Structural characterisation of nanomaterials incorporated into bioengineered samples: An electron microscopy approach

Abstract

This study analyses the properties of nanohydroxyapatite (nHA) and layered double hydroxide (LDH) materials that can be used as non‐viral gene delivery vectors. The former is a biocompatible, non‐toxic calcium phosphate mineral which makes it a suitable candidate as a delivery platform 1 . On the other hand, LDHs are composed of brucite‐like layers with a charge compensating anionic interlayer 2 . (Scanning) Transmission electron microscopy ((S)TEM) and Energy Dispersive X‐ray Spectroscopy (EDX) methods were utilised to characterize LDH and nHA nanomaterials and their respective interactions with plasmid DNA structures (pDNA). The current work also characterizes the ultrastructure of mesenchymal stem cells (MSCs), as well as investigating the uptake and localisation of the LDH gene delivery vectors at the nanoscale. Fig. 1(a) and Fig. 1(b) present the TEM images of the nHA and LDH particles respectively. The nHA particles are found to have a spherical structure of diameter ~50nm, indicating that nHA is a good candidate for cellular transfection. LDHs present a well‐defined hexagonal platelet‐like structure of ~100nm, providing promising results towards their application as a gene delivery vector. The interaction of nHA and LDH particles with pDNA was also investigated using STEM. Morphological variations are clearly evident in both the pDNA‐nHA and pDNA‐LDH composites. An annular‐like structure is presented in the case of pDNA‐nHA, whereas the DNA structures absorb to the surface of the LDH particles, displayed in Fig. 2(a) and Fig. 2(b) respectively. The application of STEM to MSCs highlight intracellular features such as membranes and nuclei, as shown in Fig. 3. The complimentary EDX techniques portray evidence of the LDH gene delivery vectors within the cell due to a presence of an Mg, Al and P peak, hence confirming uptake and localisation at the molecular level. The enhanced resolution provided by electron microscopy techniques allows for the study and understanding of cellular biological processes, such as that of pDNA‐nanoparticle delivery.