PREPARATION OF LIPOSOMES AND MATERIALS USED:
If you have the knowledge and facilities to prepare liposomes in your own laboratory, you can follow the detailed description of the technique in J.Immunol.Meth. 174 p.83-93,1994. The 'Roche' clodronate was used in a concentration of 2.5 grams per 10 ml. of aqueous solution (usually PBS). Please be aware that other industrial sources of clodronate are available and at moment we use clodronate from an industrial provider. Allthough different providers of clodronate give exactly the same specifications according to their product information sheets, there may be small differences in solubility. The industrial form that we used in our original studies (e.g. Van Rooijen, N. and Van Nieuwmegen, R. 1984. Elimination of phagocytic cells in the spleen after intravenous injection of liposome encapsulated dichloromethylene-diphosphonate: An enzyme-histochemical study. Cell Tissue Res. 238, 355-358) could be used in a maximum concentration of ca 1.9-2.0 grams per 10 ml of aqueous solution. If you do not know the origin of your clodronate, we recommend to try at first the lower concentration. At moment there is no explanation for this difference, but the resulting encapsulation efficacy of both forms of clodronate is not markedly different. The final suspension of the present clodronate liposomes does contain ca. 5 milligrams of clodronate per ml of the suspension (was 7 milligrams for the Roche clodronate). The clodronate solution remaining after the encapsulation procedure, can be reused by replenishing with fresh stock solution (only 1-2 % of the clodronate is encapsulated). To this purpose the final liposome suspension should not be diluted before the first centrifugation (washing) step. About 90% of the original clodronate solution can be recovered in this way. If as a consequence, liposomes are not spun down during the centrifuge step, they have to be collected from the surface. Keep the liposomes at a temperature 4 °C, and resuspend before administration to the animals.
For the most recent publications on the preparation of clodronate liposomes, see:
Van Rooijen, N., Van Kesteren-Hendrikx, E., 2003. 'In vivo' depletion of macrophages by liposome-mediated 'suicide'.. Methods in Enzymology. vol. 373. pp. 3-16 & Van Rooijen, N., Hendrikx, E. 2010. Liposomes for specific depletion of macrophages from organs and tissues. Methods Mol. Biol. 605; 189-203
Whether or not clodronate liposomes will be spun down (or precipitate) or will end up on top of the suspension depends on the specific gravity of the liposomes compared to the surrounding solution in which the suspension was made.
Please be aware that the specific gravities of the clodronate liposomes and their surrounding solution differ only marginally. The slightest difference can decide which of both will be on top and which of them will end up on the bottom.
Clodronate is currently purchased from an industrial provider
Materials for preparation of liposomes are:
Phosphatidylcholine (LIPOID E PC) is purchased from Lipoid GmbH, Ludwigshafen,Germany.
Cholesterol is purchased from SIGMA Chem.Co. USA.
There is only one parameter that is important. The present suspension of liposome encapsulated clodronate is containing ca. 5 mg of clodronate per 1 ml of the suspension. The dimensions and other parameters of liposomes in the suspension are varying (not between different suspensions, but in one single suspension). Given that the clodronate enters the macrophage with the liposomes as Trojan horses, their dimensions are not important, only the total amount of encapsulated clodronate injected.
Useful references: (Full titles and authors can be found under references)
Copyright (C) 2006 Dr. Nico van Rooijen - All rights reserved.