Use of liposomes as carriers for radiation synovectomy

Use of liposomes as carriers for radiation synovectomy

Nucl. Med. Biol. Vol. 15, No. 2, pp. 151-156, 1988 In:. J. Radio:. Appl. Instrum. Parr B Printed in Great Britain. All rights reserved 0883-2897/88$3...

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Nucl. Med. Biol. Vol. 15, No. 2, pp. 151-156, 1988 In:. J. Radio:. Appl. Instrum. Parr B Printed in Great Britain. All rights reserved

0883-2897/88$3.00+ 0.00 Copyright 0 1988 Pergamon Journals Ltd

Use of Liposomes as Carriers for Radiation Synovectomy M. R. ZALUTSKY, M. A. NOSKA, P. W. GALLAGHER,* S. SHORTKROFF and C. B. SLEDGE Departments

of Radiology (Nuclear Medicine) and Orthopedic Boston, MA 02115, U.S.A. (Received

Surgery, Harvard Medical

School,

26 April 1987)

Using phnTc]pertechnetate as an aqueous space marker, the permeability of liposomes composed of seven different mixtures of distearoylphosphatidylcholine (DSPC) and sphingomyelin (SM) was determined. Liposomes containing 20-33% SM were the least permeable in the presence of rheumatoid synovial fluid. Following injection of 99mTc-containing liposomes into the knee joints of rabbits, retention of *Tc in the knee was more than 200 times greater than following injection of nonencapsulated pTc]pertechnet&e. The knee clearance biologic half time of %Tc with DSPC/SM (4: 1) liposomes was 64 h. Most of the activity that had leaked from the knee was not found in extra-articular tissues, suggesting rapid excretion. When DSPC/SM (4:l) liposomes were labeled with r”In(oxine), a knee clearance biologic half time of greater than 1200 h was observed.

Introduction Radiation synovectomy is an attractive alternative to surgery in the treatment of certain patients with rheumatoid arthritis. In this procedure, a beta-

emitting isotope in colloidal or particulate form is injected directly into the affected joint in order to irradiate selectively the inflamed synovial tissue. Our results using ferric hydroxide macroaggregates (FHMA) labeled with the 2.3-h half-life beta emitter 165Dy suggest that this radiocolloid is an effective agent for radiation synovectomy (Sledge et al., 1986). For example, in knees with stage I radiographic changes, more than 70% showed improvement following treatment with 270-300 mCi 16’Dy-FHMA. At this dose level, we have estimated that 900010,000 rad are delivered to the synovial tissue (Zalutsky et al., 1986a). Unfortunately, the short half life of 165Dy limits the use of this therapy to medical centers in close proximity to a nuclear reactor. More widespread application of radiation synovectomy awaits the

Supported in part by Grant AM 23063 from NIADDKD and Department of Energy Contract Number DOE76EVO4115. For reprints contact: Michael R. Zalutsky, Ph.D., Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710, U.S.A. * Present address: Department of Radiology, Methodist Hospital, Indianapolis, IN 46202, U.S.A.

development of radioparticulates labeled with longer lived beta emitters such as v (64.0 h), i9*Au (64.7 h) and ‘*‘Re (16.9 h). A significant problem observed with inorganic colloids of these radionuclides is rapid egress of radioactivity from the injected joint which can cause significant radiation exposure of the liver, spleen and draining inguinal lymph nodes. For example, following intra-articular injection of WY and 19”Aucolloids in patients, as much as 25 and 60% of the injected dose has been found in the lymph nodes, respectively (de la Chappelle et al., 1975; Topp et al. 1975). In contrast, the extra-articular spread of j6’Dy-FHMA results in an average lymph node uptake of only 0.12% and an average liver uptake of 0.64% 24 h after injection (Zalutsky et al., 1986a). One particulate carrier that could be a suitable alternative for use in radiation synovectomy is the liposome. An advantage of lipid vesicles is that they can be labeled using radionuclides in virtually any chemical form; lipophilic compounds can be entrapped in their lipid bilayers and water-soluble molecules can be encapsulated between the bilayers. Liposomal encapsulation of a radionuclide in a chemical form which can be rapidly eliminated from the body is particularly appealing since uptake by the liver, spleen and lymph nodes following degradation of the liposome should be minimized. Although liposomes were originally thought to have wide ranging applications in drug delivery, it now appears that their properties are most suited for the treatment of 151

