FAQ

Macrophages play an important role in the immune system. They regulate functions of many non-phagocytic cells, mainly through mediation of soluble molecules as cytokines and chemokines. Macrophages are also involved in ‘homoeostasis’ of the body by ingesting and digesting microorganisms or non-self particles and macromolecules. Digestion is in turn mediated by their lysosomal enzymes.

Liposomes are artificially prepared lipid vesicles, consisting of concentric phospholipid bilayers entrapping aqueous compartments. They can be used to encapsulate strongly hydrophilic molecules solved in aqueous solutions, such as clodronate, a non-toxic bisphosphonate developed for human application.

After injection, clodronate liposomes function as Trojan horses. Macrophages ingest the clodronate liposomes and digest the liposomal membranes. Clodronate is not digested and will remain inside the macrophage. More clodronate will accumulate in the macrophage as it ingests and digests more clodronate liposomes. At a certain intracellular concentration, clodronate will eliminate the macrophage by initiating its programmed cell death, i.e. apoptosis [1].
By choosing the right administration route of clodronate liposomes, particular organs or tissues can be depleted of macrophages. Therefore, clodronate liposomes are an excellent tool to study macrophage function [2].

1. Van Rooijen, N., & Hendrikx, E. (2010). Liposomes for specific depletion of macrophages from organs and tissues. In Liposomes (pp. 189-203). Humana Press.
2. Van Rooijen, N., Sanders, A., & van den Berg, T. K. (1996). Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine. Journal of immunological methods, 193(1), 93-99.

The half-life (t1⁄2) of clodronate released from dead macrophages is in the order of minutes[3], since free clodronate is rapidly cleared from the circulation by the renal system. Free clodronate molecules will not easily enter into cells due to difficulty in passing cell membranes, including liposomal phospholipid bilayers. However, if they remain in the surrounding medium, they may slowly accumulate into cells[4].

The half-life of clodronate liposomes is difficult to determine. Liposomes are difficult to isolate from blood and their distribution in the animal is not even. Further, the half-life of clodronate liposomes depends on the time it takes to be ingested and digested by macrophages, which is determined by many factors, such as: immunocompetence of the animal model, route of administration, dose of administration.

3. Fleisch, H. (2000). Bisphosphonates in bone disease: from the laboratory to the patient. Elsevier.
4.Claassen, I., Van Rooijen, N., & Claassen, E. (1990). A new method for removal of mononuclear phagocytes from heterogeneous cell populations in vitro, using the liposome-mediated macrophage ‘suicide’ technique. Journal of immunological methods, 134(2), 153-161.

Failure of activity of clodronate liposomes can be due to various reasons:

1. The expiry date has passed: activity cannot be ensured after the expiry date has passed;

2. Storage at extreme temperatures: clodronate liposomes should be stored between 4 and 8 degrees Celsius. Freezing of liposomes or temperatures above 30 degrees Celsius can affect the membrane’s stability and reduce clodronate liposome activity;

3. Dilution of the liposomal suspension: this affects the osmotic pressure in the suspension, which may cause instability of the liposomal membrane;

4. The administration protocol is inadequate: protocols should be chosen carefully and depend on multiple factors, for instance:

  1. the type of macrophages you intend to deplete (e.g. Kupffer cells, microglia, alveolar macrophages, osteoclasts). For example, alveolar macrophages can be targeted by intratracheal administration, whilst the interstitial macrophages on the other side of the endothelial/epithelial barrier will not be targeted by this administration route;
  2. whether you intend to deplete them short- or long-term;
  3. the weight of the animal: larger animals require more clodronate liposomes in order to establish depletion.

5. The liposomal suspension was not resuspended before use: liposomes tend to precipitate during storage, make sure to resuspend by gently shaking before usage to prevent injecting only the PBS solution without the liposomes;

6. Something went wrong whilst injecting: it is important to verify whether the injection is successful. For instance, if you see a bulb forming during intravenous injection in the tail vein, it might be the case that you missed the tail vein. When injecting intraperitoneally, make sure that you inject the liposomal suspension into the intraperitoneal cavity (and not in the intestine). To verify if the liposomes have reached your desired location, fluorescent DiI liposomes can be used;

7. Inadequate ways of measuring depletion: the right markers need to be chosen to establish whether depletion has been successful. In addition, you need to take into account that depletion and repopulation kinetics of macrophages differ per administration protocol and (animal) model.

Note:
We have numerous customers worldwide who purchase our liposomal products. When we receive reports about insufficient macrophage depletion after injections of our clodronate liposomes, we always check if other clients have reported similar observations. Until now, we are glad to inform that we have not encountered this issue.

Animals have died after (clodronate) liposome injection, what can be the reason?

This is most probable caused by microbial contamination shortly before or during injection. When injecting clodronate liposomes, the depletion of macrophages can make an animal more susceptible to an infection.

Note: intraperitoneal injections (i.p) are complicated and contain a higher risk of contamination. If you inject i.p. we advice to make sure that the skin is shaved and cleaned with ethanol (100%) due to the large risk of bringing bacteria that are living on the skin into the mice along with the needle.

This is most probably due to injection of non-homogenous, precipitated liposomes. The precipitated liposomes may cause an embolism and thereby a partial or total blocking of blood flow in an artery or vein. Since liposomes tend to precipitate during storage, please make sure to resuspend by gently shaking before usage.

Another reason might be the temperature of the liposomal suspension. Liposomes should be stored between 4 and 8 degrees Celsius, but they should be administered at room temperature.

Note: If injection takes too much time, the liposomes possibly precipitate in the syringe, this may result in differential dosing when multiple animals are injected using the same syringe.

Store between 4 – 8 ºC (or 39 – 47 ºF). The liposomal suspensions should never be frozen, nor be exposed to extreme high temperatures.

On average, the entire liposome suspension has a concentration of 5 mg clodronate / mL. Clodronate is encapsulated in the liposomal vesicles in the form of CH2Na2Cl2O6P2 · 4 H2O.

All our liposomes (i.e. clodronate, PBS and fluorescent DiI liposomes) are slightly negatively charged and will therefore not clog together. The liposomes in the suspension are not sized, meaning that it contains smaller and larger liposomes as well (up to 3 micron). On average, the size of the liposomes are 1.7 micron.

All our liposomal formulations (i.e. clodronate liposomes, control liposomes and fluorescent DiI liposomes) are suspended in sterile phosphate buffered saline (PBS): 10 mM Na2HPO4, 10 mM NaH2PO4, >140 mM NaCl.

Control liposomes labeled with the fluorochrome DiI, or DiI-labeled liposomes, can be used to investigate whether the liposomes have reached the targeted macrophage population via the chosen administration protocol. The fluorescent label will show the distribution pattern of the liposomes within tissues and their uptake by macrophages. DiI-labeled liposomes can be detected either by fluorescent microscopy of flow cytometry. The DiI-molecule’s excitation and emission is 549 and 565 nm respectively (see figure).

If you have made use of our liposomal formulations for your experiments and intend to publish your findings, please refer to us as “Liposoma, the Netherlands (clodronateliposomes.com)”

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