Overview of Carbon Nanotubes for Biomedical Applications

The unique combination of mechanical, optical and electrical properties offered by carbon nanotubes has fostered research for their use in many kinds of applications, including the biomedical field. However, due to persisting outstanding questions regarding their potential toxicity when considered as free particles, the research is now focusing on their immobilization on substrates for interface tuning or as biosensors, as load in nanocomposite materials where they improve both mechanical and electrical properties or even for direct use as scaffolds for tissue engineering. After a brief introduction to carbon nanotubes in general and their proposed applications in the biomedical field, this review will focus on nanocomposite materials with hydrogel-based matrices and especially their potential future use for diagnostics, tissue engineering or targeted drug delivery. The toxicity issue will also be briefly described in order to justify the safe(r)-by-design approach offered by carbon nanotubes-based hydrogels

Control by Osmotic Pressure of Voltage-Induced Permeabilization and Gene Transfer in Mammalian Cells

AbstractCells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with β-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for β-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer

Electrical properties of double-wall carbon nanotubes nanocomposite hydrogels

The electrical behaviour of nanocomposite hydrogels and especially hydrogels containing carbon nanotubes is generally poorly understood. In this paper, we investigate the influence of double-wall carbon nanotubes (DWCNT) content on the electrical properties of agarose/DWCNT nanocomposite hydrogels. These nanocomposite hydrogels are potential candidates as electrode materials for transdermal drug delivery by electropermeabilization. Both alternating current (AC) and direct current (DC) measurements at different voltage amplitudes were performed, as well impedance spectroscopy (1 Hz–1 MHz). Data suggest a non-linear dependence of the conduction phenomena vs the applied electric field. From the current-voltage characteristics, the nanocomposite conduction phenomenon is narrowed to two possible mechanisms, a Schottky type or a Poole-Frenkel type. These findings are the first step towards the understanding of the conduction phenomena in such complex nanocomposite structures, comprising DWCNT, water and the 3D polymeric network. The work described in this work is of much wider interest because this kind of nanocomposites may have many other applications, while the fundamental questions about their electrical conductivity remain universal

Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells

AbstractElectropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells

Transdermal Delivery of Macromolecules Using Two-in-One Nanocomposite Device for Skin Electroporation

Delivery of hydrophilic molecules through the skin using electroporation is a promising alternative approach to intradermal injection. Recently, we developed a two-in-one electrode/reservoir material composed of carbon nanotubes and agarose hydrogel. In this work, we evaluated the potential of the device to achieve non-invasive transdermal drug delivery using skin electroporation. As it involved an electrode configuration different from the literature, critical questions were raised. First, we demonstrated the efficiency of the device to permeabilize the skin of hairless mice, as observed by propidium iodide (PI) uptake in the nuclei of the epidermis cells through macro fluorescence imaging and histology. Application of Lucifer yellow (LY) at different times after unipolar electroporation treatment demonstrated the partial reversibility of the skin permeabilization after 30 min, and as such, that barrier function properties tended to be restored. We uncovered, for the first time to our knowledge, an intrinsic asymmetry of permeation pathways generated in the stratum corneum during treatment. Electrophoresis was here the main driving force for macromolecule delivery, but it competed with passive diffusion through the generated aqueous pathways for smaller molecules. Finally, we validated 4 kDa dextran labelled with fluorescein isothiocyanate (FD4) as a model molecule to optimize the electrical parameters, needed to improve macromolecule deliver

Spatial mechanistic modeling for prediction of 3D multicellular spheroids behavior upon exposure to high intensity pulsed electric fields

International audienceThe objective of this work was to investigate the growth specificities of cancer cells spheroids subjected to pulsed electric field. Multicellular HCT-116-GFP spheroids were exposed to different electric field intensities and the volume of multicellular spheroids was monitored by fluorescence and bright field microscopy. Thanks to an advanced mathematical model, based on differential equations and well-adapted estimation strategies, our modeling enables us to characterize the multicellular spheroids growth after permeabilizing pulsed electric field. In particular, we identify the percentage of cells which are destroyed and the percentage of cells which exhibit an altered growth pattern for different magnitudes of the electric field. We also quantify the growth resumption upon reversible and partially irreversible electroporation. Our preliminary results provide a first quantification of the impact of electroporation on multicellular spheroids growth, and suggest a booming growth of partially irreversible electric pulses, leading to an accelerated regrowth

Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle

<p>Abstract</p> <p>Background</p> <p>Electrotransfer of plasmid DNA into skeletal muscle is a promising strategy for the delivery of therapeutic molecules targeting various muscular diseases, cancer and lower-limb ischemia. Internal Ribosome Entry Sites (IRESs) allow co-expression of proteins of interest from a single transcriptional unit. IRESs are RNA elements that have been found in viral RNAs as well as a variety of cellular mRNAs with long 5' untranslated regions. While the encephalomyocarditis virus (EMCV) IRES is often used in expression vectors, we have shown that the FGF-1 IRES is equally active to drive short term transgene expression in mouse muscle. To compare the ability of the FGF-1 IRES to drive long term expression against the EMCV and FGF-2 IRESs, we performed analyses of expression kinetics using bicistronic vectors that express the bioluminescent <it>renilla </it>and firefly luciferase reporter genes. Long term expression of bicistronic vectors was also compared to that of monocistronic vectors. Bioluminescence was quantified <it>ex vivo </it>using a luminometer and <it>in vivo </it>using a CCD camera that monitors luminescence within live animals.</p> <p>Results</p> <p>Our data demonstrate that the efficiency of the FGF-1 IRES is comparable to that of the EMCV IRES for long term expression of bicistronic transgenes in mouse muscle, whereas the FGF-2 IRES has a very poor activity. Interestingly, we show that despite the global decrease of vector expression over time, the ratio of firefly to <it>renilla </it>luciferase remains stable with bicistronic vectors containing the FGF-1 or FGF-2 IRES and is slightly affected with the EMCV IRES, whereas it is clearly unstable for mixed monocistronic vectors. In addition, long term expression more drastically decreases with monocistronic vectors, and is different for single or mixed vector injection.</p> <p>Conclusion</p> <p>These data validate the use of bicistronic vectors rather than mixed monocistronic vectors for long term expression, and support the use of the FGF-1 IRES. The use of a cellular IRES over one of viral origin is of particular interest in the goal of eliminating viral sequences from transgenic vectors. In addition, the FGF-1 IRES, compared to the EMCV IRES, has a more stable activity, is shorter in length and more flexible in terms of downstream cloning of second cistrons. Finally, the FGF-1 IRES is very attractive to develop multicistronic expression cassettes for gene transfer in mouse muscle.</p

Sphingosine Kinase-1 Is Central to Androgen-Regulated Prostate Cancer Growth and Survival

BACKGROUND: Sphingosine kinase-1 (SphK1) is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival. METHODOLOGY/PRINCIPAL FINDINGS: Short-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT) to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE)-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway--by negatively impacting SphK1 activity--could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity. CONCLUSIONS/SIGNIFICANCE: We report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a compensatory mechanism allowing prostate cancer cells to survive in androgen-depleted environment, giving support to its inhibition as a potential therapeutic strategy to delay/prevent the transition to androgen-independent prostate cancer

A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor

A monoclonal anti-CD160 antibody inhibits the growth of new vessels in pathological ocular and tumor neoangiogenesis but not in healthy tissues