1 ABSTRACT
Carbon nanotubes are either single-wall (SWCNTs) consisting of a single graphite lattice rolled into a perfect cylinder or multi-wall (MWCNTs) made up of several concentric cylindrical graphite shells (Russian doll configuration). By virtue of their ability to interact with cells and cross cell membranes, (1) they can be used as carriers for drug and DNA delivery. In recent years several groups have demonstrated that both SWCNTs and MWCNTs can be internalized by a variety of cell types and thus used to deliver therapeutic and diagnostic small molecules. (2;3;4). Conjugation of CNTs with different molecules can be used for new vaccine production and novel therapies against retrovirus infection and tumor cell proliferation. Pantarotto et al (5) reported that VP-1 protein of foot-and-mouth disease virus (FMDV) covalently linked to SWCNTs induced a specific anti-body response in vivo without any cross reactivity. Liu et al (6) transfected human T cells and peripheral blood mononuclear cells with siRNA molecules conjugated to CNTs to abrogate the expression of cell-surface receptors CD4 and co-receptors CXCR4 necessary for HIV entry and infection of T cells; moreover, McDevitt et al (7) constructed a specific CNT conjugated antibody to target the CD20 epitope on Human Burkitt lymphoma cells and simultaneously deliver a radionuclide. The physical and chemical properties of MWCNTs (e.g., high strength, electrical conductivity, flexibility, functionalisation with biomolecules) make them attractive as nano-vectors for enhancing cell and tissue growth on scaffolds in vitro (8) and for the development of neural complex prosthetic implants (9;10). For these biomedical applications MWCNTs need to be purified, coated and functionalized because in their native state they have structural and physical properties (11;12) which render them electrostatically charged and hydrophobic. Some studies have underlined the relation between the toxic effects induced by CNTs and their length (13) and degree of agglomeration (14). For biological applications it is thus necessary to make nanotubes more hydrophilic by coating them with particular surfactants (15). A non-ionic surfactant, PF-127, has been successfully used for CNTs dispersion in aqueous solutions and it exhibited low toxicity when tested in various cell types (16;17;18). In recent years, conflicting data have been reported concerning safety and biocompatibility of these nanotubes. In a study on the cytotoxicity of unrefined SWCNTs to immortalized
2 human epidermal keratinocytes (HaCaT), Shvedova et al (19) showed accelerated oxidative stress, loss in cell viability and morphological alterations of cellular structures. They concluded that those effects were the result of high content (30%) of residual iron catalyst present in the as produced SWCNTs. Other groups confirmed these toxic effects, e.g., induction of intracellular reactive oxygen species (ROS) (20), DNA damage (21) and cell apoptosis (22). Recently, Poland and colleagues (23) demonstrated that exposure of the mesothelial lining of the peritoneal cavity of mice to long multi-walled carbon nanotubes (>15 microns in length) results in asbestos-like, length-dependent, pathological inflammation and the formation of giant cell granulomas. In sharp contrast, other reports have demonstrated that CNTs do not induce toxic effects on cells. Huczko et al showed that CNTs exhibited negligible risk of skin irritation and allergy (24). Cherukuri et al (4) observed that macrophages phagocytosed SWCNTs at the rate of approximately one SWCNT per second without any apparent cytotoxicity. Kam et al (25) reported no cytotoxicity for the pristine SWCNTs and Pantarotto et al concluded that CNTs coated with DNA provided a useful vector for safe gene delivery (26). Recently, Bardi et al demonstrated that MWCNTs dispersed in PF-127 did not induce neuron apoptosis in vitro and inflammation after direct injection of CNTs into the brain substance was not observed (27). Although CNTs production method, purity and functionalisation treatments influence biocompatibility, the majority of published reports have lacked this essential information. I have extensively investigated the biocompatibility of MWCNTs in different cell lines as a result of which identification was possible of good parameters in term of length, diameter and amount of metal catalyst needed for biocompatibility and avoidance of toxicological effects.
The final aim of this PhD thesis is to design, develop and test a smart system based on MWCNTs for drug delivery and exploit the magnetic properties of these nanoparticles to improve the delivery of chemotherapeutic drugs and to guide the cell homing after cell transplantation in vivo.
In order to achieve the final goal of my thesis I considered some midterm aims which were essential for success of the project. These can be summarized as follows:
3 1) Interaction between cells and MWCNTs: in vitro toxicology studies
Concerning the toxicity study on SH-SY5Y cell line, I performed four independent assays (MTT, WST-1, Hoechst assay and oxidative stress assay) in order to identify the effect of the nanotubes on cytocompatibility. The MTT test after 72 h of incubation showed a statistically significant (p<0.001) loss of viability (12%-15%) in cells treated with MWCNTs compared to the control sample, while cells treated only with PF-127 0.01% maintained a viability of 99%. In previously reported literature, it had been demonstrated that nanotubes absorbance could influence the results of the MTT assay at high nanotube concentration. For this reason, I performed WST-1 assay as double check. In contrast with the results of the previous test, the WST-1 assay revealed that cells treated with all three MWCNTs types after 72 h of incubation maintained high level of viability compared to untreated cells. In order to confirm these results I evaluated SH-SY5Y cell death by Hoechst 33258, which enables determination of the presence of DNA condensates, a characteristic feature of apoptosis. The number of apoptotic nuclei was determined on at least five randomly selected areas from three cover slips of each experimental group, each area containing approximately 100 cells. Results are expressed as percentage of apoptotic cells and compared to the control sample. Hoechst assay results showed that after 72h of incubation there was no apoptosis in cells treated with nanotubes, confirming the results obtained by WST-1 assay.
Even if carbon nanotubes did not show acute toxicity in vitro, Karin Pulskamp et al. demonstrated that they can induce intracellular reactive oxygen species (ROS). In order to study if the MWCNTs sample could induce oxidative stress I used the Image-IT Green Reactive Oxygen Species Detection kit (Invitrogen, U.S.A.). My results showed that all three samples of MWCNTs did not induce oxidative stress in neuroblastoma cell lines after 72 h of incubation. To determine the effects of carbon nanotubes purity and surfactant on cell viability after long time of incubation I performed a set of experiments designed to measure the viability at one and two weeks of treatments (fig.5). However, long term viability assays performed after two weeks of continuous incubation with the MWCNT samples showed a significant decrease in cytocompatibility of MWCNTs HNO3-97% and MWCNTs 97% respect to MWCNTs 99% and control sample. The data obtained indicated that MWCNTs 99% of purity
4 represented the best sample for long-term treatments because it minimized accumulation of metal catalyst which is toxic to the cells.
2) Exploitation of the magnetic properties of MWCNTs: application for cell displacement in vitro; guiding of the homing of mesenchymal stem cells after their transplantation in vivo; tracking of the cells loaded with these nanoparticles by using the Magnetic Resonance Imaging (MRI).
Carbon nanotubes 97% of purity showed interesting magnetic properties due to the presence of metal impurities (Fe). In these experiments I observed that cells treated with MWCNTs 97% move towards a magnetic source. This magnetic guide could have many applications in cancer therapy and in cell transplantation. I performed some tests to observe the movements of tumor cells and mesenchymal stem cells loaded with MWCNTs towards a magnetic source. In human neuroblastoma (SH-SY5Y) and mice pheochromocytoma (PC-12) cell lines, I demonstrated that 10 µg/ml MWCNTs 97% were sufficient to promote cells displacement in magnetic field and this dose of nanotubes appears to be non toxic in vitro. In particular I observed that cells grew without damage and in presence of specific differentiation factors were able to differentiate into neurons, whilst maintaining high viability level.
