CARBON NANODOTS: BUILDING BLOCKS FOR THE PRECISE SYNTHESIS OF NANO- AND MICROSTRUCTURES

Engineering microcompartments to exhibit life-like behaviors is one of the major challenges for bottom-up synthetic biology and nanotechnology. These microcompartments are defined as protocells when mimicking key biological functions, such as compartmentalization, selective permeability, and catalytic activity. One emerging category of protocells is colloidosomes, micro compartmentalized assemblies built from amphiphilic colloidal particles. Organic colloidosomes, typically composed of protein-based nanocomposites, have drawbacks such as low stability over time and high production costs. In contrast, Carbon NanoDots (CNDs) offer significant advantages due to their unique physicochemical properties, including excellent photostability, tunable fluorescence, low toxicity, and ease of functionalization. These properties position CNDs as promising platforms for fabricating more complex structures. The objective of this Thesis is to exploit CNDs as building blocks for protocell synthesis while investigating the composition, superficial chemistry, and reactivity of these carbon nanomaterials with molecular and nanomaterial species. In the first chapter, the characterization of CNDs using multi-detector Gel Permeation Chromatography is explored. Initially, the synthesis and purification of L-arginine and ethylenediamine-derived CNDs are evaluated through various characterization techniques. Then, GPC analyses are employed to determine the molecular weight moments and distribution of different CND batches, supported by ζ-potential and Dynamic Light Scattering measurements. The investigation extends to quantify and exploit CND surface functionalities for derivatization reactions, revealing insights into their interfacial reactivity. The second chapter investigates the exploitation of interfacial reactivity of CNDs for their covalent assembly via Copper(I)-catalyzed azide-alkyne cycloaddition to form carbon-based suprastructures. Functionalized CNDs are used to construct these suprastructures and their optical and photophysical properties are examined, revealing energy transfer processes between different types of emitting CNDs. The third chapter discusses the synthesis and characterization of the amphiphilic units for colloidosomes fabrication, namely CND/polymer nanoconjugates. The chapter initially focuses on the design and synthesis of an N-isopropyl acrylamide-based copolymer through Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization and the study of its thermoresponsive properties. The CND/polymer nanoconjugates retained the key characteristics of both the CNDs and copolymers, and the study of their composition are carried out by multi detector GPC. Finally, the fourth chapter investigates the fabrication of CND-based colloidosomes via the Pickering emulsion technique. After engineering these microcapsules, they are characterized using microscopy techniques and evaluated for their enzyme-like activity, representing a significant advancement toward developing a new type of protocells.

Engineering microcompartments to exhibit life-like behaviors is one of the major challenges for bottom-up synthetic biology and nanotechnology. These microcompartments are defined as protocells when mimicking key biological functions, such as compartmentalization, selective permeability, and catalytic activity. One emerging category of protocells is colloidosomes, micro compartmentalized assemblies built from amphiphilic colloidal particles. Organic colloidosomes, typically composed of protein-based nanocomposites, have drawbacks such as low stability over time and high production costs. In contrast, Carbon NanoDots (CNDs) offer significant advantages due to their unique physicochemical properties, including excellent photostability, tunable fluorescence, low toxicity, and ease of functionalization. These properties position CNDs as promising platforms for fabricating more complex structures. The objective of this Thesis is to exploit CNDs as building blocks for protocell synthesis while investigating the composition, superficial chemistry, and reactivity of these carbon nanomaterials with molecular and nanomaterial species. In the first chapter, the characterization of CNDs using multi-detector Gel Permeation Chromatography is explored. Initially, the synthesis and purification of L-arginine and ethylenediamine-derived CNDs are evaluated through various characterization techniques. Then, GPC analyses are employed to determine the molecular weight moments and distribution of different CND batches, supported by ζ-potential and Dynamic Light Scattering measurements. The investigation extends to quantify and exploit CND surface functionalities for derivatization reactions, revealing insights into their interfacial reactivity. The second chapter investigates the exploitation of interfacial reactivity of CNDs for their covalent assembly via Copper(I)-catalyzed azide-alkyne cycloaddition to form carbon-based suprastructures. Functionalized CNDs are used to construct these suprastructures and their optical and photophysical properties are examined, revealing energy transfer processes between different types of emitting CNDs. The third chapter discusses the synthesis and characterization of the amphiphilic units for colloidosomes fabrication, namely CND/polymer nanoconjugates. The chapter initially focuses on the design and synthesis of an N-isopropyl acrylamide-based copolymer through Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization and the study of its thermoresponsive properties. The CND/polymer nanoconjugates retained the key characteristics of both the CNDs and copolymers, and the study of their composition are carried out by multi detector GPC. Finally, the fourth chapter investigates the fabrication of CND-based colloidosomes via the Pickering emulsion technique. After engineering these microcapsules, they are characterized using microscopy techniques and evaluated for their enzyme-like activity, representing a significant advancement toward developing a new type of protocells.