Depletion of alveolar macrophages by Clodrosome® (Clodronate encapsulated liposome)

Lung macrophages play a significant role in the biology of lung. They release over one hundred known secretory products such as cytokines, arachidonic acid metabolites and oxygen radicals. Lung macrophages are involved in diverse function such as the maintenance of lung sterility, immune modulation, identification and killing of cancer cells and prevention of allergy. Lung macrophages may also contribute to lung injury. As an example, cigarette smoking stimulates the generation of oxygen radicals by macrophages. Lung macrophages function as mobile unicellular endocrine and paracrine that are strategically located to monitor and modify the micro-environment of the lung.

Usually the term "lung macrophage" and "alveolar macrophage (AM)" are used synonymously, however this is not completely accurate. Pleural macrophages are also another sub-population of lung macrophages. Alveolar macrophages obtain energy predominantly by aerobic metabolism, since they reside in alveoli where the concentration of oxygen is high. Pleural macrophages, on the other hand, rely on glycolytic pathway to replenish their energy stores, because the concentration of oxygen in the pleural space is relatively low.

One way to investigate the in vivo function of a particular cell type is to deplete these cells in the laboratory animals such as mice and rats and to note alterations in response to experimental manipulations.

In this blog entry, we discuss the use of tracheal insufflation of liposome encapsulated clodronate to selectively deplete alveolar macrophages in laboratory animals. Clodronate liposomes target phagocytic macrophages, while minimizing exposure and possible injury to other cells that do not take up liposomes. Many papers have reported a significant depletion of alveolar macrophages in mice and rats lasting for more than 5 days after a single insufflation of clodronate liposomes. These alveolar macrophage depleted animals showed a markedly reduced ability to recruit neutrophils and to release tumor necrosis factor (TNF) into the alveolar space on endotoxin challenge. Insufflation of free and non-encapsulated clodronate drug also causes alveolar macrophage depletion. However free clodronate-induced alveolar macrophage depletion was not specific since ultra-structural studies revealed that it is also caused damage to the alveolar epithelial cells. Thus, encapsulation of clodronate into the liposomes is necessary to specifically target clodronate to macrophages.

The fact that clodronate causes lysis of alveolar macrophages buy only cytoplasmic edema of alveolar epithelial cells suggests that clodronate is preferentially more toxic to macrophages. Administration of clodronate liposomes leads to ingestion of liposomes by macrophages, which are then destroyed after phospholipase-mediated disruption of the liposomal membranes and release of encapsulated clodronate. Previous studies have shown that intravenous injection of free and non-encapsulated clodronate do not deplete hepatic and splenic macrophages. However, it has been observed that tracheal insufflation of free and non-encapsulated clodronate also depleted alveolar macrophages. The reason for this discrepancy is due to rapid clearance of clodronate which has a plasma half-life of a few minutes after intravenous injection. Because of the tight alveolar epithelial barrier the clearance of free and non-encapsulated clodronate from the alveolar space after tracheal insufflation is expected to be slow. As a result, alveolar macrophages are exposed to free and non-encapsulated clodronate for a much more longer period of time. To determine whether depletion of alveolar macrophages was depend on encapsulation of clodronate in liposomes, the effects of plain PBS liposomes and free and non-encapsulated clodronate on tracheal insufflation was studied by Breg et al (Journal of Applied Physiology, June 1993, vol. 74, no. 62812-2819). The study showed that insufflation of plain PBS liposomes (80 ul) had no effect on the number of rat alveolar macrophages, whereas insufflation of free and non-encapsulated clodronate at the dosage equivalent to the amount present in 80 ul of clodronate liposome caused a similar degree of alveolar macrophage depletion and PMN influx into the alveolar space as clodronate liposomes did.

Many studies have suggested that maximum depletion of alveolar macrophages occurred 3 days after tracheal insufflation of clodronate liposome. A single insufflation of clodronate liposome caused a dosed dependent reduction of alveolar macrophages reaching a maximal depletion of >70%at a dose of 120 ul (5 mg/ml encapsulated clodronate) of clodronate liposomes. A dose dependent increase in the number of PMNs also occurred. A time course study using 80 ul and 120 ul of clodronate liposomes shows that a significant depletion of alveolar macrophages occurred 1 day after insufflation and lasted for 7 days. However, by day 9, the number of alveolar macrophages was almost back to the baseline level. 120 ul of clodronate liposomes resulted in a higher PMN influx than did 80 ul of clodronate liposomes. PMN influx is not desirable and therefore the best dosage is the one that depletes the macrophages and minimizes the influx of macrophages (See figure 1).

