290
BIOCHEMICAL SOCIETY TRANSACTIONS
Properties of (99”rcj Technetium-Labelled Liposomes in Normal and Tumour-Bearing Rats VERNON J. RICHARDSON,* K. JEYASINGH,? R. F. JEWKES,t BRENDA E. RYMAN* and MARTIN H. N. TATTERSALLS
*Department of Biochemistry, Charing Cross Hospital Medical School, ?Department of Nuclear Medicine, and $Department of Medical Oncology, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, U.K. Liposomes (phospholipid vesicles) have been proposed as carriers of materials of therapeutic interest, and work in this area has been reviewed (Tyrrell et al., 1976). The possibility of utilizing such a system in tumour therapy has received some attention, and Gregoriadis et al. (1974) injected patients with liposomes using entrapped l 3 I I labelled albumin as a marker. Tissue distribution was measured post mortem, several days after administration. As a more versatile alternative we have been investigating ~ to study liposome distribution in the use of the yemitter 9 9 m T(technetium) experimental animals. This isotope has been found ideal for radioisotope imaging. Technetium, as the pertechnetate ion (99mT~04-), has previously been used as a vesicle marker to follow the short-term fate of liposomes in mice (McDougall et al., 1975), erythrocyte ‘ghosts’ in rats (Tyrrell & Ryman, 1976) and to follow liposome uptake by cells in tissue culture (Dunnick et al., 1976). We have prepared liposomes from combinations of the following purified lipids; phosphatidylcholine (egg lecithin), cholesterol, dicetyl phosphate and stearylamine. Rotary-evaporated lipid films were shaken with sterile 0.9 % NaCl to give a final suspension of 20% (w/v). These were surrounded by an ice bath and sonicated with a titanium , 30 scoolingbetween. probe( 1.Ocm diameter)for20 bursts (6,um peak-to-peak) of ~ O Swith Technetium label was attached to these pre-formed liposomes by an SnCI, method. To the liposomes (600mg in 3.0ml) was added 0.5ml of a sterile neutral solution of 3m-SnClz previously prepared in 02-free water and stored under Nz at 4°C. The mixture was shaken well and not more than 2.5ml of sodium pertechnetate solution in 0.9% NaCl was added to the suspension, drop by drop, with vigorous shaking, and left at room temperature for 15-30min before injection. The presence of free pertechnetate was estimated by dialysis and found to be less than 2%. The possibility that a labelled SnCI, colloid was formed in the preparation of liposome batches was explored. A preparation with NaCI, SnCI, and pertechnetate formed a labelled colloid under the conditions of the labelling used for the liposomes, and this label could be removed by centrifugation at 4000rev./min for 5min in an MSE Super Minor bench centrifuge. Liposomes labelled under the same conditions remained in the supernatant. The contamination of technetium-labelled liposomes with free pertechnetate and colloid was therefore minimal. Liposomes (20mg of lipid) labelled with 1 mCi of 99mTcwere injected into the tail vein of normal Wistar rats. Tissue distribution and clearance of the label from the blood was studied at various times by killing the animals and counting individual organs for radioactivity. Immediately before killing, a picture of the distribution of radioactivity in the whole animal was obtained by a y-camera-computer system (scintiscan). This provided a photographic image and a quantitative radioactivity distribution map for each rat. For the tumour studies, Wistar rats were injected in the right inguinal region with 4 x lo6 viable Walker 256 carcinoma cells obtained from an ascites-tumour cell line carried in Wistar rats. After 6 days liposomes (80mg of lipid), labelled with 1 mCi of 99mTc.were injected into the tail vein of each rat. Tissue distribution and scintiscans were obtained as in the normal rat studies. Table 1 shows the distribution of technetium-labelled liposomes 29 h after intravenous injection into normal Wistar rats. Blood clearance of labelled liposomes was much slower than either SnCI, colloid or free pertechnetate. Negatively charged liposomes, prepared phosphate (7 :2 :1 molar ratio), had the from phosphatidylcholine/cholesterol/dicetyl 1977
566th MEETING, CAMBRIDGE
29 1
Table 1. Percentage of injected dose of 99mTc-labelledliposomes in tissues after 29 h The compositions of the positively charged, neutral and negatively charged liposomes are given in the text.
-~
Percentage of injected dose Neutral
Positive
Negative
r
(Per (Perg Tissue organ) of tissue) 0.26 Blood Liver 24.24 5.33 Spleen 1.08 2.23 Stomach and 6.83 0.54 intestines* Kidneys 9.00 8.89 Lungs 0.37 0.29 Heart 0.04 0.08 Total dose 46.15 recovered/rat * Weight included contents.
