Nanogels (NGs) are nanosized three-dimensional networks of crosslinked hydrophilic polymers. Their tunable chemical structure, physico-chemical properties and potential biocompatibility make them interesting substrates for the synthesis of “nanodevices” with therapeutic and/or diagnostic functions. In this PhD project, NGs were synthesized through pulsed e-beam irradiation of Poly-N-(Vinyl-Pyrrolidone) (PVP) aqueous solutions. The manufacturing process is fast and straightforward; it does not require the employment of organic solvents, surfactants, initiators or catalyst and guarantees high yields of the desired product. In dilute aqueous polymer solutions, ionizing radiation is mainly absorbed by water that undergoes radiolysis. The radiolytically produced hydroxyl radicals (•OH) react with the polymer forming macroradicals, that will further evolve through different competing reactions (combination, disproportionation, chain scission, etc.). Despite of the apparent simplicity of the manufacturing process, the mechanism of nanogel formation is quite complex and not yet fully understood, due to the large variety of system and process parameters and the intermittent nature of initiation reactions. Various particle size and structures can be obtained by changing the irradiation conditions. The most common synthetic approaches and the main outcomes from mechanistic studies are briefly reviewed in Chapter 1. Chapter 2 concerns an experimental study, carried out on dilute PVP aqueous solutions irradiated by pulsed electron beams from an industrial-type irradiator, of the polymer molecular structure modification with the aim of elucidating some of the aspects of the reaction mechanism at the basis of nanogels formation and functionalization. The irradiated polymers were characterized in terms of molecular weight (by gel filtration chromatography), hydrodynamic size (by dynamic light scattering) and chemical structure (by FTIR analysis and colorimetric titrations). Furthermore, the production of H2O2, that can be considered an indicator of the •OH scavenging efficiency of the polymeric solute, was quantitatively measured (by the Ghormley triiodide method). These results were discussed also in the light of numerical simulations of radiation chemistry of water, taking into account the actual irradiation conditions (dose per pulse, frequency, pulse length and total dose) as in the experiments, and also in the presence of various amounts of a molecular hydroxyl radical scavenger. It is interesting to note that substantial amounts of molecular oxygen is produced in N2O-saturated systems. Finally, the application of the Ghormley triiodide method allowed for the quantification of the double bonds formed in the polymer upon irradiation. The double bond concentration build up with dose provides some hints about the type of follow-up reactions of the formed macroradicals, occurring in the various phases of the process. In Chapter 3, nanogels made of crosslinked PVP with a small amount of grafted acrylic acid (PVP-co-AA), produced with one of the two irradiation set-ups illustrated in Chapter 2, are described. These nanogels were synthetized to be used as substrate for the development of biomedical nanodevices to target two different pathologies: Alzheimer’s disease (AD) and ColoRectal cancer (CRC). The PVP-co-AA NGs were characterized in terms of hydrodynamic size, net surface charge, hydrodynamic volume distribution and chemical functionalities, that are useful for the covalent attachment of (bio)molecules of interest. PVP-co-AA NGs were evaluated in terms of in vitro and in vivo biocompatibility, biodistribution, and clearance in mice model. The engineered nanogel system shows encouraging physico-chemical properties and biological response. In fact, NGs show suitable hydrodynamic size, negative surface charge density, biocompatibility (absence of toxicity, proliferative, immunogenic, and thrombogenic responses) and clearance from the bloodstream and urines. In Chapter 4, the synthesis and characterization of an insulin-nanogel conjugate as potential neuroprotective nanodevice in the treatment of AD is presented. Conjugation protocols were developed in order to obtain the desired conjugation degrees and controlled physico-chemical properties. The insulin-nanogel conjugate was also subjected to colloidal stability tests (upon storage, after freeze-drying) and various biological evaluations (in vitro and in vivo insulin biological activity, in vivo biodistribution upon intraperitoneal and intranasal administration) that support the potential of this nanodevice for AD treatment. Concerning the development of nanodevices for the treatment and/or diagnosis of colorectal cancer, PVP-co-AA NGs were evaluated as carriers of the inhibitor of miR-31, a small RNA molecule with an important role in colorectal cancer (CRC) progression, and with Bombesin-DOTA. The latter is a difunctional ligand comprising an active targeting agent (Bombesin) and a chelating agent (DOTA) that – in combination with proper radioisotopes – could be used for either diagnosis or internal radiotherapy. This work is reported in Chapters 5 and 6. Conjugation protocols were developed in order to obtain the desired conjugation degrees and controlled physico-chemical properties. The nanogel-conjugated systems were also subjected to colloidal stability tests (upon storage, after freeze-drying and at high temperature) and to biological evaluations, that encourage to proceed with further evaluation of these nanogel-conjugated systems for the diagnosis and/or treatment of CRC. The studies carried out during this PhD project offer new insights on the mechanism of formation and functionalization of radiation-engineered PVP NGs and show how versatile these nanomaterials are. Only by varying the type of “decoration”, the same substrate can be transformed into different nanodevices that can potentially address the specific diagnostic/therapeutic challenges of a pathologic condition. These promising results encourage (1) to continue to investigate the NGs formation and functionalization mechanisms, by combining experimental and simulation approaches, with the aim of understanding how a specific set of physico-chemical properties and structural modifications can be attained by a selection of a proper set of irradiation conditions and, (2) to proceed further with the biological evaluation of the already developed NG conjugates on relevant in vivo disease models in order to evaluate their efficacy in the prospect of requesting approval for clinical trials.

Ionizing radiation synthesis of multifunctional nanogels for biomedical applications.