M. R. ZhLUTsKY et al.

152

diseases of the reticuloendothelial system and isolated body spaces such as joints (Poste ef al., 1984). Use of liposomes as a carrier for the treatment of rheumatoid arthritis was first suggested by Shaw et al. (1976) who proposed entrapping steroids within lipid vesicles as a means of increasing the local delivery of anti-inflammatory drug to arthritic joints. In a subsequent study, this group showed that cortisol palmitate-containing liposomes were rapidly taken up by the phagocytic cells lining the arthritic synovium of rabbit knees (Shaw et al., 1979). In the present paper, we have investigated several properties of liposomes that are pertinent to their potential utility in the development of radiation synovectomy agents. The in vitro permeability of phaTc]pertechnetate-containing liposomes composed of pure sphingomyelin (SM), pure distearoyiphosphatidylcholine (DSPC) and five different DSPC: SM ratios were measured in the presence of human synovial fluid. These phospholipid mixtures were selected based on the results of our previous study which showed that combining the rigidizing effects of a high phase-transition temperature phospholipid, DSPC, with one that can form hydrogen bonds, SM, can yield vesicles of decreased permeability (Zalutsky et al., 1986b). Following intra-articular injection of liposomes of three different lipid compositions, the leakage rate of %Tc from the rabbit knee was measured and the extra-articular tissue distribution of %Tc activity was determined. Properties of multilamellar DSPC/SM (4: I) liposomes containing “‘In (oxine) were also investigated.

Materials and Methods Preparation of liposomes

Multilamellar liposomes containing sodium PTcJpertechnetate were prepared as previously described (Zalutsky et al., 1986b). Vesicles were produced with the following phospholipid compositions: pure DSPC, pure SM, and DSPC: SM in molar ratios of 8:1, 4:1, 3:1, 5:2, 2:l and 1:l. Unencapsulated PTclpertechnetate and unilamellar liposomes were removed from the multilamellar liposome preparations by repeated centrifugation at IOOOg. Multilamellar liposomes composed of DSPC/SM in a 4:l molar ratio were also labeled with the lipophilic compound “‘In(oxine). Indium-111 oxine in ethanol was added to a round-bottom flask containing 20 pmol of phospholipid in deaerated chloroform and the mixture was evaporated at 64°C using a microrotary evaporator. Liposomes were then prepared and purified using our standard method. Permeability measurements

In order to differentiate between the effects of high and low molecular weight substances in synovial fluid, the permeability of each labeled liposome prep aration was determined by both dialysis and direct exposure at 37°C. Synovial fluid was aspirated under

aseptic conditions from the knees of patients suffering from active rheumatoid arthritis. The fluid was pooled and centrifuged at 1000 g for 10 min to remove infiltrating cells. The synovial fluid was diluted 1: 1 with phosphate buffered saline and stored at -20°C for future use. Details of the dialysis and direct exposure-centrifugation methods used to assess liposome leakage have previously been described (Zalutsky et al., 1986b). Leakage from the rabbit knee

The loss of radioactivity from the rabbit knee following liposome administration was determined by serial gamma camera imaging. Groups of four New Zealand white rabbits were injected through the patellar ligament of the right knee with each of the following substances: DSPC, DSPCjSM (4: 1) and DSPC/SM (5:2) liposomes containing p”Tc]pertechnetate; DSPC/SM (4: 1) liposomes labeled with “‘In(oxine); and as controls, unencapsulated P9”‘Tc]pertechnetate and “‘In(oxine). Each rabbit received approximately 3 pmol of phospholipid and 2OOpCi of either 99mTc or “‘In in a volume of 0.1-0.2 mL. Images were obtained using a Picker Dyna Camera IV interfaced to a PDP-11 computer. A high resolution collimator was used for the %Tc studies and a medium energy collimator was used to image “‘In. Animals were positioned 2 cm below the camera such that the superior pole of the right patella was directly below the center of the grid on the collimator face. The lower torso of the rabbits was shielded from the camera using a l-cm thick lead sheet. One hundred to 200 thousand count analog as well as 64 x 64 matrix digital images were collected over 2-12min. Regions of interest were set to encompass the right knee and a background area proximal to the knee but outside the body. The leakage of radioactivity from the knee at each time point was calculated by dividing the net counts in the knee to those in an injection standard of known activity. Comparison of the knee counts in each image to an image obtained immediately after injection was also performed. [9”“Tc]liposome biodistribution

