Elsevier

Analytical Biochemistry

Volume 309, Issue 2, 15 October 2002, Pages 196-199
Analytical Biochemistry

Size distribution measurement of vesicles by atomic force microscopy

https://doi.org/10.1016/S0003-2697(02)00291-9Get rights and content

Abstract

Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems. When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body. Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal. Therefore atomic force microscopy was attempted as another size measurement technique. After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure. The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle. At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate. The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle. Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies.

Section snippets

Materials and methods

Hepatitis B virus surface antigen (HBsAg) L particles were used as vesicles in our study. HBsAg L particles were overexpressed in Saccharomyces cerevisiae AH22R harboring an expression plasmid pGLDLIIP39-RcT [2]. HBsAg L particles were purified with three sequential ultracentrifugation steps without any chromatographic step [9]. An HBsAg L particle consists of transmembrane HBsAg L proteins (52 kDa) and phospholipids and forms a vacant liposome. The buffer solution used was Dulbecco’s

Results and discussion

Typical AFM images for the low concentration (10 mg/L) of HBsAg L particles are shown in Figs. 1A and B. The images show flattened structures with heights of 1.8–2.5 nm and diameters of 550–1500 nm scattered on the mica surfaces. DLS analysis indicated that these HBsAg L particles have diameters of approximately 80 and 300 nm in solution [9]. Thus, it was considered that HBsAg L particles are adsorbed onto the substrate as a flattened structure. However, the size of HBsAg L particles can be

Conclusions

Atomic force microscopy was demonstrated as a suitable method for examining the vesicle size and the size distribution directly, because AFM facilitates the imaging of molecules one by one. In this study, by using the HBsAg L particles as model vesicles, high and low concentrations of vesicles have been adsorbed onto the substrate and then subjected to AFM observation. At the low concentration, the AFM images reveal that vesicles are generally flattened on the substrate after adsorption. At the

Acknowledgements

This work has been performed under the Center Of Excellence (COE) program supported by the Ministry of education, Science, Sports and Culture, Japan.

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