A Guide for Using Transmission Electron Microscopy for Studying the Radiosensitizing Effects of Gold Nanoparticles In Vitro

Abstract

The combined effects of ionizing radiation (IR) with high-z metallic nanoparticles (NPs) such as gold has developed a growing interest over the recent years. It is currently accepted that radiosensitization is not only attributed to physical effects but also to underlying chemical and biological mechanisms’ contributions. Low- and high-linear energy transfer (LET) IRs produce DNA damage of different structural types. The combination of IR with gold nanoparticles may increase the clustering of energy deposition events in the vicinity of the NPs due to the production mainly of photoelectrons and Auger electrons. Biological lesions of such origin for example on DNA are more difficult to be repaired compared to isolated lesions and can augment IR’s detrimental effects as shown by numerous studies. Transmission electron microscopy (TEM) offers a unique opportunity to study the complexity of these effects on a very detailed cellular level, in terms of structure, including nanoparticle uptake and damage. Cellular uptake and nanoparticle distribution inside the cell are crucial in order to contribute to an optimal dose enhancement effect. TEM is mostly used to observe the cellular localization of nanoparticles. However, it can also provide valuable insights on the NPs’ radiosensitization pathways, by studying the biochemical mechanisms through immunogold-labelling of antigenic sites at ultrastructural level under high resolution and magnification. Here, our goal is to describe the possibilities, methodologies and proper use of TEM in the interest of studying NPs-based radiosensitization mechanisms.

Requirements for Designing an Effective Metallic Nanoparticle (NP)-Boosted Radiation Therapy (RT)

“…However, all chemical preparation stages, if not applied correctly, may lead to image artifacts. For a more detailed information, the reader can resort to a recent technical note about all the technicalities regarding TEM [162]. There exist several other techniques to study DNA damage, e.g., Immunocytochemistry and Gel Electrophoresis, which are considerably more timesaving, require fewer demanding skills and protocols, and are less expensive.…”

“…TEM was not used to study DNA damage induced NP radiosensitization until now, but it was used to monitor cellular uptake and distribution of NPs [77,105,165]. Moreover, recently, the technical use of TEM was thoroughly described as a means of studying NP-induced radiosensitization in vitro [162].…”

Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM)

“…TEM analysis of the intracellular localization of NM can greatly facilitate and provide useful information for understanding of the biological effects of NM [80][81][82]. Overall, NM localized in the mitochondria, lysosomes or within the nucleus are considered more hazardous than the ones localized in the cytoplasm [83][84][85][86].…”

Immune Responses to Nanomaterials for Biomedical Applications

“…Last but not least, a methodology paper on the use Transmission Electron Microscopy (TEM) to carefully study the effects of ionizing radiation (IR) combined to gold NPs has been included [ 13 ]. The authors provide valuable insights on the NPs’ radio-sensitization and the subsequent biochemical mechanisms through immunogold-labelling of antigenic sites at ultrastructural level.…”