Frequently Asked Questions
NOTE: The answers below are based upon…
- Encapsula scientists’ experience and current understanding of published work, and
- feedback from our clients which we appreciate, greatly value and encourage.
These answers will be revised whenever new information is obtained.
Question 1: 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 . A further complicating factor is that published data varies widely as to exactly when clodronate liposomes begin to induce apoptosis in macrophages. Mönkönnnen, et. al. show that macrophage death is measurable within the first hour after clodronate liposome treatment on RAW264 cells in vitro , 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.
- Mönkkönen J, Liukkonen J, Taskinen M, Heath TD, Urtti A. Studies on liposome formulations for intra-articular delivery of clodronate. Journal of Controlled Release. 1995 Aug;35(2–3):145–54.
- Polfliet MM, Goede PH, van Kesteren-Hendrikx EM, van Rooijen N, Dijkstra CD, van den Berg TK. A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system. J. Neuroimmunol. 2001 Jun 1;116(2):188–95.
Addendum 1: When monitoring monocyte uptake in vivo in normal animals, the circulating monocytes may “disappear” or show reduced counts within the first 2 h post-injection due to margination of the monocytes post-liposome phagocytosis. These cells will re-enter the circulation within a few hours. Sunderkötter, et. al. demonstrate this phenomenon and discuss the behavior in detail. Also take into account that circulating monocytes have a lifetime of about 24 h so labeled monocytes will be continually leaving the circulation, even in normal animals, due to aging of the monocytes.
- Sunderkötter C, Nikolic T, Dillon MJ, van Rooijen N, Stehling M, Drevets DA, Leenen P. Subpopulations of Mouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response. J Immunol. 2004 Apr 1;172(7):4410–7.
Question 2: Are there any precautions that should be taken before injecting Clodrosome into animals?
Answer 2: Yes. When injecting Clodrosome into live test subjects, be aware that the drug will wipe out macrophages which are a large part of your subject’s immune system. Bacteria or viruses that were once harmless to the animal become fatal. Because of this, it is very important to monitor the animal under sterile conditions. If the animal is not studied under sterile conditions, there is a high probability that it will become infected and possibly die. The proper standard operating procedure calls for washing your hands with soap and water, wearing sterile laboratory gloves, cleaning the Clodrosome vial with alcohol prior to injection, and working in a laminar flow hood. This is especially important when working with immunodeficient animals, such as NCG and SCID mice, because they have no defense mechanisms against infections. If the research involves the in vivo administration of Clodrosome® to immunodeficient mice, such as NCG and SCID mice, a high mortality rate (often more than 50%) is to be expected. In order to have statistically meaningful results, using a larger pool size of mice is recommended.