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|Title: ||Cationic Carbon Nanotubes for Nucleic Acids Delivery|
|Other Titles: ||Nanotubes de carbone cationiques pour la vectorisation d’acides nucléiques|
|Authors: ||Prato, Maurizio|
Da Ros, Tatiana
|Keywords: ||Carbon Nanotubes|
|Issue Date: ||26-Mar-2012|
|Publisher: ||Università degli studi di Trieste|
|Abstract: ||Carbon nanotubes (CNTs) are a new form of carbon discovered in the ’50/’60, but described at the atomic level only in 1991 by Iijima. CNTs are constituted by one or more rolled up graphene sheets and they can be classified in single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs). The peculiar properties of CNTs, characterized by their physical, chemical and mechanical properties, by their thermic conductivity and their large aspect ratio, rendered this material a promising candidate in several research fields, such as material science and nanotechnology. Recently, it has been discovered that CNTs have the ability to be uptaken by different types of human cells with a typical non-energy dependent mechanism called “nanoneedle”. This new property rendered CNTs a promising candidate as a vector for drugs.
In this context, during my thesis I focused on the functionalization of carbon nanotubes bearing cationic moieties, in order to study their ability to complex the genetic material for a potential gene therapy. In the first part of the thesis MWCNTs were functionalized with different cationic dendrons and their capability to interact with plasmid DNA and siRNA (small interfering RNA) was evaluated. In the second part of the thesis, I focused on the functionalization of MWCNTs with a targeting peptide for mitochondria and the consequent possibility to use these conjugates as gene delivery system in mitochondrial diseases.
Initially, a library of compounds with a ramified structure was synthesized. Dendrons of zero, first and second generation were obtained, bearing at their termini ammonium or guanidinium moieties, for the interaction with the genetic material. Two groups of dendrons were synthesized with two different functions at their core: i) an amine or ii) an azide. This was realized in order to have the possibility to link the dendrons to CNTs, exploiting two covalent approaches, the amidation reaction or the “click” reaction, respectively. In parallel a non-covalent approach was also investigated, coupling by “click” reaction a dendron of first generation to a pyrene bearing an acetylenic group. The resulting molecule was then adsorbed on the surface of MWCNTs, exploiting the known ability of pyrene to interact with nanotubes through “π-π stacking”.
The conjugates obtained in this way were characterized by complementary techniques, such as thermogravimetric analysis, Kaiser test and transmission electron microscopy, allowing in this way to assess the degree of functionalization of CNTs and to observe their morphology. The ability of these conjugates to complex plasmid DNA and siRNA was studied through agarose gel electrophoresis using different charge ratios of genetic material and cationic CNTs. Preliminary cellular studies on guanidinium derivatives, to evaluate the effective ability of the conjugates to be internalized into human cancer cells (A549 cells), were performed in collaboration with Prof. K. Kostarelos and Dr. K. T. Al-Jamal in London. Further studies are currently under development to determine the aptitude of the derivatives to carry and deliver siRNA inside cells. The efficient gene silencing and the eventual cytotoxic effects of MWCNTs conjugates will be also studied into human cells.
In the second part of the thesis, we focused on the development of a new gene delivery system able to direct the genetic material inside mitochondria, for a possible treatment of genetic diseases caused by mutation in the mitochondrial genome. To this purpose MWCNTs were functionalized with a targeting peptide (mitochondrial target sequence peptide, MTS) able to direct CNTs into mitochondria. The effective internalization of the material inside macrophages (RAW 264.7) and human cancer cells (HeLa cells) and their subcellular localization were studied through different microscopic techniques. Using confocal microscopy it was possible to observe a co-localization of CNTs and mitochondria, then confirmed also by TEM images. This latter technique permitted also to evaluate the possible mechanism used to internalize CNTs: i) “nanoneedle” mechanism and ii) phagocytosis (for macrophages) or endocytosis (for HeLa cells).
In order to shed further light on the ability of CNTs to interact with mitochondria, nanotubes were double functionalized with the targeting peptide MTS and with a mitochondrial toxic peptide, called sVpr. The toxic effects of this material, in comparison with CNTs functionalized just with the toxic peptide, were studied on isolated mitochondria. The internalization of different conjugates into isolated mitochondria was then analysed by TEM. This work has been performed in collaboration with Prof. P. Bernardi (Università degli Studi di Padova).
In order to obtain a system able to complex DNA, a double functionalization of CNTs was developed, coupling to the carbon material the targeting peptide MTS and a dendron, for the complexation of genetic material. The ability of this conjugate to interact with plasmid DNA was then verified through agarose gel electrophoresis, revealing its potentiality as efficient gene delivery system.
In conclusion, in this work MWCNTs were functionalized with cationic dendrons, showing their ability to interact with the genetic material. Moreover when incubated into cells, the aptitude of MWCNTs, functionalized with a targeting peptide, to be directed in the proximity of mitochondria and to localize in their interior was demonstrated using different microscopic techniques. These results show the potentiality of this material in the field of nanomedicine, resulting a promising vector in gene therapy.|
|Appears in Collections:||Scienze chimiche|
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