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.


van Lent PL, van den Bersselaar L, van den Hoek AE, van de Ende M, Dijkstra CD, van Rooijen N, van de Putte LB, van den Berg WB.
Reversible depletion of synovial lining cells after intra-articular treatment with liposome-encapsulated dichloromethylene diphosphonate.
Rheumatol. Int. 1993;13(1):21–30.
[toggle_content title=”Abstract”]We studied the depletion and repopulation of synovial lining cells in mice. A single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP) in the mouse knee joint caused selective elimination of synovial lining cells. Depletion of cells occurred within a few days as evidenced by light microscopic, electron microscopic and immunohistochemical studies. Maximal depletion was seen on day 7. Repopulation was observed in the following weeks, starting at the bone side of the joint. Until day 30, full recovery (60% recovery) was not observed in the lining lying adjacent to the dermis. Side effects on cartilage metabolism, such as inhibition of proteoglycan synthesis or degradation of proteoglycans from the matrix was minor but significant, 1 and 2 days after liposome treatment but thereafter full recovery was observed. Selective elimination of lining cells from the joint enabled us to study the in vivo role of these cells in the onset and subsequent pathology of experimental arthritis. An immune-complex-mediated experimental arthritis elicited in lining cell depleted joints that had received CL2MDP-liposomes 7 days earlier prevented inflammation as compared to controls.[/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/ml 21.5 mg/ml EPC/Chol 77/23 MLV PBS 75 µg/6 µl
[/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 MOMA-1+, MOMA-2+synovial , F4/80+NLDC-145+ phagocytes no N/A
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. No liposome prep method was cited.
  2. This concentration of encapsulated clodronate is atypically high although a typical encapsulation efficiency of 1% was reported.
  3. The authors report centrifuging at 100,000xg during the washing process so they may have collected more of the smaller liposomes which are not usually spun down during a 10,000xg centrifugation step.
[/toggle_content] [toggle_content title=”Results”]
  1. When when a single dose of clodronate liposomes, control liposomes, clodronate, or clodronate + control liposoomes was injected into normal mouse knee joints followed by histological examination on days 1, 3 and 7 revealed that
    • Free clodronate had no effect on the synovial lining cells nor did it elicit inflammatory cells into the synovium.
    • Control fluorescent liposomes and clodronate liposomes elicited some inflammatory cells into the synovial fluids which had disappeared by day 7.
    • Control fluorescent liposomes localized to the synovial lining and remained visible at least until day 7; no further time points investigated.
    • Clodronate liposomes caused enlargement and gradual depletion of synovial lining cells which reached a maximum at day 7, although the fibroblast-like cells just under the lining were unchanged.
  2. Continued observation of clodronate liposome treated animals showed
    • Synovial lining cells had begun to repopulate by day 9 on the femoral side of the synovium; complete repopulation at this site was accomplished by day 15.
    • Recovery of the dermal side of the lining began on day 20 and was only about 60% complete at day 30.
    • Only MOMA-2+ and F4/80+ cells were found in the synovial lining.
  3. Co-culturing of synovial macrophages and fibroblasts followed by treatment with clodronate liposomes confirmed that only the macrophages were affected.
  4. Proteoglycan synthesis was inhibited and degradation increased (collagen metabolism) on days 1-3 but returned to baseline by day 4.
  5. Control liposomes solicited PMN at a similar level to clodronate liposomes, but the control liposomes did not affect collagen metabolism.
  6. Immune-complex induced arthritis was initiated on day 7, but no joint swelling was measured in clodronate-liposome treated knees.