Spinal cord injury is a major devastating condition which leads to significant morbidity and mortality. It causes permanent neurological damage due to injury in motor neurons. Despite enormous developments in medical and surgical cares, the existing clinical treatments for SCI are limited. Exploration of probable utility of stem cells in neurological regeneration and repair has been an exciting development in neuroscience. Despite broad therapeutic benefits, numerous fundamental issues related to stem cell transplantation in SCI remains to be addressed. Stem cell tracking allows better understanding of the cell therapeutic approaches which ensures maximum therapeutic benefit with minimal amount of cells at a site while minimizing the harmful effects. Current interest in stem cell transplantation as most promising approach for repairing SCI is being added by magnetic nanoparticles and magnetic resonance imaging. We have developed bi-metal SPIO-nanoparticles by combining Gadolinium with SPIO (Gd-SPIO, <50nm) using soft-chemical approach to study particle functionalization and easy uptake by cells. It generates better contrast as compared to gadolinium and other available contrast agents and is easily detectable in 1.5T Magnetome. Hence, Gd-SPIO may be considered as one of the most effective nanoparticles for MRIbased imaging. Directed transplantation of neural stem cells labelled with Gd-SPIO nanoparticles under external magnetic field might be considered as better approach with high contrast imaging and non-invasive cell trapping at the desired site. This combinational strategy of Gd-SPIO labelled neural stem cells for the revival of chronically injured spinal cord would overcome the effects of the glial scar, inhibitory molecules, and use of tissue engineering strategies to bridge the lesion more effectively.
Keywords: Gd-SPIO nanoparticles; Stem Cell Therapy; Spinal Cord Injury, Magnetic Targeting; Regenerative Strategy
Pathophysiology of Spinal Cord Injury (SCI)
SCI is a major devastating condition which leads to significant morbidity and mortality. It causes permanent neurological damage due to injury in motor neurons. The lumber and cervical regions are most commonly affected areas during SCI. Functional decline in SCI are attributed by both mechanical as well as secondary pathological defects. Further, these mechanisms results in hemorrhage at the injury site followed by reactive gliosis which result in loss of conductivity and axonal regrowth [1-3]. The instant consequences involve swollen spinal cord with disrupted blood vessels, severed axonal network and injured neuronal cells. Edema and ischemic cell death continue, which elicit devastating inflammation accompanied by infiltration
of cells through conciliated blood-spinal cord barrier.
Available treatment strategies for SCI
Despite enormous developments in medical and surgical cares, the existing clinical treatments for SCI are limited. Endogenous components such as residing cells and growth factors in central nervous system do not support axonal regeneration and very limited recovery is achieved . There are vast confines about the existing treatment modalities for spinal cord repair and regeneration. The foremost rationale of existing treatment is to prevent the extent of secondary consequences during SCI, which frequently involves high dose of steroids . But alas, even with this consideration, very rare choices for clinical applications exist.
Stem cell-based therapies in SCI
Exploration of probable utility of stem cells in neurological regeneration and repair has been an exciting development in neuroscience. Stem cell therapy could be potentially used to either stimulate the endogenous stem cells or supplementing stem cells to repair the damage and replace neuronal and glial cells that undergo cell death soon after injury [6-7]. Different sources of stem cells have been used to address the fundamental issues involved in SCI regenerative mechanisms . A detailed view of potential role of different types of stem cells in the treatment of SCI has been provided in our earlier studies [3,8].
Major challenges in developing stem cell-based therapies for SCI
Despite broad therapeutic benefits, numerous fundamental issues related to stem cell transplantation in SCI remain to be addressed. Few of them includes, selection of appropriate cell source, quantity of cells (cell dosing) required for transplantation, reproducibility of cells after transplantation, route of cell delivery, unwanted proliferation and differentiation of cells, graft rejection and cell fate post-transplantation. In order to resolve these facets real-time and long-term monitoring of stem cells post-transplantation is a major area of interest as it relates with safety, and efficacy of cell therapeutic benefits and mechanisms involved in cellular regeneration. Hence, tracking of stem cells post-transplantation is crucial for identifying their bio-distribution, quantity, viability, site of engraftment and final differentiation.
