Int J CARS (2012) 7 (Suppl 1):S219–S221 DOI 10.1007/s11548-012-0718-1
NANOTECHNOLOGY
MSCs transplantation and imaging by using magnetic multiwall carbon nanotubes O. Vittorio1, P. Parchi2, N. Funel3, M. Masini3, A. Cuschieri4, M. Cecchini1, M. Lisanti2 1 Istituto Nanoscienze-CNR and Scuola Normale Superiore, Nest Lab, Pisa, Italy 2 University of Pisa, Orthopedic Deptartment, Pisa, Italy 3 University of Pisa, Department of Experimental Pathology, Pisa, Italy 4 Scuola Superiore S.Anna, Medical Science Lab, Pisa, Italy Keywords Carbon nanotubes Mesenchymal stromal cells MRI Osteo-chondral disease Purpose MSCs (mesenchymal stromal cells) are bone marrow-derived cells capable of replication and differentiation in vitro into several tissues including bone, cartilage, stroma, fat, muscle and tendon. MSCs can be isolated by relatively simple procedures and then expanded without losing the ability to differentiate into multiple lineages. As such, these cells have immense clinical potential in regenerative medicine, especially in orthopaedics for repair or replacement of damaged tissues. In particular our interest is focused in the repair of bone and cartilage defects for the treatment of osteo-chondral disease. In this paper, we investigated the interaction between magnetic multiwall carbon nanotubes (MWCNTs) and MSCs and their ability to guide these cells by using an external magnetic field. Moreover, we investigated the potential of MWCNTs as magnetic resonance imaging (MRI) contrast agents. Methods MSCs were cultured in a MWCNT-containing medium. We investigated if MWCNTs and magnetic field can alter cell viability, proliferation rate and the ability of cells to differentiate in osteocytes and chodrocytes. We also studied the effect of interaction with MWCNTs in terms of cell phenotype and cytoskeletal conformation. We investigated the ability of MSCs labelled with MWCNTs to move towards the magnetic source in vitro (Fig. 1) and the possibility to be guided into target organ in vivo. Finally, MSCs were labelled with MWCNTs to demonstrate the effectiveness of MWCNTs as MRI contrast agents for cellular MRI.
Fig. 1 Cells magnetization and movement towards the magnetic source
Results MWCNTs did not affect cell viability and their ability to differentiate in osteocytes and chondrocytes. Both the MWCNTs and the magnetic field did not alter cell growth rate, phenotype and cytoskeletal conformation. MWCNTs, when exposed to magnetic fields, are able to shepherd MSCs towards the magnetic source in vitro and in vivo [1]. Our studies also showed the potential use of multiwall carbon nanotubes as MRI contrast agents. The r2 relaxivity of MWCNTs in 1 % agarose gels at 19 °C was 348 s-1 mM-1. The r2 relaxivity of nanotubes was attributed to both the presence of iron oxide impurities and also to the carbon MWCNT structure itself. MSCs were labelled with MWCNTs to demonstrate their effectiveness as labels for cellular MR imaging without impairment of cell viability and proliferation [2]. Conclusion MWCNTs hold the potential for use as nano-devices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. We demonstrated that MSCs after the incubation with MWCNTs maintained all the normal cellular activity, such as the differentiation in osteocytes and chondrocytes. Our results also suggest that the MRI contrast agent properties of the MWCNTs could be used in vivo for stem cell imaging. We believe that our studies could pave the way for the development of new strategies for manipulation/guidance of MSCs in regenerative medicine and cell transplantation. References [1] Vittorio O, Duce S, Pietrabissa A, and Cuschieri A. (2011) Multiwall Carbon Nanotubes as MRI contrast agents can track stem cells. Nanotechnology Journal 22 095706. [2] Vittorio O, Quaranta P, Raffa V, Funel N, Campani D, Pelliccioni S, Longoni B, Mosca F, Pietrabissa A and Cuschieri A. (2011) Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by magnetic fields. Nanomedicine Future Medicine. 6:1:43–54.
