van Lent PLEM, van den Bersselaar LAM, Holthuyzen AEM, van Rooijen N, van de Putte LBA, van den Berg WB.
Phagocytic synovial lining cells in experimentally induced chronic arthritis: down-regulation of synovitis by CL2MDP-liposomes.
Rheumatology International. 1994;13(6):221–8.
[toggle_content title=”Abstract”]Chronic inflammation of the joint is characterized by the long-term presence of macrophage-like cells in the multilayered synovium. We examined whether synovial phagocytic cells which have settled in the inflamed lining layer play a role in perpetuating synovitis by selectively eliminating them from chronically arthritic murine knee joints. For this purpose we used liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP, Clodronate). Injection of CL2MDP-liposomes into acutely inflamed knee joints (6h, 1 and 3 days) had no significant effect on late chronic synovitis (14 and 21 days after arthritis induction) as observed in haematoxylin and eosin-stained total knee joint sections. Liposomes did not reach the lining layer, as seen with fluorescent liposomes. Additional in vitro studies revealed that activated polymorphs were not affected by CL2MDP-liposomes within 16 h of incubation. Liposomes formed clusters, however, in the presence of intact polymorphs or extracts of polymorphs. In contrast, a significant down-regulation of late synovitis was observed if CL2MDP-liposomes were given during the chronic phase (day 7). Phosphate-buffered saline (PBS) alone or PBS-liposomes had no effect on synovitis. A single injection of CL2MDP-liposomes eliminated many of the phagocytic lining cells and deeper lying inflammatory cells for at least 4 weeks. Free CL2MDP had a minor but significant effect. This study indicates that phagocytic synovial lining cells play an important role in propagating chronic synovitis. To eliminate them from inflamed knee joints, CL2MDP-liposomes should be injected in the chronic and not in the early arthritic phase.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes Control Free Clodronate
12.5 mg/ml1 23.5 mg/ml EPC/Chol 84/16 MLV PBS ND

1This number is very high for the amount of lipid and the prep method cited. Typically this prep would contain around 5 mg/ml clodronate.

[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6 mice, male, 8-12 w, 25-30 g 75 µg/6 µl intra-articular/knee synovial no N/A
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Liposome prep method cited—
    van Rooijen N. The liposome-mediated macrophage “suicide” technique. J. Immunol. Methods. 1989 Nov 13;124(1):1–6.
  2. As stated above, this clodronate concentration in the liposomes is about 2.5 higher than that usually reported. The authors do not report measuring the clodronate and lipid concentrations in the final prep
[/toggle_content] [toggle_content title=”Results”]
  1. When when a single dose of clodronate liposomes was administered 7 d, but not 0.25, 1 or 3 d, post-induction of arthritis, synovitis was alleviated for at least 4 weeks.
  2. A single dose of free clodronate at 7 d also provided a significant reduction in synovitis, but was only about 50% as effective as liposomal clodronate.
  3. When fluorescent clodronate liposomes were injected at the earlier time points (0.25, 1, 3 d), the liposomes did not localize to the phagocytic cells of the synovial lining, while liposomes injected at 7 d post-induction of arthritis were almost exclusively found in the phagocytes.
  4. When incubated in vitro (5% FCS), clodronate liposomes and activated neutrophils aggregate; free clodronate nor PBS causes any aggregation and control liposomes cause slight aggregation under similar conditions.
    • Do the clodronate liposomes alone aggregate in the incubation media?
    • Does the incubation/aggregation result in release of clodronate from the liposomes?
    • Would a mixture of free clodronate and control liposomes result in aggregation?
    • How similar in size and morphology are the clodrosome and control liposomes?
    • Would clodronate liposomes which were pre-incubated in serum cause neutrophil aggregation?
    • Would the aggregation occur in serum-free media?
    • Would clodronate liposomes which were pre-incubated in serum cause neutrophil aggregation in serum-free media?

    Liposomes are known to aggregate in serum containing media and some believe that the mechanism of enhanced circulation times of small liposomes containing PEG lipids is related to the fact that the pegylated liposomes do not aggregate in serum. As to the authors’ speculation that this aggregation in the presence of  neutrophils prevents liposomes from interacting with the phagocytes in the acute phase of arthritis, this question could be easily answered.

    • Since this aggregation phenomenon must be a saturable process, multiple injections of clodronate liposomes timed closely together will saturate the binding sites on the neutrophils and/or temporarily deplete serum components which contribute to the aggregation. Once saturation is complete, clodronate liposomes will be available to the phagocytes.
    • The difference in behaviors between the clodronate and control liposomes is less clear, however we know that it is not uncommon for a drug-laden liposome to have a different size distribution and morphology when compared to “empty” liposomes of the same lipid composition. This factor may be at work here.
  5. Neutrophil lysates cause aggregation and destruction of liposomes; not surprising since phospholipases are among the most predominant destructive enzymes in granules.
  6. Under the same incubation conditions, activated neutrophils die at about 4 hours independent of the additive (clodronate liposomes, free clodronate, control liposomes, PBS).
  7. Clodronate liposomes did not affect Ag removal from the synovial fluid.