Carbon nanotube and graphene cellular impact towards their possible use as nanovectors for anticancer therapy and cell targeting

In the last two decades, lots of progresses have been made in the application of nanomaterials in biology, giving rise to a new revolutionary field of the biomedical research that is called nanomedicine. Carbon nanotubes (CNTs) and graphene are among the most attractive candidates under investigation in this context. Such carbon-based nanomaterials possess outstanding chemico-physical properties and their high surface area provides multiple attachment sites for almost all the molecules of biological interest. In fact, chemical strategies allow for their conjugation with DNA, proteins, peptides and small drugs. The so-functionalized materials have shown great promises in several biomedical contexts, from diagnosis to tissue regeneration. In addition, the ability of CNTs and graphene to enter and accumulate inside the cells makes them good candidates as nanovectors for drugs. In vitro and in vivo studies showed how these nanomaterials could enhance the drug accumulation inside the cells and its bioavailability. CNTs and graphene are also able to passively target tumor tissues exploiting the so-called enhanced permeability and retention effect, and actively, following the conjugation with targeting moieties. On the other hand, the use of these materials in nanomedicine will be approved only after the demonstration of their safety in terms of tissue damage, carcinogenicity and proinflammatory response. Different approaches have been used to make these materials biocompatible and recently, it has been demonstrated that functionalized CNTs and graphene oxide can be degraded by oxidative enzymes. However, the results obtained on CNT and graphene toxicity are often conflicting; thus, further investigations are needed. In the first introductive chapter of this thesis, an overview of the general features of nanomaterials is given. The focus goes then on CNTs and graphene. First, we will see how their biocompatibility, biodegradability and in vivo fate strictly depend on several factors. The mechanisms by which the two nanomaterials are able to enter the cells are also illustrated. In the second part of the introduction, the potential of CNTs and graphene in targeted drug delivery is described, focusing on anticancer therapy. The three following chapters illustrate the results obtained by three related studies carried out during my PhD internship. Results described in the second chapter shed more light on the impact of CNTs on living cells. CNTs having single walls (SWCNTs) were dispersed with the biocompatible protein bovine serum albumin (BSA). BSA protein showed to be a good dispersant agent for the CNTs and was able to enhance their biocompatibility. The impact of the protein-coated materials on cell vital parameters, such as viability, activation and interaction/internalization mechanisms, is described. In addition the effect of nanotubes on the plasma membrane dielectric characteristics and ion flux, very poorly 2 Abstract investigated up to date, is presented. In the following chapter, a cisplatin prodrug was encapsulated within the inner cavity of two types of multi-walled CNTs (MWCNTs) having different diameters, in order to allow a controlled release inside the cells. The efficacy of the complexes was investigated on human cervix cancer cells and compared to murine macrophages. The latter were also used to evaluate the possibility of a proinflammatory effect. Results on cell viability, cell activation and cell uptake show that CNTs are promising nanocarriers to improve the accumulation of a chemotherapeutic drug inside the cells, without inducing a high proinflammatory response. In addition, by tuning CNT diameter it is possible to control the time of release of the drug prolonging its anticancer efficacy. The fourth chapter gives more insights about the impact of different types of graphene on cells, focusing on macrophages. Results evidenced a specific cytotoxic effect of graphene oxide (GO) towards this cell population among the other murine immune cells. The mechanisms by which macrophages internalize GO are also elucidated. In addition, GO was conjugated with lysozyme protein and was tested for its ability to selectively target a B cell model overexpressing a lysozyme-specific B cell receptor. Results showed that graphene is able to target B cells in a selective manner, thus suggesting its possible application in therapies where the specific elimination of B lymphocytes is required. Finally, a conclusive chapter summarizes the main findings obtained during my doctoral work, focusing, in particular, on future perspectives.