF), or alginate containing VEGF₁₆₅ combined with PLG microspheres containing PDGF-BB (VEGF/PDGF) at 2, 4, and 6 weeks, ×200.

Fig. 7

Capillary vessel density from thigh muscle at 2, 4, and 6 weeks.

Fig. 8

Dual delivery of VEGF plus PDGF induced larger vessel formation. Cross-sectional areas of vessels were measured from α-SMA-stained thigh muscle of the mice treated by intramuscular injections of alginate (Blank), alginate containing VEGF₁₆₅ (VEGF), or alginate containing VEGF₁₆₅ combined with PLG microspheres containing PDGF-BB (VEGF/PDGF) at 2, 4, and 6 weeks. For each condition, at least 100 blood vessels from 6–10 tissue sections were counted.

Fig. 9

VEGF concentration from thigh muscle of apoE^–/– mice treated by alginate containing VEGF₁₆₅ (VEGF), or alginate containing VEGF₁₆₅ combined with PLG microspheres containing PDGF-BB (VEGF/PDGF); or PDGF concentration from thigh muscle treated by alginate containing VEGF₁₆₅ combined with PLG microspheres containing PDGF-BB (VEGF/PDGF).