A comparitive study of ferumoxide and multiwall carbon nanotubes for the labeling and tracking of human pancreatic islets F. Syed1, M. Bugliani1, M. Masini2, C. Riggio3, O. Vittorio4, F. Filipponi5, U. Boggi6, P. De Simone5, L. Marselli1, V. Battaglia6, V. Raffa3, P. Marchetti1 1 University of Pisa, Department of Endocrinology And Metabolism, Metabolic Unit, Pisa, Italy 2 University of Pisa, Department of Experimental Pathology, Medical Biotechnology, Infectious Disease And Epidemiology, Pisa, Italy 3 Scuola Superiore Sant’anna, Institute of Life Science, Pisa, Italy 4 Nest Scuola Normale Superiore and Istituto Nanoscienze-Cnr, Pisa, Italy 5 University of Pisa, Department of Surgery, Pisa, Italy 6 University of Pisa, Department of Oncology, Transplants and Advanced Technologies In Medicine, Pisa, Italy Keywords Human pancreatic islets Ins-1e cells Endorem Carbon nanotubes
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S220 Purpose Limited tools are available for the non-invasive monitoring of transplanted islets. In this study we have compared the widely used superparamagnetic iron oxide (SPIO) nanoparticle ferumoxide (EndoremÒ) and multiwall carbon nanotubes (MWCNTs) for islet cell labeling. Methods INS-1E cells and human pancreatic islets isolated from 11 non-diabetic cadaveric organ donors(age: 62 ± 16 years, BMI: 24.6 ± 3.3 kg/m2) were incubated with 50 lg/ml EndoremÒ or 15 lg/ml MWCNTs and studied after 3 and 7 days in terms of beta cell morphology, ultra structure, function, cell survival and in vitro magnetic resonance imaging (MRI). Results Light microscopy showed well maintained INS-1E and human islets during the 7-day incubation. Electron microscopy (EM) revealed preserved beta cell ultra structure and the presence of EndoremÒ in lipid fusion bodies and MWCNT particles across the plasma membrane of human pancreatic betacell, Figs. 1 and 2. Glucose-stimulated insulin secretion from human islets, expressed as stimulation index (SI), was similar in control condition (3 days: 3.8 ± 1.6; 7 days: 5.2 ± 2.9) as in the presence of either EndoremÒ (3 days: 3.9 ± 1.5; 7 days: 5.5 ± 4.5) orMWCN (3 days: 4.1 ± 1.2; 7 days: 5.4 ± 3.4). Vital staining and EM analysis showed approximately C90 % cell survival at any incubation condition. INS-1E containing EndoremÒ or MWCNTs were successfully visualized by MRI in vitro.
Fig. 1 Electron microscopy image showing endorem in lipid fusion bodies of human pancreatic islets
Fig. 2 Electron microscopy image showing carbon nanotubes near and across the b cell plasma membrane of human pancreatic islets
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Int J CARS (2012) 7 (Suppl 1):S219–S221 Conclusion These results show similar effects of EndoremÒ and MWCNTs on INS-1E cells and human islets for up to 7 days of incubation, proposing MWCNTs as an innovative tool, to be further explored for labeling pancreatic islet cells. Supported by CariPisa foundation.
