Vera Neves

Higher Dispersion Efficacy of Functionalized Carbon Nanotubes in Chemical and Biological Environments

Aqueous dispersions of functionalized carbon nanotubes (CNTs) are now widely used for biomedical applications. Their stability in different in vitro or in vivo environments, however, depends on a wide range of parameters, such as pH and salt concentrations of the surrounding medium, and length, aspect ratio, surface charge, and functionalization of the applied CNTs. Although many of these aspects have been investigated separately, no study is available in the literature to date, which examines these parameters simultaneously. Therefore, we have chosen five types of carbon nanotubes, varying in their dimensions and surface properties, for a multidimensional analysis of dispersion stability in salt solutions of differing pH and concentrations. Furthermore, we examine the dispersion stability of oxidized CNTs in biological fluids, such as cellular growth media and human plasma, and their toxicity toward cancer cells. To enhance dispersibility and biocompatibility, the influence of different functionalization schemes is studied. The results of our investigations indicate that both CNT dimensions and surface functionalization have a significant influence on their dispersion and in vitro behavior. In particular, factors such as a short aspect ratio, presence of oxidation debris and serum proteins, low salt concentration, and an appropriate pH are shown to improve the dispersion stability. Furthermore, covalent surface functionalization with amine-terminated polyethylene glycol (PEG) is demonstrated to stabilize CNT dispersions in various media and to reduce deleterious effects on cultured cells. These findings provide crucial data for the development of biofunctionalization protocols, for example, for future cancer theranostics, and optimizing the stability of functionalized CNTs in varied biological environments.

AFM imaging of functionalized carbon nanotubes on biological membranes

Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

Design of double-walled carbon nanotubes for biomedical applications

Double-walled carbon nanotubes (DWNTs) prepared by catalytic chemical vapour deposition were functionalized in such a way that they were optimally designed as a nano-vector for the delivery of small interfering RNA (siRNA), which is of great interest for biomedical research and drug development. DWNTs were initially oxidized and coated with a polypeptide (Poly(Lys:Phe)), which was then conjugated to thiol-modified siRNA using a heterobifunctional cross-linker. The obtained oxDWNT-siRNA was characterized by Raman spectroscopy inside and outside a biological environment (mammalian cells). Uptake of the custom-designed nanotubes was not associated with detectable biochemical perturbations in cultured cells, but transfection of cells with DWNTs loaded with siRNA targeting the green fluorescent protein (GFP) gene, serving as a model system, as well as with therapeutic siRNA targeting the survivin gene, led to a significant gene silencing effect, and in the latter case a resulting apoptotic effect in cancer cells.

Cellular localization, accumulation and trafficking of double-walled carbon nanotubes in human prostate cancer cells

AbstractCarbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT-RNA) is presented. Fixed cells, previously exposed to oxDWNT-RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT-RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT-RNA with the small GTP-binding protein (Rab 11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network.

Carbon Nanotubes Loaded with Anticancer Drugs: A Platform for Multimodal Cancer Treatment

Approximately every fourth person in the world currently dies of cancer. Although many efficient anticancer drugs have been developed over the last 60 years or more, most therapeutic approaches still lack specificity for their intended site of action in the body, resulting in reduced effectiveness and severe side effects. The emerging field of nanomedicine provides a whole range of materials and techniques to develop customizable drug delivery vehicles that assist the targeting of therapeutic agents to the desired site of action. Amongst these, carbon nanotubes have emerged as promising candidates, being capable of penetrating mammalian cell membranes and allowing for the attachment of high loads of drugs and targeting agents on their surface or the inner cavity. This chapter will discuss the principles of targeted, anticancer chemotherapies and introduce carbon nanotubes as novel tools for vector-based, targeted drug delivery.

Uptake, Intracellular Localization and Biodistribution of Carbon Nanotubes

Carbon nanotubes (CNTs) exhibit unique size, shape and physical properties, which make them promising candidates for biomedical applications. In particular, carbon nanotubes have been intensively studied for conjugation with pre-existing therapeutic agents for more effective targeting, as a result of their ability to cross cell membranes. However, to utilise them effectively in biological systems it is extremely important to understand how they behave at the cellular level and their distribution in vivo. Additionally, in order to consider carbon nanotubes as candidate delivery systems of therapeutic agents it is important to ascertain their fate in vivo, but also take into account many factors, such as solubility, stability and clearance. Issues surrounding their short term and long term safety are currently the subject of toxicology testing. Herein, we propose to summarize the main findings on the uptake, trafficking and biodistribution of carbon nanotubes, with special focus on functionalized carbon nanotubes for delivery of therapeutic agents.

Abstract 3701: A targeted delivery system for anticancer drugs based on functionalized carbon nanotubes

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Carbon nanotubes have emerged as a promising nanomaterial for the development of customizable drug delivery system due to their ability to penetrate cell membranes and to attach high loads of drugs and targeting agents on their surface. In the field of oncology, this promotes the development of therapies with improved effectivity and fewer side effects by assisting the targeting of therapeutic agents to the desired site of action. Our work focuses on the targeted delivery of the anthracycline and anthracenedione anticancer drugs doxorubicin and mitoxantrone to human colon cancer cells (WiDr) by means of a carbon nanotube-based drug delivery system. The drugs are attached to the nanotube surface in a non-covalent manner, resulting in a weight ratio (drug: CNTs) of 4:1 owing to the high surface area of CNTs. As drug binding is higher at physiological pH than at a slightly acidic pH, this method allows for the release of the drug from the nanotubes in such environments, for example late endosomes and lysosomes. In a previous study, we have shown that doxorubicin-loaded carbon nanotubes are are taken up by WiDr colon cancer cells and release their drug payload upon cellular internalization, which then translocates to the nucleus [1]. Current work is focusing on the targeting aspect: to increase the specificity of our system, we further attach a monoclonal antibody to the nanotube carrier that recognizes/binds to carcinoembryonic antigen (CEA), a target structure overexpressed by WiDr colon cancer cells. In vitro cytotoxicity studies show that the targeted drug-CNT complexes indeed have a greater therapeutic effect on WiDr cells than on MCF7 breast cancer cells, which do not express CEA. Future work will include in vivo studies on mice bearing tumor xenographs to evaluate the feasibility and behavior of our system in a setting of clinical relevance. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3701.