The project also included the investigation of the interaction between magnetic carbon nanotubes and mesenchymal stem cells and their ability to guide these cells injected intravenously in living mice by using an external magnetic field. The data obtained from these experiments showed that MWCNTs did not affect cell viability and their ability to differentiate into osteocytes and adipocytes. Furthermore MWCNTs and the magnetic field used did not alter cell growth rate, phenotype and cytoskeletal conformation. These magnetic nanotubes, when exposed to magnetic fields, were able to shepherd MSCs towards the magnetic source in vitro. Moreover, the application of a magnetic field alters the bio distribution of CNT-labelled MSCs after intravenous injection into rats, increasing the accumulation of cells into the target organ (liver). These studies enabled the conclusion that MWCNTs hold a distinct potential for use as nano-devices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. This could pave the way for the development of new strategies for manipulation/guidance of MSCs in regenerative medicine and cell transplantation.
5 In a series of other experiments, the effectiveness of cellular imaging using 97% MWCNTs as a MRI contrast agent was investigated by labelling mesenchymal stem cells. Specifically, the potential of multiwall carbon nanotubes (MWCNTs 97%) with low metal impurities (2.57% iron) as MRI contrast agents was investigated. The results showed that the r2 relaxivity of MWCNTs in 1% agarose gels at 19 °C was 564 ± 41 s-1mM-1. This is attributed to both the presence of iron oxide impurities and to the carbon structure of the MWCNTs. Moreover, in these studies we were able to label stem cells with MWCNTs to demonstrate their effectiveness as labels for cellular MR imaging. These results suggest that the MRI contrast agent properties of the MWCNTs could be used in vivo for stem cell tracking/ imaging and during MWCNT-mediated targeted electro-chemotherapy of tumours.
3) Design and Development of magnetic MWCNTs as carrier for the guided delivery of the innovative chemotherapeutic agent GDC-0941 produced by Astra Zeneca in collaboration with the Life Science Dept. of the University of Dundee.
GDC-0941 is a specific inhibitor of the PI3- I kinase and is very effective in the inhibition of the pathway of mTor, which is important for the regulation of the cellular cycle. It is well established that mTor deregulation pathway is involved in many types of tumours. The GDC-0941, which is still in phase I clinical trial, showed very good results in killing cancer cells. In particular, it has been shown to be very effective for the treatment of different types of lymphoma. Unfortunately, this drug exhibited significant side effects on the healthy tissues. The toxicity and associated side effects showed of the current chemotherapy regimens remain one of the major problems in clinical oncology. As a possible solution to this problem, I investigated the binding of GDC-0941 drug to magnetic nanotubes, as this would enable the administration of low doses of the drug to patients by driving the drug towards the tumours mass with the use of a magnetic field. For this purpose, I functionalized MWCNTs with Poly-Lysine which is a biocompatible molecule, conferring many NH2 group to the nanotubes. These amino groups can bind by non covalent interaction with the COO- group of the GDC-0941. With this strategy I was able to obtain and characterize the compound
MWCNT-GDC-6 0941. I tested the ability of this compound to inhibit the mTor pathway in 293 cells derived by human kidney cancer. In vitro results showed that the MWCNTs-GDC compound was stable in physiological solution and the drug not only maintained its antitumor activity, but this increased by 30% after the conjugation with the nanoparticles.
7 1. Introduction
The first use of the concepts found in 'nano-technology' was in "There's Plenty of Room at the Bottom", a talk given by physicist Richard Feynman at an American Physical Society meeting at Caltech on December 29, 1959. Feynman described a process by which the ability to manipulate individual atoms and molecules might be developed, using one set of precise tools to build and operate another proportionally smaller set, and so on down to the scale. In the course of this, he noted, scaling issues would arise from the changing magnitude of various physical phenomena: gravity would become less important, surface tension and van der Waals attraction would become increasingly more significant, etc. This basic idea appeared plausible, and exponential assembly enhances it with parallelism to produce a useful quantity of end products. The term "nanotechnology" was defined by Tokyo Science University Professor Norio Taniguchi in a 1974 paper as follows: "'Nano-technology' mainly consists of the processing of, separation, consolidation, and deformation of materials by one atom or by one molecule." Thirty-seven years later, nanotechnology has been shown to impact on all sectors of human life: electronics and computers, mobile phones, food and agriculture industry, cosmetics, paints and, of course, healthcare. Today in the market there are more than 1000 products based on nanotechnology produced by 485 companies worldwide (30). In essence nanotechnology entails the manufacturing and manipulation of matter at a scale ranging from single atoms to micron-sized objects. From the biological standpoint, nanomaterials match the typical size of naturally occurring functional units or components of living organisms and, for this reason, enable more effective interaction with biological systems. It was clear that nanotechnology could have an immense potential impact in many aspects of the healthcare, and this has paved the way at a new era labeled as ‘Nanomedicine’ (31). Since nanomaterials‘ safety is yet to be fully demonstrated, there are still some doubts about the use of nanoparticles. Although it is prudent to investigate the adverse effects on health both during manufacture and use of nanomaterials, with adequate safe guards and standards emanating from nano-safety and toxicity studies, there is no reason to doubt the overriding benefit which nanomedicine will provide (32). The EU Technology platform on Nanomedicine (33) defines nanomedicine as ‘the application of nanotechnology to achieve breakthroughs in healthcare’. It exploits the improved and often novel physical,
8 chemical and biological properties of materials at the nanometer scale (34). Nanomedicine has the potential to enable early detection and prevention, and to improve diagnosis, treatment and follow-up of many diseases. It has already enabled miniaturization of many current devices resulting in faster operation or enhanced integration of several operations.
1.1 Nanoscale and nanomaterials
A nanometre (nm) equals one thousand millionth of a meter and for comparison, one single human hair measures 80,000 nm across and a red blood corpuscle has a diameter of 7,000 nm. By definition the nanoscale ranges from 100 nm down to the size of atoms (approximately 0.2 nm). At this scale the properties of materials are very different from their bulk equivalents. Thus nanomaterials have a relatively larger surface area when compared to bulk material and for this reason, are more chemically reactive. Indeed some materials are inert and become reactive only in their nano-scale form. Additionally, the nano-scale has a marked effect on the strength and electrical properties as the quantum effects dominate the behavior of materials with respect to their optical, electrical and magnetic properties. Nanomaterials fall into three categories: Uni-dimensional – nano-scale in one dimension, e.g. very thin surface coatings, two-dimensional, e.g., nanowires, nanotubes and three-two-dimensional, e.g., nanoparticles and buckey balls. The latter were first produced and reported by Richard Smalley, Robert Curl, and Harold Kroto in 1985 (35) for which they received the Nobel prize for chemistry in 1996. Buckey balls are spherical fullerenes (made entirely of carbon), whereas carbon nanotubes (CNT, also known as buckey tubes) are cylindrical with closed ends and may be single or multi-walled (SWCNT, MWCNT). Carbon nanotubes were discovered by S. Iijima in 1991 (36) and have generated considerable interest and a vast body of research during the last decade on their potential biomedical applications (37;38). In the recent years many different nanomaterials have been produced and they are investigated especially for biomedical application:
- Liposomes are vesicles that consist of one to several, chemically-active lipid bilayers. The lipid molecules posses heads which are hydrophilic and thus line up to form a surface facing aqueous fluids (interstitial fluid in the case of the
bi-9 phospholipid cell membrane); whereas the tails are hydrophobic and face the other way. In the case of cell membranes, the heads of the inner layer face the intracellular fluid with the tails being orientated away from it. Thus the hydrocarbon tails of one layer face the hydrocarbon tails of the outer layer forming the normal bilipid cell membrane. The structure of lysosomes is exactly the same and they can be composed of natural phospholipids with mixed lipid chains (e.g. phosphatidylethanolamine), or of pure surfactant components such as dioleoylphosphatidylethanolamine. Liposomes are biocompatible, nontoxic, biodegradable, and able to act as carriers for hydrophobic, hydrophilic, and amphiphilic molecules. They have received considerable attention during the past 30 years as pharmaceutical carriers with various applications: drug delivery vehicles, adjuvant in vaccination, signal enhancers/carriers in medical diagnostics and analytical biochemistry, solubilizers for various materials as well as support matrix for chemical ingredients and as penetration enhancers in cosmetic products.