(Per organ)
24.18 0.79 7.91
(Per 8 of tissue) 0.28 2.99 1.63 0.70
11.88 2.00 4.76
(perg of tissue) 0.51 1.62 3.96 0.40
9.24 0.45 0.04
7.27 0.29 0.07
11.06 0.49 0.09
8.72 0.39 0.16
(Per organ) -
52.28
-
41.92
slowest blood clearance, followed by positively charged liposomes containing phosphatidylcholine/cholesterol/stearylamine (7 :2 :1 molar ratio). The neutral liposomes, prepared from phosphatidyIcholine/cholesterol (8 :2, molar ratio), were cleared most rapidly. Both liver and spleen took up the label, and in the liver the affinity for the label was greatest for positively charged liposomes and least for the negatively charged liposomes. The spleen showed greatest affinity for negatively charged liposomes and least affinity for the neutral liposomes. The distribution of 99mTc-labelledliposomes in tumour-bearing rats was similar to that in normal rats. However, variation of the charge on the liposomes resulted in a different uptake of the label by the tumours, the greatest uptake being with negatively charged liposomes where values of approx. 1.3 %/g of tissue were obtained. Positively charged liposomes and neutral liposomes had values between 0.30 and 0.66 %/g of tissue. For negatively charged liposomes extensive sonication before labelling increased the uptake of the label by the tumour 7-fold. Tumour detection, by using the y-camera-computer system, was possible after 12h with positive and negative liposomes, but not with neutral liposomes. Our method of preparing 99mTc-labelledliposomes has allowed us to test the feasibility of using these vesicles to deliver anti-tumour drugs selectively. Evidence has been obtained which suggests that the 99mTc-labellednegatively charged liposomes were localized in the Walker tumour after intravenous injection. Positively charged liposomes and neutral liposomes appeared to be localized to a much lesser extent in the tumour tissue. We thank the Cancer Research Campaign for financial support of this work. Also, we thank Dr. T. A. Connors for helpful discussion and for providing the Walker tumour. Dunnick, J. K., Rooke, J. D., Aragon, S. & Kriss, J. P. (1976) Cancer Res. 36,2385-2389 Gregoriadis, G., Swain, C. P., Wills, E. J. & Tavill, A. S. (1974) Lancet i, 1313-1316 McDougall, R.,Dunnick, J. K., Goris, M. L. & Kriss, J. P. (1975)J. Nucl. Med. 16,488-491 Tyrrell, D. A. & Ryman, B. E. (1976) Biochem. SOC.Trans. 4, 677-680 Tyrrell, D. A., Heath, T. D., Ryman, B. E. & Colley, C. M. (1976) Biochim. Biophys. Actu Rev. 457,259-302
VOl. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
Properties of (99”rcj Technetium-Labelled Liposomes in Normal and Tumour-Bearing Rats VERNON J. RICHARDSON,* K. JEYASINGH,? R. F. JEWKES,t BRENDA E. RYMAN* and MARTIN H. N. TATTERSALLS
*Department of Biochemistry, Charing Cross Hospital Medical School, ?Department of Nuclear Medicine, and $Department of Medical Oncology, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, U.K. Liposomes (phospholipid vesicles) have been proposed as carriers of materials of therapeutic interest, and work in this area has been reviewed (Tyrrell et al., 1976). The possibility of utilizing such a system in tumour therapy has received some attention, and Gregoriadis et al. (1974) injected patients with liposomes using entrapped l 3 I I labelled albumin as a marker. Tissue distribution was measured post mortem, several days after administration. As a more versatile alternative we have been investigating ~ to study liposome distribution in the use of the yemitter 9 9 m T(technetium) experimental animals. This isotope has been found ideal for radioisotope imaging. Technetium, as the pertechnetate ion (99mT~04-), has previously been used as a vesicle marker to follow the short-term fate of liposomes in mice (McDougall et al., 1975), erythrocyte ‘ghosts’ in rats (Tyrrell & Ryman, 1976) and to follow liposome uptake by cells in tissue culture (Dunnick et al., 1976). We have prepared liposomes from combinations of the following purified lipids; phosphatidylcholine (egg lecithin), cholesterol, dicetyl phosphate and stearylamine. Rotary-evaporated lipid films were shaken with sterile 0.9 % NaCl to give a final suspension of 20% (w/v). These were surrounded by an ice bath and sonicated with a titanium , 30 scoolingbetween. probe( 1.Ocm diameter)for20 bursts (6,um peak-to-peak) of ~ O Swith Technetium label was attached to these pre-formed liposomes by an SnCI, method. To the liposomes (600mg in 3.0ml) was added 0.5ml of a sterile neutral solution of 3m-SnClz previously prepared in 02-free water and stored under Nz at 4°C. The mixture was shaken well and not more than 2.5ml of sodium pertechnetate solution in 0.9% NaCl was added to the suspension, drop by drop, with vigorous shaking, and left at room temperature for 15-30min before injection. The presence of free pertechnetate was estimated by