Immediately after obtaining the 24 h images, the rabbits that had received the three different *Tccontaining liposome preparations were euthanized by i.v. sodium pentobarbital injection for the biodistribution measurements. An additional rabbit was injected intravenously with %Tc-containing DSPC liposomes and the distribution of 99mTcactivity was determined 9 h later. This time was selected because, as discussed below, it approximated the average time at which activity leaked from the knees during the first 24 h after an intra-articular liposome injection. Tissues of interest were removed, washed with saline, blotted and weighed. The following tissues were obtained: liver, spleen, lungs, heart, stomach, intestines, kidneys, bladder/urine, fat, muscle, bone and

Liposomes for radiation synovectomy

DSPC SM

0.2 0.4 0.6 0.8 0.6 0.6 0.4 0.2

0.2 0.4 0.6 0.6 0.6 0.6 04 0.2

153

0.2 04 0.6 0.6 06 0.6 04 0.2

Mole Fraction

Fig. 1. Leakage of phnTc]pertechnetate from multilamellar liposomes in the presence of human rheumatoid synovial fluid as a function of DSPC and SM content. Measured by dialysis (solid line) and direct exposure (dashed line).

blood. The tissues were assayed for 99mTc activity. using an automated gamma counter. In order to calculate whole body recovery of activity, it was assumed that the fat, muscle, bone (without marrow) and blood constituted 20, 40, 10 and 6% of total body weight, respectively.

weight substances. After only a 1 h exposure to synovial fluid, liposomes composed of high mole fractions of either DSPC or SM lost more than 75% of encapsulated *Tc activity. The cumulative leakages measured at 6 h were 94-100% of those at 48 h. Liposomes composed of 25-33% SM were the least permeable. After 48 h, approximately 52% leakage of 99mTc was observed, a value which is about 25% Results higher than measured by dialysis. The leakage of 99mTcactivity from each of seven The leakage of “‘In activity from DSPC/SM (4: 1) different molar ratios of DSPC and SM. Preparation liposomes containing “‘In(oxine) was also measured was measured 1, 2, 6, 24, and 48 h after exposure to by both dialysis and direct exposure. In the dialysis 50% synovial fluid at 37°C. In Fig. 1, leakage data determinations, a correction for binding of undetermined by both dialysis (solid lines) and direct encapsulated “‘In(oxine) to dialysis tubing was reexposure (dashed lines) are shown. Control measurequired for the 16 h points. Binding of free “‘In(oxine) to the dialysis tubing ranged from 90% ments using nonencapsulated pmTc]pertechnetate demonstrated that greater than 98% of the 99mT~ at 1 h to 20% at 6 h. In the dialysis studies, the activity was in non-particulate form at all time points. fraction of “‘In activity which was in nonparticulate The permeability of liposomes in the presence of form increased from 2.3% at 1 h to 11.2% at 48 h. 50% synovial fluid was dependent on the molar ratio The loss of “‘In activity from the liposomes deterof DSPC to SM which was utilized. The cumulative mined in the direct exposure experiments ranged leakages measured at 6 h were 35-60% of those seen from 21% at 1 h to 37.5% at 48 h. at 48 h. High mole fractions of either DSPC or SM The phospholipid mixtures used for the in vim resulted in liposomes with the highest leakage rates studies were pure DSPC, DSPC/SM (4:l) and while those composed of 20-33% SM appeared to DSPCjSM (5:2) representing compositions of relabe the least permeable. In the dialysis measuretively high, moderate and low in vitro permeability, ments, the DSPC/SM (3: 1) preparation was the most respectively. The size distribution of the three lipostable, leaking 39.3 + 1.5% after 48 h; in comparison, some preparations as determined by optical microscopy was similar; the majority of the vesicles were 89.2+4.5% of the 99mT~activity had leaked from liposomes composed of DSPC/SM (1: 8). I-4 pm diameter. The clearance of *Tc activity In the direct exposure measurements, the relationfrom the rabbit knee following the intra-articular ship between liposome permeability and the cominjection of the three different %Tc-containing lipoposition of the lipid bilayer was more pronounced, some formulations and PgmTc]pertechnetate are comparticularly at early time points. The leakage rates pared in Fig. 2. Encapsulation of the PgmTclperwere significantly higher than those determined in the technetate in liposomes considerably decreased the dialysis measurements, suggesting an additional effect leakage of 99mT~activity from the rabbit knee. For due to exposure of the liposomes to high molecular example, use of liposomes as a carrier for *Tc

