In the Western World, pathologies related to cardiovascular apparatus as coronary and peripheral arterial represent one of the prevalent causes of mortality. Atherosclerosis is an inflammatory and progressive disease characterized by the presence of an accumulation of low-density lipoprotein. The accumulation is commonly called plaque and it can occlude the blood flow in the arteries. The evolution of the plaque can represent a target in all stages of its progression. In the early-stage of atherosclerotic plaque, nanocarriers can be used as a system for targeted drug deliver, and during its progression it could be necessary to replace the vessel by using innovative engineered biodegradable vascular grafts. Nanoparticles can inhibit the progression of the plaque when correctly functionalized on the surface to target the atheromatous site. The first part of this Ph.D. thesis is focused on the production of nanoparticles with a comparative study between liposomes produced trough the thin layer hydration technique and polylactic co glycolic acid (PLGA)-based nanoparticles produced using the double emulsion technique. In this preliminary work, both carriers were loaded with different concentrations of albumin as model protein. A study of morphology, dimension, size, entrapment efficiency, cytocompatibility, and hemocompatibility was performed. Both particles analyzed present positive points however, the studied polymeric nanocarriers can be considered as a good model to be engineered with antibodies on their surface in order to be employed in the vascular field as a nanosystem for protein drug delivery. A second work on PLGA nanoparticles was performed at the Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S) at the University of Porto, under the supervision of Professor Bruno Sarmento. Here, it was optimized the encapsulation of bevacizumab, a commercial antibody, Avastin®, active against the vascular endothelial growth factor (VEGF) receptor commonly expressed at the atheromatic site. The functionalization process with immunouteroglobin-1 (IUG1) a human recombinant antibody (scFv) specific for extra-domain B (EDB) of fibronectin, a well-known marker of angiogenesis was considered and tested. These polymeric and functionalized nanoparticles were analyzed in terms of morphology, size, ζ-potential, entrapment efficiency, conjugation efficiency, release studies at 37° C, FTIR, CD, biocompatibility with endothelial cells (EA.hy926), macrophage (RAW 246.7), and hemocompatibility. This part of the work involves an innovative therapeutic approach for the treatment of the early-stage of atherosclerosis, the most common disease in cardiovascular field. The second part of this Ph.D. thesis is focused on the development of synthetic grafts for vessels with a diameter smaller than 6 mm. In the clinical practice, when the vessel is obstructed by the atheromatous plaque is essential to reconstruct the blood flow, maintaining all the functions of the downstream tissue, is necessary to introduce a bypass. It is preferable to use autologous veins but, if not available, the use of synthetic grafts is required. Non-biodegradable vascular prostheses such as poly (ethylene terephthalate fibers and expanded poly (tetrafluoroethylene), commercially known as Dacron® and Gore-Tex®, respectively) have been successfully used as vascular grafts for aortic and iliac replacement. These materials are not used to substitute small caliber vessels (inner diameter < 6 mm) because the longer contact of the blood with the wall of the vessels due to the lower pressure can induce thrombogenic event, there is the absence of endothelialization after the implantation, it can occur infection of the synthetic grafts and related inflammatory response to the foreign materials. Nowadays, it looks essential to fabricate vessel grafts to replace these small vessels, and several approaches have been used to fabricate small-caliber blood vessel grafts as 3D printing, film casting, electrospinning, decellularization matrices from animals, and cell-seeding. An ideal vascular graft should be biodegradable acting as a scaffold to help the regeneration of a native vessel. The mechanical properties result to be essential to mimic the native arteries, being resistant to thrombosis, dilatation, good flexibility, and no kinking, showing good suture retention, hemocompatibility and have an appropriate degradation kinetic. Given these problems in the clinical procedure, small caliber vascular prostheses made of biodegradable and bioabsorbable polymers through electrospinning have been developed through electrospinning and then functionalize with bioactive compounds. The electrospinning technique is considered one of the most promising methods in the tissue engineering area, in particular for the