dialysis and found to be less than 2%. The possibility that a labelled SnCI, colloid was formed in the preparation of liposome batches was explored. A preparation with NaCI, SnCI, and pertechnetate formed a labelled colloid under the conditions of the labelling used for the liposomes, and this label could be removed by centrifugation at 4000rev./min for 5min in an MSE Super Minor bench centrifuge. Liposomes labelled under the same conditions remained in the supernatant. The contamination of technetium-labelled liposomes with free pertechnetate and colloid was therefore minimal. Liposomes (20mg of lipid) labelled with 1 mCi of 99mTcwere injected into the tail vein of normal Wistar rats. Tissue distribution and clearance of the label from the blood was studied at various times by killing the animals and counting individual organs for radioactivity. Immediately before killing, a picture of the distribution of radioactivity in the whole animal was obtained by a y-camera-computer system (scintiscan). This provided a photographic image and a quantitative radioactivity distribution map for each rat. For the tumour studies, Wistar rats were injected in the right inguinal region with 4 x lo6 viable Walker 256 carcinoma cells obtained from an ascites-tumour cell line carried in Wistar rats. After 6 days liposomes (80mg of lipid), labelled with 1 mCi of 99mTc.were injected into the tail vein of each rat. Tissue distribution and scintiscans were obtained as in the normal rat studies. Table 1 shows the distribution of technetium-labelled liposomes 29 h after intravenous injection into normal Wistar rats. Blood clearance of labelled liposomes was much slower than either SnCI, colloid or free pertechnetate. Negatively charged liposomes, prepared phosphate (7 :2 :1 molar ratio), had the from phosphatidylcholine/cholesterol/dicetyl 1977
566th MEETING, CAMBRIDGE
29 1
Table 1. Percentage of injected dose of 99mTc-labelledliposomes in tissues after 29 h The compositions of the positively charged, neutral and negatively charged liposomes are given in the text.
-~
Percentage of injected dose Neutral
Positive
Negative
r
(Per (Perg Tissue organ) of tissue) 0.26 Blood Liver 24.24 5.33 Spleen 1.08 2.23 Stomach and 6.83 0.54 intestines* Kidneys 9.00 8.89 Lungs 0.37 0.29 Heart 0.04 0.08 Total dose 46.15 recovered/rat * Weight included contents.
(Per organ)
24.18 0.79 7.91
(Per 8 of tissue) 0.28 2.99 1.63 0.70
11.88 2.00 4.76
(perg of tissue) 0.51 1.62 3.96 0.40
9.24 0.45 0.04
7.27 0.29 0.07
11.06 0.49 0.09
8.72 0.39 0.16
(Per organ) -
52.28
-
41.92
slowest blood clearance, followed by positively charged liposomes containing phosphatidylcholine/cholesterol/stearylamine (7 :2 :1 molar ratio). The neutral liposomes, prepared from phosphatidyIcholine/cholesterol (8 :2, molar ratio), were cleared most rapidly. Both liver and spleen took up the label, and in the liver the affinity for the label was greatest for positively charged liposomes and least for the negatively charged liposomes. The spleen showed greatest affinity for negatively charged liposomes and least affinity for the neutral liposomes. The distribution of 99mTc-labelledliposomes in tumour-bearing rats was similar to that in normal rats. However, variation of the charge on the liposomes resulted in a different uptake of the label by the tumours, the greatest uptake being with negatively charged liposomes where values of approx. 1.3 %/g of tissue were obtained. Positively charged liposomes and neutral liposomes had values between 0.30 and 0.66 %/g of tissue. For negatively charged liposomes extensive sonication before labelling increased the uptake of the label by the tumour 7-fold. Tumour detection, by using the y-camera-computer system, was possible after 12h with positive and negative liposomes, but not with neutral liposomes. Our method of preparing 99mTc-labelledliposomes has allowed us to test the feasibility of using these vesicles to deliver anti-tumour drugs selectively. Evidence has been obtained which suggests that the 99mTc-labellednegatively charged liposomes were localized in the Walker tumour after intravenous injection. Positively charged liposomes and neutral liposomes appeared to be localized to a much lesser extent in the tumour tissue. We thank the Cancer Research Campaign for financial support of this work. Also, we thank Dr. T. A. Connors for helpful discussion and for providing the Walker tumour. Dunnick, J. K., Rooke, J. D., Aragon, S. & Kriss, J. P. (1976) Cancer Res. 36,2385-2389 Gregoriadis, G., Swain, C. P., Wills, E. J. & Tavill, A. S. (1974) Lancet i, 1313-1316 McDougall, R.,Dunnick, J. K., Goris, M. L. & Kriss, J. P. (1975)J. Nucl. Med. 16,488-491 Tyrrell, D. A. & Ryman, B. E. (1976) Biochem. SOC.Trans. 4, 677-680 Tyrrell, D. A., Heath, T. D., Ryman, B. E. & Colley, C. M. (1976) Biochim. Biophys. Actu Rev. 457,259-302
VOl. 5
