16 research outputs found

    A comparative study on the enzymatic biodegradability of covalently functionalized double- and multi-walled carbon nanotubes

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    The assessment of the biodegradability potential of carbon nanotubes (CNTs) is a fundamental point towards their applications in materials science and biomedicine. Due to the continuous concerns about the fate of such type of nanomaterials, it is very important to understand if they can undergo degradation under certain conditions and if the morphology and structure of the nanotubes play a role in this process. For this purpose we have decided to undertake a comparative study on the enzymatic degradation of CNTs with concentric multilayers. Double-walled (DW) and multi-walled (MW) CNTs of various lengths, degrees of oxidation and functionalizations using different methods were treated with horseradish peroxidase (HRP). While all tested DWCNTs resulted resistant to the biodegradation, some of the MWCNTs were partially degraded by the enzyme. We have found that short oxidized multi-walled CNTs functionalized by amidation were reduced in length and presented a high amount of defects at the end of the period of treatment with HRP. This comparative study holds its importance in the understanding of the structural changes of different types of nanotubes towards the catalytic enzymatic degradation and will help to design safer CNTs for future applications

    Nanotubes de carbone cationiques pour la vectorisation d’acides nucléiques

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    2010/2011Carbon 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.XXIV Ciclo198

    Nanotubes de carbone cationiques pour la vectorisation d'acides nucléiques

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    Les nanotubes de carbone (CNTs) sont une nouvelle forme allotropique du carbone, décrits pour la première fois à l échelle atomique en 1991 par Iijima. Dans ce travail de thèse, les MWCNTs portant des charges cationiques ont été fonctionnalisés, avec pour objectif d étudier leur aptitude à complexer des acides nucléiques pour obtenir un système de délivrance génétique. Initialement, nous avons fonctionnalisé les MWCNTs avec des dendrons portant à leur extrémité des groupes ammonium ou guanidinium et leur aptitude à complexer des acides nucléiques a été évaluée par électrophorèse en gel d agarose. En outre, nous avons fonctionnalisé et caractérisé les MWCNTs avec un peptide ciblant les mitochondries et leur habilité à se localiser à l intérieur de ces dernières a été étudié par différentes techniques microscopiques. Ensuite, nous avons doublementfonctionnalisé les CNTs avec un dendron de deuxième génération et avec le peptide de ciblage. La capacité de ce conjugué à complexer l ADN a finalement été confirmée par électrophorèse en gel d agarose.Carbon nanotubes (CNTs) are a new allotropic form of carbon described at the atomic level in 1991 by Iijima. During my thesis, carbon nanotubes bearing cationic moieties have been functionalized, in order to study their ability to complex the genetic material to obtain a gene delivery system. Initially we have functionalized MWCNTs with dendrons bearing at their termini ammonium or guanidinium groups. Their ability to complex the genetic material has been evaluated through agarose gelelectrophoresis. Moreover, we have functionalized and characterized MWCNTs with a targeting peptide for mitochondria and their ability to localize inside this organelle was studied by different microscopic techniques. Then, we have double-functionalized MWCNTs with a dendron of second generation and with the targeting peptide and the ability of this conjugate to complex DNA was confirmed by agarose gel electrophoresis.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

    Carbon nanomaterials as new tools for immunotherapeutic applications

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    Carbon-based nanomaterials, including carbon nanotubes and graphene, have gained great attention in the scientific community due to their unique physico-chemical properties, which could be also promising in many biomedical-related fields. In particular, their low cytotoxicity, achieved when properly functionalized, along with the possibility to link multiple bioactive molecules, realistically allows envisaging their potential use as a therapeutic platform. In this context, the immune system and immune responses play an important role in our organism, as they are involved either directly or indirectly in many diseases. Therefore, the possibility to prevent or block a disease by controlling and/or modulating the immune responses has become an important task in nanomedicine. In this feature article the advantages of using carbon-based materials in immunotherapy are presented. Important goals achieved using carbon nanotubes and graphene are described, highlighting the promising use of these nanomaterials in cancer treatment, imaging and vaccine development. The capacity of functionalized carbon nanotubes to modulate the immune responses is also discussed, highlighting the current state of the art and the future developments on this subject

    Improved chemical and mechanical stability of peptoid nanosheets by photo-crosslinking the hydrophobic core

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    Peptoid nanosheets can be broadly functionalized for a variety of applications. However, they are susceptible to degradation when exposed to chemical or mechanical stress. To improve their strength, photolabile monomers were introduced in order to crosslink the nanosheet interior. Photo-crosslinking produced a more robust material that can survive sonication, lyophilization, and other biochemical manipulations

    Peptide-based carbon nanotubes for mitochondrial targeting

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    In the present study, we report the design and synthesis of peptide-based-multi-walled carbon nanotubes (MWCNTs) to target mitochondria. Targeting these intracellular organelles might open the way to develop alternative systems to address diseases related to genetic mutations in mitochondrial (mt)-DNA, by delivering therapeutic oligonucleotides. The first step towards mitochondrial delivery of this type of nucleic acid was to target MWCNTs to mitochondria by covalent functionalization with a well-known endogenous mitochondrial targeting sequence (MTS). The subcellular localization of the conjugates, which were fluorescently labeled, in murine RAW 264.7 macrophages and human HeLa cells was then studied using different microscopy techniques, such as wide-field epifluorescence microscopy, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). The localization of the MTS-MWCNT conjugates into mitochondria was further confirmed by analyzing the isolated organelles using TE
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