AbstractCancer nanotechnology emerged in the last two decades as an extremely promising approach to fight cancer Cancer nanotechnology encompasses the exploitation of the properties of different kinds of nanoparticules (NPs) for the diagnosis and the treatment (gathered in the term “theranostics”) of cancer. Amid those NPs, carbon nanotubes (CNTs) and magnetic nanoparticles (MNPs) both emerged as very potent theranostics agents. Indeed, the exceptional structural, physical and chemical properties of CNTs made them excellent scaffold for drug delivery, fluorescence imaging, light-induced hyperthermia, and so on. On their side, MNPs have proven to be excellent materials for complementary applications such as magnetic resonance imaging (MRI) and magnetic fluid hyperthermia (MFH). The combination of these two types of nanoparticles affords hybrids with properties coming from both components but also from a synergy between MNPs and CNTs. In this respect, the aim of this thesis is the evaluation of functionalized endohedral MNPs@CNTs hybrids for cancer theranostics applications. Before addressing the detailed investigations of this thesis work, a general introduction about the state-of-the-art of MNPs-CNTs hybrids syntheses and applications will be given in Chapter 1.
The Chapter 2 describes the design of novel Fe-filled CNTs (Fe@CNTs) conjugated to different targeting units for selective cell recognition and subsequent magnetically-induced extraction from a medium or induce cell mortality by the application of an alternating magnetic field. A brief introduction about the importance of chosen targets, the epidermal growth factor receptor (EGFR) and the adenosine receptor A3 (A3AR), in cancer therapy and the different current strategies employed to target them will be given. The second section of this chapter will develop the results obtained during our exploration of the impact of Fe loading and crystallinity onto in-vitro MFH and cell shepherding responses of monoclonal antibody-conjugated Fe@CNTs (Fe@CNTs-Ab). Our experimental findings reveal that an optimal antibody/Fe weight ratio of 1.2 is needed for efficient magnetic cell shepherding, whereas enhanced MFH-induced mortality (70 vs. 15 %) can be reached with hybrids enriched in the coercive Fe3C phase. These results suggest that a synergistic effect between the Ab loading and the Fe distribution in each nanotube exists, for which the maximum shepherding and hyperthermia effects are observed when higher density of Fe@CNTs featuring the more coercive phase are interfaced with the cells. Based on these results, we then aimed at extending the scope of Fe@CNTs by conjugating them with a pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-pyrimidine (PTP) ligand (13) for the selective recognition of adenosine receptors. Through production of PEGylated Fe@CNTs, we successively achieved the synthesis of two different materials Fe@CNTs-TEG-PTP and Fe@CNTs-HEG-PTP which showed a promising ability at targeting A3ARs as assayed through radioactive agonist displacement binding constant measurement. These materials were then tested on A3AR-overexpressing CHO cells to perform a selective magnetic shepherding. However, we found out that a large degree of non-specific electrostatic interactions between positively-charged Fe@CNTs-H-PTP and negatively-charged cell membranes hampered any specific recognition of A3AR-overexpressing cells over other cells.
The Chapter 3 makes the link between in-vitro assessment of Fe@CNTs described in Chapter 2 and their use for in-vivo cancer theranostics applications described in the final Chapter 4. In particular, we aimed at the solubilisation of Fe@CNTs and regular CNTs by exploiting the supramolecular scaffolding effect offered by CNTs to wrap around dynamic polymers. After an introduction about the different existing supramolecular polymers that were employed to solubilize CNTs, the second section describes the synthesis and the self-assembly of H-bonded supramolecular polymers formed by complementary uracil and di(acetylamino)pyridine (DAP) monomers 57 and 58. This supramolecular polymer enabled solubilisation and functionalization of various CNTs in different solvents, including H2O. By different means, we proved that the formation of an H-bonding network is crucial to achieve an efficient solubilisation of CNTs. Moreover, H2O does not seem to affect the H-bonding ability of our supramolecular polymer. Once the concept proved, we developed new functionalized monomers, allowing the addition of new functions to CNTs through the supramolecular polymer. For instance, we successfully appended Au NPs onto thioacetate-monomers wrapped CNTs, as shown by TEM. As a perspective, another type of dynamic bond was also explored based on acylhydrazone covalent dynamic bonds. This kind of link has been previously employed in literature to form polymers in H2O that can be reversible or not according to the pH conditions. The synthesis of new aldhedyde and acylhydrazine monomers was then carried out and afforded the desired complementary building blocks. This new polymer is currently under investigation for CNTs solubilisation.
Finally, the Chapter 4 describes the evaluation of the potential of Fe@CNTs for in-vivo cancer theranostics. Firstly, an introduction about the different applications of CNTs for in-vivo biological imaging and cancer therapy is proposed to the reader. The following section describes the assessment of two different imaging modalities based on the Fe@CNTs scaffold: firstly, we envisaged the possibility to track in-vivo administered CNTs through their conjugation to a fluorescent indocyanine green (ICG) label, producing Fe@CNTs-ICG. This material was tested on a fluorescence recorder dedicated at in-vivo imaging. However, the presence of strong π-π stacking interactions and subsequent energy transfer from the dye to CNT surface leading to fluorescence quenching did not allow to obtain a sufficient signal to follow Fe@CNTs by this technique. On the other hand, the intrinsic magnetism of Fe@CNTs allowed them to act as efficient contrast agent in magnetic resonance imaging (MRI). This property was used in a further in-vivo studies employing Fe@CNTs-Ab wrapped by our supramolecular polymer, which confers them enhanced stability in water. Specifically, Fe@CNTs-Ab were administered to mice bearing tumour xenographs of EGFR+ A431 cancer cells and followed by MRI. This technique allowed us to observe the apparition of Fe@CNTs in tumour tissues but also predominantly in liver and spleen. In order to get a quantification of the organ biodistribution, we developed a new ex-vivo spectroscopic quantification methodology based on the high resistance of our Fe@CNTs towards alkaline treatments, allowing a selective dissolution of living tissues, and their ability to be magnetically extracted to perform their efficient isolation from complex homogenized tissues. After this recovery, the high absorbance of Fe@CNTs in the near-IR was employed to quantify them. Application of this methodology to mice administered with Fe@CNTs allowed us to get a precise biodistribution profile showing a preferential accumulation in lungs, liver and spleen.
|Date of Award||8 Apr 2016|
|Sponsors||Fund for Research Training in Industry and Agriculture (FRIA)|
|Supervisor||Davide BONIFAZI (Supervisor), Carine MICHIELS (President), Stephane VINCENT (Jury), Tatiana Da Ros (Jury) & Jean Marie Raquez (Jury)|
- carbon nanotubes
- Magnetic nanoparticles
Attachment to an Research Institute in UNAMUR
Functionalized Fe-filled Carbon Nanotubes for Cancer Theranostics
Pineux, F. (Author). 8 Apr 2016
Student thesis: Doc types › Doctor of Sciences