Wireless remote real-time monitoring of multiple neurotransmitters using nanoarrays R. Andrews1, K. Bennet2, S. Chang2, I. Kim2, C. Kimble2, J. Koehne3, K. Lee2, M. Marsh2, M. Meyyappan3, P. Min2 1 NASA Ames Research Center, Nanotechnology & Smart Systems, Los Gatos, United States 2 Mayo Clinic, Rochester, United States 3 NASA Ames Research Center, Nanotechnology, Moffett Field, United States Keywords Deep brain stimulation Neurotransmitters Nanotechnology Dopamine+serotonin Purpose Although the brain is composed of 90 % glial cells versus 10 % neurons, and recent evidence suggests that neurotransmitter release by astrocytes is important in neuronal firing, our monitoring of brain function has been largely limited to either electrical activity (e.g. electroencephalography, evoked potentials) or blood flow (e.g. functional MRI). It is likely that variations in brain neurotransmitters are crucial to the control of brain function. Deep brain stimulation (DBS) is a state-of-the-art neurosurgical therapy for many patients with movement disorders such as Parkinson’s disease and dystonia, epilepsy, and psychiatric disorders such as depression and obsessive compulsive disorder. Despite its wellestablished clinical efficacy, the mechanism of DBS remains poorly understood. One hypothesis is that the electrical stimulation of DBS evokes release of neurotransmitters. Monitoring of these released neurotransmitters is likely to improve both our understanding of, and patient outcome in, these disorders. The Wireless Instantaneous Neurotransmitter Concentration System (WINCS) is being developed to measure the DBS release of neurotransmitters. WINCS has proved successful in monitoring neurotransmitters in vitro and in vivo, and has led to better understanding of the clinical efficacy of DBS (Fig. 1). Multiplexed carbon nanofiber (CNF) arrays can be used to detect with high sensitivity many small biomolecules including DNA, proteins, and neurotransmitters. Integration of the CNF array with WINCS (WINCSnanotrode) has the advantage of sensing at multiple locations for better spatial resolution of neurotransmitter release, and monitoring more than one neurotransmitter simultaneously. Methods The WINCS uses fast-scan cyclic voltammetry (FSCV) and Bluetooth wireless communication to allow free-roaming animals, with the device implanted in the skull, to have remote in vivo monitoring of neurotransmitters (Fig. 1). FSCV customarily uses a carbon microfiber electrode (CMF). For CNF arrays, a multiplexed chip is fabricated on a 4-inch silicon wafer using standard lithographic techniques. For the WINCSnanotrode, CNFs are grown directly on the wafer from lithographically defined nickel catalyst. Silicon dioxide is added to insure that only the CNF tips are exposed as sensing electrodes. Electrode performance is evaluated using FSCV and electrochemical impedance spectroscopy. The present 393 CNF array has pads 200 mm in diameter (Fig. 1). The CNF array and WINCS are integrated as WINCSnanotrode to perform neurotransmitter detection using FSCV. The WINCS base-station software (WINCS-ware) controls the wireless module, filters and processes the received data stream, and displays the results
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Fig. 2 Top FSCV triangle wave for dopamine, N-shape wave for serotonin. Middle Comparison of CMF and CNF raw cyclic voltammogram (CV), background subtracted CV, and calibration curves for the triangle and N-shape waveforms. Bottom Pseudo-color plots during simultaneous decoupled application of triangle and N-shape waveforms (dopamine and serotonin at 5 micromolar concentrations)
Fig. 1 Top WINCS circuit board, FSCV microcontroller, and Bluetooth. Middle Scanning electron microscopy of the 393 CNF array. Bottom Flow cell to house the 393 CNF array in nearly real time. The WINCS hardware, WINCS software and WINCSnanotrode are used in a flowcell system to detect the neurotransmitters dopamine and serotonin simultaneously (Fig. 1). By using a standard triangle-shape FSCV wave, dopamine (or dopamine plus serotonin) are detected; by using an N-shape wave only serotonin is detected (Fig. 2). The same protocol is employed to compare neurotransmitter detection with the CMF and CNF WINCSnanotrode. Results The CNF electrodes have been successfully integrated with WINCS to detect dopamine and serotonin in vitro, as well as to detect each separately. In each case, the CNF electrode displays similar detection
performance and sensitivity as the CMF electrode (Fig. 2). Our results suggest that CNF electrodes may detect neurotransmitters at various electrode locations (on pads across the array surface). Further testing is underway to use the WINCSnanotrode for in vivo neurotransmitter monitoring, with a much smaller needle-shape CNF electrode (with pads approximately 1/10 the size of the present 393 CNF array) being fabricated. Conclusions The present study has demonstrated the integration of CNF electrode arrays with the WINCS for the simultaneous monitoring of two neurotransmitters dopamine and serotonin. The CNF electrode’s multiple sensing pads can be used for precise localized recordings of two neurotransmitters in various regions of the brain. The increased spatial resolution of neurotransmitter release events will improve our understanding of both the pathophysiology of various disorders of the nervous system and the mechanism of DBS efficacy. Combining DBS stimulation with WINCSnanotrode is leading to an implantable ‘‘smart’’ DBS system that can utilize neurotransmitter (as well as electrical) feedback control of DBS. Such a ‘‘smart’’ DBS system should result in improved efficacy in treating various nervous system disorders.
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