Elastin-like polypeptide mediated proangiogenic and anti-inflammatory gene therapy for critical limb ischemia

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been modest success in reducing the rate of amputations in these patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this thesis was to develop a controlled delivery system capable of delivering multiple therapeutic genes in an extended manner. Initially, a Chitosan/Polyglutamic acid hollow sphere system was tested in vitro as a model system to elucidate the combinatorial effects of physicochemical properties such as size and surface charge on cell viability and, most importantly, on cellular internalization. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and in situ scaffold of elastin-like polypeptide. The hollow spheres and in situ scaffold were independently capable of carrying gene complexes and released the gene complexes in an extended manner. Furthermore, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. Initially, a subcutaneous dose study was performed in the mouse model to determine a therapeutic dose of hIL-10 and heNOS. In the injectable ELP system, hIL-10 was loaded inside the scaffold and heNOS inside the ELP hollow spheres. Human eNOS(20 µg) and hIL-10(10 µg)/heNOS(20 µg) showed comparatively more blood vessel density than others and hIL-10(10 µg) showed comparatively reduced the amount of inflammatory cells. These groups were then selected for the hind limb ischemic study including control groups: saline, injectable system only. The treatment groups that showed higher blood perfusion measured using laser doppler perfusion imaging were the groups with heNOS treatment groups. Saline group showed signs of sever necrosis. Human IL-10 treatment groups showed reduction in the level of inflammation cells. Furthermore, a mechanistic study showed proangiogenic activity of eNOS by up-regulating major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B and fibroblast growth factor 1. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediated non-viral delivery of IL10-eNOS is a promising therapy towards treating limb ischemia

The influence of size and charge of chitosan/polyglutamic acid hollow spheres on cellular internalization, viability and blood compatibility

Polymeric hollow spheres can be tailored as efficient carriers of various therapeutic molecules due to their tunable properties. However, the entry of these synthetic vehicles into cells, their cell viability and blood compatibility depend on their physical and chemical properties e.g. size, surface charge. Herein, we report the effect of size and surface charge on cell viability and cellular internalization behaviour and their effect on various blood components using chitosan/polyglutamic acid hollow spheres as a model system. Negatively charged chitosan/polyglutamic acid hollow spheres of various sizes 100, 300, 500 and 1000 nm were fabricated using a template based method and covalently surface modified using linear polyethylene glycol and methoxyethanol amine to create a gradient of surface charge from negative to neutrally charged spheres respectively. The results here suggest that both size and surface charge have a significant influence on the sphere¿s behaviour, most prominently on haemolysis, platelet activation, plasma recalcification time, cell viability and internalization over time. Additionally, cellular internalization behaviour and viability was found to vary with different cell types. These results are in agreement with those of inorganic spheres and liposomes, and can serve as guidelines for tailoring polymeric solid spheres for specific desired applications in biological and pharmaceutical fields, including the design of nanometer to submicron-sized delivery vehicles

An injectable elastin-based gene delivery platform for dose-dependent modulation of angiogenesis and inflammation for critical limb ischemia

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 rig) and IL-10 (10 mu g)/eNOS (20 mu g) and reduced inflammation with IL-10 (10 mu g) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELF injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia. (C) 2015 Elsevier Ltd. All rights reserved.Science Foundation Ireland grant no. 07/SRC/B1163peer-reviewed2017-10-3

Mannosylated Polyethyleneimine-Hyaluronan Nanohybrids for Targeted Gene Delivery to Macrophage-Like Cell Lines

Journal articleNonviral gene delivery systems have a number of limitations including low transfection efficiency, specificity, and cytotoxicity, especially when the target cells are macrophages. To address these issues, the hypothesis tested in this study was that mannose functionalized nanohybrids composed of synthetic and natural polymers will improve transfection efficiency, cell viability, and cell specificity in macrophages. Robust nanohybrids were designed from hyaluronic acid (HA) and branched polyethyleneimine (bPEI) using carbodiimide chemistry. The reaction product, i.e., branched polyethyleneimine-hyaluronic acid (bPEI-HA) copolymer was subsequently functionalized with mannose at the terminal end of the copolymer to obtain mannosylated-bPEI-HA (Man-bPEI-HA) copolymer. UV spectroscopy and gel retardation studies confirmed the formation of polyplexes at polymer to DNA weight ratio >= 2. Alamar Blue and MTT assay revealed that the cytotoxicity of the developed nanohybrids were significantly (P < 0.05) lower than that of unmodified bPEI. Mannose functionalization of these nanohybrids showed specificity for both murine and human macrophage-like cell lines RAW 264.7 and human acute monocytic leukemia cell line (THP1), respectively, with a significant level (P < 0.05) of expression of gaussia luciferase (GLuc) and green fluorescent reporter plasmids. Internalization studies indicate that a mannose mediated endocytic pathway is responsible for this higher transfection rate. These results suggest that hyaluronan-based mannosylated nanohybrids could be used as efficient carriers for targeted gene delivery to macrophages

TNF-α Preconditioning Promotes a Proangiogenic Phenotype in hiPSC-Derived Vascular Smooth Muscle Cells.