- Dendrimers are nanometre-sized polymer macromolecules consisting of a central core, branching units and terminal functional groups. The core chemistry determines the solubilizing properties of the cavity within the core, whereas the external chemical groups determine the solubility and chemical behavior of the dendrimer itself. Targeting is achieved by attaching specific ligands to the external surface of the dendrimer enabling its binding to disease sites (39), while its stability and protection from phagocytes is achieved by ‘decorating’ the dendrimers with polyethylene glycol chains (40). They are able to bind and/or encapsulate a drug, thus protecting it against chemical and enzymatic degradation. Polymeric delivery systems have the potential to maintain therapeutic levels of drugs, reduce side effects and facilitate the delivery of drugs with short in vivo half-lives (41). In environmentally sensitive controlled drug delivery systems, the release of the drug from the carrier (capsule or matrix) at the intended site is achieved by an external trigger including change in pH or heating (which opens the polymer) by exposure to ultrasound In recent years, biodegradable polymeric nanoparticles have attracted considerable attention in the controlled release of drugs targeting particular organs/ tissues, as
10 carriers of DNA in gene therapy and in their ability to deliver proteins, peptides and genes by the oral route (42).
- Quantum dots discovered by Louis E Brus (Bell Labs) constitute an important class of nanomaterials (43). They are semiconductor nanocrystals whose excitons (electrons bound to electron holes) are confined in all three spatial dimensions. The crystals are composed of periodic groups of II-VI, III-V, or IV-VI materials and thus they have properties that are intermediate between those of bulk semiconductors and discrete molecules. Different sized quantum dots (5–100 nm depending on fabrication) emit different color light due to quantum confinement. They are used in computing, photovoltaic devices (nanocrystal solar cells) in biological research (for intracellular tracking of materials including carbon nanotubes) and in light emitting diodes. Colloidal quantum dots can be attached to a variety of molecules via functional groups (thiol, amine, nitrile, carboxylic acid or other ligands) but for life science research non-heavy metal InP water stabilized quantum dots exemplified by Evident’s T2-MP EviTags (44) are preferred in view of their cytocompatibility. These have lattice structure with three components: An inner core of InGaP crystals surrounded by a ZnS shell and an external functional lipid coating. The matched pattern of atoms results in a brighter quantum dot with increased stability for longer lasting fluorescence. The outer lipid coating ensures stability in aqueous solution, can be easily functionalised for biomedical applications, and is less toxic to cells than quantum dots based on heavy metals. Silica multifunctional QD microspheres are used in studies of the microcirculation (45) and for sentinel lymph node mapping.
- Boron nitride nanotubes (BNNT) have the identical structure to CNTs with the carbon molecules being replaced by B and N. They have generated considerable interest in the last five years in view of their piezo-electric properties through which they are able to acquire an electric charge on exposure to ultrasound and polarized light (46).
- Superparamagnetic iron oxide (SPIOs) nanoparticles coated with dextran are the only nanomaterials in established clinical use. They have been the standard
11 contrast agents for magnetic resonance imaging of tumours since the early 1990s. More recent MRI contrast agents include FePt nanoparticles (47) and nanoparticulate agents based on liposomes (48) and lipidic nanoparticles containing Ln III. Engineered magnetic nanoparticles (MNP) are being actively researched by several groups for cell therapies and targeted concentration of trophic factors needed for regeneration of specialized tissues after injury.
1.2 Carbon nanotubes
CNTs are usually produced by catalytic chemical vapor deposition and contain metals, mainly Fe at their closed ends (65). For this reason they are paramagnetic – a useful property for certain biomedical applications. CNTs vary in diameter (from a few nm to 100 nm) and widely in length (up to several mm). Their molecular structure accounts for their unique properties: high tensile strength, high electrical conductivity, heat resistance and efficient thermal conduction and relative chemical inactivity (constituent atoms not easily displaced). The exact structure of CNT especially their n – m chirality determines their electric properties. When n-m is a multiple of 3 (armchair type), the CNTs have static dielectric properties, i.e. exhibit a metallic longitudinal and an insulator transverse response. In practice all multiwalled CNTs behave in this way. By virtue of their nano-scale, electron transport in CNTs occurs through quantum effects and thus only propagates uni-dimensionally along the axis of the tube. One of the problems which have impeded the use of CNTs for biomedical applications which has since been resolved is their insolubility in aqueous solution. This is a essential requirement for biological interactions and biocompatibility. The problem has been resolved by studies on protocols for covalent and non-covalent polymer coating which has enabled in-vitro cell viability assays and in-vivo studies on biocompatibility (49;16). Carbon nanotubes exhibit a good ability to interact with cells and cross cell membranes (1). By virtue of this characteristic, they can be used as carriers for drug and DNA delivery. In recent years several groups have demonstrated that both SWCNTs and MWCNTs can be internalized by a variety of cell types and thus used to deliver therapeutic and diagnostic small molecules (2;3;4). Conjugation of CNTs with different
12 molecules can be used for improving new vaccine production, novel therapies against retrovirus infection and tumor cell proliferation.
1.3 Open issue: CNTs toxicity
In recent years, conflicting data have been reported concerning safety and biocompatibility of these nanotubes. In a study on the cytotoxicity of unrefined SWCNT to immortalized human epidermal keratinocytes (HaCaT), Shvedova et al (19) showed accelerated oxidative stress, loss in cell viability and morphological alterations of cellular structures. They concluded that those effects were the result of high content (30%) of residual iron catalyst present in the unrefined SWCNTs. Other groups confirmed these toxic effects, e.g., induction of intracellular reactive oxygen species (ROS) (20), DNA damage (21) and cell apoptosis (22). Recently, Poland et al (23) demonstrated that exposure of the mesothelial lining of the peritoneal cavity of mice to long (10-15mm) multi-walled carbon nanotubes results in asbestos-like, length-dependent, pathological inflammation and the formation of giant cell granulomas. In sharp contrast, other reports have demonstrated that CNTs do not induce toxic effects on cells. Huczko et al showed that CNTs exhibited negligible risk of skin irritation and allergy (24). Cherukuri et al (4) observed that macrophages phagocytosed SWCNTs at the rate of approximately one SWCNT per second without any apparent cytotoxicity. Kam et al (25) reported no cytotoxicity for the pristine SWCNTs and Pantarotto et al concluded that CNTs coated DNA provided a useful vector for safe gene delivery (26).
