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Efficient clodronate entrapment within multilamellar and unilamellar liposomes
JPM Vol 27. No. 3 May 1992:185-189
Efficient Clodronate Entrapment Within Multilamellar and Unilamellar Liposomes William G. L o v e , J e r e m y P. Camilleri, and B r y a n D. Williams
Welsh School of Pharmacy (W.G.L.), and Rheumatology Department, (J.P.C., B.D. W.) University of Wales College of Medicine, Cardiff, Wales, Great Britain
Clodronate (dichloromethylene bisphosphonate) encapsulated within liposomes and administered intravenously eliminates resident macrophages within the liver and spleen. Macrophage depletion in the rat requires 20 mg of the encapsulated drug, and so far this has only been achieved using large multilamellar vesicles (MLV). Recent studies have shown that small unilamellar vesicles (SUV) when injected intravenously accumulate at inflamed joint sites in both animal models of arthritis and patients with rheumatoid arthritis; multilamellar vesicles were not able to do so. If phagocytic cells, such as macrophages, are responsible for SUV sequestration, then SUV containing clodronate may be targeted to the inflamed joint and may eliminate the macrophage population leading to reduction in the state of inflammation. We have adapted an existing technique to radiolabel clodronate with 99mTechnetium to use as a tracer to determine its encapsulation within liposomes, a technique that has advantages over other current methods. We have achieved a high-encapsulation efficiency of the drug within MLV and produced SUV containing sufficient clodronate to deplete macrophages in rats in a small enough volume to administer it intravenously as a single dose.
Keywords: Clodronate; Entrapment; Liposomes; Macrophage
Introduction An existing technique for 99mTechnetium (Tc) radiolabelling methylene diphosphonate was utilized to label clodronate (dichloromethylene bisphosphonate). The 99mTc-clodronate is stable in buffer and plasma, and the technique is simple to execute and 97% efficient in attachment of the isotope to the drug. The radiolabelled drug can then be used as a tracer to determine its encapsulation within liposomes, a technique that has advantages over other methods currently used such as spectroscopy and competitive calcium binding assay. Available techniques have been utilized to achieve a high-encapsulation efficiency up to 18% (w,w) of the drug within multilamellar vesicles. For the production of clodronate entrapped within small, unilamellar vesicles, an extrusion technique proved superior to probe
Address reprint requests to Dr. J.P. Camilleri, Department of Rheumatology, University Hospital of Wales, Heath Park, Cardiff CF4 4XW, Wales, Great Britain. Received November 1991; accepted February 1992. Journal of Pharmacologicaland Toxicological Methods 27, 185-189 (1992) © 1992 Elsevier Science PublishingCo., Inc., 655 Avenue of the Americas, New York, NY 10010
sonication with 7% compared to 2.8% encapsulated drug. Liposomes, injected intravenously, accumulate in the reticuloendothelial system (RES) due to uptake by fixed tissue macrophages (Roerdink et al., 1981), After a single dose, liver uptake accounts for 40%-50% of the dose with 8%-20% going to the spleen, 5% to the kidneys, and 1%-4% to the lungs. The size and lipid composition of the liposomes can be manipulated to alter the organ distribution (Patel et al., 1983). Studies have demonstrated that clodronate (dichloromethylene bisphosphonate) encapsulated within liposomes and administered intravenously eliminates resident macrophages within the liver and spleen (Van Rooijen and Van Nieuwmegen, 1984; Van Rooijen et al., 1990). Macrophage depletion in the rat is rapid, achieved between 24 and 48 hr following a single liposome-clodronate dose containing 20 mg of the drug. There is no increase in mortality in the macrophage-depleted rats, implying that the defensive function of reticuloendothelial system is not significantly depressed. The macrophages return to pretreatment levels by 4-6
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JPM Vol 27. No. 3 May 1992:185-189
weeks (Van Rooijen 1989, Van Rooijen et al., 1990). There is some experimental evidence of altered immune function after macrophage elimination in the liver and spleen. Supression of antibody responses after immunization with thymus-independent Type 2 antigen (TNP-Ficoll) has been demonstrated (Claassen et al., 1986a). Reduced uptake of radiolabelled liposomes by liver and spleen after macrophage depletion has also been demonstrated (Love et al., 1991). The study of the functional and biochemical mechanisms of macrophages is important in the understanding of inflammatory disease, and therefore, the macrophage depleting ability of liposome-associated clodronate is an important tool for investigators. Recent studies have shown that small unilamellar vesicles (SUV) when injected intravenously accumulate at inflamed joint sites in both animal models of arthritis (Love et al., 1989, 1990), and patients with rheumatoid arthritis (Williams et al., 1987 and O'Sullivan et al., 1988); multilamellar vesicles (MLV) were not able to do so. Macrophages are known to be intimately involved in the inflammatory response and are found in large numbers in a state of activation within inflamed joints (Zvaifler, 1973; Johnson et al., 1986). If phagocytic cells, such as macrophages, are responsible for SUV sequestration, then SUV-containing clodronate may be targeted to the inflamed joint and eliminate the macrophage population leading to a reduction in the state of inflammation. Due to the reduced encapsulation ability of SUV compared to MLV, techniques have been used to improve upon previous studies involving clodronate encapsulation (Claassen and Van Rooijen, 1986b). The effect of liposome size, (MLV and SUV), and techniques for liposome formulation on the efficiency of clodronate encapsulation have been studied. We also present investigations on the use of a simple, efficient, and stable radiolabelling method for attachment of 99mTechnetium to clodronate to allow measurement of the compound.
