Nanoceria-GO-intercalated multicellular spheroids revascularize and salvage critical ischemic limbs through anti-apoptotic and pro-angiogenic functions

Abstract

Critical limb ischemia (CLI) is a serious form of peripheral arterial disease that involves severe blockage of blood flow in lower extremities, often leading to foot necrosis and limb loss. Lack of blood flow and high pro-inflammation with overproduced reactive oxygen species (ROS) in CLI aggravate the degenerative events. Among other therapies, cell delivery is considered potential for restoring regenerative capacity, and preservation of cell survival under high oxidative stress has been challenging and prerequisite to harness cellular functions. Here, we introduce a multicellular delivery system that is intercalated with nanoceria-decorated graphene oxide (CeGO), which is considered to have high ROS scavenging ability while providing cell-matrix interaction signals. The CeGO nano-microsheets (8-nm-nanoceria/0.9-μm-GO) incorporated in HUVEC/MSC (7/3) could form cell-material hybrid spheroids mediated by cellular contraction. Under in vitro oxidative-stress-challenge with H₂O₂, the CeGO-intercalation enhanced the survival and anti-apoptotic capacity of cellular spheroids. Pro-angiogenic events of cellular spheroids, including cell sprouting and expression of angiogenic markers (HIF1α, VEGF, FGF2, eNOS) were significantly enhanced by the CeGO-intercalation. Proteomics analysis also confirmed substantial up-regulation of a series of angiogenesis-related secretome molecules. Such pro-angiogenic events with CeGO-intercalation were proven to be mediated by the APE/Ref-1 signaling pathway. When delivered to ischemic hindlimb in mice, the CeGO-cell spheroids could inhibit the accumulation of in vivo ROS rapidly, preserving high cell survival rate (cells were more proliferative and less apoptotic vs. those in cell-only spheroids), and up-regulated angiogenic molecular expressions. Monitoring over 28 days revealed significantly enhanced blood reperfusion and tissue recovery, and an ultimate limb salvage with the CeGO-cell delivery (∼60% salvaged vs. ∼29% in cell-only delivery vs. 0% in ischemia control). Together, the CeGO intercalated in HUVEC/MSC delivery is considered a potential nano-microplatform for CLI treatment, by scavenging excessive ROS and enhancing transplanted cell survival, while stimulating angiogenic events, which collectively help revascularization and tissue recovery, salvaging critical ischemic limbs.

Keywords: Angiogenesis; Anti-apoptosis; Cell delivery; Critical limb ischemia; Graphene oxide; Nanoceria; ROS scavenging.