Cryogenic Electron Microscopy Methodologies as Analytical Tools for the Study of Self-Assembled Pharmaceutics.

Na’Ama Koifman,Yeshayahu Talmon

doi:10.3390/pharmaceutics13071015

Abstract

Many pharmaceutics are aqueous dispersions of small or large molecules, often self-assembled in complexes from a few to hundreds of molecules. In many cases, the dispersing liquid is non-aqueous. Many pharmaceutical preparations are very viscous. The efficacy of those dispersions is in many cases a function of the nanostructure of those complexes or aggregates. To study the nanostructure of those systems, one needs electron microscopy, the only way to obtain nanostructural information by recording direct images whose interpretation is not model-dependent. However, these methodologies are complicated by the need to make liquid systems compatible with high vacuum in electron microscopes. There are also issues related to the interaction of the electron beam with the specimen such as micrograph contrast, electron beam radiation damage, and artifacts associated with specimen preparation. In this article, which is focused on the state of the art of imaging self-assembled complexes, we briefly describe cryogenic temperature transmission electron microscopy (cryo-TEM) and cryogenic temperature scanning electron microcopy (cryo-SEM). We present the principles of these methodologies, give examples of their applications as analytical tools for pharmaceutics, and list their limitations and ways to avoid pitfalls in their application.

Highlights

Cryogenic temperature electron microscopy is a set of sophisticated tools for the imaging of nanostructured liquids, and, as such, is most useful for the nanostructural characterization of a wide range of drug delivery systems
There are issues related to the interaction of the electron beam with the specimen such as micrograph contrast, electron beam radiation damage, and artifacts associated with specimen preparation
In this article, which is focused on the state of the art of imaging self-assembled complexes, we briefly describe cryogenic temperature transmission electron microscopy and cryogenic temperature scanning electron microcopy

Summary

Introduction

Cryogenic temperature electron microscopy (cryo-EM) is a set of sophisticated tools for the imaging of nanostructured liquids, and, as such, is most useful for the nanostructural characterization of a wide range of drug delivery systems. As long as cryo-EM specimen preparation preserves molecules in their native state, it enables high-resolution structural research of a variety of proteins in a straightforward manner It allows the imaging of large complexes, e.g., several proteins or proteins and drugs, and of pure isolated proteins, which is very valuable in structure-based drug discovery. Another type of complex aggregates with high potential in drug delivery in whole and in medical theranostic application in particular are cubosomes [39,40] For those systems, cryo-TEM has been proven most useful in the characterization of the preparation, showing the coexistence of simple vesicles with liposomes in the system, as well as the existence of an intermediate state of aggregation known as “interlamellar attachments,”. Cryo-TEM single particle analysis used for drug discovery is an important topic, it is outside the scope of this publication

Cryogenic-Temperature Electron Microscopy

Examples

Concluding Remarks