Abstract Biomimetic and bioinspired materials have clearly conquered a special position in the fi... more Abstract Biomimetic and bioinspired materials have clearly conquered a special position in the field of drug delivery. Due to their similarities with biologically relevant structures, either at the chemical, physical, or morphological level, they are expected to solve caveats of the current existing solutions and even outperform many of the currently available materials. Here we present the design and preparation of five of the most promising classes of bioinspired and biomimetic materials for application as drug-delivery systems. Particular focus was given to nucleic acid-based and dendrimer-based systems. These constitute newer classes of building blocks and their potential has been contributing to the growing interest in their use.
Cells are continuously exposed to physical forces and the central nervous system (CNS) is no exce... more Cells are continuously exposed to physical forces and the central nervous system (CNS) is no exception. Cells dynamically adapt their behavior and remodel the surrounding environment in response to forces. The importance of mechanotransduction in the CNS is illustrated by exploring its role in CNS pathology development and progression. The crosstalk between the biochemical and biophysical components of the extracellular matrix (ECM) are here described, considering the recent explosion of literature demonstrating the powerful influence of biophysical stimuli like density, rigidity and geometry of the ECM on cell behavior. This review aims at integrating mechanical properties into our understanding of the molecular basis of CNS disease. The mechanisms that mediate mechanotransduction events, like integrin, Rho/ROCK and matrix metalloproteinases signaling pathways are revised. Analysis of CNS pathologies in this context has revealed that a wide range of neurological diseases share as h...
Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and r... more Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and represent a growing concern worldwide. Despite the high social and clinical interest in trying to solve these pathologies, there are many challenges to bridge in order to achieve an effective therapy. One of the main obstacles to advancements in this field that has hampered many of the therapeutic strategies proposed to date is the presence of the CNS barriers that restrict the access to the brain. However, adequate brain biodistribution and neuronal cells specific accumulation in the targeted site also represent major hurdles to the attainment of a successful CNS treatment. Over the last few years, nanotechnology has taken a step forward towards the development of therapeutics in neurologic diseases and different approaches have been developed to surpass these obstacles. The versatility of the designed nanocarriers in terms of physical and chemical properties, and the possibility to func...
After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment ... more After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favorable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration-RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture...
The ability to design nanoparticle delivery systems capable of selectively target their payloads ... more The ability to design nanoparticle delivery systems capable of selectively target their payloads to specific cell populations is still a major caveat in nanomedicine. One of the main hurdles is the fact that each nanoparticle formulation needs to be precisely tuned to match the specificities of the target cell and route of administration. In this work, molecular recognition force spectroscopy (MRFS) is presented as a tool to evaluate the specificity of neuron-targeted trimethyl chitosan nanoparticles to neuronal cell populations in biological samples of different complexity. The use of atomic force microscopy tips functionalized with targeted or non-targeted nanoparticles made it possible to assess the specific interaction of each formulation with determined cell surface receptors in a precise fashion. More importantly, the combination of MRFS with fluorescent microscopy allowed to probe the nanoparticles vectoring capacity in models of high complexity, such as primary mixed culture...
To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor ... more To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor cells (NSPCs) and its capacity to support NSPC migration and neurite extension, we explored the tethering of synthetic peptides engaging integrin α6β1, a cell receptor enriched in NSPCs. Six α6β1 integrin ligands were tested for their ability to support integrin α6β1-mediated adhesion of embryonic stem cell-derived NSPCs (ES-NSPs) and sustain ES-NSPC viability, migration, and neuronal differentiation. Due to their better performance, peptides T1, HYD1, and A5G81 were immobilized into fibrin and functionalized gels characterized in terms of peptide binding efficiency, structure and viscoelastic properties. Tethering of T1 or HYD1 successfully enhanced cell outgrowth from ES-NSPC neurospheres (up to 2.4-fold increase), which exhibited a biphasic response to peptide concentration. Inhibition assays evidenced the involvement of α6β1 and α3β1 integrins in mediating radial outgrowth on T1-/HYD...
Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we p... more Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerve...
Journal of tissue engineering and regenerative medicine, Jan 29, 2016
In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibr... more In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% βIII-tubulin(+) cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic...
Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-b... more Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-based culture system to provide insight into vector performance and safety. Using compartmentalized neuron cultures we dissected nanoparticle bioactivity upon delivery taking advantage of (quantitative) bioimaging tools. Targeted and nontargeted nanoparticles were internalized at axon terminals and retrogradely transported to cell bodies at similar average velocities but the former have shown an axonal flux 2.7-times superior to nontargeted nanoparticles, suggesting an improved cargo-transportation efficiency. The peripheral administration of nanoparticles to axon terminals is nontoxic as compared with their direct administration to the cell body or whole neuron. A neuron-targeted nanoparticle system was put forward. Microfluidic-based neuron cultures are proposed as a powerful tool to investigate nanoparticle bio-performance.
Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid ... more Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid fundamental knowledge on the etiology of many of the major neurodegenerative diseases has made it difficult to obtain effective therapies to treat these conditions. Scientists have been looking to carry out more-human-relevant studies, with strong statistical power, to overcome the limitations of preclinical animal models that have contributed to the failure of numerous therapeutics in clinical trials. Here, we identify currently existing platforms to mimic central nervous system tissues, healthy and diseased, mainly focusing on cell-based platforms and discussing their strengths and limitations in the context of the high-throughput screening of new therapeutic targets and drugs.