M. R.

154

ZALUTSKY et al.

Table 1. Clearance half times for radioactive substances from the rabbit knee joint Substance pTc]DSpc pkpspcjsM

. 25-

[PO.TC]DSPC/SM~~

. [-'Tel OSPC

'

8

42 (4:i) MLv

(5:2) MLv [9R”Tc]pertechnetate [“‘In(oxine)]DSPC/SM (4: 1) MLV “‘In(oxine) ph"T~pSpcjSM

IIML"

MLV

0 [99"Tt] DSPC/SMiS

Halftime (h)

mv

PIMLV

64

34 0.25 >I200 315

G .E

I \

I



I ‘\

‘\\ \.

Fig. 2. Comparison

,022%

t

‘\

I

24

8

of the clearance

of%Tc

activity from the

knee following intra-articular injection of mTc-containing liposomes or PTclpertechnetate.

resulted in the retention of 250-350 times more activity in the knee 24 h after injection. In Fig. 3, loss of “‘In activity from the knee following intra-articular injection of DSPC/SM (4: 1) liposomes containing “‘In(oxine) and unencapsulated “‘In(oxine) are compared. Liposome encapsulation increased the retention of “‘In activity in the

knee at 24 h by only a factor of 1.25; however, in both forms, the clearance of activity from the knee was significantly slower than that observed following *Tc-containing liposome injection. The biologic half times for knee clearance following intra-articular administration of the various radioactive substances are summarized in Table 1. Following the knee clearance studies, the tissue distribution of the 99mT~activity which had leaked from the knee was determined for the three different liposome compositions. These data are compared in Table 2 to the distribution of activity observed 9 h after iv. injection of 99”Tc-containing DSPC liposomes. This time was chosen because it represents the approximate midpoint for 99mTc leakage from the knee during the 24 h period. Following i.v. administration, more than 70% of the injected dose was recovered from the liver, spleen and lungs accounting for more than 80% of the total activity in the body. In contrast, only 0.38, 0.79 and 1.43% of the injected activity was found in the liver, spleen and lungs after intra-articular administration of DSPC/SM (5:2), DSPC/SM (4: 1) and pure DSPC liposomes, respectively. Activity in these tissues accounted for 12, 14 and 9% of the total activity in the body at 24 h. Comparison of the total body activity calculated from the tissue distribution experiments with the knee activity determined with the gamma camera immediately prior to necropsy suggests that the majority of the 99mTcactivity which had leaked from the knee had been excreted during the 24 h period. This is most apparent for the DSPC/SM (5:2) preparation; although 47% of the injected activity had leaked from the knee over 24 h, only 3.2% of the injected dose was recovered in extra-articular tissues.

Discussion

I 24

48

Hours

72

96

120

Fig. 3. Comparison of the clearance of “‘In activity from the knee following intra-articular injection of [“‘In(oxine)]DSPC/SM (4: 1) liposomes or free “‘In(oxine).

