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Gd-Labeled Liposomes for Monitoring Liposome-Encapsulated Chemotherapy
Gd-Labeled Liposomes for Monitoring Liposome-encapsulated Chemotherapy: Quantification of Regional Uptake in Tumor and Effect on Drug Delivery1 Erika Rubesova, MD, Ferco Berger, MS, Michael F. Wendland, PhD, Keelung Hong, PhD Kathryn J. Stevens, MD, Charles A. Gooding, MD, Philipp Lang, MD
RATIONALE AND OBJECTIVES Recently developed sterically stabilized liposomes are used clinically for delivery of anticancer chemotherapy (currently the drug doxorubicin, Doxil®). In investigational drug development they are studied as targeted (by surface attached antibody or receptor ligands) and nonspecific drug delivery strategies for a variety of pathologies (cancer, arthritis, AIDS). Liposomes have also been considered as potential MRI contrast media vehicles, primarily for blood pool enhancement (1) and for contrast enhancement in the diagnosis and characterization of tumors (2–7). However, little work has been aimed at using liposomal MRI contrast material for purposes of image-based tracking of drug delivery of liposome-drug formulations. Intravenously administered liposomes are distributed passively by flowing blood and extravasate, usually slowly, in territories where vascular endothelium is either (a) very leaky due to large intracellular gaps such as in inflammatory zones, tumors and healing wounds; or (b) fenestrated such as liver or spleen. Selective concentration of liposomes in tumors is slow-typically requiring day(s) for maximal accumulation—which imposes certain limitations on strategies for external image-based detection, as well as upon the liposome itself. Non-sterically stabilized liposomes are cleared from the blood within a few hours Acad Radiol 2002; 9(suppl 2):S525–S527 1From the Department of Radiology, Stanford University School of Medicine, Stanford, Calif (E.R., F.B., K.J.S., P.L.); Department of Radiology, University of California, San Francisco (M.F.W., C.A.G.); and Liposome Research Lab, California Pacific Medical Center, San Francisco, Calif (K.H.). Address correspondence to E.R., Department of Radiology, CHU SaintPierre, Rue Haute 322, 1000 Brussels, Belgium.
©
AUR, 2002
via the reticuloendothelial system and accumulation in tumor is obviated kinetically. Sterically stabilized liposomes are successful for antitumor application because they remain in the circulation much longer, with plasma half-lives on the order of days, and substantial accumulation in tumor is possible. Similarly for image-based external detection strategies, the use of PET or SPECT tracers bound to liposomes would fail to the short radioactive half-lives of acceptable radionuclides. Consequently, only liposomal-bound CT or MRI contrast media would be viable candidates for this purpose. In the current study, sterically stabilized liposomes labeled with encapsulated GdDTPA-BMA were examined as probes for quantitative tracking of liposomal drug delivery in implanted osteogenic sarcoma model. The purpose of the study was to evaluate dose-response in the context of intravenously administered dose versus the response of ⌬R1 accumulation in the tumor. MATERIALS AND METHODS GdDTPA-BMA-liposomes (80 nm dia) were prepared from egg phosphatidyl choline (EPC), cholesterol, and polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) (ratio 3:2:0.3) and 0.25 M GdDTPA-BMA by the reverse phase evaporation method followed by extrusion (8). Inclusion of PEG-PE provided steric stabilization. Each batch was characterized by assay of phosphate content and MR determination of Gd content (spectroscopic inversion recovery measurement of ⌬R1 of intact and detergent solubilized liposomes, with and without GdDTPABMA). Encapsulated volume was calculated using assumption that all Gd was encapsulated at 0.25 M concentration.