Abstract Biomimetic and bioinspired materials have clearly conquered a special position in the fi... more Abstract Biomimetic and bioinspired materials have clearly conquered a special position in the field of drug delivery. Due to their similarities with biologically relevant structures, either at the chemical, physical, or morphological level, they are expected to solve caveats of the current existing solutions and even outperform many of the currently available materials. Here we present the design and preparation of five of the most promising classes of bioinspired and biomimetic materials for application as drug-delivery systems. Particular focus was given to nucleic acid-based and dendrimer-based systems. These constitute newer classes of building blocks and their potential has been contributing to the growing interest in their use.
Cells are continuously exposed to physical forces and the central nervous system (CNS) is no exce... more Cells are continuously exposed to physical forces and the central nervous system (CNS) is no exception. Cells dynamically adapt their behavior and remodel the surrounding environment in response to forces. The importance of mechanotransduction in the CNS is illustrated by exploring its role in CNS pathology development and progression. The crosstalk between the biochemical and biophysical components of the extracellular matrix (ECM) are here described, considering the recent explosion of literature demonstrating the powerful influence of biophysical stimuli like density, rigidity and geometry of the ECM on cell behavior. This review aims at integrating mechanical properties into our understanding of the molecular basis of CNS disease. The mechanisms that mediate mechanotransduction events, like integrin, Rho/ROCK and matrix metalloproteinases signaling pathways are revised. Analysis of CNS pathologies in this context has revealed that a wide range of neurological diseases share as h...
Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and r... more Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and represent a growing concern worldwide. Despite the high social and clinical interest in trying to solve these pathologies, there are many challenges to bridge in order to achieve an effective therapy. One of the main obstacles to advancements in this field that has hampered many of the therapeutic strategies proposed to date is the presence of the CNS barriers that restrict the access to the brain. However, adequate brain biodistribution and neuronal cells specific accumulation in the targeted site also represent major hurdles to the attainment of a successful CNS treatment. Over the last few years, nanotechnology has taken a step forward towards the development of therapeutics in neurologic diseases and different approaches have been developed to surpass these obstacles. The versatility of the designed nanocarriers in terms of physical and chemical properties, and the possibility to func...
After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment ... more After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favorable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration-RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture...
The ability to design nanoparticle delivery systems capable of selectively target their payloads ... more The ability to design nanoparticle delivery systems capable of selectively target their payloads to specific cell populations is still a major caveat in nanomedicine. One of the main hurdles is the fact that each nanoparticle formulation needs to be precisely tuned to match the specificities of the target cell and route of administration. In this work, molecular recognition force spectroscopy (MRFS) is presented as a tool to evaluate the specificity of neuron-targeted trimethyl chitosan nanoparticles to neuronal cell populations in biological samples of different complexity. The use of atomic force microscopy tips functionalized with targeted or non-targeted nanoparticles made it possible to assess the specific interaction of each formulation with determined cell surface receptors in a precise fashion. More importantly, the combination of MRFS with fluorescent microscopy allowed to probe the nanoparticles vectoring capacity in models of high complexity, such as primary mixed culture...
To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor ... more To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor cells (NSPCs) and its capacity to support NSPC migration and neurite extension, we explored the tethering of synthetic peptides engaging integrin α6β1, a cell receptor enriched in NSPCs. Six α6β1 integrin ligands were tested for their ability to support integrin α6β1-mediated adhesion of embryonic stem cell-derived NSPCs (ES-NSPs) and sustain ES-NSPC viability, migration, and neuronal differentiation. Due to their better performance, peptides T1, HYD1, and A5G81 were immobilized into fibrin and functionalized gels characterized in terms of peptide binding efficiency, structure and viscoelastic properties. Tethering of T1 or HYD1 successfully enhanced cell outgrowth from ES-NSPC neurospheres (up to 2.4-fold increase), which exhibited a biphasic response to peptide concentration. Inhibition assays evidenced the involvement of α6β1 and α3β1 integrins in mediating radial outgrowth on T1-/HYD...
Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we p... more Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerve...
Journal of tissue engineering and regenerative medicine, Jan 29, 2016
In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibr... more In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% βIII-tubulin(+) cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic...
Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-b... more Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-based culture system to provide insight into vector performance and safety. Using compartmentalized neuron cultures we dissected nanoparticle bioactivity upon delivery taking advantage of (quantitative) bioimaging tools. Targeted and nontargeted nanoparticles were internalized at axon terminals and retrogradely transported to cell bodies at similar average velocities but the former have shown an axonal flux 2.7-times superior to nontargeted nanoparticles, suggesting an improved cargo-transportation efficiency. The peripheral administration of nanoparticles to axon terminals is nontoxic as compared with their direct administration to the cell body or whole neuron. A neuron-targeted nanoparticle system was put forward. Microfluidic-based neuron cultures are proposed as a powerful tool to investigate nanoparticle bio-performance.
Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid ... more Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid fundamental knowledge on the etiology of many of the major neurodegenerative diseases has made it difficult to obtain effective therapies to treat these conditions. Scientists have been looking to carry out more-human-relevant studies, with strong statistical power, to overcome the limitations of preclinical animal models that have contributed to the failure of numerous therapeutics in clinical trials. Here, we identify currently existing platforms to mimic central nervous system tissues, healthy and diseased, mainly focusing on cell-based platforms and discussing their strengths and limitations in the context of the high-throughput screening of new therapeutic targets and drugs.
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Papers by Ana Pêgo