Ionizing radiation synthesis of multifunctional nanogels for biomedical applications

Ditta, Lorena Anna

Abstract

Nanogels (NGs) are nanosized three-dimensional networks of crosslinked hydrophilic polymers. Their tunable chemical structure, physico-chemical properties and potential biocompatibility make them interesting substrates for the synthesis of “nanodevices” with therapeutic and/or diagnostic functions. In this PhD project, NGs were synthesized through pulsed e-beam irradiation of Poly-N-(Vinyl-Pyrrolidone) (PVP) aqueous solutions. The manufacturing process is fast and straightforward; it does not require the employment of organic solvents, surfactants, initiators or catalyst and guarantees high yields of the desired product. In dilute aqueous polymer solutions, ionizing radiation is mainly absorbed by water that undergoes radiolysis. The radiolytically produced hydroxyl radicals (•OH) react with the polymer forming macroradicals, that will further evolve through different competing reactions (combination, disproportionation, chain scission, etc.). Despite of the apparent simplicity of the manufacturing process, the mechanism of nanogel formation is quite complex and not yet fully understood, due to the large variety of system and process parameters and the intermittent nature of initiation reactions. Various particle size and structures can be obtained by changing the irradiation conditions. The most common synthetic approaches and the main outcomes from mechanistic studies are briefly reviewed in Chapter 1. Chapter 2 concerns an experimental study, carried out on dilute PVP aqueous solutions irradiated by pulsed electron beams from an industrial-type irradiator, of the polymer molecular structure modification with the aim of elucidating some of the aspects of the reaction mechanism at the basis of nanogels formation and functionalization. The irradiated polymers were characterized in terms of molecular weight (by gel filtration chromatography), hydrodynamic size (by dynamic light scattering) and chemical structure (by FTIR analysis and colorimetric titrations). Furthermore, the production of H2O2, that can be considered an indicator of the •OH scavenging efficiency of the polymeric solute, was quantitatively measured (by the Ghormley triiodide method). These results were discussed also in the light of numerical simulations of radiation chemistry of water, taking into account the actual irradiation conditions (dose per pulse, frequency, pulse length and total dose) as in the experiments, and also in the presence of various amounts of a molecular hydroxyl radical scavenger. It is interesting to note that substantial amounts of molecular oxygen is produced in N2O-saturated systems. Finally, the application of the Ghormley triiodide method allowed for the quantification of the double bonds formed in the polymer upon irradiation. The double bond concentration build up with dose provides some hints about the type of follow-up reactions of the formed macroradicals, occurring in the various phases of the process. In Chapter 3, nanogels made of crosslinked PVP with a small amount of grafted acrylic acid (PVP-co-AA), produced with one of the two irradiation set-ups illustrated in Chapter 2, are described. These nanogels were synthetized to be used as substrate for the development of biomedical nanodevices to target two different pathologies: Alzheimer’s disease (AD) and ColoRectal cancer (CRC). The PVP-co-AA NGs were characterized in terms of hydrodynamic size, net surface charge, hydrodynamic volume distribution and chemical functionalities, that are useful for the covalent attachment of (bio)molecules of interest. PVP-co-AA NGs were evaluated in terms of in vitro and in vivo biocompatibility, biodistribution, and clearance in mice model. The engineered nanogel system shows encouraging physico-chemical properties and biological response. In fact, NGs show suitable hydrodynamic size, negative surface charge density, biocompatibility (absence of toxicity, proliferative, immunogenic, and thrombogenic responses) and clearance from the bloodstream and urines. In Chapter 4, the synthesis and characterization of an insulin-nanogel conjugate as potential neuroprotective nanodevice in the treatment of AD is presented. Conjugation protocols were developed in order to obtain the desired conjugation degrees and controlled physico-chemical properties. The insulin-nanogel conjugate was also subjected to colloidal stability tests (upon storage, after freeze-drying) and various biological evaluations (in vitro and in vivo insulin biological activity, in vivo biodistribution upon intraperitoneal and intranasal administration) that support the potential of this nanodevice for AD treatment. Concerning the development of nanodevices for the treatment and/or diagnosis of colorectal cancer, PVP-co-AA NGs were evaluated as carriers of the inhibitor of miR-31, a small RNA molecule with an important role in colorectal cancer (CRC) progression, and with Bombesin-DOTA. The latter is a difunctional ligand comprising an active targeting agent (Bombesin) and a chelating agent (DOTA) that – in combination with proper radioisotopes – could be used for either diagnosis or internal radiotherapy. This work is reported in Chapters 5 and 6. Conjugation protocols were developed in order to obtain the desired conjugation degrees and controlled physico-chemical properties. The nanogel-conjugated systems were also subjected to colloidal stability tests (upon storage, after freeze-drying and at high temperature) and to biological evaluations, that encourage to proceed with further evaluation of these nanogel-conjugated systems for the diagnosis and/or treatment of CRC. The studies carried out during this PhD project offer new insights on the mechanism of formation and functionalization of radiation-engineered PVP NGs and show how versatile these nanomaterials are. Only by varying the type of “decoration”, the same substrate can be transformed into different nanodevices that can potentially address the specific diagnostic/therapeutic challenges of a pathologic condition. These promising results encourage (1) to continue to investigate the NGs formation and functionalization mechanisms, by combining experimental and simulation approaches, with the aim of understanding how a specific set of physico-chemical properties and structural modifications can be attained by a selection of a proper set of irradiation conditions and, (2) to proceed further with the biological evaluation of the already developed NG conjugates on relevant in vivo disease models in order to evaluate their efficacy in the prospect of requesting approval for clinical trials.
Biomedical nanomaterials
Ionizing radiation
Ionizing radiation synthesis of multifunctional nanogels for biomedical applications.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10447/265691
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