The exact fate of carbon nanotubes inside the cells and in the animals remains controversial. In vitro studies showed that SWCNTs can be degraded by living cells which cause their complete biodegradation (50); however the degradation of MWCNTs has not been demonstrated.
Only few studies have been published on the bio-distribution of carbon nanotubes in vivo. Singh et al. (51) studied CNT bio-distribution following intraperitoneal administration in mice and they observed rapid blood clearance from systemic blood circulation through renal excretion. Moreover, urinary excretion studies using both f-SWNT and functionalized MWCNT followed by electron microscopy analysis of urine
13 samples revealed that nanotubes were excreted as intact nanotubes after three hours. More work is needed to understand the clearance of nanotubes and the characteristics which influence their biodistribution, degradation or excretation.
It is our view that many of the published studies on CNT toxicity are flawed for one of two reasons. The first is that some studies used CNT purchased from various sources without characterization of length, purity, metal content and carbon soot. Secondly other studies have used in-vitro cell viability studies in which the CNTs react with the reagent used in the assay; as shown in the work of Wörle-Knirsch et al. (52) on the interferences of carbon nanotubes with the MTT proliferation assay. For this reason, the use of different and independent assays to study toxicological effects of nanoparticles has been proposed and generally accepted (53).
In order to define valid guidelines for the toxicological studies on nanomaterials, the EU has recently established the Nano-safety Cluster – a network of researchers and toxicologists to address the existing controversies and develop appropriate guidelines. There is also an urgent need for the definition of ‘medical grade’ CNT in terms of length, purity and metal content.
1.4 Properties of CNTs exploited for biomedical applications: the future of Nanomedicine
In the last ten years, several groups have shown that SWCNTs and MWCNTs can be used as excellent intracellular transporters to deliver therapeutic and diagnostic small molecules and macromolecules to cells (54). Different uptake mechanisms (phagocytosis, diffusion and endocytosis) have been reported in the literature. Some physico-chemical characteristics of the carbon nanotubes (e.g., the nanotube dispersion, the formation of supramolecular complexes, and the nanotube length) drive the uptake pathway (55). Both SWCNTs and MWCNTs can be internalized by a variety of cell types and thus used to deliver therapeutic and diagnostic molecules. Conjugation of CNTs with different molecules can be used for new vaccine production, novel therapies against retrovirus infection and tumor cell proliferation. Pantarotto et al (5) reported that VP-1 protein of the foot-and-mouth disease virus (FMDV) covalently linked to SWCNT
14 induced a specific anti-body response in vivo without any cross reactivity. Liu et al (6) transfected human T cells and peripheral blood mononuclear cells with siRNA molecules conjugated to CNTs to abrogate the expression of cell-surface receptors CD4 and co-receptors CXCR4 necessary for HIV entry and T cells infection. Additionally McDevitt et al (7) constructed a specific CNT conjugated antibody to target the CD20 epitope on Human Burkitt lymphoma cells and simultaneously deliver a radionuclide. The physical and chemical properties of MWCNTs (e.g., high strength, electrical conductivity, flexibility, functionalization with biomolecules) make them attractive as nano-vectors for enhanced cell and tissue growth on scaffolds in vitro (8) and for the development of complex neural prosthetic implants (9-10).
CNTs also possess intriguing magnetic properties which derive from the metal catalyst impurities entrapped at the CNT extremities during their manufacture, enabling them to react to external magnetic fields. This property has been utilised by Cai et al. to develop a physical technique for in-vitro and ex-vivo gene transfer known as ‘nanotube spearing’, capable of effective cell transfection with plasmid DNA (55). Similarly, we have recently demonstrated that MWCNTs are able to interact with cells and, when exposed to a magnetic field, induce their migration towards the magnetic source (55;56). This control of cell movement has important medical applications both in cell therapy for regeneration, cell transplantation and in cancer therapy (anti-metastasis). Moreover, we recently have demonstrated that MWCNTs with low metal impurities (2.57 % iron) can be used as MRI (magnetic resonance imaging) contrast agents even at concentrations of tens of µg/ml, as the MWCNTs have a significant effect on the observed 1H transverse (1/T2) relaxation rate of water. Consequently, cells labelled with MWCNTs exhibit a reduced image intensity in T2-weighted MR images compared to cells without internalized MWCNTs. The 3D MRI cellular study suggests that it should be possible to track stem cells injected in vivo by labeling cells with these low Fe MWCNTs. It is possible that MRI could be used in the studies to better understand the biodistrudution and the fate of carbon nanotubes in vivo. Another important property of CNTs is their strong absorbance of near infra red (NIR) light with heating and release of this heat for destruction of cells – nanohyperthermic ablation of tumours (57). Shi Kam and colleagues achieved selective cancer cell destruction in vitro by using folate functionalized nanotubes heating by continuous NIR radiation. Additionally CNTs
15 acquire and release heat on application of radio-frequency current (58). Gannon et al. induced efficient heating of aqueous suspensions of SWNTs by using RF fields. In particular they produced a noninvasive, selective, and SWNT concentration dependent thermal destruction in vitro of human cancer cells that contained internalized SWNTs; moreover they observed that intratumoral in vivo injection of SWNTs in the liver followed by immediate RF was able to induce necrosis in the tumor mass without evident side effects into the healthy tissues. Only recently discovered (and patented by the NINIVE consortium) is the ability of CNTs to act as a dipole antenna and acquire a charge on exposure to electromagnetic radiation in the microwave range. This is the basis of an exciting new technology for remote wireless low voltage electro-chemotherapy and gene transfection and novel forms of electro-stimulation therapies for neurodegenerative disease (advanced Parkinson’s disease unresponsive to medication), electrostimulation for skeletal and visceral muscle palsies and for cardiac arrhythmias.
Several groups have investigated different nanoparticles in order to exploit nanotechnology strategies for neurological applications. As carbon nanotubes/fibers have excellent electrical conductivity, strong mechanical properties and similar nano-scale dimensions to neurites, they have been explored for their ability to guide axonal regeneration and to improve neural activity by acting as biomimetic scaffolds at sites of nerve injury. In particular, Mattson et al were the first group to demonstrate that neurons grow on MWCNTs (59); moreover they reported an increase of over 200% in total neurite length and nearly a 300% increase in the number of branches and neurites on MWCNTs coated with 4-hydroxynonenal compared to uncoated MWCNTs. Hu et al reported that positively charged MWCNTs significantly increased the number of growth cones and neurite branches compared to negatively charged MWCNTs (60). Gheith et al investigated the biocompatibility of a free-standing positively charged single wall carbon nanotubes (SWCNT)/polymer thin-film membrane prepared by layer-by-layer assembly (61); they observed a 94-98% viability of neurons on the SWCNT/polymer films after 10 days of incubation and this induced neuronal cell differentiation, guided neuron extension and directed more elaborate branches than controls. Moreover, Lovat et al demonstrated that purified MWCNTs have the potential to boost electrical signal transfer of neuronal networks (62). Recently Cellot et al investigated the nature of CNT-neuron interactions and proposes a mechanism in which carbon nanotubes
16 (CNTs) can boost neuronal activity by providing a shortcut for electrical coupling between somatic and dendritic neuronal compartments (63).
