30 www.microscopy-today.com • 2010 March doi: 10.1017/S155192951000012X Introduction A liposome is a spherical particle formed by a lipid bilayer enclosing an aqueous compartment at its center. ese particles can be comprised of a variety of lipids, particularly phospholipids. ey have been long considered as potential delivery devices in the medical and pharmaceutical industries because of their ability to encapsulate different compounds as the lipids form into liposomes. e biocompatibility of many liposomes has been widely studied, and this has led to the development of a number of drug formula- tions [1]. e ability of liposomes to be stored in the body and to be taken up by cells makes them ideal for drug delivery. Our research work aims to explore the variations in mineral deposits that can form inside liposomes by preparing liposomes in modified calcifying buffers. e overall aim of the work is to prepare calcium-loaded liposomes. is article deals with the characterization of liposomes, the investigation of the types of minerals that develop within liposomes, and the identification via x-ray mapping (XRM) of different phases forming within liposomes. Methods e liposomes were produced using L-α-phosphatidycholine (PC) and cholesterol, both obtained from Sigma-Aldrich (#61755 and #C3045, respectively). PC is a phospholipid with a polar head and non-polar tail. Cholesterol is used to strengthen the membrane of the liposomes and reduce the possibility of rupturing of the liposomes during processing and storage. e liposomes were prepared in three different calcified aqueous solutions, one containing a CaCl 2 -based solution (First Solution), the second containing CaCl 2 in addition to NaHCO 3 (Second solution), and the third containing CaCl 2 in addition to K 2 HPO 4 -based solution (ird Solution). Encapsulation of calcium or mineral is believed to occur either because a preformed mineral deposit in the solution gets trapped as the liposome lamellar are forming or because the ions, which have an attraction to the phosphate of the lipid layers, build up on the surface of the liposomes before the second layer is placed on top. Portions of samples were collected and imaged via TEM to ensure the formation of liposomes. e remaining samples were washed in triplicate by re-suspending in 200 µl of Milli Q water and spun for one hour at 1200 rpm. e liposomes were re-suspended in 50 µl of Milli Q water. e ion concentration differences between the inside and outside of the liposome cause rupturing at this point, and the liposomes can no longer be visualized. However, because of the wash steps, it can be determined that all remaining mineral was bound inside the liposome. e solution was placed onto a silicon wafer to concentrate and dry. e samples were carbon coated and characterized using a Philips XL-30 ESEM and a JEOL 35CF with a Moran Scientific x-ray microanalysis mapping system. Results Quantitative x-ray mapping (QXRM) is able to show subtle changes in the distribution of elements, suggesting the phases present, which can then be positively identified using x-ray diffraction [2]. is makes QXRM particularly useful for identifying the location of individual elements and chemical phases within liposomes. Figures 1A–1C show primary-color pseudocoloring x-ray maps and reveal the types of phases produced. With primary coloring, each of three elements selected from the QXRM is assigned a color of red, green, or blue [3, 4]. In this example, for all three primary-color images in Figures 1A–1C, red codes for P, green is Ca, and blue is Si. When comparing these primary-color maps, it can be seen in Figure 1C that there is a concentration of yellow, indicating precipitates of a Ca-P phase, which was confirmed through further analysis by x-ray diffraction. Whereas, Figure 1B shows no mixing of colors (that is, no yellow structure), and, therefore, Ca and P have little association with each other. By rotating the color coding for various elements, features that could be missed, such as hairline cracks, fine precipitates, and small boundary interfaces, can be seen. X-Ray Mapping of Mineral Phases Incorporated into Liposomes K. Lewis 1 , R. Wuhrer 2 *, B. Ben-Nissan 1 , S.M. Valenzuela 3 , and K. Moran 4 1 Faculty of Science, University of Technology, Sydney, P.O. Box 123, Broadway, NSW, Australia 2 Microstructural Analysis Unit, University of Technology, Sydney, Australia 3 Department of Medical and Molecular Biosciences, University of Technology, Sydney, Australia 4 Moran Scientific Pty Ltd, P.O. Box 651, Goulburn, NSW 2580, Australia * richard.wuhrer@uts.edu.au Figure 1: Primary color x-ray maps for (A) Ca solution (First Solution), (B) Ca with CO 3 solution (Second Solution), and (C) Ca with and PO 4 (Third Solution). X-ray maps were collected at 20 keV, 512 × 512 pixels, 100 msec/pixel, and 7 kcps. Width of field (WOF) = 300 μm. https://doi.org/10.1017/S155192951000012X Published online by Cambridge University Press