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RUBESOVA ET AL
Thirty nude athymic rats (NCI, 80 –110 gm, 5-weeks old) were implanted with osteogenic sarcoma (cell line UMR 106 E IV) by subcutaneously injecting 3–5 million cells while under ketamine-xylazine anesthesia. When palpable tumors were approximately 0.6 – 0.8 cm diameter (6 to 10 days later) animals were imaged. MRI was conducted at 2 Tesla using a Bruker Omega system (Bruker Instruments, Inc., Fremont, CA). Animals were anesthetized by exposing to isoflurane (5% to induce and 1.7–2% to maintain) mixed into 100% O2. Animals were placed supine on a holder covered by a heated water pad (38 ⫾ 1°C), inserted into a 5 cm birdcage rf coil and placed in the magnet. A tail vein was catheterized for administration of liposomes. GdDTPA-BMA liposome was infused (5–10 min) at Gd doses of 0.025, 0.05, 0.1 and 0.2 mmol/kg body weight (n ⫽ 6). MRI was conducted before and 24 and 48 hr after liposome administration. Contrast enhancement was measured from standard multislice T1 weighted spin echo (TR/TE ⫽ 500/13 ms) images. Tumor size and morphology were determined from magnetization transfer (3D spoiled GRE, TR/TE ⫽ 100/3 ms, flip ⫽ 30 deg) and/or diffusion weighted images (TR/TE ⫽ 2500/44 ms with diffusion gradients set to 1, 5, 8 and 10 G/cm, b ⫽ 14, 366, 938, and 1465 s/mm2). Regional T1 values were measured before and after contrast administration by either IR EPI (TR/TE ⫽ 10 s/10 ms, single slice) or IR snapshot FLASH (TR/ TE ⫽ 3/1.5 ms, flip ⫽ 10 deg, centric phase ordering, 6 –10 slices) with TI’s ranging from 50 –3000 ms. The remaining six rats were administered GdDTPABMA (0.1mmol/kg bolus, 0.004 mmol/kg/hr infusion) and imaged as described above. Regional T1 values were measured using IR snapshot flash MRI after 30 min of infusion. Blood samples were taken before and during infusion and plasma R1 was measured by spectroscopy. Distribution volume of GdDTPA-BMA was calculated from the ratio of ⌬R1tumor/⌬R1plasma.
RESULTS AND CONCLUSIONS Maximum contrast enhancement was observed at 24 hours after injection of Gd-liposomes. The ⌬R1 values of central tumor regions measured at 24 hours increased with dose until 0.1 mmol/kg. Higher dose of Gd-liposomes produced no further increase in ⌬R1. Significantly greater ⌬R1 was measured in the tumor periphery for all doses of Gd-liposomes, and the peripheral region showed no plateau at the higher doses (Table).
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Academic Radiology, Vol 9, Suppl 2, 2002
⌬R1 value (ⴞSEM) at 24 Hours, in the Tumor, after Injection of Different Doses of Gd-Liposomes. Fractional Volume is 100 ⴛ Liposome-encapsulated Volume Divided by the Distribution Volume of GdDTPA-BMA Measured in Similar Regions of Different Animals Central Region of the Tumor
Peripheral Region of the Tumor
Dose (mmol/kg)
䡠 R1 ⫾ SEM (sec⫺1)
Fractional Volume
䡠 R1 ⫾ SEM (sec⫺1)
Fractional Volume
0.025 0.05 0.1 0.2
0.08 ⫾ 0.02 0.16 ⫾ 0.03 0.36 ⫾ 0.04 0.36 ⫾ 0.02
0.11% 0.24% 0.53% 0.53%
0.22 ⫾ 0.04 0.28 ⫾ 0.03 0.61 ⫾ 0.06 0.78 ⫾ 0.06
0.11% 0.15% 0.33% 0.42%
The liposomal Gd relaxivity among batches was between 3.4 –3.7 s⫺1 mM⫺1. These values led to calculated values of liposome-encapsulated volume within tumor pixels of 0.09 to 0.43 microL/mL in the central region and 0.25 to 0.88 microL/mL in the periphery. The distribution volume of GdDTPA-BMA was 79 ⫾ 1 microL/mL in the central region and 213 ⫾ 2 microL/mL in the peripheral region of the tumor. Thus, although the high liposome dose produced a saturation of the ⌬R1 response in the central region, the volume occupied by the liposome (estimated from the encapsulated volume) was very small in comparison to available extracellular space, as estimated from the distribution volume of GdDTPA-BMA (Table 1). This suggests that the liposome poorly penetrates the tumor interstitium. In comparison to liposome doses used in clinics, the saturation dose of 0.1mmol/kg found in this study is approximately 100 times a single dose of Doxil® (liposomeencapsulated doxorubicin). From this it can be concluded that typical clinical doses can be increased greatly with commensurate increased accumulation in the tumor. Moreover, this study supports the proposition that Gdliposomes are a powerful tool for probing the distributional behavior of liposome-based therapies, and may be very useful in clinical applications. ACKNOWLEDGMENTS
Supported by NIH grant 1R 21 CA 79825-01. E.R. supported by Belgian American Educational Foundation and Royal Belgian Society of Radiology. K.S. supported by Doctor Karol Sicher Cancer Research fellowship and Stanford University Dean’s fellowship.
