Electron Microscopy studies of magnetic nanoparticles inside neuronal cells
Scanning Transmission Electron Microscopy (STEM), Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM) and Energy Dispersive X-Ray Spectroscopy (EDS) have been used to assess the validation of a non-invasive approach for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles (MNPs) and magnetic fields. The concept is that the application of a tensile force to a neuronal cell can stimulate neurite initiation or axon elongation in the desired direction, the MNPs being used to generate this tensile force under the effect of a static external magnetic field providing the required directional orientation.
The combination of different Electron Microscopy techniques is essential for a good understanding of the biodistribution of the MNPs inside the cells, their morphology, size, structure, chemical composition and degree of aggregation before and after interacting with the living cell.
Nerve regeneration mediated by magnetic nanoparticles: MNPs bind the injured nerve; a magnetic field is thus applied. MNPs create a mechanical tension which stimulates nerve regeneration in a direction imposed by the magnetic force. This physical guidance should direct more efficiently the regeneration of the injured nerve from the proximal toward the distal stump. Additionally, MNP binding to the neuronal cells can be enhanced with biological molecules, e.g., neural binders and neurotrophic factors.
For STEM observations, the preparation of the cells was made by seeding PC12 cells (precursors of the actual neuronal cells) on 6 well plate previously treated with Poly-L-Lisine. Subsequently, the growth medium was removed and replaced with the reduced medium containing MNPs. After 24 hours of incubation the cells were detached and fixed with a glutaraldehyde and treated with potassium ferrocianate and osmium tetraoxide. After washing, cells were dehydrated with increasing concentrations of acetone. After drying, samples were embedded in a solution of EPOXI resin and acetone overnight, and then for 4-5 hours in resin EPOXI Samples were dried for 2 days at 60°C and then 70 nm slices were cut at the ultra-microtome. For FIB/SEM studies cells were also detached, fixated and dehydrated, and sputtered with gold after drying.
Electron Microscopy studies
The trafficking of the particles inside the cells was monitored by TEM analysis. Particles appeared to enter the cells by an endocytic-like pathway. A cluster of particles was localized on the cell membrane which engulfs them forming vesicles which internalize the particles in the. No particles were detected in nuclei and no damages at the cyto-plasmatic organelles were found.
The presence of Fe inside the cells was confirmed by High Angle Annular Dark Field (HAADF) images and EDS spectra in STEM mode. Due to the contrast enhancement of heavier elements, Iron-based nanoparticles appear with a much brighter contrast in the HAADF images. The collection of EDS spectra proved the presence of iron material inside the cells. All STEM-EDS analyses were performed at liquid nitrogen temperature with a Gatan Cryo-holder. Low temperature decreases direct beam damage on the sample, allowing us to work at higher magnifications for HAADF imaging, as well as increasing the signal/noise ratio in the EDS spectra.
SEM analysis allowed studying the internal distribution of particles in adherent cells. EDS spectra performed on differentiated PC12 cells cross-sectioned by FIB/SEM confirmed the particle localization also in the growth cone of the cells.
Cellular localization of Magnetic nanoparticles. (A-B) TEM analysis of PC12 cells incubated 24 h with MNPs. White arrows: particles in the cytoplasm, yellow arrows: particles entering and membrane invagination; (C) STEM-HAADF image (?173 °C) of PC12 cells incubated 24 h with f-MNPs. Inset: EDS spectrum of cytoplasmatic MNPs; (D) SEM/FIB of PC12 cells incubated 72 h with MNPs: cross section of a single cell. Inset: EDS analysis of MNPs in the growth cone of the cell.
This work has proved that MNPs and external magnetic fields can influence the growth direction of neurites, aligning them preferentially to the direction of the magnetic force, without affecting their length and number. The mechanism involved in the re-direction of the neurites can be related to the magnetic force acting on the MNP-bound neuritis, which allows to manipulate axons in order to orient and to overcome inhibitory substrates.