100 p~l of saline was added to 0.5 mL of a 10 mg/ml Clodronate solution in normal saline. Simultaneously, 40 ~1 of a 10 mg/ml solution of SnClz in deoxygenated water was added to the mixture. This was allowed to stand for 30 min at room temperature and then used. 99mTc-tin colloid (a possible contaminant of the reaction mixture) was formed as a control by the addition of 99mTC to an aliquot of the SnClz solution.
Sephadex G-25 Chromatographic Analysis of 99mTc-Clodronate A Sephadex G-25, PD-10, column (Pharmacia), fractionation range 1-5 kDa was used to determine 99mTC attachment to the clodronate. The column was run in saline at a flow rate of 2 ml/min, and 1-ml fractions were collected. Column void and bed volumes were determined by the passage of 125I-bovine serum albumin and free 99mTC,respectively, through the gel. Sample volumes were between 10 and 100 ~1.
Sephadex G-IO0 Chromatographic Analysis of 99mTc-Clodronate Encapsulated within Small Unilamellar Vesicles A Sephadex G-100 column, fractionation range 4-150 kDa was used to determine the encapsulation of 99mTc-clodronate within SUV. The column was eluted with saline at a flow rate of 200 ~l/min, and 0.5-ml fractions were collected. Column void and bed volumes wee determined by elution of ~zsI-IgM and free 99mTC,respectively. Unlabelled clodronate (300 mg/ml) was also run on the column, and the absorbance of the fractions were collected read at 210 nm.
TSK G3000SW Ultrogel Chromatographic Analysis of Molecular Size of Clodronate
Egg phosphatidylcholine (EPC) was obtained from Lipid products U.K., and cholesterol and stannous chloride from Sigma U.K. 99mTechnetium pertechnetate (99mTc) was obtained from a 99mTc generator, Amersham U.K. Dichloromethylene bisphosphonate (clodronate) was a kind gift from Dr. N. Van Rooijen, Vrije university, The Netherlands. All other chemicals were obtained from BDH U.K.
A TSK G3000SW high-performance liquid chromatography column (8 x 300 mm) was used to determine the molecular size range of 99mTc-labelled/unlabelled clodronate. The fractionation range of the column is 1-300 kDa and was run at a flow rate of 1 ml/min in saline with 0.5-ml fractions collected and sample volumes of 20-100 p.1 used. Unlabelled clodronate was applied to the column, and the fractions were read using a flow through variable ultraviolet (UV) detector set at 210 nm. For radiolabelled clodronate, the counts in each fraction were determined using an LKB Compugamma counter. Free 99mTC and dextran 10 kDa were also run on the column as a molecular markers.
Clodronate Radiolabelling Technique
Liposome Entrapment of 99mTc-Clodronate
The method of Subramanian et al., 1975 was used to 99mTc label the clodronate. 1-10 M Bq of 99mTc in
Typically, 100 mg of EPC and 20 mg of cholesterol were dissolved in 5 ml of methanol/chloroform (1:1)
Methods and Materials
LOVE ET AL. CLODRONATE ENTRAPMENT WITHIN LIPOSOMES
and dried to a thin, lipid film in a round-bottom flask using a rotary evaporator at 50°C. To the lipid, 1.2 ml of a 300-mg/ml clodronate solution in saline (containing at tracer of the 99mTc-labelled clodronate) was added and hand shaken until all the lipid was removed from the flask wall and it formed the multilamellar vesicles (MLV). The MLV were left at room temperature for 30 min and then washed in 10 ml of saline by centrifugation at 17000 g for 15 min, the pellet was washed twice to ensure removal of all unencapsulated clodronate. The amount of radioactivity within the MLV pellet and supernatants from the three washes were counted, and the percentage clodronate encapsulation was calculated. Controls were also set up where empty MLV were incubated with free 99rnTc-clodronate, and free 99mTc-clodronate itself was washed and centrifuged. For the freeze/thaw technique (Mayer et al., 1985) the MLV were frozen in liquid nitrogen and thawed in a water bath at 50°C, this process was repeated 10 times prior to the washing and centrifugation steps.
Sonication and Extrusion Techniques for SUV Production SUV were formed using two methods: probe sonication and high-pressure extrusion through polycarbonate filters (Mayer et al., 1986). MLV containing radiolabelled clodronate were formed as described above. After standing for 30 min, the volume was made up to 2 ml with saline and transferred to a small glass vessel. For the sonication method, the MLV were exposed to three 5-min bursts of sonication at 6-¢m amplitude using a 9.5-mm titanium probe (Soniprep 150, MSE). The vessel containing the liposomes was immersed in an ice bath throughout the sonication steps. Titanium fragments produced during sonication were removed from the SUV by centrifugation at 5000 g for 5 min. The extrusion technique utilized an Extruder apparatus (Lipex biomembranes, Vancouver Canada); the MLV were passed under high pressure (800 psi) through 0.4 I~m polycarbonate filters 10 times followed by passage through 0.1-1xm filters again 10 times. The diameter of the SUV produced was 101 _+ I nm as determined by photon correlation spectroscopy, (Malvern, U.K.). Once formed by either technique, an aliquot (10-100 i~1) of the SUV was run on the Sephadex G-100 column in order to separate unencapsulated 99mTc-clodronate from that remaining encapsulated within the liposomes. The efficiency of encapsulation using both SUV techniques could then be calculated.