Academic Radiology, Vol 9, Suppl 2, 2002
REFERENCES 1. Unger E, Needleman P, Cullis P and Tilcock C. Gadolinium-DTPA liposomes as a potential MRI Contrast Agent. Invest Rad 1988; 23:928 – 932. 2. Unger E, Winocur T, Mac Dougall P, Rosenblum J, Clair M, Gatenby R et al. Hepatic Metastases: Liposomal Gd-DTPA-enhanced MRI Imaging. Radiology 1989; 171:81– 85. 3. Unger E, Mac Dougall, Cullis P and Tilcock C. Liposomal Gd-DTPA: Effect on enhancement of hepatoma model by MRI. Magn Res Imaging 1989; 7:417– 423. 4. Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, Dewhirst MW. Increased microvascular permeability contributes to preferential accumulation of stealth liposomes in tumor tissue. Cancer Res 1993; 53:3765– 3770.
GD-LABELED LIPOSOMES
5. Papahadjopoulos D, Allen TM, Gabizon , Mayhew E, Matthay K, Huang SK et al. Sterically stabilized liposomes: Improvements in phermacokinetics and antitumor therapeutic efficacy; Proc Natl Acad Sci 1991; 88: 11460 –11464. 6. Polo L, Segalla A, Jori G, Bocchiotti G, Verna G, Franceschini R et al. Liposome-delivered 131I-labeled Zn(II)-phtalocianine as radiodiagnostic agent for tumors. Cancer letters 1996; 109:57– 61. 7. Huang SK, Lee KD, Hong K, Friend DS and Papahadjopoulos D. Microscopic localization of sterically stabilized liposomes in Colon Carcinoma-bearing mice; Cancer Res 1992; 52:5135–5143. 8. Szoka F, Olson F, Heath T, Vail W, Mayhew E, Papahadjopoulos. Preparation of unillamellar liposomes of intermediate size by a combination of reverse phase evaporation and extrusion through polycarbonate membranes. Biochim Biophys Acta 1980; 601:559 –571.
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RATIONALE AND OBJECTIVES Recently developed sterically stabilized liposomes are used clinically for delivery of anticancer chemotherapy (currently the drug doxorubicin, Doxil®). In investigational drug development they are studied as targeted (by surface attached antibody or receptor ligands) and nonspecific drug delivery strategies for a variety of pathologies (cancer, arthritis, AIDS). Liposomes have also been considered as potential MRI contrast media vehicles, primarily for blood pool enhancement (1) and for contrast enhancement in the diagnosis and characterization of tumors (2–7). However, little work has been aimed at using liposomal MRI contrast material for purposes of image-based tracking of drug delivery of liposome-drug formulations. Intravenously administered liposomes are distributed passively by flowing blood and extravasate, usually slowly, in territories where vascular endothelium is either (a) very leaky due to large intracellular gaps such as in inflammatory zones, tumors and healing wounds; or (b) fenestrated such as liver or spleen. Selective concentration of liposomes in tumors is slow-typically requiring day(s) for maximal accumulation—which imposes certain limitations on strategies for external image-based detection, as well as upon the liposome itself. Non-sterically stabilized liposomes are cleared from the blood within a few hours Acad Radiol 2002; 9(suppl 2):S525–S527 1From the Department of Radiology, Stanford University School of Medicine, Stanford, Calif (E.R., F.B., K.J.S., P.L.); Department of Radiology, University of California, San Francisco (M.F.W., C.A.G.); and Liposome Research Lab, California Pacific Medical Center, San Francisco, Calif (K.H.). Address correspondence to E.R., Department of Radiology, CHU SaintPierre, Rue Haute 322, 1000 Brussels, Belgium.