Figure 1- Effect of tracheal insufflation of clodronate liposomes on alveolar macrophages and neutrophils on rats (data was reproduced from the study done by Berg et al published in Journal of Applied Physiology, June 1993, vol. 74, no. 62812-2819)

How does Clodrosome® (Clodronate encapsulated liposome) kill macrophages?

Mechanism of action

After Clodrosome® has been dosed to the animal by the chosen route, the clodronate liposomes will come into contact with macrophages and other phagocytic cells. The phagocyte immediately recognizes the liposomes as foreign particles and proceeds with destroying these invading particles. The first step in this destruction is phagocytosis in which the liposomes are engulfed by the cell into an internal vesicle known as a phagosome as shown in the figure below. Lysosomes, which contain many types of destructive enzymes, including phospholipases, fuse with the phagosome forming a phagolysosome. The lysosomal membrane also contains proton pumps which will lower the internal pH of the phagolysosome. The low pH, phospholipases and other macromolecular interactions all contribute to compromising the liposomal membrane thus releasing the encapsulated clodronate. The low internal pH of the phagolysosome may contribute to the ability of the clodronate to cross the phagolysosomal membrane into the macrophage’s cytosol.

Once in the cytosolic medium, clodronate is mistakenly recognized as cellular pyrophosphate and used by several Class II aminoacyl-tRNA synthetases to produce a non-hydrolyzable ATP analog, adenosine 5?-(β, γ-dichloromethylene) triphosphate (AppCCl₂p)^1–3. The exact mechanism by which AppCCl₂p causes cell death remained elusive for some time until Lehenkari and coworkers generated data which supported the hypothesis described in the lower figures. Basically, their hypothesis involves cytosolic AppCCl₂p crossing the mitochondrial outer membrane and irreversibly binding to the ATP/ADP translocase which transverses the mitochondrial inner membrane. Inhibiting this enzyme initiates pore openings in the mitochondrial inner membrane. Loss of mitochondrial inner membrane integrity, in turn, results in depolarization and allows molecular signals to be released from the mitochondrion which initiate cell death via apoptosis⁴.

Note that the ability to internalize clodronate from the external medium is unique to osteoclasts, therefore free clodronate is toxic only to these cells in the bone when dosed to animals. The only proven effective method for delivering clodronate intracellularly to other cell types in vivo is via liposomes as described above.

Figure 1- Phagocytosis and degradation of Clodronate liposomes

Figure 2-In a functional mitochondrion, the TCA cycle generates high-energy protons in its matrix which are pumped into the inner membrane space along the electron transport chain (requires cytochrome C as a cofactor). The resulting proton gradient provides energy for the f₁f₀ ATP synthase to generate ATP from ADP and P_i. ATP is transported to the cytosol via ATP/ADP translocase. The translocase only functions when both ATP and ADP are bound to the enzyme, therefore one ADP is removed from the cytosolic ADP pool for each ATP leaving the mitochondrial matrix.

Figure 3- However, when AppCCl₂p diffuses from the cytosol into the inner membrane space, it binds to an ATP-binding site on the ATP/ADP translocase inhibiting the transport activity. This inhibition initiates a cascade which eventually opens the PT pore depleting the electrochemical and proton gradients as well as releasing mitochondrial proapoptotic factors, such as cyt C, into the cytosol. See reference 4 for further detail.

References: 

1. Rogers, M.J., Russell, R.G.G., Blackburn, G.M., Williamson, M.P. & Watts, D.J. Metabolism of halogenated bisphosphonates by the cellular slime mould dictyostelium discoideum. Biochemical and Biophysical Research Communications 189, 414-423 (1992).
2. Rogers, M.J. et al. Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime mold dictyostelium discoideum. Journal of Bone and Mineral Research 9, 1029-1039 (2009).
3. Frith, J.C., Mönkkönen, J., Blackburn, G.M., Russell, R.G.G. & Rogers, M.J. Clodronate and Liposome-Encapsulated Clodronate Are Metabolized to a Toxic ATP Analog, Adenosine 5′-(β,γ-Dichloromethylene) Triphosphate, by Mammalian Cells In Vitro. Journal of Bone and Mineral Research 12, 1358-1367 (1997).
4. Lehenkari, P.P. et al. Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Mol. Pharmacol. 61, 1255-1262 (2002).