Results The 99mTcradiolabelling of clodronate was successful yielding a 97% labelling efficiency as assessed by
187
50 ¸ 40 _= 30
:
20
/
# 10 # 0 0
2
4
6
8
10
12
14
Elution volume (ml) o- -o 99mTc-Clodronate
=
= Free
99mTc
Figure 1. Elution profiles of 99mTc-labelled clodronate and f r e e 99mTCon a Sephadex G-25 column. V, void volume; B, bed volume.
Sephadex G-25 gel permeation chromatography (Figure 1). 99mTc-Tin colloid could be eliminated as a potential contaminant from the radiolabelled clodronate as 100% of the isotope was recovered from the column when the 99mTc-clodronate was run, whereas when the 99mTc-tin colloid was applied to the column it bound to the filter at the top of the column (due to its large size), and 0% isotope was eluted. An additional step for the removal of free 99m-Tc from the radiolabelled clodronate was deemed unnecessary as only 3% was present; even after incubation at 37°C for 15 hr in saline and normal human plasma there was still only 3.5% free isotope detected by Sephadex G-25 chromatography. Clodronate is ionized in solution and has high affinity for bivalent cations and can form complexes. Chromatographic analysis of the clodronate complex using the TSK G3000SW column shows a molecular size range of (1-10 kDa) (Figure 2). The absorbance profile of unlabelled clodronate was similar to that produced when the radioactivity counts were plotted from the 99mTc-clodronate run suggesting that the radiolabelling technique did not effect the size of the clodronate complex. The efficiency of clodronate encapsulation within MLV showed that up to 20% was entrapped as assessed by pelleting of the MLV after centrifugation. Incubation of radiolabelled clodronate with preformed empty MLV demonstrated that only 3% of the drug was liposome associated presumably due to association with the surface of the MLV, also the control of free radiolabelled clodronate showed that the centrifugation did not cause any pelleting of the free drug. The freeze/thawing technique significantly increased the entrapment of clodronate within MLV, from 12% to 18%, p < 0.002 Student's t test.
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JPM Vol 27. No. 3 May 1992:185-189
IOKDa marker
1KDa Marker
1
40~
Discussion
I Q 'b
30"
'i
~ 20. c
2
~. 10-
1
" - 0--,'~
0 10
o
15
I0 20
Elution vol {ml) ¢
:
~ O
o---o
% Counts
Figure 2. Elution profiles of 99mTc-labelled clodronate (expressed as % counts eluted) and unlabelled clodronate (absorbance at 210 nm) obtained from a TSK G3000SW Ultrogel column, (10-kDa marker is dextran and 1-kDa bed volume cutoff is marked using free 99mTC).
The Sephadex G100 column was used in order to separate SUV-encapsulated clodronate fromunencapsulated drug. The second peak at 9.5 ml of the SUV99mTc-clodronate profile coincides with the profiles of free 99mTc-clodronate and unlabelled clodronate on the the Sephadex G-100 column (Figure 3). SUV containing encapsulated 99mTc-clodronate eluted in the void volume of the Sephadex G-100 column at 3 ml (Figure 3). The two techniques for the production of SUV containing clodronate gave significantly different encapsulation efficiencies. SUV produced by sonication contained 2.8% of the original drug dose, whereas SUV produced by extrusion contained 7%, p < 0.001 Student's t test.
