Carbon nanotubes in nanomedicine

The development of nanomedicine is based primarily on the development of smart and multifunctional nanomaterials that can serve under the different clusters including drug delivery systems, diagnostics, and regenerative medicine. Recently, carbon nanotubes (CNTs) have received enormous attention due to their extraordinary properties. CNTs have a wide range of applications and are used in a variety of products thus exposure to CNTs has become unavoidable, which may prompt an inflammatory response. The present Thesis is focused on studying CNTs especially addressing the key challenges highlighted by the Food and Drug Administration and the Alliance for Nano-Health, including imaging, biodistribution, interaction with biological environment, and predictive modeling.

For imaging in order to evaluate biodistribution we were able, for the first time, to use thermostable Luciferase from Luciola cruciate (LcL) as a qualitative imaging modality. LcL offers an alternative approach for following the biodistribution of CNTs over time. The biodistribution profile of CNTs was found to be similar to the majority of nanoparticles, falling in the same size criteria, and predominantly accumulate in the liver. This raised the question whether CNTs could interfere with the liver functionality explicitly the metabolizing activity of drugs and other xenobiotics by phase I metabolizing enzymes CYP450.

We therefore studied the ability of single wall carbon nanotubes (oxSWNTs) on inhibiting enzymatic capacity of CYP3A4. We found that oxSWNTs inhibit mediated conversion of testosterone (as a model compound), to its major metabolite 6_-hydroxy testosterone in a dose dependent manner. When oxSWNTs is pre-coated with bovine serum albumin, the enzymatic activity of CYP3A4 was restored. Also, the covalent functionalization of oxSWNTs with polyethylene glycol (PEG) has shown to have no influence on the enzymatic activity of CYP3A4. Further understanding of the molecular interactions was obtained by computational modeling and simulations (MD). MD simulations revealed that the inhibition of CYP3A4 catalytic activity is mainly due to blocking of the exit channel for substrate/products through a complex binding mechanism. CYP3A4 is a well-recognized isozyme accountable for the metabolization of various endogenous and exogenous xenobiotics by means of the monooxygenase cycle.

In the Thesis, we also studied the degradation of pristine and oxidized SWNTs (p-SWNTs, oxSWNTs) by CYP3A4, by Raman spectroscopy. We found that both p-SWNTs and oxSWNTs were degraded as evidenced by the increase of D-band, which corresponds to the increase of the structural defects. Surprisingly, CYP3A4 bactosomes were more proficient in degrading p-SWNTs more than oxSWNTs under similar incubation conditions. MD simulations suggested that CYP3A4 has a higher affinity for p-SWNTs (minimal MolDock Score of _186.34 kcal/mol) compared to oxSWNTs which bind in a weaker manner (MolDock Score = _111.47 kcal/mol). Pulmonary accumulation of CNTs has shown to be critical. We therefore studied the biodegradation of oxSWNTs by Lactoperoxidase (LPO), a secreted peroxidase enzyme present in the mucus of the airways. We also investigated whether pulmonary surfactants can play a role in the biodegradation of oxSWNTs. Biodegradation was monitored using Raman spectroscopy, scanning electron microscopy, and UVĐVisĐNIR spectroscopy. The biodegradation of oxSWNTs was not impeded by the formation of protein corona formed in the presence of lung surfactant (Curosurf¨). Moreover, cell-free digestion of oxSWNTs was observed ex vivo in murine bronchoalveolar lavage fluid in the presence of peroxidase cofactors.

Since CNTs is studied as a theranostic agent, we therefore studied the biodegradation of PEGylated oxSWNTs. PEG is acknowledged as the gold standard for extending blood circulation times for many biological molecules. OxSWNTs functionalized with PEG of different molecular weight (MW) were incubated with myeloperoxidase (MPO). Biodegradation was noted only for oxSWNTs chemically functionalized with PEG. There was no sign of degradation of oxSWNTs with physically adsorbed PEG, nor for p-SWNTs. The extent of oxSWNT biodegradation by MPO was inversely proportional to the molecular weight of the PEG chains. Ex vivo biodegradation using isolated primary human neutrophils revealed that both chemically and physically PEGylated oxSWNTs undergo biodegradation independently of the PEG chain MW. These ex vivo findings suggest that, in a cell system, a combined process of stripping and biodegradation of PEGylated oxSWNTs might occur.

List of scientific papers

I. Ramy El-Sayed , Mohamed Eita , sa Barrefelt , Fei Ye , Himanshu Jain , Mona Fares, Arne Lundin , Mikael Crona , Khalid Abu-Salah , Mamoun Muhammed , and Moustapha Hassan, ŇThermostable Luciferase from Luciola cruciate for Imaging of Carbon Nanotubes and Carbon Nanotubes Carrying Doxorubicin Using in Vivo Imaging SystemÓ Nano Letters, 2013, 13 (4), pp 1393Đ1398
https://doi.org/10.1021/nl304123u

II. Ramy El-Sayed, Zonglin Gu, Zaixing Yang, Jeffrey K. Weber, Hu Li, Klaus Leifer, Yichen Zhao, Muhammet S. Toprak, Ruhong Zhou, and Bengt Fadeel, ŇSingle-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4Ó, 2016. [Accepted]
https://doi.org/10.1038/srep21316

III. Ramy EL-Sayed, kariem Ezzat, Sergey Shityakov, Birgit Brandner, Mamoun Muhammed, and Moustapha Hassan, ŇDegredation of pristine and oxidised single wall carbon nanotubes by cytochrome P450; CYP 3A4Ó [Manuscript]

IV. Kunal Bhattacharyaa, Ramy El-Sayed, Fernando T. And—na, Sourav P. Mukherjeea, Joshua Gregoryb, Hu Lid, Yinchen Zhaoe, Wanji Seof, Andrea Fornara, Birgit Brandner, Muhammet S. Toprak, Klaus Leifer, Alexander Star, and Bengt Fadeel ŇLactoperoxidase-mediated degradation of single-walled carbon nanotubes in the presence of pulmonary surfactantÓ. Carbon, 2015, 91, pp 506-517
https://doi.org/0.1016/j.carbon.2015.05.022

V. Kunal Bhattachary, Cristiano Sacchetti, Ramy El-Sayed, Andrea Fornara, Gregg P. Kotchey, James A. Gaugler, Alexander Star,Massimo Bottinib,and Bengt Fadel, ŇEnzymatic ÔstrippingŐ and degradation of PEGylated carbon nanotubesÓ, Nanoscale, 2014, 6, pp 14686-14690.
https://doi.org/10.1039/c4nr03604b