Cytotoxicity of Bisphosphonates such as Clodronate, Pamidronate and Zelodronate

Many experiments have shown that the bisphosphonates ,such as clodronate, pamidronate and zoledronate have cytotoxic effect on endothelial as well as on human and mouse tumor cells, all representing component of the tumor stroma. The IC 50 values of all bisphosphonates are in the mM range. Clodronate shows the lowest efficiency in all cases. The nitrogen containing bisphosphonates such as pamidronate and zoledronate have higher activity compare to non-nitrogen containing bisphosphonates such as clodronate. Human Umbilical Vein Endothelial Cells (HUVEC) have shown the highest sensibility for all bisphosphonates tested, especially for zoledronate the cytotoxic activity is twice as high as for tumor cells. The enhanced cytotoxicity of zoledronate on HUVECs compared to other bisphosphonates has already been reported by several groups. This is due to the difference in mechanism of action of nitrogen containing bisphosphonates (e.g. zelodronate and pamidronate) compare to non-nitrogen containing bisphosphonate such as Clodronate.

Both groups of bisphosphonates (non-nitrogen containing and nitrogen containing) inhibit bone resorption by induction of osteoclast apoptosis. However, at the molecular level the mechanism of action of these molecules differ. Non-nitrogen containing bisphosphonates such as Clodronate are metabolically incorporated into analogues of ATP that are resistant to hydrolysis by ATP-dependent metabolic enzymes. On the other hand, nitrogen containing bisphosphonates such as pamidronate and zoledronate are inhibitors of the mevalonate pathway and thereby they prevent prenylation of small GTPase signaling protein required for osteoclast function.

The table below summarizes the toxicity of free and encapsulated bisphosphonates that were reported in various scientific papers.

Table 1- IC50 values (after 4 hours of incubation) of free and liposomal encapsulated clodronate, pamidronate and zoledronate in human rhabdomyosarcoma (A673), murine teratocarcinoma cells (F9) and human umbilical vein endothelial cells (HUVECs) and non activated and activated mouse peritonial macrophages. (Source: Comparison of cytotoxic properties of free and liposomal bisphosphonates in vitro, Renate Frei, Thesis, Swiss Federal Institute of Technology Zurich, 2005)

** The IC50 unit is mM.

The Future of Nano-Medicine

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Lipid movements in liposomes

Liposomes are dynamic systems. The lipids in the liposome bilayers move in various ways. 


Lateral diffusion refers to the lateral movement of lipids in the membrane. Lipids are generally free to move laterally if they are not restricted by certain interactions. Lateral diffusion is a fairly quick and spontaneous process.  The first part of the animation shows the lateral movement of a red color lipid.


Lipid rotation around its axis:  The whole lipid molecule is free to rotate around its vertical axis. This motion is slower: it takes few microseconds to complete one rotation.


Lipid tail wagging: The non-polar tails undergo a "wagging" motion due to the rotation around the C-C single bonds. These motions are rapid (several times in a nanosecond) because the barrier for internal rotation around the C-C bond is low. However, the configuration around the cisdouble bond remains unchanged. 


Flip-Flop from one half of the bilayer to the other half of the bilayer is normally very slow. Flip-flop would require the polar head-group of a lipid to traverse the hydrophobic core of the membrane. The last part of the animation shows the flip-flop of a red color lipid from one layer to another. 

Doxorubicin Liposomes: A journey through nano-particle engineering

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Immunoliposomes

Immunoliposomes are generated by conjugating antibodies either directly to lipid bilayer of liposomes in presence or absence of PEG chains (type I immunoliposomes) or to the distal end of the PEG chain (type II immunoliposomes).

Conjugation of antibodies directly to the lipid bilayer of a PEG containing liposomes (type I immunoliposomes) can result in reduced or even diminishes antigene bonding, depending on the amount of incorporated PEG and the length of the PEG chains. However antigen binding properties of immunoliposomes can be restored by conjugating the antibody to the terminus of the PEG chain and therefore most of the recently developed immunoliposomes are based on type II immunoliposomes.

Target cell recognition by immunoliposomes is influenced by two factors:

1) The type of the antibody molecule (ie, whole antibody or antibody fragments)
2) The chemistry of conjugation (eg, random coupling vs directional coupling)

It has been extensively shown that whole antibodies coupled to liposomes are highly immunogenic. These liposomes are rapidly eliminated through Fc-mediated phagocytosis by macrophages of the liver and spleen, and also by tumor localized macrophages.

