Controlled delivery of platelet lysate by polymer nanoparticles in ischemic tissue

Purpose: Platelet lysate (PL) contains growth factors, cytokines and chemokines useful for angiogenesis and tissue repair. Polymer-based nanoparticles (NP) could be used as PL carriers for a controlled release in ischemia. Our aim was to characterize the in vitro (viability, tissue repair and inflammatory response activation) and in vivo (injection in rat ischemic hindlimb and Laser Doppler Perfusion Imaging, LDPI) effect of PL-loaded NP. Methods: Human umbilical vein endothelial cells (HUVEC) were obtained from healthy donors. Experiments were performed at p3, with a pool from 3 donors. PL obtained by thrombapheresis from healthy donors was resuspended (6x108 thrombocytes/ml), subjected to 3 freeze-thaw cycles and stored at -80°C. Two polymers, Poly(Gly-co-HPMA) (fast drug release, e.g. few days) and PLGA (slow drug release, e.g. several weeks), were used to obtain NP for different application. HUVEC were incubated for 48h at 37°C with PL-loaded NP (corresponding to 1.5% PL), with 5% FBS and EGF to assess cell viability. ERK1/2 and NFkB were detected by FACE ELISA, monitoring proteins activated by phosphorylation. HUVEC were treated with PL-loaded NP for 30 and 60' (ERK 1/2) or 1, 24 and 48h (NFkB). Unilateral hindlimb ischemia was induced in adult male Wistar rats (n=24) by surgical excision of the femoral vessels. Blood flow was monitored by LDPI at different time points, up to 21d. After 7d, intramuscular injections of PL, PL-loaded fast NP and saline were performed using fibrin for enhancing engraftment; 6 rats received no treatment. Blood flow was monitored by LDPI. Results: PL-loaded NP did not affect cell viability. PL-loaded fast NP induced ERK1/2 activation at 60', unlike PL-loaded slow NP. PL-loaded slow NP activated NFkB at 48 hours, unlike PL-loaded fast NP. After a short postoperative period of depressed perfusion (p<0.005), the rats showed moderate hindlimb ischemia from d7 onwards, with constant decreased perfusion. Rats receiving only fibrin were comparable to untreated rats. At 7d after intramuscular injection, PL-loaded fast NP significantly increased blood flow (1.01±0.15 ischemic/contralateral hind limb perfusion ratio) in the ischemic hindlimb vs. PL (0.78±0.12, p<0.05). Conclusions: The nanosystems showed cytocompatibility and a differential capability to activate important inflammatory pathways such as ERK 1/2 and NFkB, involved in angiogenesis and tissue repair. Release of PL from loaded NP was effective for blood flow enhancement in a model of chronic, moderate hindlimb ischemia, underlining the advantages of using such drug-controlled release in regenerative medicine.