The EU Technology Platform advocates that nanomedicine should aim for meaningful improvements ‘in areas that contain the most severe challenges in future healthcare appropriate to the technology’ and has identified six disease categories which share the following three characteristics:
- reduce the patient’s quality of life - have a very high prevalence
- impose a high socio-economical burden on society.
Within this framework, nanotechnology is expected to have a high impact on the care process for the following disorders identified by the EU Technology Platform:
- cardiovascular disease, - cancer,
- musculoskeletal disorders,
- neuro-degenerative and psychiatric disorders, - diabetes mellitus and
- bacterial and viral infections.
This outlines the priorities of nanomedicine and indicates to the nano-scientists and nano-engineers the direction of their research and development programs which is most likely to benefit healthcare in terms of improved outcome from life-threatening disorders for which current therapies are only partially effective. Nanotechnology has ushered a new era of nanomedicine and life nanoscience with very significant potential for more effective therapy of life-threatening or disabling disorders. Currently nanomedicine is at the first generation (passive) stage of nanotechnology but progress has now reached the threshold of the 2nd generation (active nanomaterials) with early clinical trials, certainly in the field of controlled drug delivery and cell therapies. Substantial progress has been made in technologies and devices for wireless
electro-17 chemotherapy and electro-stimulation therapies but further R&D is needed as well as pre-clinical safety and efficacy studies. Certainly both basic and applied life science research has benefited from the biological application of nanomaterials and indirectly, this will help to accelerate the advance of nanomedicine. However before the widely recognized potential of nanomedicine is realized, the safety aspects both during production and in application of clinical therapies based on nanomaterials have to be addressed.
Fig. 1 CNT properties and their application in the biomedical field.
2. Material and Methods
2.1 MWCNT preparation.
The MWNT production method used here is based on the synthesis of carbon nanostructures by catalytic chemical vapour deposition (CCVD or CVD) of hydrocarbon sources on substrates of metal oxides (Al2O3) impregnated with metal catalysts (Fe). MWCNTs were obtained from Nanothinx S.A. (Rio Patras, Greece). The solutions of MWCNTs were created with polyoxyethylene-polyoxypropylene block
18 copolymer (PF-127; Sigma), a water-soluble surfactant with molecular weight = 12,600. PF127 was dissolved in Dulbecco's modified Eagle's medium (DMEM; Lonza, Milan, Italy) at a concentration of 0.1% (wt/vol); 0.5 mg/mL of MWCNTs were subsequently added, and then the solutions were heated over an hot plate at 70°C with magnetic stirring for 4 hours. The resulting mixture was sonicated with a sonicator (Bransonic 2510; Bransonic, Danbury, Connecticut) at 20 W for 16 hours. The mixture was then centrifuged at 1100g for 10 minutes to remove no dispersed CNTs. The average concentration of the CNT solution measured by spectrophotometric analysis at 280 nm (64) was 50 µg/mL. PF127 used at a concentration below 0.1% is not cytotoxic. With the same procedure we prepared a solution of carbon nanotubes dispersed in Poli-L-Lisina 0.01% (w/v), which confers positive charge on the surface of the nanotubes. MWCNT samples were fully characterized by SEM, Raman spectroscopy, transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and FIB microscopy as previously reported. (56;66) The purity of CNTs was assessed by a postdeposition TGA treatment, which also provided a measure of the residual metal catalysts percentage. SEM and TEM were used for the morphology and structure examination of CNTs; moreover, Raman spectroscopy was used for the determination of the type of CNTs, as well as the presence of defects and amorphous carbon content. The measurement of the MWCNTs length was performed by a FIB system: FEI 200 (FEI, Hillsboro, Oregon, U.S.A.) (FIB localized milling and deposition) delivering a 30-keV beam of gallium ions (Ga+). We used three different samples of MWCNTs: 97% purity, 99% purity, and 97% purity treated with nitric acid (HNO3; 97% acid-treated [a.t.]) (Figure 2).The carbon content of the 97% pure MWCNTs produced was 97.02% with less than 3% amorphous carbon content. TGA analysis of the 99% pure MWCNTs revealed a carbon content of 99.2% and a minimal (~1%) amount of carbon soot. We obtained 97% a.t. MWCNTs by treating 97% MWCNTs with 65% HNO3 in 100 mL/g of CNTs for 10 minutes under ultrasonication. This solution was mechanically stirred at 220°C for 20 minutes and then cooled to room temperature, filtered, and finally dried at 25°C for a few hours; the dried sample was then dispersed in aqueous solution. Table 1 outlines the details of the three MWCNT samples. The magnetic properties of the MWCNTs 97% utilized in these experiments were previous analyzed by the use of a SQUID magnetometer (MPMSXL-7, Quantum Design). The magnetization curve was recorded for solid samples of 5 mg of CNTs. Measurements were performed at 37 °C with an
19 applied magnetic field up to 20,000 Oe. The analysis showed a magnetization curve typical of paramagnetic materials, with a coercivity of about -400 Oe and a remanence of about 0.44 emu/g. Saturation magnetization (ms) was about 1.6 emu/g for a field of about 5,000 Oe. The MWCNT-modified culture medium was obtained by mixing the MWCNT solutions with the complete culture medium in a ratio of 1:10 (vol/vol), corresponding to a MWCNT concentration of 5 µg/mL and 10 µg/mL and a PF127 concentration of 0.01% (w/v). The complete culture medium and the MWCNT modified culture medium were used as control and test groups, respectively; moreover, to study the effects of PF127 surfactant we studied an additional experimental group (cells incubated with complete culture medium supplemented by PF127 0.01%). For short-term assays we incubated cells with the MWCNT-modified culture medium for 24, 48 and 72 hours; for long-term assays we continuously cultured cells with the MWCNT-modified culture medium for 2 weeks, replacing the MWCNT-MWCNT-modified culture medium with fresh MWCNT-modified culture medium twice per week.
2.2 Preparation of MWCNTs functionalized with GDC-0941
With the same procedures utilized for PF-127, we functionalized MWCNTs with Poly-L-Lisina (PLL, Sigma-aldrich) 0.01%, which is a biocompatible molecule conferring many NH2 group to the nanotubes. These amino groups can bind by non covalent interaction with the COO- group of the GDC-0941. We incubate for 30 minutes a RT 30µg of MWCNTs functionalized with PLL with a solution of GDC-0941 300mM. After that we washed this compound three times with 500µl of PBS1X by ultracentrifugation at 3*104 xg. Finally, we dissolved the compound in DMSO (Dimethylsulfoxide, Sigma-aldrich). Following this strategy we obtained and characterized the compound MWCNT-GDC-0941.
2.3 Cell line culture
The human neuroblastoma cell line SH-SY5Y, was obtained from American Type Culture Collection (ATCC, Rockville, MD). SH-SY5Y cells were grown in a complete culture medium consisting of 1:1 mixture of Hams F12 (Lonza, Milan, Italy) DMEM
20 supplemented with 2mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10% heat inactivated fetal bovine serum (FBS, Lonza, Milan, Italy).
Immortalized mouse hippocampal cells HN9.10e (donated by Dr. Mercedes Garcia, University of Pisa, Italy) were cultured in DMEM with 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM L glutamine.