Figure 3. Elution profiles of 99mTc-clodronate, unlabelled clodronate, and SUV-entrapped 99mTc-clodronate obtained from a Sephadex G-100 column. Expressed as % counts eluted and absorbance at 210 nm. V
B
1
1
10-
"0.8
8"
E .0.6
'~ .>,
oJ
6" 0.4 4
ae
0.2
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2
4
6
8
10
12
14
Elution v o l u m e (ml)
o - - - o 99mTc-Clodronate Clodronate n o - l a b e l
-"
-" S U V - 9 9 m T c - C l o d r o n a t e
0 16
The 99mTc radiolabelling method of Subramanian et al., 1975 has been successfully used for the labelling of clodronate. A high efficiency of binding of the 99mTC to the clodronate was achieved (97%), and the stability of the radiolabel after 15 hr in saline and normal human plasma at 37°C showed only a 0.5% loss. Free 99myc was not removed as only 3% was present and this would be removed later during the centrifugation and chromatography steps. Chromatographic analysis of the labelled drug confirmed that no 99mTc-tin colloid was formed and that the drug existed in a complex of varying molecular size (1-10 kDa). The radiolabelling technique did not alter the distribution of complex size formation as the absorbance profile of unlabelled clodronate was similar to that of the radioactivity profile. As the radiolabelled clodronate was stable in buffer, it was then used as a tracer added to unlabelled drug in order to estimate encapsulation efficiency within liposomes. This technique has advantages over those presently used, (UV spectrophotometery and competitive calcium binding assay) in that it is simple to carry out using an isotope with a short half-life, encapsulation and clodronate loss from the liposomes can be studied in plasma, and in vivo tissue distribution studies could be contemplated. By utilizing clodronate at its maximum solubility level (300 mg/ml) in normal saline and keeping the volume added to the dry lipid film to a minimum, the encapsulation efficiency achieved in MLV was 12%, over sixfold greater than that achieved by others (Claassen and Van Rooijen, 1986b). A further enhancement of encapsulation was achieved by freeze/thawing the MLV, (18% encapsulation efficiency, a ninefold increase on previous reports), this technique is reported to increase interlamellar space and distribute aqueous soluble compounds evenly between all the phospholipid bilayers of the liposome (Mayer et al., 1985). MLV, when injected intravenously into adjuvantarthritic rats, do not have the ability to gain access to the inflamed joint tissues, however, up to 7% of an injected dose of SUV was found to accumulate in rats with the inflammatory condition (Love et al., 1989). So, if one contemplates the use of liposomal clodronate to remove inflamed tissue macrophages, then SUV would be required. It was, therefore, important not only to achieve an efficient clodronate entrapment within MLV but also have technique(s) for SUV production in which a minimum drug loss from the liposome occurs during the size reduction process. The two SUV-forming techniques tested both led to a loss of clodronate from the liposomes, however, extrusion proved superior over probe sonication with
LOVE ET AL. CLODRONATEENTRAPMENTWITHIN LIPOSOMES
7% compared to 2.8%, respectively, encapsulated. The extrusion method caused much less disruption to the liposome structure than the high-frequency bursts of ultrasonic energy associated with the sonication technique, and this may explain the differences in loss of the encapsulated clodronate. Efficient clodronate entrapment within SUV is also of importance to avoid the necessity of a huge lipid dose formulation that would be needed to administer the required clodronate dosage if only a poor entrapment were achieved. In conclusion, a simple, efficient, and stable radiolabel for clodronate has been demonstrated, and procedures are described for the efficient trapping of the compound within both MLV and SUV. These procedures should be of help to those with an interest in the macrophage-depleting ability of liposome-encapsulated clodronate. The authors thank the Arthritis and Rheumatism Council for their financial support.
References Claassen E, Kors N, Van Rooijen N, (1986a) Influence of carriers on the development and localisation of anti-2,4,6-trinitrophenyl (TNP) antibody-forming cells in the murine spleen. II. Suppressed antibody response to TNP-ficoll after elimination of marginal zone cells. Eu J Irnmunol 16:492-497. Claassen E, Van Rooijen N, (1986b) Preparation and characteristics of dichloromethylene disphosphonate containing liposomes. J Microencap 3:109-114. Johnson WJ, Dimartino MJ, Hanna N (1986) Macrophage activation in rat models or inflammation and arthritis: Determination of markers of stages of activation. Cell Immunol 103:54-56. Love WG, Amos N, KeUaway IW, Williams BD (1989) Specific accumulation of 99"Technetium radiolabelled, negative liposomes
189
in the inflamed paws of adjuvant-induced arthritic rats: Effect of liposome size. Ann Rheum Dis 48:144-149. Love WG, Amos N, Kellaway IW, Williams BD (1990) Specific accumulation of cholesterol-rich liposomes in the inflammatory tissue of rats with adjuvant arthritis. Ann Rheum Dis 49:611-614. Love WG, Camilleri JP, Williams BD (1991) Macrophage depletion using clodronate encapsulated within small unilamellar liposomes. Arth Rheum 34:Suppl 9:166. Mayer LD, Hope MJ, Cullis PR, Janoff AS (1985) Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles. Biochirn Biophys Acta 817:193-196. Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161-168. O'Sullivan MM, Powell N, French AP, Morgan JR, Williams BD (1988) Inflammatory joint disease: A comparison of liposome scanning, bone scanning, and radiography. Ann Rheum Dis 47:485-49t. Patel HM, Nilden ST, Ryman BE (1983) Inhibitory effect of cholesterol on uptake of liposomes by liver and spleen. Biochem Biophys Acta 761:142-151. Roerdink F, Dijkstra J, Hartman G, Bolscher B, Scherphof G (1981) The involvement of parenchymal, kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes. Effects of lanthanum and gadolinum salts. Biochem Biophys Acta 667:79-89. Subramanian G, McAfee JG, Blair RJ, Kallfelz FA, Thomas FD (1975) Technetium-99m-methylene diphosphonate: A superior agent for skeletal imaging: Comparison with other technetium compelxes. J Nucl M e d 16:744-755. Van Rooijen N, Van Neuwmegen R (1984) Elimination of phagocytic cells in the spleen after i.v. injection of liposome-encapsulated DMDP. Cell Tissue Res 238:355-388. Van Rooijen N (1989) The liposome mediated macrophage suicide technique. J Immunol Methods 124:1-6. Van Rooijen N, Kors N, Ende Mvd, Dijkstra CD (1990) Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated DMDP. Cell Tissue Res 260:215-222. Williams BD, O'Sullivan MM, Saggu GS, Williams KE, Williams LA, Morgan JR (1987) Synovial accumulation of 99mTechnetium labelled liposomes in Rheumatoid Arthritis. Ann Rheum Dis 46:314-318. Zvaifler NJ (1973) The immunopathology of joint inflammation in rheumaotid arthritis. Adv Immunol 16:265-336.