Available strategies for stem cell labeling and tracking
Stem cell tracking allows better understanding about the cell therapeutic approaches which ensures maximum therapeutic benefit with minimal amount of cells at a site while minimizing the harmful effects. Since last few years’ enormous efforts have been made for efficient labeling and non-invasive tracking of stem cells both in vitro and in vivo. For decades, one of the most widely known methods of cell tracking has been the utility of fluorescent markers. Several studies have reported use of florescent markers for tracking movement and differentiation of stem cells due to their strong signals [9-11]. But unfortunately, these markers are difficult to detect in vivo as the emission and/or excitation signals may not be able to infiltrate the body at the niche of stem cells. Other methods such as multi-photon imaging and stimulated emission depletion (STED) microscopy that utilize long wavelength laser beams seem more effective than traditional fluorescence-based approaches but again lack deep penetration into all types of tissues [12-13]. In recent techniques radio-labeled tracking of stem cells has been employed due to its strong signal, deep tissue penetration and relatively easy detection . However, the signal demises over the time as cells divide and the radio-labeled elements get metabolized. Another drawback is its radioactive nature which may damage the DNA.
Nanomedicine unlocks a new-fangled approach for stem cell tracking
Merging of interdisciplinary areas in the physical, chemical and biological sciences have accelerated the progress in nanoparticles application in medicine. Nanotechnology is an important meadow of current research and is rapidly gaining remarkable applications in the area of cellular and molecular medicine. It has resulted in a range of innovative nano-bioengineering approaches for repair and regeneration of damaged tissues and/ or organs. In addition to available methods for stem cell tracking, many scanning techniques such as X-ray computed tomography, PET scans and magnetic resonance imaging (MRI) scans have also been used with the help of contrast agents [15- 17]. Among these methods, MRI-based tracking and imaging of stem cells have proved its potential for non-invasive and high contrast imaging in vivo. One of the most commonly used contrast agents in MRI has been nanoparticles. This is one of the most promising and widespread methods used in stem cell tracking as nanoparticles allow a good visualization due to their strong signal. Current interest in stem cell transplantation as most promising approach for repairing SCI is being added by nanotechnology and MRI.
Super paramagnetic iron oxide nanoparticles (SPIO-NPs) as better choice for more effective and targeted delivery of cells
Cell therapy using stem cells has emerged as most promising solution for the treatment of many devastating diseases and tissue regeneration that can’t be achieved by conventional strategies. However, complete mechanisms and therapeutic potential of stem cells post-transplantation need to be clearly understood. Various stem cell labeling and tracking strategies by using nanotechnology have been developed, among them SPIO-NPs have attracted major attention not only for labeling and tracking of stem cells but also efficient drug/gene delivery and high contrast imaging. It passes magnetic properties and has considered most biocompatible and biodegradable [18-19]. We have developed bi-metal SPIO by combining Gadolinium with SPIO (Gd-SPIO, <50nm) using soft-chemical approach to study particle functionalization and easy uptake by cells (Unpublished). Recently this approach is being used for efficient synthesis of nanoparticles with significantly reduced cytotoxicity which is highly suitable for biological applications .
Synthesis and characterization of Gd-SPIO nanoparticles
Gadolinium trichloride hexahydrate and anhydrous ferric tri-chloride were purchased from Sigma Aldrich. FeCl3 (12.31mmol) was taken in de-ionized water with continuous stirring by adding 4.57g of GdCl3.6H2O (12.31mmol) and refluxed for 6h. After cooling, it was filtered to remove un-reacted impurities. After drying the filtrate at 100°C for 6h in lab oven, dark brown solid was obtained in good yield. After calcinations in dry air with a heating rate of 10°C per minute from room temperature to 1000°C, the dark brown bimetallic oxide nanoparticle was obtained in good yield. The FeGdO3 nanomaterial (Gd-SPIO) was characterized by XRD, SEM, TEM, thermal analysis and FT-IR. The X-ray diffraction patterns indicate the formation of square planar crystallite size of 41.65 nm after annealing (data not shown).
Cells and Gd-SPIO nanoparticles
Cell is typically ~10-100μm in size having much smaller cell components in the sub-micron size. Generally proteins inside the cells are very small (i. e. approximately 5nm) and are almost equal to the nanoparticles size. Considering size as a valuable factor for cell labeling and tracking will allow us to discover the cellular machinery without much hindrance . Gd-SPIO nanoparticles interact with cells and its components without interfering with biological properties for their safe biological applications . More precise labeling and monitoring of stem cells in SCI is crucial needs to investigate further to comprehensively understand cellular proliferation, differentiation and migration dynamics. Gd-SPIO nanoparticles have unique property for producing optical rings around them and are easily detectable in light microscope (Figures. 1,2).
Figure 1. Identification of Gd-SPIO nanoparticles by using a conventional microscope in dark field. Nanoparticles are well dispersed in water without agglutination. Single nanoparticle produces an optical ring around itself and increases the resolution 1000 times larger than its nominal size (yellow arrow, scale bar: 20μm).