NIH-3T3-L1 cells obtained from ATCC, were grown in DMEM supplemented with 10% fetal bovine serum, 200 U/mL penicillin, and 50 µg/mL streptomycin (standard medium) to confluence (hereafter referred to as day 0). For differentiation, confluent NIH-3T3-L1 cells were stimulated to differentiate by addition of hormonal cocktail (0.5 mM 1-methyl-3- isobutylmethyl-xanthine, 0.5 µM dexamethasone, and 170 nM insulin [MDI]) to the standard medium for 2 days and were maintained thereafter in standard medium with 170 nM insulin. Typically, by day 6 more than 95%of the cells fully differentiate into adipocytes.
The Crandell feline kidney fibroblasts (CRFK) cell line was obtained from the ATCC. Cells were maintained in DMEM containing 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.
Rat pheochromocytoma PC12 cell line obtained from ATCC, were maintained in DMEM supplemented with 2mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 5% heat inactivated fetal bovine serum and 10 % horse serum (HS, Lonza, Italy). The rat pheochromocytoma PC12 cell line is a model system for neuronal differentiation. In the presence of nerve growth factor (NGF), PC12 cells change their shape from round cells to neurite-extending cells and acquire properties of sympathetic neurons.
PC12 cells when cultured in serum-free conditions cannot proliferate and most die within a few days. However, in the presence of NGF (50 ng/ml) cells remained viable, and after 5 - 7 days developed neuritis, thus differentiating into neurons.
Human embryonic kidney cancer cells (HEK 293), were gently donated by Prof. Dario Alessi from the MRC of the University of Dundee. Cells were maintained in DMEM supplemented with 2mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10% heat inactivated foetal bovine serum.
21 All the cell lines were maintained at 37°C in a saturated humidity atmosphere of 95% air and 5% CO2.
2.4 SH-SY5Y and HN9.10e preparation for the experiments of in vitro magnetic translocation mediated by MWCNTs.
A fixed cell number ( 25x103cells/cm2 ) was seeded in a 24-well plate and maintained at 37 C in a saturating humidity atmosphere containing 95% air/5% CO . Cells were incubated for 2 h to allow cell attachment and CNTs-modified medium was added to the culture at a ratio 1 : 10. After 1 h of incubation, a magnet was placed in the well. The magnet was a Neodymium disk magnet (Eclipse Magnetics Ltd, N703-RB), with 10 mm in diameter and 5 mm in height. This magnet has a lifting strength of 2.5 kg when in contact and produces a magnetic flux density of 0.0374 T in the A1 area and 0.305 T in the A2 area, with reference to Fig.13. The growth of the cells was monitored for 72 h, and digital image acquisition was carried out using a Nikon TE2000U fluorescent microscope with a 20 X objective equipped with Nikon DS-5MC USB2 cooled CCD camera. Two lines were drawn on the bottom of the plate dish as a reference point to collect pictures in the same position (A1 and A2) on different days. The cell density in the A1 and A2 area has been monitored each day, and the number of cells per area unit (cm2) have been counted with the image analysis software “ImageJ”
2.5 PC12 preparation for the experiments of in vitro magnetic translocation mediated by MWNCTs.
Cells used for experiments were washed with phosphate-buffered saline (PBS, Lonza, Italy), removed from the plastic dishes by scraping, pelleted and re-suspended in complete medium. A fixed cell number (25x10³cells/cm2) was seeded in a 24–well plate and maintained at 37 °C in a saturated humidity atmosphere containing 95% air / 5% CO2. Cells were incubated for 6 hours to allow cell attachment and MWCNTs-modified medium was added to the culture. MWCNTs modified medium was prepared by mixing PF127-MWCNT solution and cell culture medium at ratio 1:10 (v/v), containing 10 µg/ml of MWCNTs and 0.01% of PF127. After 12 hours of incubation, a magnet was
22 placed in the well; the magnet was a Neodymium cube magnet (Eclipse Magnetics Ltd, Atlas, UK), with a lifting strength of 2,5 kg when in contact (Residual Induction 1.41 T) . The growth and movements of the cells was monitored for 168 hours, and digital image acquisition was carried out using a Nikon TE2000U microscope with a 20x objective equipped with Nikon DS-5MC USB2 cooled CCD camera.WCNTs .
2.6 Primary cells
2.6.1 MSC isolation and culture
Rat bone marrow cells were collected from tibia and femur of Wistar Furth animals following the Dobson procedure (67). Total nucleated cells were cultured in Dulbecco modified culture medium (Sigma-Aldrich, Italy) supplemented with 10% fetal bovine serum (Eurobio, Italy), 1% L-glutamine (Sigma-Aldrich, Italy), penicillin (Eurobio, 50U/mL), streptomycin (Eurobio, 50µg/mL), amphoterycin B (Sigma-Aldrich 0.2µg/mL) and incubated at 37°C in fully humidified atmosphere containing 95% air and 5% carbon dioxide. After seven days half of the culture medium was changed. On reaching confluence, the adherent cells were detached by 0.05% trypsin and 0.02% EDTA for 5–10 min at 37°C, harvested and washed with HBSS and 10% FBS and finally re-suspended in complete medium (primary culture, P0). Cells were re-seeded at 104 cells/cm2 in 100-mm dishes (P1) for in-vitro differentiation assessment, and for further cellular expansion achieved by successive cycles of trypsinization and re-seeding. The frequency of Colony Forming Units-Fibroblasts (CFU-F) was measured using the method of Castro-Malaspina. Visible colonies with 50 or more cells (the conventional value for defining a colony) were counted and referred to 106 plated cells (no. of CFU-F/106 TNC).
2.6.2 Immunophenotyping and differentiation of MSC and MSC-CNT sample
The morphologically homogeneous population of untreated MSC and of MSC cultured respectively with PF127-CNTs and PF127 alone for 5 days were analysed for the expression of cell surface molecules using flow cytometry procedures: MSC were recovered from flasks by trypsin–EDTA treatment and washed in HBSS and 10% FBS.
23 Aliquots (1.5×105 cells/100µl) were incubated with the following conjugated monoclonal antibodies: CD34, CD45, CD11b (in order to quantify hemopoietic– monocytic contamination), CD90, CD106, CD73. Non-specific fluorescence and morphologic parameters of cells were determined by incubation of the same cell aliquot with isotype-matched mouse monoclonal antibodies. Incubations were performed for 20 min, thereafter the cells were washed and 7-AAD was added in order to exclude dead cells from the analysis. Flow cytometric acquisition was performed by collecting 104 events on a FACScan (argon laser equipped; Becton Dickinson) instrument, and data were analysed on DOT-PLOT bi-parametric diagrams using CELL QUEST software (Becton Dickinson, Franklin Lakes, NJ, USA). The ability of untreated MSC and MSC cultured for 5 days respectively with PF127-CNTs and PF127 alone to differentiate along osteogenic and adipogenic lineages was assayed, as described previously by Pittenger et al (68). Osteogenic and adipogenic differentiation were evaluated by cytochemical analysis. Petri dishes were stained to assess extracellular matrix mineral bound by Alizarin Red-S. Adipogenic differentiation was evaluated by Oil Red O stain for lipid-rich vesicles.