Efficient Clodronate Entrapment Within Multilamellar and Unilamellar Liposomes William G. L o v e , J e r e m y P. Camilleri, and B r y a n D. Williams
Welsh School of Pharmacy (W.G.L.), and Rheumatology Department, (J.P.C., B.D. W.) University of Wales College of Medicine, Cardiff, Wales, Great Britain
Clodronate (dichloromethylene bisphosphonate) encapsulated within liposomes and administered intravenously eliminates resident macrophages within the liver and spleen. Macrophage depletion in the rat requires 20 mg of the encapsulated drug, and so far this has only been achieved using large multilamellar vesicles (MLV). Recent studies have shown that small unilamellar vesicles (SUV) when injected intravenously accumulate at inflamed joint sites in both animal models of arthritis and patients with rheumatoid arthritis; multilamellar vesicles were not able to do so. If phagocytic cells, such as macrophages, are responsible for SUV sequestration, then SUV containing clodronate may be targeted to the inflamed joint and may eliminate the macrophage population leading to reduction in the state of inflammation. We have adapted an existing technique to radiolabel clodronate with 99mTechnetium to use as a tracer to determine its encapsulation within liposomes, a technique that has advantages over other current methods. We have achieved a high-encapsulation efficiency of the drug within MLV and produced SUV containing sufficient clodronate to deplete macrophages in rats in a small enough volume to administer it intravenously as a single dose.
Keywords: Clodronate; Entrapment; Liposomes; Macrophage
Introduction An existing technique for 99mTechnetium (Tc) radiolabelling methylene diphosphonate was utilized to label clodronate (dichloromethylene bisphosphonate). The 99mTc-clodronate is stable in buffer and plasma, and the technique is simple to execute and 97% efficient in attachment of the isotope to the drug. The radiolabelled drug can then be used as a tracer to determine its encapsulation within liposomes, a technique that has advantages over other methods currently used such as spectroscopy and competitive calcium binding assay. Available techniques have been utilized to achieve a high-encapsulation efficiency up to 18% (w,w) of the drug within multilamellar vesicles. For the production of clodronate entrapped within small, unilamellar vesicles, an extrusion technique proved superior to probe
Address reprint requests to Dr. J.P. Camilleri, Department of Rheumatology, University Hospital of Wales, Heath Park, Cardiff CF4 4XW, Wales, Great Britain. Received November 1991; accepted February 1992. Journal of Pharmacologicaland Toxicological Methods 27, 185-189 (1992) © 1992 Elsevier Science PublishingCo., Inc., 655 Avenue of the Americas, New York, NY 10010
sonication with 7% compared to 2.8% encapsulated drug. Liposomes, injected intravenously, accumulate in the reticuloendothelial system (RES) due to uptake by fixed tissue macrophages (Roerdink et al., 1981), After a single dose, liver uptake accounts for 40%-50% of the dose with 8%-20% going to the spleen, 5% to the kidneys, and 1%-4% to the lungs. The size and lipid composition of the liposomes can be manipulated to alter the organ distribution (Patel et al., 1983). Studies have demonstrated that clodronate (dichloromethylene bisphosphonate) encapsulated within liposomes and administered intravenously eliminates resident macrophages within the liver and spleen (Van Rooijen and Van Nieuwmegen, 1984; Van Rooijen et al., 1990). Macrophage depletion in the rat is rapid, achieved between 24 and 48 hr following a single liposome-clodronate dose containing 20 mg of the drug. There is no increase in mortality in the macrophage-depleted rats, implying that the defensive function of reticuloendothelial system is not significantly depressed. The macrophages return to pretreatment levels by 4-6
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JPM Vol 27. No. 3 May 1992:185-189
weeks (Van Rooijen 1989, Van Rooijen et al., 1990). There is some experimental evidence of altered immune function after macrophage elimination in the liver and spleen. Supression of antibody responses after immunization with thymus-independent Type 2 antigen (TNP-Ficoll) has been demonstrated (Claassen et al., 1986a). Reduced uptake of radiolabelled liposomes by liver and spleen after macrophage depletion has also been demonstrated (Love et al., 1991). The study of the functional and biochemical mechanisms of macrophages is important in the understanding of inflammatory disease, and therefore, the macrophage depleting ability of liposome-associated clodronate is an important tool for investigators. Recent studies have shown that small unilamellar vesicles (SUV) when injected intravenously accumulate at inflamed joint sites in both animal models of arthritis (Love et al., 1989, 1990), and patients with rheumatoid arthritis (Williams et al., 1987 and O'Sullivan et al., 1988); multilamellar vesicles (MLV) were not able to do so. Macrophages are known to be intimately involved in the inflammatory response and are found in large numbers in a state of activation within inflamed joints (Zvaifler, 1973; Johnson et al., 1986). If phagocytic cells, such as macrophages, are responsible for SUV sequestration, then SUV-containing clodronate may be targeted to the inflamed joint and eliminate the macrophage population leading to a reduction in the state of inflammation. Due to the reduced encapsulation ability of SUV compared to MLV, techniques have been used to improve upon previous studies involving clodronate encapsulation (Claassen and Van Rooijen, 1986b). The effect of liposome size, (MLV and SUV), and techniques for liposome formulation on the efficiency of clodronate encapsulation have been studied. We also present investigations on the use of a simple, efficient, and stable radiolabelling method for attachment of 99mTechnetium to clodronate to allow measurement of the compound.