development of synthetic vascular tissue producing structures similar to the extracellular matrix. In this process, the solvent evaporates immediately, and the applied electrical fields induces the deposition of the polymeric solution on a tubular collector. The prostheses thus produced, are composed of a blend of two different polymers: poly (ε-caprolactone) (PCL) and poly (glycerol sebacate) (PGS) in a ratio 1:1 both 20 % m/v. These constructs, due to the high permeability, need a coating with gelatin which significantly reduces the water permeability. One of the aims of this project is to produce grafts that can influence the cells inducing the regeneration process. Furthermore, the post-surgical inflammation is able to negatively affect the cellular attachment and can induce several damages to the structure of the biomaterials compromising it’s the characteristics. In order to modulate the inflammation, scaffolds were functionalized with antioxidants. Quercetin (3, 3’, 4’, 5, 7-pentahydroxyflavone) is an antioxidant popular in different kinds of fruit and vegetables. The addition of quercetin to the studied polymeric vascular grafts did not change both their morphology and their overall performances. Also, the presence of quercetin counteracted the activity of TNF-α inducing a decrease in MMP-9 expression level, showing the capability to modulate the inflammatory process. To develop a suitable construct is important to study its behavior in different conditions; for this reason, a study in static-conditions, dynamic-conditions and a study in bioreactor have been performed. The latter was fundamental to understand the behavior under different pression regimes mimicking the peripherical district. During or at the end of the experimentations vascular grafts were always by characterize at different point of view: morphological (SEM, fiber distribution and dimensions), chemical physical (release of bioactive compounds, degradation , fluid uptake and degradation at different pH ), mechanical (tensile strength, young modulus and elongation percentage). The novelty of this second part is the fabrication of small-diameter biodegradable and bioabsorbable vascular graft and its characterization in several conditions (Static, dynaminc and bioreactor) showing promising results.

Innovative tools for vascular disease

DE NEGRI ATANASIO, GIULIA
2021-06-10

Abstract

In the Western World, pathologies related to cardiovascular apparatus as coronary and peripheral arterial represent one of the prevalent causes of mortality. Atherosclerosis is an inflammatory and progressive disease characterized by the presence of an accumulation of low-density lipoprotein. The accumulation is commonly called plaque and it can occlude the blood flow in the arteries. The evolution of the plaque can represent a target in all stages of its progression. In the early-stage of atherosclerotic plaque, nanocarriers can be used as a system for targeted drug deliver, and during its progression it could be necessary to replace the vessel by using innovative engineered biodegradable vascular grafts. Nanoparticles can inhibit the progression of the plaque when correctly functionalized on the surface to target the atheromatous site. The first part of this Ph.D. thesis is focused on the production of nanoparticles with a comparative study between liposomes produced trough the thin layer hydration technique and polylactic co glycolic acid (PLGA)-based nanoparticles produced using the double emulsion technique. In this preliminary work, both carriers were loaded with different concentrations of albumin as model protein. A study of morphology, dimension, size, entrapment efficiency, cytocompatibility, and hemocompatibility was performed. Both particles analyzed present positive points however, the studied polymeric nanocarriers can be considered as a good model to be engineered with antibodies on their surface in order to be employed in the vascular field as a nanosystem for protein drug delivery. A second work on PLGA nanoparticles was performed at the Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S) at the University of Porto, under the supervision of Professor Bruno Sarmento. Here, it was optimized the encapsulation of bevacizumab, a commercial antibody, Avastin®, active against the vascular endothelial growth factor (VEGF) receptor commonly expressed at the atheromatic site. The functionalization process with immunouteroglobin-1 (IUG1) a human recombinant antibody (scFv) specific for extra-domain B (EDB) of fibronectin, a well-known marker of angiogenesis was considered and tested. These polymeric and functionalized nanoparticles were analyzed in terms of morphology, size, ζ-potential, entrapment efficiency, conjugation efficiency, release studies at 37° C, FTIR, CD, biocompatibility