2.6.3 Neurons culture
Primary mouse cortical cultures were prepared as follows: Brains were removed from mice at postnatal days 0-2 and placed in dissection medium(16mMglucose, 22mMsucrose, 10mMHEPES, 160mMNaCl, 5mMKCl, 1mMNa2HPO4, 0.22 mM KH2PO4; pH 7.4; 320-330 mOsm). Cortices were dissected and meninges removed. Chopped small pieces of cortex were triturated with a Pasteur pipette in the presence of 0.25% trypsin and DNase to reach a single-cell suspension. After 5 to 10 minutes a small volume of suspension at the top was placed in an equal volume of DMEM supplemented with10% horse serum. Cells were then counted and directly cultured on coverslips or Petri dishes previously coated with polylysine. After 24 hours DMEM– horse serum was replaced with Neurobasal-A medium (Invitrogen, Carlsbad, California) supplemented with B27, 2 mM glutamine, and antibiotics. After 10 days fully differentiated neurons lay on glia cells growing in a layer underneath the neurons.
24 2.7 Biocompatibility assays
2.7.1 Trypan blue assay
Trypan blue (TB, Sigma-aldrich) is a vital stain that is used to color dead cells. After three days of culture, for each sample, cells have been detached from the substrate and 200 µl of cell suspension were placed in a tube and an equal volume of 0.4% Trypan blue was added. Then 10 µl of stained cells was placed in a Burker chamber and the number of viable (unstained) and dead (stained) cells was counted. The repeatability of the method was assessed and confirmed by six assays.
2.7.2 MTT and WST-1 assays
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-aldrich) and 2-(4-iodophenyl)-3-(4-nitophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium salt (WST-1; BioVision, Mountain View, California) cell proliferation assays were used to investigate the effect of the three types of MWCNTs and the surfactant on cell viability.
2.7.3 Short-term assays (24, 48 and 72 hours)
For MTT assay 25 × 103 cells were seeded into each well of a 96-well plate and then incubated with the culture medium. This was then replaced with 100 µL of medium containing 0.5 mg/mL of MTT, and cells were incubated for 2 hours at 37°C and 5% CO2. In this assay the mitochondrial dehydrogenase of viable cells reduces the water-soluble MTT to water-inwater-soluble formazan crystals. The MTT-containing medium was then replaced with 100 µL of dimethylsulfoxide and left for 10 minutes on a platform shaker to solubilize the converted formazan. The absorbance was measured on a Versamax microplate reader (Molecular Devices, Sunnyvale, California) at a wavelength of 570 nm with background subtracted at 690 nm. As previous literature states, the CNTs' absorbance can influence the results of the MTT assay at high CNT concentration (51); we also performed a WST-1 assay as a check measure. Again 25 × 103 cells were seeded into each well of a 96-well plate and then incubated with the culture medium. After the cell incubation period with MWCNTs samples, we added 10 µL of WST-1 solution (as per instructions supplied with this cell proliferation assay kit) before incubation for 2 hours. The mitochondrial dehydrogenase of viable cells reduce WST-1 tetrazolium salt, which is water-soluble, avoiding the need for
25 dimethylsulfoxide treatment. The absorbance of test and control assays was performed using a Versamax microplate reader (Molecular Devices) at a wavelength of 450 nm with the reference wavelength 650 nm. The results of both assays are expressed as percentage of the control assays.
2.7.4 Long-term assays (1–2 weeks)
Cells were seeded in a six-well plate (200,000 cells/well) and incubated with the CNT-modified medium for 1–2 weeks (maintained at 37°C in a saturated-humidity atmosphere containing 95% air and 5% CO2). For each sample, cells were passaged every 72 hours of incubation by trypsinization, and one fourth of the cells were again seeded in the six-well plate. Samples were analyzed with WST-1 as described above.
2.7.5 Dose response curve
In order to identify the effects of the nanotube concentration on cells viability, we performed WST-1 assay in cells by using the following nanotubes concentration: 5, 10, 50, 100 and 500 µg/ml. Ten ml of CNT solutions (initial concentration 50 µg/ml) were ultra-centrifuged at 30000g for 30 min at 4° C (Optima L-70 K Ultracentrifuge, Beckman Coulter, Milan, Italy). At this speed, unbounded surfactant do not precipitate and the resulting pellet is composed only by PF127-CNT complexes. Removing the supernatant and re-suspending the pellet in 1 ml of DMEM, a final concentration of 500 µg/ml is obtained. 25x10³ cells were seeded into each well of a 96-well plate and then incubated with the culture complete media for 24 h, after that we added MWCNT samples. Cells were incubated for 6 h and then washed three times with phosphate buffered saline; cells were then incubated with 100 µl of complete cell culture medium supplemented by WST-1 solution and cells viability was measured for each concentration of three different MWCNT samples as described before. To verify that the absorbance of this assay was not influenced by the high nanotubes concentration, we performed the WST-1 with the same tested MWCNT solution but without cells.
2.7.6 Oxidative stress evaluation.
ROS production in cells treated with CNTs was detected using the Image-IT Green Reactive Oxygen Species Detection kit (Invitrogen, Milan, Itay). The assay is based on 5-(and-6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), a
26 fluorogenic marker for ROS in viable cells (68). Tert-butyl hydroperoxide (TBHP, Invitrogen, Milan, Italy) was used as a positive control because it is a known inducer of ROS production (68). In addition, we co-stained the cells with red fluorescent Evans-blue (Sigma, St. Louis, MO, U.S.A.) (70) to distinguish oxidatively-stressed from non-stressed cells by fluorescence microscopy.
2.7.7 Cell staining and evaluation of apoptotic cell death.
25x103 cells cultured on cover slips were exposed for 24, 48 and 72 h to MWCNT modified culture media; control cells were incubated in PF-127 0.01% modified culture medium without MWCNTs.
Cell death was evaluated by Hoechst 33258 (Invitrogen, Milan, Italy) 0.5ug/ml staining of cell nuclei (excitation 346 nm; emission 460 nm), which enables determination of the presence of DNA condensates indicative of apoptosis (69;70;71;73;74). After washing with PBS, cells were fixed with 40 g/l para-formaldehyde in PBS, incubated for 30 min at 4°C and then washed three times with PBS before further incubation for 20 minutes at 37C with 0.5 µg/ml Hoechst 33258 in PBS. The cells were then washed again with PBS and dried and then each cover slip was inverted onto a slide containing 10 µl of mounting media (VECTASHIELD mounting media, Vector Laboratories, Burlingame, CA, U.S.A.); the excess mounting medium was removed with fibre-free paper and each cover slip was sealed with regular transparent nail polish and allowed to dry for 3 min. The pyknotic cells were examined with a Zeiss epifluorescence microscope (Carl Zeiss, Oberkochen, Germany). The number of apoptotic nuclei was counted on five randomly selected areas from three cover slips from each experimental group. Results are expressed as number of apoptotic cells as a percentage of the total number of cells and compared to the control sample.
To study the apoptosis in the brain of mice injected with MWCNTs AnnexinVApoAlert Kit (Clontech Laboratories, Mountain View, California) were used to evaluate differences between normal and apoptotic cells after treatments. Anti-mouse NeuN antibodies to stain neurons were used at a concentration of 20 µg/mL, and Alexa 488 secondary antibodywere used at 2.5 µg/mL. Phase contrast and fluorescence cell images were acquired by a camera-mounted Zeiss Axioskop microscope (Carl Zeiss MicroImaging Inc., Thornwood, New York) and by a Leica TCS NT confocal microscope (Leica Microsystem, Wetzlar, Germany).
27 2.8 Flow cytometry.