100 p~l of saline was added to 0.5 mL of a 10 mg/ml Clodronate solution in normal saline. Simultaneously, 40 ~1 of a 10 mg/ml solution of SnClz in deoxygenated water was added to the mixture. This was allowed to stand for 30 min at room temperature and then used. 99mTc-tin colloid (a possible contaminant of the reaction mixture) was formed as a control by the addition of 99mTC to an aliquot of the SnClz solution.
Sephadex G-25 Chromatographic Analysis of 99mTc-Clodronate A Sephadex G-25, PD-10, column (Pharmacia), fractionation range 1-5 kDa was used to determine 99mTC attachment to the clodronate. The column was run in saline at a flow rate of 2 ml/min, and 1-ml fractions were collected. Column void and bed volumes were determined by the passage of 125I-bovine serum albumin and free 99mTC,respectively, through the gel. Sample volumes were between 10 and 100 ~1.
Sephadex G-IO0 Chromatographic Analysis of 99mTc-Clodronate Encapsulated within Small Unilamellar Vesicles A Sephadex G-100 column, fractionation range 4-150 kDa was used to determine the encapsulation of 99mTc-clodronate within SUV. The column was eluted with saline at a flow rate of 200 ~l/min, and 0.5-ml fractions were collected. Column void and bed volumes wee determined by elution of ~zsI-IgM and free 99mTC,respectively. Unlabelled clodronate (300 mg/ml) was also run on the column, and the absorbance of the fractions were collected read at 210 nm.
TSK G3000SW Ultrogel Chromatographic Analysis of Molecular Size of Clodronate
Egg phosphatidylcholine (EPC) was obtained from Lipid products U.K., and cholesterol and stannous chloride from Sigma U.K. 99mTechnetium pertechnetate (99mTc) was obtained from a 99mTc generator, Amersham U.K. Dichloromethylene bisphosphonate (clodronate) was a kind gift from Dr. N. Van Rooijen, Vrije university, The Netherlands. All other chemicals were obtained from BDH U.K.
A TSK G3000SW high-performance liquid chromatography column (8 x 300 mm) was used to determine the molecular size range of 99mTc-labelled/unlabelled clodronate. The fractionation range of the column is 1-300 kDa and was run at a flow rate of 1 ml/min in saline with 0.5-ml fractions collected and sample volumes of 20-100 p.1 used. Unlabelled clodronate was applied to the column, and the fractions were read using a flow through variable ultraviolet (UV) detector set at 210 nm. For radiolabelled clodronate, the counts in each fraction were determined using an LKB Compugamma counter. Free 99mTC and dextran 10 kDa were also run on the column as a molecular markers.
Clodronate Radiolabelling Technique
Liposome Entrapment of 99mTc-Clodronate
The method of Subramanian et al., 1975 was used to 99mTc label the clodronate. 1-10 M Bq of 99mTc in
Typically, 100 mg of EPC and 20 mg of cholesterol were dissolved in 5 ml of methanol/chloroform (1:1)
Methods and Materials
LOVE ET AL. CLODRONATE ENTRAPMENT WITHIN LIPOSOMES
and dried to a thin, lipid film in a round-bottom flask using a rotary evaporator at 50°C. To the lipid, 1.2 ml of a 300-mg/ml clodronate solution in saline (containing at tracer of the 99mTc-labelled clodronate) was added and hand shaken until all the lipid was removed from the flask wall and it formed the multilamellar vesicles (MLV). The MLV were left at room temperature for 30 min and then washed in 10 ml of saline by centrifugation at 17000 g for 15 min, the pellet was washed twice to ensure removal of all unencapsulated clodronate. The amount of radioactivity within the MLV pellet and supernatants from the three washes were counted, and the percentage clodronate encapsulation was calculated. Controls were also set up where empty MLV were incubated with free 99rnTc-clodronate, and free 99mTc-clodronate itself was washed and centrifuged. For the freeze/thaw technique (Mayer et al., 1985) the MLV were frozen in liquid nitrogen and thawed in a water bath at 50°C, this process was repeated 10 times prior to the washing and centrifugation steps.
Sonication and Extrusion Techniques for SUV Production SUV were formed using two methods: probe sonication and high-pressure extrusion through polycarbonate filters (Mayer et al., 1986). MLV containing radiolabelled clodronate were formed as described above. After standing for 30 min, the volume was made up to 2 ml with saline and transferred to a small glass vessel. For the sonication method, the MLV were exposed to three 5-min bursts of sonication at 6-¢m amplitude using a 9.5-mm titanium probe (Soniprep 150, MSE). The vessel containing the liposomes was immersed in an ice bath throughout the sonication steps. Titanium fragments produced during sonication were removed from the SUV by centrifugation at 5000 g for 5 min. The extrusion technique utilized an Extruder apparatus (Lipex biomembranes, Vancouver Canada); the MLV were passed under high pressure (800 psi) through 0.4 I~m polycarbonate filters 10 times followed by passage through 0.1-1xm filters again 10 times. The diameter of the SUV produced was 101 _+ I nm as determined by photon correlation spectroscopy, (Malvern, U.K.). Once formed by either technique, an aliquot (10-100 i~1) of the SUV was run on the Sephadex G-100 column in order to separate unencapsulated 99mTc-clodronate from that remaining encapsulated within the liposomes. The efficiency of encapsulation using both SUV techniques could then be calculated.
