Liposomes and Clodronate: A Short History

Hydrophilic molecules solved in the aqueous solution used for preparation of the liposomes could be encapsulated in the water compartments between the phospholipid bilayers, whereas hydrophobic molecules could be associated with the phospholipid bilayers themselves, and it was GREGORY GREGORIADIS (Greece / UK), who proposed and initiated the application of liposomes as carriers of drugs in biology and medicine (drug carrier concept).

An important paper by Allison and Gregoriadis in 1974 (Liposomes as immunological adjuvants, Nature, 252; 252-252, only one page, but nevertheless for me the most important paper of the decade) did attract my attention since I was studying the extracellular trapping of antigen-antibody complexes on the cell processes of the follicular dendritic cells (FDC’s) in germinal centers, a process thought to play a role in the affinity maturating of B-lymphocytes and the related forming of immunological memory.

Given that nearly all protein antigens are cleared from the body by macrophages in the form of antigen-antibody complexes, as soon as the production of specific antibodies has been started, the question was: What about the immunological memory, if the antigen is administered entrapped in liposomes and will not be able to form a complex with its specific antibody, since it will be quickly degraded after ingestion by macrophages in that liposome entrapped form.

However, it appeared that a very small amount of antigen, presented on the outer surface of the liposomes was responsible for the immune response, since empty liposomes coated afterwards with the antigen did elicit a similar response as liposomes exhibiting antigen on their surface and having in addition excess of it, entrapped in their aqueous inner compartments. (Van Rooijen, N., Van Nieuwmegen, R. , 1980. Liposomes in immunology: Evidence that their adjuvant effect results from surface exposition of the antigens. Cell. Immunol.. vol. 49. pp. 402-407). Consecutively we combined the application of liposomes as drug carriers and liposomes as antigen carriers in our studies on functional aspects of lymphoid and non-lymphoid cells in the spleen. Immune responses against freely soluble protein antigens and the same antigens in a liposome-associated (particulate) form were given to normal animals and animals depleted from macrophages by liposome encapsulated clodronate (Su, D., Van Rooijen, N., 1989. The role of macrophages in the immunoadjuvant action of liposomes: Effects of elimination of splenic macrophages on the immune response against intravenously injected liposome associated albumin antigen. Immunology. vol. 66. pp. 466-470).

Clodronate (dichloromethylene bisphosphonate) is a member of the family of bisphosphonates developed for the treatment of osteolytic bone diseases (Roche Diagnostics GmbH). It shows high affinity for calcium and as a consequence adheres to bone when administered to vertebrates. Osteoclasts, play a role in the physiology of bone by breaking it down, opposed to osteoblasts who are involved in its reconstruction. It appeared that the activity of osteoclasts could be affected by bisphosphonate molecules sticked to the bone.

Given that both osteoclasts and macrophages belong to the mononuclear phagocyte system (MPS), we decided to try clodronate as one of the first effector molecules in our planned “liposome mediated macrophage suicide technique”. This approach, in which liposomes are used as a Trojan horse for the intracellular introduction and accumulation of effector molecules was developed in the early 1980's in order to deplete macrophages from organs and tissues in vivo. Allthough subsequently we found several other hydrophilic molecules, that were also suitable as effector molecules in the approach, clodronate may still be considered the best choice because it shows maximum efficacy and minimal toxicity.

Moreover, both liposomes and clodronate have been introduced in the clinic and it may be anticipated that their combination in the transient suppression of macrophage activity for human application would be easier to achieve than for any of the other candidate effector molecules which are known up to now.