One approach to the development of improved radiopharmaceuticals for radiation synovectomy is to encapsulate radionuclides in liposomes. Because of the isolated nature of the synovium and its phagocytic character, the rheumatoid knee could present an ideal target for the radiotherapeutic application of labeled liposomes. A critical factor affecting the utility of liposomes for the treatment of arthritis is their stability and permeability after exposure to synovial fluid. Although liposomes are known to degrade more rapidly in the presence of serum than in buffers (Allen and

Liposomes for radiation synovectomy Cleland, 1980; Zalutsky et al., 1986b), their behavior after exposure to rheumatoid synovial fluid is largely unknown. For a number of reasons, synovial fluid could be a particularly deleterious environment for liposomes. Synovial fluid contains hyaluronic acid (McCarty, 1979), a protein-polysaccharide which could interact with the lipid bilayer of the liposome. In addition, the synovial fluid from patients with rheumatoid arthritis has been shown to contain elevated levels of lysosomal (in particular, proteolytic) enzymes (Weissmann, 1972; Fell and Dingle, 1963) as well as protein (Ruddy, 1974). In the present study, liposomes composed of DSPC and 20-33% SM were the least permeable in synovial fluid. In the dialysis studies, the leakage of *Tc from DSPCjSM liposomes in synovial fluid was quite similar to that observed previously in serum (Zalutsky et al., 1986b). However, in the direct exposure experiments, synovial fluid induced 24 times greater leakage after a l-24 h exposure with the difference decreasing to about 10% at 48 h. Observation of accelerated liposomal degradation in synovial fluid compared to serum in the direct exposure but not the dialysis measurements suggests the action of a substance which is more abundant in synovial fluid which is larger than the 14,000 molecular weight pore size of the dialysis tubing. Increased loss of lipid-soluble substances from liposomes in the presence of synovial fluid has been reported. Shaw and Dingle (1980) have shown that the loss of [3H]cortisol palmitate and octanoate from dipalmitoylphosphatidylcholine (DPPC) vesicles was about two-fold greater in synovial fluid than in either serum albumin or phosphate buffered saline. Similarly, Kellaway and Chawla (1982) have reported that the efflux rate constant for the loss of chloroquine from DPPC liposomes is three times greater in synovial fluid than in water. It is interesting to note that the leakage of “‘In from DSPC/SM (4:1) liposomes containing “‘In(oxine) in their lipid bilayer was about 50% less than the leakage of 99”T~ encapsulated in the aqueous

155

layers. This suggests that liposome degradation is not only a result of the creation of channels in the bilayer permitting the release of aqueous solute, but can also be attributed to loss of lipid from the vesicle. Similar results have been reported for the relative loss of aqueous and lipid markers from liposomes in the presence of serum (Senior and Gregoriadis, 1984). Encapsulation of PTclpertechnetate in multilamellar liposomes composed of three different mixtures of DSPC and SM resulted in a more than lOO-fold increase in retention of %Tc activity in the rabbit knee. Liposomes formulated from DSPC/SM (4: 1) exhibited the lowest leakage of %Tc, an observation that is consistent with their relatively low permeability in vitro. With this preparation, 20-37% of the injected dose of 99mTchad cleared from the knee 24 h after intra-articular injection. In comparison, leakage rates of 34.6 + 2.2% and 65.1 k 4.3% were measured at 24 h in vitro by dialysis and direct exposure to synovial fluid, respectively. It is possible that the greater leakage of 99mT~observed in the direct exposure measurements relative to the in vivo study may be related to a higher concentration of degradative substances such as proteolytic enzymes in human rheumatoid compared to normal rabbit synovial fluid. The biologic half time for knee clearance of 99mTc, approximately 64 h, following injection of ~“Tc]DSPC/SM (4: 1) liposomes is longer than the 33 h half time reported by Kellaway and Chawla (1982) for ‘311-labeled DPPC liposomes of similar size and the 8.8 and 10.5 h half lives measured for 99mTc-labeled antimony sulfide and sulphur colloids, respectively. Unfortunately, even using our optimal liposome formulation, the loss of activity from the knee was an order of magnitude greater than that observed previously with ferric hydroxide macroaggregates (Noble et al., 1983). One of the potential advantages of liposomes is that the clearance of extra-articular activity can be expedited by encapsulating radionuclides in a chemical form which can be eliminated rapidly from the body. Technetium-99m pertechnetate was used in the

