In this Ph.D. thesis several kinds of metal-based NPs were tested in Xenopus laevis oocytes, in order to understand the metal NP related toxicity. The mechanism of the metals entrance in the cell and the passive permeation hypothesis were investigated. The results can be categorized as follows: • The metallic NPs tested (Co, Fe, Ni) dissolve more than their oxide counterparts; however, they are unstable in suspension and their aggregation rate is fast. All these particles are not able to induce modifications in membrane biophysical parameters and in intracellular metal concentration, thereby excluding a direct membrane crossing mechanism for their internalization. • The metal oxide NPs which retain a population below 200 nm (Fe3O4, Co3O4) in suspension in experimental medium induced significant modifications in membrane biophysical parameters and in intracellular metal concentration. The effects are recorded only when the population of NPs smaller than 200 nm is present, namely only within 5 min from the particles addition to the medium; after this time range, effects on membrane become milder until they disappear, stating the complete fast recovery of the membrane. • The metal oxide NPs that never go below 200 nm (NiO, Fe2O3) in suspension in the experimental medium do not cross the membrane and do not modify neither intracellular metal concentration nor membrane biophysical parameters. • The surface coating modifies particles behavior in suspension in the experimental medium. The non-covalent coating by BSA stabilized the 200 nm population for Co3O4 and Fe3O4 NPs but abolished the modifications in intracellular metal concentration and in membrane biophysical parameters. The covalent coating by APTES of Fe2O3 NPs partially stabilized particles around 100 nm but failed to induce membrane crossing. The role of particle surface is thus crucial to achieve direct membrane crossing. Many of the studies conducted nowadays on direct membrane crossing rely on models, both in silico as well as in vitro, that are limited in replicating the biological complexity of the cells. Using voltage clamp and fluorescent probe on Xenopus Laevis oocytes may lay the foundation for an innovative screening platform, allowing to study the internalization and the effects on membrane. Xenopus laevis oocytes are easy to collect, maintain and prepare. They are flexible and adaptable to different culture conditions, allowing to study interactions in controlled environment. Moreover, Xenopus laevis oocytes can also be a useful tool to study and optimize targeted internalization. Their historical use as heterologous expression system is well known, selectively express target proteins to study specific internalization pathways of new modified nanoparticles will be one of the new goals for the therapeutic use of nanomaterial.

Metal nanoparticle permeation through the plasma membrane: Xenopus laevis oocytes as novel tools for membrane permeability evaluation and physico-chemical characterization of particle properties / Zanella, Daniele. - (2019).

Metal nanoparticle permeation through the plasma membrane: Xenopus laevis oocytes as novel tools for membrane permeability evaluation and physico-chemical characterization of particle properties.

Zanella, Daniele
2019-01-01

Abstract

In this Ph.D. thesis several kinds of metal-based NPs were tested in Xenopus laevis oocytes, in order to understand the metal NP related toxicity. The mechanism of the metals entrance in the cell and the passive permeation hypothesis were investigated. The results can be categorized as follows: • The metallic NPs tested (Co, Fe, Ni) dissolve more than their oxide counterparts; however, they are unstable in suspension and their aggregation rate is fast. All these particles are not able to induce modifications in membrane biophysical parameters and in intracellular metal concentration, thereby excluding a direct membrane crossing mechanism for their internalization. • The metal oxide NPs which retain a population below 200 nm (Fe3O4, Co3O4) in suspension in experimental medium induced significant modifications in membrane biophysical parameters and in intracellular metal concentration. The effects are recorded only when the population of NPs smaller than 200 nm is present, namely only within 5 min from the particles addition to the medium; after this time range, effects on membrane become milder until they disappear, stating the complete fast recovery of the membrane. • The metal oxide NPs that never go below 200 nm (NiO, Fe2O3) in suspension in the experimental medium do not cross the membrane and do not modify neither intracellular metal concentration nor membrane biophysical parameters. • The surface coating modifies particles behavior in suspension in the experimental medium. The non-covalent coating by BSA stabilized the 200 nm population for Co3O4 and Fe3O4 NPs but abolished the modifications in intracellular metal concentration and in membrane biophysical parameters. The covalent coating by APTES of Fe2O3 NPs partially stabilized particles around 100 nm but failed to induce membrane crossing. The role of particle surface is thus crucial to achieve direct membrane crossing. Many of the studies conducted nowadays on direct membrane crossing rely on models, both in silico as well as in vitro, that are limited in replicating the biological complexity of the cells. Using voltage clamp and fluorescent probe on Xenopus Laevis oocytes may lay the foundation for an innovative screening platform, allowing to study the internalization and the effects on membrane. Xenopus laevis oocytes are easy to collect, maintain and prepare. They are flexible and adaptable to different culture conditions, allowing to study interactions in controlled environment. Moreover, Xenopus laevis oocytes can also be a useful tool to study and optimize targeted internalization. Their historical use as heterologous expression system is well known, selectively express target proteins to study specific internalization pathways of new modified nanoparticles will be one of the new goals for the therapeutic use of nanomaterial.
metal NPs, plasma membrane, Xenopus laevis oocytes, membrane permeability
Metal nanoparticle permeation through the plasma membrane: Xenopus laevis oocytes as novel tools for membrane permeability evaluation and physico-chemical characterization of particle properties / Zanella, Daniele. - (2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11383/2090679
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