Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease.

Cell therapy for treatment of peripheral arterial disease (PAD) is a promising approach but is limited by poor cell survival when cells are delivered using saline. The objective of this study was to examine the feasibility of aligned nanofibrillar scaffolds as a vehicle for the delivery of human stromal vascular fraction (SVF), and then to assess the efficacy of the cell-seeded scaffolds in a murine model of PAD. Flow cytometric analysis was performed to characterize the phenotype of SVF cells from freshly isolated lipoaspirate, as well as after attachment onto aligned nanofibrillar scaffolds. Flow cytometry results demonstrated that the SVF consisted of 33.1 ± 9.6% CD45+ cells, a small fraction of CD45-/CD31+ (4.5 ± 3.1%) and 45.4 ± 20.0% of CD45-/CD31-/CD34+ cells. Although the subpopulations of SVF did not change significantly after attachment to the aligned nanofibrillar scaffolds, protein secretion of vascular endothelial growth factor (VEGF) significantly increased by six-fold, compared to SVF cultured in suspension. Importantly, when SVF-seeded scaffolds were transplanted into immunodeficient mice with induced hindlimb ischemia, the cell-seeded scaffolds induced a significant higher mean perfusion ratio after 14 days, compared to cells delivered using saline. Together, these results show that aligned nanofibrillar scaffolds promoted cellular attachment, enhanced the secretion of VEGF from attached SVF cells, and their implantation with attached SVF cells stimulated blood perfusion recovery. These findings have important therapeutic implications for the treatment of PAD using SVF.

Delivery of hepatocyte growth factor mRNA from nanofibrillar scaffolds in a pig model of peripheral arterial disease.

Background: Chemical modification of mRNA (mmRNA) substantially improves their stability and translational efficiency within cells. Nanofibrillar collagen scaffolds were previously shown to enable the spatially localized delivery and temporally controlled release of mmRNA encoding HGF both in vitro and in vivo. Materials & methods: Herein we developed an improved slow-releasing HGF mmRNA scaffold and tested its therapeutic efficacy in a porcine model of peripheral arterial disease. Results & conclusion: The HGF mmRNA was released from scaffolds in a temporally controlled fashion in vitro with preserved transfection activity. The mmRNA scaffolds improved vascular regeneration when sutured to the ligated porcine femoral artery. These studies validate the therapeutic potential of HGF mmRNA delivery from nanofibrillar scaffolds for treatment of peripheral arterial disease.

Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA.

RNA-based vector delivery is a promising gene therapy approach. Recent advances in chemical modification of mRNA structure to form modified mRNA (mmRNA or cmRNA or modRNA) have substantially improved their stability and translational efficiency within cells. However, mmRNA conventionally delivered in solution can be taken up nonspecifically or become cleared away prematurely, which markedly limits the potential benefit of mmRNA therapy. To address this limitation, we developed mmRNA-incorporated nanofibrillar scaffolds that could target spatially localized delivery and temporally controlled release of the mmRNA both in vitro and in vivo. To establish the efficacy of mmRNA therapy, mmRNA encoding reporter proteins such as green fluorescence protein or firefly luciferase (Fluc) was loaded into aligned nanofibrillar collagen scaffolds. The mmRNA was released from mmRNA-loaded scaffolds in a transient and temporally controlled manner and induced transfection of human fibroblasts in a dose-dependent manner. In vitro transfection was further verified using mmRNA encoding the angiogenic growth factor, hepatocyte growth factor (HGF). Finally, scaffold-based delivery of HGF mmRNA to the site of surgically induced muscle injury in mice resulted in significantly higher vascular regeneration after 14 days, compared to implantation of Fluc mmRNA-releasing scaffolds. After transfection with Fluc mmRNA-releasing scaffold in vivo, Fluc activity was detectable and localized to the muscle region, based on noninvasive bioluminescence imaging. Scaffold-based local mmRNA delivery as an off-the-shelf form of gene therapy has broad translatability for treating a wide range of diseases or injuries.