Comparison of sample preparations for
TEM
observations of lipid nanoparticles (Lipidots®)
Authors: Arnould Amandine , Bacia Maria , Caputo Fanny , Couffin Anne‐Claude , Gallet Benoit , Matei Constantin , Soulas Romain , Damlencourt Jean‐François 
Published: December 2016

Lipid nanoparticles (lipidots or liposomes for example) can be used in medicine as drug nanocarriers. Their core‐shell structure allows the functionalization of these nanoparticles using antibodies, peptides or proteins. The main aim of this targeting is to reduce the drug toxicity occurring during treatment. Such nanoparticles are currently used to treat breast cancer [1]. Further applications as fluorescent agent carriers for in‐vitro and in‐vivo diagnostics are also considered [2]. To have a better understanding of the effectiveness of these nanoparticles, characterization of these objects is required. Classical imaging in TEM mode leads to many issues as dehydrating, agglomeration and lack of contrast. Thus, optimal sample preparation is mandatory.
In this study, several preparation methods have been tested to determine the most appropriate one for TEM observations and 3D electron tomography acquisition. F80‐Lipidots (expected diameter 80nm) have been synthetized at CEA‐Grenoble using ultrasound or high pressure homogenization technologies [3]. TEM micrographs were taken using a FEI Tecnai Osiris having a cryo operating mode and working at 200kV. It is also equipped with four EDX detectors and a GIF for chemical analysis.
These particles were firstly observed by classical imaging (droplet drying) with and without negative staining. It appeared that the staining remarkably improved the contrast of the nano‐objects which appeared to have a circular shape. Nevertheless, Lipidot size determination was difficult due to aggregation phenomena during drying procedure. This method needed to be complemented by cryo‐microscopy.
Cryo‐techniques were used to ensure the preservation of the samples in their native state. Rapid freezing in ethane on C‐Flat grids after blotting was performed thanks to a FEI Vitrobot. Its settings (blot force and blot time) were adjusted to obtain vitrified films with a controlled thickness.Ice thickness was firstly determined thanks to EELS and EFTEM [4]. It showed that the ice is thinner in the center of the grid holes (Fig. 1). This could be a problem if this thickness is thinner than the particle size. Indeed, in that case, the particles tend to concentrate next to the carbon film of the grid (Fig. 2) and make it difficult to record tomography images. To avoid these artifacts, high pressure freezing (EM PACT2, LEICA) was also performed. It was then followed by cryo‐substitution, polymer resin embedding (EM AFS2, LEICA) and ultramicrotomy; or cryo‐ultramicrotomy (EM‐FC7, LEICA) directly after freezing (CEMOVIS). The constant thickness of the slices is thus an asset for tomogram acquisition. The main difficulty lies in maintaining the “cold chain” from the preparation to the observation.
The pros and cons of different sample methods (simplicity, reproducibility and reliability) will be discussed and results in what concerns the shape and the size of the particles will be compared. These results will be correlated with other techniques for size measurement at the nanometric scale such as DLS.