Random coupling methods (eg, using thiolated antibody coupled to maleimide PEG lipids or using modified amino reactive PEG lipids) risk antibody inactivation and liposome aggregation by cross linking. The disadvantages of using whole antibody can be circumvented by the use of antibody fragments such as fragment antigen binding (Fab') or single chain fragment variable (scFv).

 Fab' fragments


Fab' fragments are generated by pepsin digestion of IgG molecules and subsequent mild reduction. Fab' fragments can also be produced in recombinant form by being expressed in prokaryotic systems. Fab' fragments have an average molecular weight of 50 kDa and expose one or several free sulfhydryl groups, depending on the method of production. Fab' type II immunoliposomes have reduced immunogenicity compared to IgG type II immunoliposomes. IgG type II immunoliposomes are cleared faster than Fab' type II immunoliposomes. A study by Pastorino et al published in Cancer Res (2003) 63 (1):86-92 shows that Fab' type II immunoliposomes have approximately two fold reduced immunogenicity compared with IgG type II immunoliposomes. The rate of elimination was three fold faster for IgG type II immunoliposomes compared with Fab' type II immunoliposomes.

scFv fragments


scFv fragments are the smallest fragments to contain the entire antigen binding site of an antibody. They are formed by connecting the variable heavy and light chain domains with a short peptide linker with 15-20 aminoacids. scFv molecules have an average molecular weight of 25 kDa and can be produced in bacteria.

In order to conjugate scFv fragments to liposomes, one or more additional cysteine residues are attached to the C terminus of scFv fragments. This allows for site-directed conjugation with the reactive sulfhadryl groups located opposite the antigen binding sites and therefore similar to conjugation of Fab' fragments, conjugation of scFv' fragments does not interfere with target cell recognition.

Expression of scFv' fragments in bacteria normally results in a mixture of monomeric and dimeric molecules. The dimeric molecules are the oxidation products of two monomeric molecules.  In order to achieve efficient coupling, scFv' preparations have to be reduced under mild conditions prior to conjugation.

Coupling of antibodies to preformed liposomes


In direct coupling method the liposomes containing reactive groups are formed, and then antibodies and other chemicals are added in order to achieve conjugation. However, direct coupling can result in lower coupling efficiencies. Coupling efficiencies of 20-80 percent was reported when scFv' molecules were directly conjugated to Mal-PEG liposomes.

Post insertion method


In post insertion method antibodies are first conjugated to Mal-PEG micelles. A study by Nielsen et al. published in Biochim Biophys Acta (2002) 159 (1-3): 109-118 has shown coupling efficiencies up to 95% and preserving more than 80% of immunoreactivity using this method.


Factors influencing therapeutic efficacy


Drug targeting with immunoliposomes is highly complex and influenced by various parameters and both the target site and the liposome.

The targeting efficiency of immunoliposomes is influenced by antigen density on the target cells. A high density of target antigens increases drug delivery and anti-tumor activity. However, antigen density on target cells is often low.

Drug loaded type II immunoliposomes containing antibodies that target an internalized antigen such as CD19 showed much more potency compared to immunoliposomes containing antibodies that target a non internalized antigen such as CD20. This study shows that internalization of immunoliposomes is a prerequisite for the induction of efficient cytotoxicity.

Therapeutic efficacy of immunoliposomes similar to regular liposomes is also dependent on the rate of release of drug and the lipid composition of the liposomes.

Unfortunately, the major problem with immunoliposomes is their poor extravasation properties. Extravasation is antibody independent and it is the rate limiting step in the tumor cell targeting of solid tumors. This will restrict the applications of immunoliposomes to those cancers in which tumor cells are readily accessible such as hematological malignancies or minimal residual diseases.

To circumvent the problems associated with poor extravasation of immunoliposomes new approaches are developed to combine immunoliposomes with vascular targeting. In this approach liposomes are targeted to cells associated with neovascularization in tumor tissue. This is a clever strategy because all solid tumors depend on neovascularization in order to grow and also tumor blood vessels are easily accessible for immunoliposomes.  Endothelial cells are genetically stable and should not become resistant to immunoliposomes therapy. Several antibodies and antibody fragments recognize antigens associated with endothelial cell activation and proliferation. These antigens include E-selectin, vascular endothelial growth factor receptor-2, the fibronectin splice variant ED-B, endoglin (CD105), vascular cell adhesion molecule-1 and tumor endothelial marker 1.  In vivo study of anti-ED-B scFv immunoliposomes showed 62-90% reduction of tumor growth in F9 teratocarcinoma bearing mice in comparison to animals treated with untargeted control liposomes.

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