with endothelial cells (EA.hy926), macrophage (RAW 246.7), and hemocompatibility. This part of the work involves an innovative therapeutic approach for the treatment of the early-stage of atherosclerosis, the most common disease in cardiovascular field. The second part of this Ph.D. thesis is focused on the development of synthetic grafts for vessels with a diameter smaller than 6 mm. In the clinical practice, when the vessel is obstructed by the atheromatous plaque is essential to reconstruct the blood flow, maintaining all the functions of the downstream tissue, is necessary to introduce a bypass. It is preferable to use autologous veins but, if not available, the use of synthetic grafts is required. Non-biodegradable vascular prostheses such as poly (ethylene terephthalate fibers and expanded poly (tetrafluoroethylene), commercially known as Dacron® and Gore-Tex®, respectively) have been successfully used as vascular grafts for aortic and iliac replacement. These materials are not used to substitute small caliber vessels (inner diameter < 6 mm) because the longer contact of the blood with the wall of the vessels due to the lower pressure can induce thrombogenic event, there is the absence of endothelialization after the implantation, it can occur infection of the synthetic grafts and related inflammatory response to the foreign materials. Nowadays, it looks essential to fabricate vessel grafts to replace these small vessels, and several approaches have been used to fabricate small-caliber blood vessel grafts as 3D printing, film casting, electrospinning, decellularization matrices from animals, and cell-seeding. An ideal vascular graft should be biodegradable acting as a scaffold to help the regeneration of a native vessel. The mechanical properties result to be essential to mimic the native arteries, being resistant to thrombosis, dilatation, good flexibility, and no kinking, showing good suture retention, hemocompatibility and have an appropriate degradation kinetic. Given these problems in the clinical procedure, small caliber vascular prostheses made of biodegradable and bioabsorbable polymers through electrospinning have been developed through electrospinning and then functionalize with bioactive compounds. The electrospinning technique is considered one of the most promising methods in the tissue engineering area, in particular for the development of synthetic vascular tissue producing structures similar to the extracellular matrix. In this process, the solvent evaporates immediately, and the applied electrical fields induces the deposition of the polymeric solution on a tubular collector. The prostheses thus produced, are composed of a blend of two different polymers: poly (ε-caprolactone) (PCL) and poly (glycerol sebacate) (PGS) in a ratio 1:1 both 20 % m/v. These constructs, due to the high permeability, need a coating with gelatin which significantly reduces the water permeability. One of the aims of this project is to produce grafts that can influence the cells inducing the regeneration process. Furthermore, the post-surgical inflammation is able to negatively affect the cellular attachment and can induce several damages to the structure of the biomaterials compromising it’s the characteristics. In order to modulate the inflammation, scaffolds were functionalized with antioxidants. Quercetin (3, 3’, 4’, 5, 7-pentahydroxyflavone) is an antioxidant popular in different kinds of fruit and vegetables. The addition of quercetin to the studied polymeric vascular grafts did not change both their morphology and their overall performances. Also, the presence of quercetin counteracted the activity of TNF-α inducing a decrease in MMP-9 expression level, showing the capability to modulate the inflammatory process. To develop a suitable construct is important to study its behavior in different conditions; for this reason, a study in static-conditions, dynamic-conditions and a study in bioreactor have been performed. The latter was fundamental to understand the behavior under different pression regimes mimicking the peripherical district. During or at the end of the experimentations vascular grafts were always by characterize at different point of view: morphological (SEM, fiber distribution and dimensions), chemical physical (release of bioactive compounds, degradation , fluid uptake and degradation at different pH ), mechanical (tensile strength, young modulus and elongation percentage). The novelty of this second part is the fabrication of small-diameter biodegradable and bioabsorbable vascular graft and its characterization in several conditions (Static, dynaminc and bioreactor) showing promising results.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/1047397
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