Figure 2. Dark field imaging of Gd-SPIO nanoparticles using a conventional microscope [A] Neural cells without nanoparticles did not show any signature in conventional microscope and were found difficult to track due to low resolution in closed aperture [B] Internalization of nanoparticles in neural cells (after 2h of incubation) was clearly observed under 40X objective of a conventional inverted microscope in closed 0.4 μm aperture. Yellow arrows indicate internalized Gd-SPIO nanoparticles within the cell.
Gd-SPIO as dual-MRI contrast agent for non-invasive stem cell labeling and tracking
MRI provides high temporal and spatial resolution, long effective imaging window, fine signal intensity and contrast, rapid in vivo image acquisition and lack of long-term radiation exposure. Due to these extensive potential, MRI has shown most promising future in tracing stem cells in vivo . Nowadays MRI is the most commonly used for non-invasive imaging modality to provide a high contrast of soft tissues and enough penetration to visualize anatomy, physiology and even molec-ular information within the entire human body. However, it is limited to 1mm spatial resolution due to lack of suitable contrast agent. Gd-SPIO nanoparticles overcome such limitations in their potential application in MRI-based imaging and tracking. They generate better contrast as compared to gadolinium and other available contrast agents which is easily detectable in 1.5T Magnetome (Figure 3). Hence, Gd-SPIO may be considered as one of the most effective nanoparticles for MRI-based imaging.
Figure 3. T1 and T2 MRI of Gd-SPIO nanoparticles in control cells (without nanoparticles) did not reveal contrast while neural cells labelled with Gd-SPIO nanoparticles (100μg/ml) produced high contrast image in 1.5T Magnetome.
We have used Gd-SPIO as MRI contrast agent in 1.5T Magnetome where it produces enhanced sensitivity by quenching the relaxation time of the proton longitudinally (T1) or transversally (T2). When we injected Gd-SPIO nanoparticles in a wister rat through tail vein to observe its biological distribution, high contrast image was captured using low power (1.5T) MRI throughout the body. Both T1 and T2 contrasts were generated which provided an evidence for its role in high contrast imaging (Figure 4).
Figure 4. Axial view of a wister rat showing high contrast T1 and T2 imaging in 1.5T Magnetome using Gd-SPIO nanoparticles at very low concentration (100μg/ml).
The extreme biological compatibility of Gd-SPIO nanoparticles with cells allows its wide application in MRI-based imaging. Gd-SPIO is a dual contrast imaging molecule due to the presence of Gadolinium and SPIO. Hence it provides both T1 and T2 contrast. Besides contrast property, it also passes paramagnetic activity.
Gd-SPIO NPs-based stem cell mobilization and trapping of stem cells in SCI
Use of SPIO-NPs has shown a better path for efficient delivery of stem cells at target location . Recently, Tukmachev et al. (2015) has demonstrated the potential of SPIO-NPs in homing of mesenchymal stem cells (MSCs) at lesion site in rat model of SCI . This study accomplished trapping of SPIO-labeled MSCs into the vicinity of lesion site from 10cm distance away of injury using an external magnetic system of 1.2T. This study provides a better hope for future magnetic nanoparticles- based delivery of stem cells at the desired site. Though, formation of glial scar and regeneration of neurons and glia that undergo cell death soon after injury in SCI remains a major issue for cell-based therapeutic approaches. Hence, choice of suitable stem cell type needs to be addressed to overcome the SCI pathogenesis.
The magnetic nanoparticles developed at our centre by soft chemical approach have proved its high biocompatibility and paramagnetic behaviour . Directed transplantation of NSCs labelled with Gd-SPIO NPs might be considered as better approach with high contrast imaging and non-invasive cell trapping at the desired site . This combinational strategy of Gd-SPIO labeled-NSCs for the revival of chronically injured spinal cord would overcome the effects of the glial scar, inhibitory molecules, and use of tissue engineering strategies to bridge the lesion more effectively (Figure 5).
Figure 5. Magnetic sheet with coil designed for magnetic trapping of neural cells labeled with Gd-SPIO nanoparticles for their non-invasive and targeted homing at the lesion site after infusion away from injury.
Conclusion and Perspectives
Available treatment modalities for SCI have serious limitations. Gd-SPIO nanoparticles tagged NSCs transplantation holds great promise for targeted homing under external magnetic field to bridge the lesion created by SCI and replace the damaged functional neurons. This combinatorial strategy would overcome the effects of glial scar and help to develop more effective tissue engineering approaches in other disorders. However, more studies are required to prove its potential in the treatment of SCI and other disorders.
This study was financially supported by Indian Council of Medical Research (ICMR), New Delhi, India.