Results The 99mTcradiolabelling of clodronate was successful yielding a 97% labelling efficiency as assessed by
187
50 ¸ 40 _= 30
:
20
/
# 10 # 0 0
2
4
6
8
10
12
14
Elution volume (ml) o- -o 99mTc-Clodronate
=
= Free
99mTc
Figure 1. Elution profiles of 99mTc-labelled clodronate and f r e e 99mTCon a Sephadex G-25 column. V, void volume; B, bed volume.
Sephadex G-25 gel permeation chromatography (Figure 1). 99mTc-Tin colloid could be eliminated as a potential contaminant from the radiolabelled clodronate as 100% of the isotope was recovered from the column when the 99mTc-clodronate was run, whereas when the 99mTc-tin colloid was applied to the column it bound to the filter at the top of the column (due to its large size), and 0% isotope was eluted. An additional step for the removal of free 99m-Tc from the radiolabelled clodronate was deemed unnecessary as only 3% was present; even after incubation at 37°C for 15 hr in saline and normal human plasma there was still only 3.5% free isotope detected by Sephadex G-25 chromatography. Clodronate is ionized in solution and has high affinity for bivalent cations and can form complexes. Chromatographic analysis of the clodronate complex using the TSK G3000SW column shows a molecular size range of (1-10 kDa) (Figure 2). The absorbance profile of unlabelled clodronate was similar to that produced when the radioactivity counts were plotted from the 99mTc-clodronate run suggesting that the radiolabelling technique did not effect the size of the clodronate complex. The efficiency of clodronate encapsulation within MLV showed that up to 20% was entrapped as assessed by pelleting of the MLV after centrifugation. Incubation of radiolabelled clodronate with preformed empty MLV demonstrated that only 3% of the drug was liposome associated presumably due to association with the surface of the MLV, also the control of free radiolabelled clodronate showed that the centrifugation did not cause any pelleting of the free drug. The freeze/thawing technique significantly increased the entrapment of clodronate within MLV, from 12% to 18%, p < 0.002 Student's t test.
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JPM Vol 27. No. 3 May 1992:185-189
IOKDa marker
1KDa Marker
1
40~
Discussion
I Q 'b
30"
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~ 20. c
2
~. 10-
1
" - 0--,'~
0 10
o
15
I0 20
Elution vol {ml) ¢
:
~ O
o---o
% Counts
Figure 2. Elution profiles of 99mTc-labelled clodronate (expressed as % counts eluted) and unlabelled clodronate (absorbance at 210 nm) obtained from a TSK G3000SW Ultrogel column, (10-kDa marker is dextran and 1-kDa bed volume cutoff is marked using free 99mTC).
The Sephadex G100 column was used in order to separate SUV-encapsulated clodronate fromunencapsulated drug. The second peak at 9.5 ml of the SUV99mTc-clodronate profile coincides with the profiles of free 99mTc-clodronate and unlabelled clodronate on the the Sephadex G-100 column (Figure 3). SUV containing encapsulated 99mTc-clodronate eluted in the void volume of the Sephadex G-100 column at 3 ml (Figure 3). The two techniques for the production of SUV containing clodronate gave significantly different encapsulation efficiencies. SUV produced by sonication contained 2.8% of the original drug dose, whereas SUV produced by extrusion contained 7%, p < 0.001 Student's t test.