Table 2. Tissue distribution of %Tc in the rabbit following injection of ~Tc] %

lniected

multilamellar liposomes

dose Intravenous injection, 9 hb

Intra-articular injection, 24 ha Tissue

DSPC/SM (5:2)

DSPC/SM (4: I )

DSPC

DSPC

Liver Spleen Lungs Kidneys Bladder/urine Blood Lymph nodes: Inguinal (L) Inguinal (R) Whole body ex&dina

0.36 + 0.07 (1.0 f 0.5) x 10-j (2.1 f 0.7) x 10-j 0.25 f 0.07 0.31 kO.10 0.53 * 0.21

0.7 f 0.28 (2.6+1.5)x 10~’ (8 f 4) x IO-’ 0.20 f 0.05 0.47 f 0.23 0.93 + 0.24

1.35 + 0.42 (6+3)x 10-l (7 F I) x 10-3 0.42 + 0.18 4.8 It. I .O 2.6 + 0.2

44.2 3.0 23.4 1.8 0.7 6.1

15.9 f 1.8

87.4

knee

(4.7 + 2.3) x IO-’ (4.7 f 2.1) x 10-4 3.2 + 0.8

5.8 k 2.1

‘Mean *standard deviation of four animals. One rabbit. Since @“‘Tcdoes not leave the knee as a bolus, an i.v. injection at 9 h, the approximate halftime for leakage from the knee during the 24 h period was used for comparison.

156

M. R. ZALUTSKYer al.

present study because of convenience; more rapid excretion of leaked activity could probably be achieved, for example, by using a DTPA complex of the isotope of interest. Even with liposomes containing [99mTc]pertechnetate, most of the extra-articular activity had been excreted by 24 h with less than 1% of the dose taken up by the liver, spleen and lungs. Lack of uptake by these tissues suggests that loss of activity from the knee is not a result of the egress of intact liposomes but rather, is due to the leakage of 99mT~from the liposome followed by rapid clearance from the knee. After evaluating the relative stability and permeability of the different ‘“Tc-containing liposome preparations, the DSPC/SM (4:l) mixture was chosen for further evaluation with a lipophilic marker, “‘In(oxine). A potential disadvantage of “‘In(oxine) is its slow clearance in nonencapsulated form, particularly from the liver and blood (Espinola et al., 1979). An advantage of using a lipid-soluble tracer is that higher encapsulation efficiencies can generally be obtained (70 vs 11% in this study). No statistically significant loss of “‘In activity from the rabbit knee was observed over 72 h. It is interesting to note that in the direct exposure measurements, this same liposome preparation exhibited 37.5% nonparticulate “‘In activity after only a 48 h incubation in synovial fluid. In addition to the environmental differences mentioned above, the slow clearance (315 h biologic half time) of “‘In activity following the intra-articular injection of unencapsulated “‘In(oxine) must also be considered in comparing the in vitro and in vivo results. Because “‘In(oxine) is a relatively weak complex, it is possible that some of the “‘In that leaks from the liposomes dissociates and is retained in the knee bound to transferrin or ferritin. In summary, we have shown that the permeability of liposomes in the presence of rheumatoid synovial fluid is dependent on the phospholipid composition of the vesicles. When labeled with a water soluble radionuclide, loss of activity from the knee is higher than that observed for some inorganic colloids, however most of the activity which leaks from the knee is excreted rapidly. Following injection of DSPC/SM multilamellar liposomes containing “‘In(oxine), no significant loss of activity from the knee was observed. If lipophilic complexes of potentially useful radionuclides for radiation synovectomy can be synthesized and if the label clears relatively slowly from the knee in nonencapsulated form, then liposomes labeled with these complexes could offer a potentially useful approach for the development of radiation synovectomy agents. For example, using the biologic half time for the knee clearance of “‘In observed in this study, we estimate that a therapeutic dose of 9000-10,000 rad could be delivered to the synovial tissue using liposomes containing either 6 mCi of 9oY or 24 mCi of ‘**Re.

References Allen T. M. and Cleland L. C. (1980) Serum induced leakage of liposome contents. Biochem. Biophys. Acta 597, 418.

