Carbon nanotubes are either single-wall (SWCNTs) consisting of a single graphite lattice rolled into a perfect cylinder or multi-wall (MWCNTs) made up of several concentric cylindrical graphite shells (Russian doll configuration). By virtue of their ability to interact with cells and cross cell membranes, they can be used as carriers for drug and DNA delivery. In recent years several groups have demonstrated that both SWCNTs and MWCNTs can be internalized by a variety of cell types and thus used to deliver therapeutic and diagnostic small molecules. Conjugation of CNTs with different molecules can be used for new vaccine production and novel therapies against retrovirus infection and tumor cell proliferation. Pantarotto et al reported that VP-1 protein of foot-and-mouth disease virus (FMDV) covalently linked to SWCNTs induced a specific anti-body response in vivo without any cross reactivity. Liu et al transfected human T cells and peripheral blood mononuclear cells with siRNA molecules conjugated to CNTs to abrogate the expression of cell-surface receptors CD4 and co-receptors CXCR4 necessary for HIV entry and infection of T cells; moreover, McDevitt et al constructed a specific CNT conjugated antibody to target the CD20 epitope on Human Burkitt lymphoma cells and simultaneously deliver a radionuclide. The physical and chemical properties of MWCNTs (e.g., high strength, electrical conductivity, flexibility, functionalisation with biomolecules) make them attractive as nano-vectors for enhancing cell and tissue growth on scaffolds in vitro and for the development of neural complex prosthetic implants. For these biomedical applications MWCNTs need to be purified, coated and functionalized because in their native state they have structural and physical properties which render them electrostatically charged and hydrophobic. Some studies have underlined the relation between the toxic effects induced by CNTs and their length and degree of agglomeration. For biological applications it is thus necessary to make nanotubes more hydrophilic by coating them with particular surfactants. A non-ionic surfactant, PF-127, has been successfully used for CNTs dispersion in aqueous solutions and it exhibited low toxicity when tested in various cell types. In recent years, conflicting data have been reported concerning safety and biocompatibility of these nanotubes. In a study on the cytotoxicity of unrefined SWCNTs to immortalized human epidermal keratinocytes (HaCaT), Shvedova et al showed accelerated oxidative stress, loss in cell viability and morphological alterations of cellular structures. They concluded that those effects were the result of high content (30%) of residual iron catalyst present in the as produced SWCNTs. Other groups confirmed these toxic effects, e.g., induction of intracellular reactive oxygen species (ROS), DNA damage and cell apoptosis. Recently, Poland and colleagues demonstrated that exposure of the mesothelial lining of the peritoneal cavity of mice to long multi-walled carbon nanotubes (>15 microns in length) results in asbestos-like, length-dependent, pathological inflammation and the formation of giant cell granulomas. In sharp contrast, other reports have demonstrated that CNTs do not induce toxic effects on cells. Huczko et al showed that CNTs exhibited negligible risk of skin irritation and allergy. Cherukuri et al observed that macrophages phagocytosed SWCNTs at the rate of approximately one SWCNT per second without any apparent cytotoxicity. Kam et al reported no cytotoxicity for the pristine SWCNTs and Pantarotto et al concluded that CNTs coated with DNA provided a useful vector for safe gene delivery. Recently, Bardi et al demonstrated that MWCNTs dispersed in PF-127 did not induce neuron apoptosis in vitro and inflammation after direct injection of CNTs into the brain substance was not observed. Although CNTs production method, purity and functionalisation treatments influence biocompatibility, the majority of published reports have lacked this essential information. I have extensively investigated the biocompatibility of MWCNTs in different cell lines as a result of which identification was possible of good parameters in term of length, diameter and amount of metal catalyst needed for biocompatibility and avoidance of toxicological effects.
The final aim of this PhD thesis is to design, develop and test a smart system based on MWCNTs for drug delivery and exploit the magnetic properties of these nanoparticles to improve the delivery of chemotherapeutic drugs and to guide the cell homing after cell transplantation in vivo.
In order to achieve the final goal of my thesis I considered some midterm aims which were essential for success of the project. These can be summarized as follows:
1) Interaction between cells and MWCNTs: in vitro toxicology studies
Concerning the toxicity study on SH-SY5Y cell line, I performed four independent assays (MTT, WST-1, Hoechst assay and oxidative stress assay) in order to identify the effect of the nanotubes on cytocompatibility. The MTT test after 72 h of incubation showed a statistically significant (p<0.001) loss of viability (12%-15%) in cells treated with MWCNTs compared to the control sample, while cells treated only with PF-127 0.01% maintained a viability of 99%. In previously reported literature, it had been demonstrated that nanotubes absorbance could influence the results of the MTT assay at high nanotube concentration. For this reason, I performed WST-1 assay as double check. In contrast with the results of the previous test, the WST-1 assay revealed that cells treated with all three MWCNTs types after 72 h of incubation maintained high level of viability compared to untreated cells. In order to confirm these results I evaluated SH-SY5Y cell death by Hoechst 33258, which enables determination of the presence of DNA condensates, a characteristic feature of apoptosis. The number of apoptotic nuclei was determined on at least five randomly selected areas from three cover slips of each experimental group, each area containing approximately 100 cells. Results are expressed as percentage of apoptotic cells and compared to the control sample. Hoechst assay results showed that after 72h of incubation there was no apoptosis in cells treated with nanotubes, confirming the results obtained by WST-1 assay.