This was carried out by a BD FACSCanto flow cytometer (BD Bioscience San Jose, California). The data were acquired and analyzed with BD FACSDiva software. After seven days of incubation with MWCNTs, PC12 cells were suspended at 50*104 ml-1 prior to acquisition. A blank was first used as a reference for adjusting controlling voltage of forward scatter signal (FSC) and side scatter signal (SSC).
2.9 Staining of cell cytoskeletal actin.
The cytoskeletal rearrangement of MSC-CNT cells in the presence of a magnetic source was studied by means of actin staining. MSCs were seeded in a 12 well plate, equipped with cover slips, at concentration of 5*104 cells/ well and incubated for 6 h in DMEM culture medium to allow cell attachment. Thus the medium was changed and the samples were cultured for 5 days in media added with PF127-CNTs. We used for the comparison cells in complete DMEM and cells incubated in PF-127 0.01% modified culture medium. A magnet was placed close to the wells 6 hours after the addition of CNTs to the cells. The actin staining was performed 5 days after incubation of cells with the MWCNTs and exposed to the magnetic field. The culture medium was removed and the cells were washed with PBS 1x at 4°C and then fixed/permeabilized with methanol at -20° C for 30 min. At the end of incubation cells were washed 4 times with PBS 1x at 4°C and incubated overnight at 4°C with the primary Actin antibody (mouse monoclonal, C-2: sc-8432 Santa Cruz Biotechnologies, Germany). Subsequently the sample was washed again 4 times with PBS at 4°C and incubated 1 h at room temperature with the secondary antibody (goat anti-mouse IgG-TR, sc-2781 Santa Cruz Biotechnologies, Germany). After incubation the cells were washed with PBS (4 times) and dried; each cover slip was removed from each well and was inverted onto a slide containing 10 µl of mounting media (VECTASHIELD); the excess mounting medium was removed with fiber-free paper and each cover slip was sealed with regular transparent nail polish and allowed to dry for 3 min. The images were analyzed by confocal microscopy.
2.10 Sample preparation for FIB imaging.
After three days of culture, the culture medium have been gently removed and samples washed two times with 500 µl of deionized sterile water; in each well 10 µl
28 paraformaldeyde 2% were added and samples have been stored at 4ºC for 15 minutes. The samples, with the adherent cells, were allowed to dry under a laminar flow cabinet immersed in ice. After 1 hour, dried samples were covered with a conductive layer (20 nm of gold) via chemical vapour deposition (Sputtering Sistec, model DCC 150) in order to perform the imaging with the FIB.
2.11 Preparation of samples for MRI.
The MRI samples were dispersed in low melting agarose (Sigma-Aldrich) gels to avoid settling of the MWCNTs. The 1% agarose (w/v) gels were prepared by dissolving agarose powder in deionized water. The MRI relaxation times were measured for gels containing 97% MWCNTs with concentrations of 10, 18, 50 and 180 µg/ml. A control sample without MWCNTs was measured. The mesenchymal stem cells were re-suspended in 1x106 cells/ml in agarose solution with preparation of three samples (one test and two controls); (a) test: MSC labelled with surfactant coated- 97% MWCNTs and held in agarose gels; (b) control i: MSC treated with surfactant and held in agarose gels; (c) control ii: cells incubated in normal medium, held in agarose gels.
2.11.1 Micro-MRI.
Micro-MRI data were acquired on a Bruker Avance FT NMR spectrometer with a wide bore 7.1 Tesla magnet resonating at 300.15 MHz for H. A birdcage radio frequency resonator with an internal diameter of 30 mm was used. All acquisitions were made at 19ºC. Two acquisition sequences were collected and averaged to improve the signal-to-noise ratio and reduce artefacts. Relaxation measurements were determined from 128 x 128 axial planes across the samples with slice thickness of 1 mm. Up to 9 samples were studied simultaneously, gels with no contrast agent acted as reference. A recycle time (TR) of 15 s was used to avoid saturation. The longitudinal (T1) relaxation times were measured using inversion recovery (180º-TI-90º) imaging pulse sequence; 8 different inversion times (TI) that ranged from 100 to 15000 ms were applied and the echo time (TE) was 4 ms. The transverse (T2) relaxation times were measured using CPMG spin echo imaging pulse sequence; a train of 16 echoes were acquired with delay (τ) between 180º pulses of 12 ms. Single exponential relaxation times were calculated from relaxation data. Each sample was studied at least three times and average T1 and T2
29 relaxation times calculated. Transverse relaxivity (r2) was calculated using the following equation: 2o 2 2 1 1 ] CA [ T T r = − (1)
where [CA] is the concentration of contrast agent such as Fe ions, 1/T2 is the transverse relaxation rate in the presence of a known concentration of contrast agent; 1/T2o is the transverse relaxation rate of the gel in the absence of contrast agent. The 128 x 128 x 128 three-dimensional (3D) RARE-4 (Rapid Acquisition with Relaxation Enhancement) experiments were acquired with recycle time (TR) of 250 ms and effective echo time (TE) of 40 ms. Scanning times were 34 min. The field of view was 30 mm and in-plane resolution was 0.234 mm/pixel. 3D surface representation of the MRI image data sets were produced using Amira® software (Visage Imaging, Inc. San Diego, CA USA).
2.12 Western Blotting.
The Western blot is an extremely useful analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are detected using antibodies specific to the target protein. We analyzed proteins extracted from cells after their incubation with the drug alone or attached on the MWCNTs. We employed a specific buffer to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors were added to prevent the digestion of the sample by its own enzymes. We used polyacrylamide gels 1% and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure and thus allows separation of proteins by their molecular weight. Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size. In order to make the proteins
30 accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose (transfer). We performed an electroblotting which uses an electric current to pull proteins from the gel into the nitrocellulose membrane. The protein move from within the gel onto the membrane while maintaining the organization they had within the gel. During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour. In this work we used a two-step process for incubation with the antibodies. We incubate the membrane with the primary antibody at 4 °C overnight against the following target proteins: AKT-T, AKT-S473-P, AKT-T308-P, Pras40-T, Pras40-T246-P, S6K-T, S6K-T389-P, S6K-T229-P, S6P-T, S6P-P.
After rinsing the membrane to remove unbound primary antibody, the membrane was exposed to the secondary antibody for 2 h at room temperature.. A horseradish peroxidase-linked secondary was used to cleave a chemiluminescent agent, and the reaction product produces luminescence in proportion to the amount of protein. A sensitive sheet of photographic film was placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot.
2.13 Statistical analysis.
Each experiment was carried out in triplicate, five random microscopic fields per cover slip were counted and three cover slips/ treatment were used for each test. The percentage of apoptotic or ROS positive cells was compared with the corresponding control. Values are expressed as means ± standard error of the mean (S.E.M.). Statistical significance was assessed by one way analysis of variance (ANOVA) followed by post-hoc comparison test (Tukey). Significance was set at 5%.
2.14 In vivo experiments.
2.14.1 MSC-CNT cells transplantation
Inbred male Wistar-Furth (WF) and Lewis rats, weighing 275-300g, were purchased from Charles River Laboratories (Italy). The animals were fed on standard rodent chow (Rieper, Italy) and water ad libitum, and were kept under a 12 h light/dark cycle. All the experimental procedures were carried out with the approval of the Ethical Committee for Animal Experimentation of the University of Pisa. Inbred male Lewis (230-250 g)