Figure 3. Elution profiles of 99mTc-clodronate, unlabelled clodronate, and SUV-entrapped 99mTc-clodronate obtained from a Sephadex G-100 column. Expressed as % counts eluted and absorbance at 210 nm. V
B
1
1
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ae
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14
Elution v o l u m e (ml)
o - - - o 99mTc-Clodronate Clodronate n o - l a b e l
-"
-" S U V - 9 9 m T c - C l o d r o n a t e
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The 99mTc radiolabelling method of Subramanian et al., 1975 has been successfully used for the labelling of clodronate. A high efficiency of binding of the 99mTC to the clodronate was achieved (97%), and the stability of the radiolabel after 15 hr in saline and normal human plasma at 37°C showed only a 0.5% loss. Free 99myc was not removed as only 3% was present and this would be removed later during the centrifugation and chromatography steps. Chromatographic analysis of the labelled drug confirmed that no 99mTc-tin colloid was formed and that the drug existed in a complex of varying molecular size (1-10 kDa). The radiolabelling technique did not alter the distribution of complex size formation as the absorbance profile of unlabelled clodronate was similar to that of the radioactivity profile. As the radiolabelled clodronate was stable in buffer, it was then used as a tracer added to unlabelled drug in order to estimate encapsulation efficiency within liposomes. This technique has advantages over those presently used, (UV spectrophotometery and competitive calcium binding assay) in that it is simple to carry out using an isotope with a short half-life, encapsulation and clodronate loss from the liposomes can be studied in plasma, and in vivo tissue distribution studies could be contemplated. By utilizing clodronate at its maximum solubility level (300 mg/ml) in normal saline and keeping the volume added to the dry lipid film to a minimum, the encapsulation efficiency achieved in MLV was 12%, over sixfold greater than that achieved by others (Claassen and Van Rooijen, 1986b). A further enhancement of encapsulation was achieved by freeze/thawing the MLV, (18% encapsulation efficiency, a ninefold increase on previous reports), this technique is reported to increase interlamellar space and distribute aqueous soluble compounds evenly between all the phospholipid bilayers of the liposome (Mayer et al., 1985). MLV, when injected intravenously into adjuvantarthritic rats, do not have the ability to gain access to the inflamed joint tissues, however, up to 7% of an injected dose of SUV was found to accumulate in rats with the inflammatory condition (Love et al., 1989). So, if one contemplates the use of liposomal clodronate to remove inflamed tissue macrophages, then SUV would be required. It was, therefore, important not only to achieve an efficient clodronate entrapment within MLV but also have technique(s) for SUV production in which a minimum drug loss from the liposome occurs during the size reduction process. The two SUV-forming techniques tested both led to a loss of clodronate from the liposomes, however, extrusion proved superior over probe sonication with
LOVE ET AL. CLODRONATEENTRAPMENTWITHIN LIPOSOMES
7% compared to 2.8%, respectively, encapsulated. The extrusion method caused much less disruption to the liposome structure than the high-frequency bursts of ultrasonic energy associated with the sonication technique, and this may explain the differences in loss of the encapsulated clodronate. Efficient clodronate entrapment within SUV is also of importance to avoid the necessity of a huge lipid dose formulation that would be needed to administer the required clodronate dosage if only a poor entrapment were achieved. In conclusion, a simple, efficient, and stable radiolabel for clodronate has been demonstrated, and procedures are described for the efficient trapping of the compound within both MLV and SUV. These procedures should be of help to those with an interest in the macrophage-depleting ability of liposome-encapsulated clodronate. The authors thank the Arthritis and Rheumatism Council for their financial support.
References Claassen E, Kors N, Van Rooijen N, (1986a) Influence of carriers on the development and localisation of anti-2,4,6-trinitrophenyl (TNP) antibody-forming cells in the murine spleen. II. Suppressed antibody response to TNP-ficoll after elimination of marginal zone cells. Eu J Irnmunol 16:492-497. Claassen E, Van Rooijen N, (1986b) Preparation and characteristics of dichloromethylene disphosphonate containing liposomes. J Microencap 3:109-114. Johnson WJ, Dimartino MJ, Hanna N (1986) Macrophage activation in rat models or inflammation and arthritis: Determination of markers of stages of activation. Cell Immunol 103:54-56. Love WG, Amos N, KeUaway IW, Williams BD (1989) Specific accumulation of 99"Technetium radiolabelled, negative liposomes
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in the inflamed paws of adjuvant-induced arthritic rats: Effect of liposome size. Ann Rheum Dis 48:144-149. Love WG, Amos N, Kellaway IW, Williams BD (1990) Specific accumulation of cholesterol-rich liposomes in the inflammatory tissue of rats with adjuvant arthritis. Ann Rheum Dis 49:611-614. Love WG, Camilleri JP, Williams BD (1991) Macrophage depletion using clodronate encapsulated within small unilamellar liposomes. Arth Rheum 34:Suppl 9:166. Mayer LD, Hope MJ, Cullis PR, Janoff AS (1985) Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles. Biochirn Biophys Acta 817:193-196. Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161-168. O'Sullivan MM, Powell N, French AP, Morgan JR, Williams BD (1988) Inflammatory joint disease: A comparison of liposome scanning, bone scanning, and radiography. Ann Rheum Dis 47:485-49t. Patel HM, Nilden ST, Ryman BE (1983) Inhibitory effect of cholesterol on uptake of liposomes by liver and spleen. Biochem Biophys Acta 761:142-151. Roerdink F, Dijkstra J, Hartman G, Bolscher B, Scherphof G (1981) The involvement of parenchymal, kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes. Effects of lanthanum and gadolinum salts. Biochem Biophys Acta 667:79-89. Subramanian G, McAfee JG, Blair RJ, Kallfelz FA, Thomas FD (1975) Technetium-99m-methylene diphosphonate: A superior agent for skeletal imaging: Comparison with other technetium compelxes. J Nucl M e d 16:744-755. Van Rooijen N, Van Neuwmegen R (1984) Elimination of phagocytic cells in the spleen after i.v. injection of liposome-encapsulated DMDP. Cell Tissue Res 238:355-388. Van Rooijen N (1989) The liposome mediated macrophage suicide technique. J Immunol Methods 124:1-6. Van Rooijen N, Kors N, Ende Mvd, Dijkstra CD (1990) Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated DMDP. Cell Tissue Res 260:215-222. Williams BD, O'Sullivan MM, Saggu GS, Williams KE, Williams LA, Morgan JR (1987) Synovial accumulation of 99mTechnetium labelled liposomes in Rheumatoid Arthritis. Ann Rheum Dis 46:314-318. Zvaifler NJ (1973) The immunopathology of joint inflammation in rheumaotid arthritis. Adv Immunol 16:265-336.