Endothelial progenitor cell-conditioned medium delivery by polymer nanoparticles in an ischemic hindlimb model

PURPOSE: to characterize the in vivo effect of hypoxic EPC-CM-loaded NPs, comparing it to hypoxic EPC-CM. METHODS: EPCs were obtained from peripheral blood of healthy donors and cultured for 24 h at 1%O2 in growth factor- and serum-free medium to obtain CM. 36 rats were divided into 3 groups: control (vehicle); EPC-CM; EPC-CM NPs. For each animal, 500 μL injections were performed at 3 sites into the ischemic hindlimb, immediately after ischemia and after 1 week. The effect of ischemia was evaluated both using Laser Doppler Blood Flow imaging (ratio between ischemic and contralateral limb), histology (hematoxylin/eosin, H/E, staining) to evaluate the inflammatory reaction and immunohistochemistry to evaluate capillary (CD31+) and artery (alpha-smooth muscle actin, alpha-SMA+) number per mm2. RESULTS: A significant increase of perfusion was observed at 2 weeks in CM-NP vs. both CM and control (p<0.05). In control, tissues showed a normal morphology, while in both CM and CM-NP, at 1 week, we observed myocyte coagulative necrosis with enlarged interstitium mainly populated by inflammatory infiltrates and thin-walled new vessels, more extensively in CM-NP vs. CM. At 2 weeks, fibrosis replacement of necrotic myocytes was found together with new vessels. Immunohistochemistry evidenced a significant increase of capillaries in rats treated with both CM and CM-NPs. Treatment with CM-NP significantly increased capillary number at both 1 (p<0.05 vs. control) and 2 weeks (p<0.0005 vs. control and p<0.005 vs. CM), while CM treatment had a significantly higher effect than control only at 2 weeks (p<0.005). No significant difference in the numbers of arteries among different groups was observed, suggesting a more pronounced effect on angiogenesis rather than arteriogenesis. CONCLUSIONS: Novel therapeutic strategies based on EPC paracrine factors may replace cell transplantation, as "cell-free" therapy could overcome the risk of adverse immunological reactions and the problem of heterologous rejection. Release of EPC-CM from loaded NPs was effective for blood flow and capillary enhancement in an in vivo model of hindlimb ischemia, underlining the advantages of using controlled release in regenerative medicine.