Even if carbon nanotubes did not show acute toxicity in vitro, Karin Pulskamp et al. demonstrated that they can induce intracellular reactive oxygen species (ROS). In order to study if the MWCNTs sample could induce oxidative stress I used the Image-IT Green Reactive Oxygen Species Detection kit (Invitrogen, U.S.A.). My results showed that all three samples of MWCNTs did not induce oxidative stress in neuroblastoma cell lines after 72 h of incubation. To determine the effects of carbon nanotubes purity and surfactant on cell viability after long time of incubation I performed a set of experiments designed to measure the viability at one and two weeks of treatments. However, long term viability assays performed after two weeks of continuous incubation with the MWCNT samples showed a significant decrease in cytocompatibility of MWCNTs HNO3-97% and MWCNTs 97% respect to MWCNTs 99% and control sample. The data obtained indicated that MWCNTs 99% of purity represented the best sample for long-term treatments because it minimized accumulation of metal catalyst which is toxic to the cells.
2) Exploitation of the magnetic properties of MWCNTs: application for cell displacement in vitro; guiding of the homing of mesenchymal stem cells after their transplantation in vivo; tracking of the cells loaded with these nanoparticles by using the Magnetic Resonance Imaging (MRI).
Carbon nanotubes 97% of purity showed interesting magnetic properties due to the presence of metal impurities (Fe). In these experiments I observed that cells treated with MWCNTs 97% move towards a magnetic source. This magnetic guide could have many applications in cancer therapy and in cell transplantation. I performed some tests to observe the movements of tumor cells and mesenchymal stem cells loaded with MWCNTs towards a magnetic source. In human neuroblastoma (SH-SY5Y) and mice pheochromocytoma (PC-12) cell lines, I demonstrated that 10 µg/ml MWCNTs 97% were sufficient to promote cells displacement in magnetic field and this dose of nanotubes appears to be non toxic in vitro. In particular I observed that cells grew without damage and in presence of specific differentiation factors were able to differentiate into neurons, whilst maintaining high viability level.
The project also included the investigation of the interaction between magnetic carbon nanotubes and mesenchymal stem cells and their ability to guide these cells injected intravenously in living mice by using an external magnetic field. The data obtained from these experiments showed that MWCNTs did not affect cell viability and their ability to differentiate into osteocytes and adipocytes. Furthermore MWCNTs and the magnetic field used did not alter cell growth rate, phenotype and cytoskeletal conformation. These magnetic nanotubes, when exposed to magnetic fields, were able to shepherd MSCs towards the magnetic source in vitro. Moreover, the application of a magnetic field alters the bio distribution of CNT-labelled MSCs after intravenous injection into rats, increasing the accumulation of cells into the target organ (liver). These studies enabled the conclusion that MWCNTs hold a distinct potential for use as nano-devices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. This could pave the way for the development of new strategies for manipulation/guidance of MSCs in regenerative medicine and cell transplantation.
In a series of other experiments, the effectiveness of cellular imaging using 97% MWCNTs as a MRI contrast agent was investigated by labelling mesenchymal stem cells. Specifically, the potential of multiwall carbon nanotubes (MWCNTs 97%) with low metal impurities (2.57% iron) as MRI contrast agents was investigated. The results showed that the r2 relaxivity of MWCNTs in 1% agarose gels at 19 °C was 564 ± 41 s-1mM-1. This is attributed to both the presence of iron oxide impurities and to the carbon structure of the MWCNTs. Moreover, in these studies we were able to label stem cells with MWCNTs to demonstrate their effectiveness as labels for cellular MR imaging. These results suggest that the MRI contrast agent properties of the MWCNTs could be used in vivo for stem cell tracking/ imaging and during MWCNT-mediated targeted electro-chemotherapy of tumours.
3) Design and Development of magnetic MWCNTs as carrier for the guided delivery of the innovative chemotherapeutic agent GDC-0941 produced by Astra Zeneca in collaboration with the Life Science Dept. of the University of Dundee.
GDC-0941 is a specific inhibitor of the PI3- I kinase and is very effective in the inhibition of the pathway of mTor, which is important for the regulation of the cellular cycle. It is well established that mTor deregulation pathway is involved in many types of tumours. The GDC-0941, which is still in phase I clinical trial, showed very good results in killing cancer cells. In particular, it has been shown to be very effective for the treatment of different types of lymphoma. Unfortunately, this drug exhibited significant side effects on the healthy tissues. The toxicity and associated side effects showed of the current chemotherapy regimens remain one of the major problems in clinical oncology. As a possible solution to this problem, I investigated the binding of GDC-0941 drug to magnetic nanotubes, as this would enable the administration of low doses of the drug to patients by driving the drug towards the tumours mass with the use of a magnetic field. For this purpose, I functionalized MWCNTs with Poly-Lysine which is a biocompatible molecule, conferring many NH2 group to the nanotubes. These amino groups can bind by non covalent interaction with the COO- group of the GDC-0941. With this strategy I was able to obtain and characterize the compound MWCNT-GDC-0941. I tested the ability of this compound to inhibit the mTor pathway in 293 cells derived by human kidney cancer. In vitro results showed that the MWCNTs-GDC compound was stable in physiological solution and the drug not only maintained its antitumor activity, but this increased by 30% after the conjugation with the nanoparticles.