Engineering of Graphene Materials for Biomedical Applications - From graphite to new functionalized graphene by top down-approaches

The discovery of graphene (G) and its functionalized derivatives has been accompanied by increasing research attention to explore these new materials for biomedical applications due to their unique structure and excellent mechanical, optical and electrical properties. Generally, G synthetic methods are classified in two categories: top-down and bottom-up. The former approach entails exfoliation of G layers from graphitic materials. The latter approach involves the building up of G using carbon-based materials. The bottom-up approach is simple, although it produces material with relatively more defects than the top-down approach. Top-down strategies separate the stacked sheets by disrupting the Van der Waals forces that hold the sheets together. Damaging of the sheets during the exfoliation process and re-agglomeration of the separated sheets are some of the disadvantages of the top-down techniques. The biocompatibility of G is the most critical issue for the applications in the biomedical/pharmaceutical field and its chemical modification is the key factor for designing stable and safe drug delivery nanodevices. It is known that size, shape, morphology, thickness, degree of functionalization and dispersibility of G play critical roles in regulating biological behaviours and toxicity. Therefore, the development of adequate G preparation methods represents a key factor to achieve good G materials for biomedical applications. The thesis describes the synthesis and the characterization by complementary techniques of new functionalized G materials obtained by two different top-down approaches. Moreover, some biomedical applications of newly synthesized G materials have been investigated. The thesis is structured in four chapters. The first chapter reports an overview of the main characteristics of G materials and describes the currently available procedures for their fabrication and summarizes the main biological applications. The chapter II reports the functionalization of G materials with the terminal alkyne units and their synthetic applications by click assembly with azido-functionalized compounds (Fig. 1). The alkyne-terminated graphene platform (G-Alk) was obtained in good yield by reduction of graphene oxide (GO) with hydrazine followed via grafting of p-(2-propynyloxy)-benzene units by reaction with diazonium salts. The click assembly of G-Alk with the azido flavonoid Silibinin (Sil) provided a new drug delivery nanoplatform (herein called G-Sil). The cytotoxicity of the new platform has been evaluated on human mesenchymal stem cells and the anticancer effects have been studied on human osteosarcoma cell lines. Our G nanoplatform did not show any cytotoxicity even at high concentration (1000 μg/ml) and Sil grafted onto G maintained its antiproliferative activity. The click reaction of G-Alk with the oxazolone (APDMA) containing an azido moiety has been used to build the reactive G platform (RAGP). The RAGP has been employed for the silylation of G surfaces and for the selective conjugation with compounds of biological interest such as glutathione (GSSG) and catalase (C). The chapter III reports the direct delamination and functionalization of graphite into G, exploiting the reactivity of mesoionic compounds in solvent-free conditions. For the first time we have demonstrated that the solvent-free 1,3-DC reaction of mesoionic compounds is an effective tool for the direct functionalization and delamination of graphite flakes into few layers of G nanosheets. The procedure has been tested by employing two differently substituted oxazolones. For both substrates under mild conditions (70–120 °C) a high degree of functionalization (2.1–4.6%@700 °C) was obtained. Moreover, the graphite exfoliation efficiency depends upon the oxazolone substitution pattern. The exfoliation and functionalization was confirmed by micro-Raman and X-ray photoelectron spectroscopies, scanning transmission electron microscopy (STEM) and thermogravimetric analysis (TGA). Furthermore computational studies showed that the reaction proceed mainly on the corner and on the edge of graphenic system. These data were confirmed by decoration of the surface G layers with gold nanoparticles (Au NPs). STEM analysis showed that Au NPs are mainly located on edge of G layers. Finally, the properties of G-NH2/Au nanocomposites as SERS materials have been investigated using Rhodamine 6G (R6G) as a probe. The final chapter (chapter IV) is focused on the development of a coiled-coil peptide system for gene-delivery. This section reports the results of the research project carried out within the Biotechnology group at the National Physical Laboratory of London (NPL, UK), under the supervision of Dr Max Ryadnov and Dr Emiliana De Santis. This chapter reports the characterization studies of peptide (SD1). TEM analysis has been used to confirm the formation of peptide sphere structures by self-assembling process; qualitative tritations have been employed to prove the formation of SD1/siRNA complex. From these studies, SD1 emerged as a good candidate for gene-delivery; actually, SD1/siRNA complex is under biological evaluation.