INTRODUCTION: hiPSC-VSMCs have been suggested as therapeutic agents for wound healing and revascularization through the secretion of proangiogenic factors. However, methods of increasing cell paracrine secretion and survivability have thus far yielded inconsistent results. This study investigates the effect of pre-conditioning of hiPSC-VSMCs with TNF-α and their integration into 3D collagen scaffolds on cellular viability and secretome. METHODS: hiPSC-VSMCs were dual-plated in a 2D environment. TNF-α was introduced to one plate. Following incubation, cells from each plate were divided and added to type-I collagen scaffolds. TNF-α was introduced to two sets of scaffolds, one from each 2D plate. Following incubation, scaffolds were harvested for their media, tested for cell survivability, cytotoxicity, and imaged. Intra-media VEGF and bFGF levels were evaluated using ELISA testing. RESULTS: hiPSC-VSMCs exposed to TNF-α during collagen scaffold proliferation and preconditioning showed an increase in cell viability and less cytotoxicity compared to non-exposed cells and solely-preconditioned cells. Significant increases in bFGF expression were found in pre-conditioned cell groups with further increases found in cells subsequently exposed during intra-scaffold conditioning. A significant increase in VEGF expression was found in cell groups exposed during both pre-conditioning and intra-scaffold conditioning. Fibroblasts treated with any conditioned media demonstrated increased migration potential. CONCLUSIONS: Conditioning hiPSC-VSMCs embedded in scaffolds with TNF-α improves cellular viability and increases the secretion of paracrine factors necessary for wound healing mechanisms such as migration. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-023-00764-0

TNF-α Preconditioning Promotes a Proangiogenic Phenotype in hiPSC-Derived Vascular Smooth Muscle Cells.

INTRODUCTION: hiPSC-VSMCs have been suggested as therapeutic agents for wound healing and revascularization through the secretion of proangiogenic factors. However, methods of increasing cell paracrine secretion and survivability have thus far yielded inconsistent results. This study investigates the effect of pre-conditioning of hiPSC-VSMCs with TNF-α and their integration into 3D collagen scaffolds on cellular viability and secretome. METHODS: hiPSC-VSMCs were dual-plated in a 2D environment. TNF-α was introduced to one plate. Following incubation, cells from each plate were divided and added to type-I collagen scaffolds. TNF-α was introduced to two sets of scaffolds, one from each 2D plate. Following incubation, scaffolds were harvested for their media, tested for cell survivability, cytotoxicity, and imaged. Intra-media VEGF and bFGF levels were evaluated using ELISA testing. RESULTS: hiPSC-VSMCs exposed to TNF-α during collagen scaffold proliferation and preconditioning showed an increase in cell viability and less cytotoxicity compared to non-exposed cells and solely-preconditioned cells. Significant increases in bFGF expression were found in pre-conditioned cell groups with further increases found in cells subsequently exposed during intra-scaffold conditioning. A significant increase in VEGF expression was found in cell groups exposed during both pre-conditioning and intra-scaffold conditioning. Fibroblasts treated with any conditioned media demonstrated increased migration potential. CONCLUSIONS: Conditioning hiPSC-VSMCs embedded in scaffolds with TNF-α improves cellular viability and increases the secretion of paracrine factors necessary for wound healing mechanisms such as migration. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-023-00764-0

Mannosylated Polyethyleneimine–Hyaluronan Nanohybrids for Targeted Gene Delivery to Macrophage-Like Cell Lines

Nonviral gene delivery systems have a number of limitations including low transfection efficiency, specificity, and cytotoxicity, especially when the target cells are macrophages. To address these issues, the hypothesis tested in this study was that mannose functionalized nanohybrids composed of synthetic and natural polymers will improve transfection efficiency, cell viability, and cell specificity in macrophages. Robust nanohybrids were designed from hyaluronic acid (HA) and branched polyethyleneimine (bPEI) using carbodiimide chemistry. The reaction product, i.e., branched polyethyleneimine-hyaluronic acid (bPEI-HA) copolymer was subsequently functionalized with mannose at the terminal end of the copolymer to obtain mannosylated-bPEI-HA (Man-bPEI-HA) copolymer. UV spectroscopy and gel retardation studies confirmed the formation of polyplexes at polymer to DNA weight ratio ≥2. Alamar Blue and MTT assay revealed that the cytotoxicity of the developed nanohybrids were significantly (<i>P</i> < 0.05) lower than that of unmodified bPEI. Mannose functionalization of these nanohybrids showed specificity for both murine and human macrophage-like cell lines RAW 264.7 and human acute monocytic leukemia cell line (THP1), respectively, with a significant level (<i>P</i> < 0.05) of expression of <i>gaussia</i> luciferase (GLuc) and green fluorescent reporter plasmids. Internalization studies indicate that a mannose mediated endocytic pathway is responsible for this higher transfection rate. These results suggest that hyaluronan-based mannosylated nanohybrids could be used as efficient carriers for targeted gene delivery to macrophages

An in situ collagen‐HA hydrogel system promotes survival and preserves the proangiogenic secretion of hiPSC‐derived vascular smooth muscle cells

Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration

Human iPSC-Derived Vascular Smooth Muscle Cells in a Fibronectin Functionalized Collagen Hydrogel Augment Endothelial Cell Morphogenesis

Tissue-engineered constructs have immense potential as autologous grafts for wound healing. Despite the rapid advancement in fabrication technology, the major limitation is controlling angiogenesis within these constructs to form a vascular network. Here, we aimed to develop a 3D hydrogel that can regulate angiogenesis. We tested the effect of fibronectin and vascular smooth muscle cells derived from human induced pluripotent stem cells (hiPSC-VSMC) on the morphogenesis of endothelial cells. The results demonstrate that fibronectin increases the number of EC networks. However, hiPSC-VSMC in the hydrogel further substantiated the number and size of EC networks by vascular endothelial growth factor and basic fibroblast growth factor secretion. A mechanistic study shows that blocking αvβ3 integrin signaling between hiPSC-VSMC and fibronectin impacts the EC network formation via reduced cell viability and proangiogenic growth factor secretion. Collectively, this study set forth initial design criteria in developing an improved pre-vascularized construct