©
AUR, 2002
via the reticuloendothelial system and accumulation in tumor is obviated kinetically. Sterically stabilized liposomes are successful for antitumor application because they remain in the circulation much longer, with plasma half-lives on the order of days, and substantial accumulation in tumor is possible. Similarly for image-based external detection strategies, the use of PET or SPECT tracers bound to liposomes would fail to the short radioactive half-lives of acceptable radionuclides. Consequently, only liposomal-bound CT or MRI contrast media would be viable candidates for this purpose. In the current study, sterically stabilized liposomes labeled with encapsulated GdDTPA-BMA were examined as probes for quantitative tracking of liposomal drug delivery in implanted osteogenic sarcoma model. The purpose of the study was to evaluate dose-response in the context of intravenously administered dose versus the response of ⌬R1 accumulation in the tumor. MATERIALS AND METHODS GdDTPA-BMA-liposomes (80 nm dia) were prepared from egg phosphatidyl choline (EPC), cholesterol, and polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) (ratio 3:2:0.3) and 0.25 M GdDTPA-BMA by the reverse phase evaporation method followed by extrusion (8). Inclusion of PEG-PE provided steric stabilization. Each batch was characterized by assay of phosphate content and MR determination of Gd content (spectroscopic inversion recovery measurement of ⌬R1 of intact and detergent solubilized liposomes, with and without GdDTPABMA). Encapsulated volume was calculated using assumption that all Gd was encapsulated at 0.25 M concentration.
S525
RUBESOVA ET AL
Thirty nude athymic rats (NCI, 80 –110 gm, 5-weeks old) were implanted with osteogenic sarcoma (cell line UMR 106 E IV) by subcutaneously injecting 3–5 million cells while under ketamine-xylazine anesthesia. When palpable tumors were approximately 0.6 – 0.8 cm diameter (6 to 10 days later) animals were imaged. MRI was conducted at 2 Tesla using a Bruker Omega system (Bruker Instruments, Inc., Fremont, CA). Animals were anesthetized by exposing to isoflurane (5% to induce and 1.7–2% to maintain) mixed into 100% O2. Animals were placed supine on a holder covered by a heated water pad (38 ⫾ 1°C), inserted into a 5 cm birdcage rf coil and placed in the magnet. A tail vein was catheterized for administration of liposomes. GdDTPA-BMA liposome was infused (5–10 min) at Gd doses of 0.025, 0.05, 0.1 and 0.2 mmol/kg body weight (n ⫽ 6). MRI was conducted before and 24 and 48 hr after liposome administration. Contrast enhancement was measured from standard multislice T1 weighted spin echo (TR/TE ⫽ 500/13 ms) images. Tumor size and morphology were determined from magnetization transfer (3D spoiled GRE, TR/TE ⫽ 100/3 ms, flip ⫽ 30 deg) and/or diffusion weighted images (TR/TE ⫽ 2500/44 ms with diffusion gradients set to 1, 5, 8 and 10 G/cm, b ⫽ 14, 366, 938, and 1465 s/mm2). Regional T1 values were measured before and after contrast administration by either IR EPI (TR/TE ⫽ 10 s/10 ms, single slice) or IR snapshot FLASH (TR/ TE ⫽ 3/1.5 ms, flip ⫽ 10 deg, centric phase ordering, 6 –10 slices) with TI’s ranging from 50 –3000 ms. The remaining six rats were administered GdDTPABMA (0.1mmol/kg bolus, 0.004 mmol/kg/hr infusion) and imaged as described above. Regional T1 values were measured using IR snapshot flash MRI after 30 min of infusion. Blood samples were taken before and during infusion and plasma R1 was measured by spectroscopy. Distribution volume of GdDTPA-BMA was calculated from the ratio of ⌬R1tumor/⌬R1plasma.
RESULTS AND CONCLUSIONS Maximum contrast enhancement was observed at 24 hours after injection of Gd-liposomes. The ⌬R1 values of central tumor regions measured at 24 hours increased with dose until 0.1 mmol/kg. Higher dose of Gd-liposomes produced no further increase in ⌬R1. Significantly greater ⌬R1 was measured in the tumor periphery for all doses of Gd-liposomes, and the peripheral region showed no plateau at the higher doses (Table).