Chappelle A. de la, Oka M., Rekonen A. and Ruotsi A. (1972) Chromosome damage after intra-articular injection of radioactive yttrium. Ann. Rheum. Dis. 31, 508. Espinola L. G., Beaucaire J., Gottschalk A. and Caride V. J. (1979) Radiolaheled liposomes as metabolic and scanning tracers in mice II. In-l 11 oxine compared with Tc-99m DPTA, entrapped in multilamellar lipid vesicles. J. Nucl. Med. 20, 434. Fell H. B. and Dingle J. T. (1963) Studies on the mode of action of excess vitamin A G. Lysosomal protease and the degradation of cartilage matrix. Biochem. J. 87, 403. Kellaway I. W. and Chawla R. S. (1982) Factors influencing the clearance rates of colloidal particles from the rabbit knee joint. In Radionuclide Imaging in Drug Research (Eds Wilson C. G. and Hardy J. G.). p. 261. Croom Helm, London. McCarty D. J. (1979) Synovial fluid. In Arthritis and Allied Conditions (Ed. McCarty D. J.) p. 51. Lea and Febiger, Philadelphia. Noble J., Jones A. G., Davis M. A., Sledge C. B., Kramer R. I. and Livni E. (1983) Leakage of radioactive particle systems from a synovial joint studied with a gamma camera; its application to radiation synovectomy. J. Bone Joint Surg. 65A, 381. Poste G., Kirsh R. and Koestler T. (1984) The challenge of liposome targeting in vivo. In Liposome Technology (Ed. Gregoriadis G.) Vol. III, p. 1. CRC Press, Boca Raton,

Florida. Ruddy S. (1974) Synovial fluid: mirror of the inflammatory lesion in rheumatoid arthritis. In Rheumatoid Arthritis (Ed. Harris E. D.) p. 58. Medicom Press, New York. Senior J. and Gregoriadis G. (1984) Methodology in assessing liposomal stability in the presence of blood, clearance from the circulation of injected animals, and uptake by tissues. In Liposome Technology (Ed. Gregoriadis G.) Vol. III. D. 263. CRC Press. Boca Raton. Florida. Shaw I. H., Knight C. G.’ and Dingle’ J. T. (1976) Liposomal retention of a modified anti-inflammatory steroid Biochem. J. 158, 473. Shaw I. H., Knight C. G., Page Thomas D. P., Phillips N. C. and Dingle J. T. (1979) Liposome-incorporated corticosteroids: I. The interaction of liposomal cortisol palmitate with inflammatory synovial membrane. Br. J. Exp. Path. 60, 142. Shaw I. H. and Dingle J. T. (1980) Liposomes as steroid carriers in the intra-articular therapy of rheumatoid arthritis. In Liposomes in Biological Systems (Eds Greaoriadis G. and Allison A. C.) p. 299. John Wiley, Chiihester. Sledge C. B., Zuckerman J. D., Zalutsky M. R., Atcher R. W.. Shortkroff S., Lionberger D. R., Rose H. A., Hurson B. J., Lankenner P. A., Anderson R. J. and Bloomer W. A. (1986) Treatment of rheumatoid synovitis with intraarticular injection of dysprosium- 165 ferric hydroxide macroaggregates. Arth. Rheum. 29, 153. Topp J. R., Cross E. G. and Fam A. G. (1975) Treatment of persistent knee effusions with intra-articular radioactive gold. Can. Med. Assoc. J. 112, 1085. Weissmann G. (1972) Lysosomal mechanisms of tissue injury in arthritis. New Engl. J. Med. 286, 141. Zalutsky M. R., Ventatesan P. P., English R. J., Shortkroff S., Sledge C. B. and Adelstein S. J. (1986a) Radiation synovectomy with ‘6sDy-FHMA: Lymph node uptake and radiation dosimetry calculations. Inr. J. Nucl. Med. Biol. 12, 457. Zalutsky M. R., Noska M. A. and Gallagher P. W. (1986b) Properties of multilamellar liposomes containing %TcO,: Effect of distearoylphosphatidylcholine to sphingomyelin ratio. Nucl. Med. Biol. 13, 269.