Friday, April 20, 2012

Why doesn’t Encapsula offer fluorescent clodronate liposomes in addition to fluorescent control liposomes?

Answer 1: The issue with fluorescent Clodrosome has to do with the potential for inaccurate and/or uninterpretable data being generated by labelled Clodrosome. When Clodrosome induces macrophage apoptosis, the fluorescent lipid incorporated into the Clodrosome that is disrupted and metabolized in the phagolysosome will be dispersed among the residual apoptotic bodies which are subsequently phagocytosed by other macrophages. Therefore, fluorescent lipid may be detected in phagocytic cells which never phagocytosed Clodrosome especially when FACS or fluoroscopy are utilized to detect fluorescent cells (FACS) or fluorescence levels in a tissue homogenate (fluoroscopy). Another potential artifact arises from fluorescent lipid remaining in the extracellular “garbage”, which has not yet been cleared by other phagocytes, generating a high background fluorescence. However, experienced confocal microscopists may be able to differentiate between the punctate fluorescence resulting from fluorescent intact liposomes versus the more diffuse fluorescence characteristic of disrupted liposomes and some have successfully used fluorescent clodronate liposomes to visualize the cellular location of these liposomes by confocal microscopy in vivo [1]. A further complicating factor is that published data varies widely as to exactly when clodronate liposomes begin to induce apoptosis in macrophages. Mönkönnnen, et. that macrophage death is measurable within the first hour after clodronate liposome treatment on RAW264 cells in vitro [2], while many report no signs of macrophage apoptosis until several hours after treatment in vivo. The variability in the data is likely due to different liposomal formulations of clodronate as well as the vastly different experimental conditions. Therefore, as with most biological studies, especially those involving liposomes, the amount of time between treating the animal or cells with clodronate liposomes and the onset of apoptosis will need to be established in each experimental model. If the nature of the research demands that Clodrosome be tracked rather than the control, Encapsula can provide diI-labelled Clodrosome upon request, and assuming that the Clodrosome distribution can definitively be assessed prior to the onset of apoptosis, clear and valid data on the biodistribution of fluorescent Clodrosome should be obtainable. Still, for most purposes, Fluoroliposome™ (fluorescent control liposomes) will provide the required data with far fewer potential artifacts.
  1. M.M. Polfliet, P.H. Goede, E.M. van Kesteren-Hendrikx, N. van Rooijen, C.D. Dijkstra, T.K. van den Berg, A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system, Journal of Neuroimmunology, 116 (2001) 188–195.
  2. J. Mönkkönen, J. Liukkonen, M. Taskinen, T.D. Heath, A. Urtti, Studies on liposome formulations for intra-articular delivery of clodronate, Journal of Controlled Release, 35 (1995) 145–154.

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