S526
Academic Radiology, Vol 9, Suppl 2, 2002
⌬R1 value (ⴞSEM) at 24 Hours, in the Tumor, after Injection of Different Doses of Gd-Liposomes. Fractional Volume is 100 ⴛ Liposome-encapsulated Volume Divided by the Distribution Volume of GdDTPA-BMA Measured in Similar Regions of Different Animals Central Region of the Tumor
Peripheral Region of the Tumor
Dose (mmol/kg)
䡠 R1 ⫾ SEM (sec⫺1)
Fractional Volume
䡠 R1 ⫾ SEM (sec⫺1)
Fractional Volume
0.025 0.05 0.1 0.2
0.08 ⫾ 0.02 0.16 ⫾ 0.03 0.36 ⫾ 0.04 0.36 ⫾ 0.02
0.11% 0.24% 0.53% 0.53%
0.22 ⫾ 0.04 0.28 ⫾ 0.03 0.61 ⫾ 0.06 0.78 ⫾ 0.06
0.11% 0.15% 0.33% 0.42%
The liposomal Gd relaxivity among batches was between 3.4 –3.7 s⫺1 mM⫺1. These values led to calculated values of liposome-encapsulated volume within tumor pixels of 0.09 to 0.43 microL/mL in the central region and 0.25 to 0.88 microL/mL in the periphery. The distribution volume of GdDTPA-BMA was 79 ⫾ 1 microL/mL in the central region and 213 ⫾ 2 microL/mL in the peripheral region of the tumor. Thus, although the high liposome dose produced a saturation of the ⌬R1 response in the central region, the volume occupied by the liposome (estimated from the encapsulated volume) was very small in comparison to available extracellular space, as estimated from the distribution volume of GdDTPA-BMA (Table 1). This suggests that the liposome poorly penetrates the tumor interstitium. In comparison to liposome doses used in clinics, the saturation dose of 0.1mmol/kg found in this study is approximately 100 times a single dose of Doxil® (liposomeencapsulated doxorubicin). From this it can be concluded that typical clinical doses can be increased greatly with commensurate increased accumulation in the tumor. Moreover, this study supports the proposition that Gdliposomes are a powerful tool for probing the distributional behavior of liposome-based therapies, and may be very useful in clinical applications. ACKNOWLEDGMENTS
Supported by NIH grant 1R 21 CA 79825-01. E.R. supported by Belgian American Educational Foundation and Royal Belgian Society of Radiology. K.S. supported by Doctor Karol Sicher Cancer Research fellowship and Stanford University Dean’s fellowship.
Academic Radiology, Vol 9, Suppl 2, 2002
REFERENCES 1. Unger E, Needleman P, Cullis P and Tilcock C. Gadolinium-DTPA liposomes as a potential MRI Contrast Agent. Invest Rad 1988; 23:928 – 932. 2. Unger E, Winocur T, Mac Dougall P, Rosenblum J, Clair M, Gatenby R et al. Hepatic Metastases: Liposomal Gd-DTPA-enhanced MRI Imaging. Radiology 1989; 171:81– 85. 3. Unger E, Mac Dougall, Cullis P and Tilcock C. Liposomal Gd-DTPA: Effect on enhancement of hepatoma model by MRI. Magn Res Imaging 1989; 7:417– 423. 4. Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, Dewhirst MW. Increased microvascular permeability contributes to preferential accumulation of stealth liposomes in tumor tissue. Cancer Res 1993; 53:3765– 3770.
GD-LABELED LIPOSOMES
5. Papahadjopoulos D, Allen TM, Gabizon , Mayhew E, Matthay K, Huang SK et al. Sterically stabilized liposomes: Improvements in phermacokinetics and antitumor therapeutic efficacy; Proc Natl Acad Sci 1991; 88: 11460 –11464. 6. Polo L, Segalla A, Jori G, Bocchiotti G, Verna G, Franceschini R et al. Liposome-delivered 131I-labeled Zn(II)-phtalocianine as radiodiagnostic agent for tumors. Cancer letters 1996; 109:57– 61. 7. Huang SK, Lee KD, Hong K, Friend DS and Papahadjopoulos D. Microscopic localization of sterically stabilized liposomes in Colon Carcinoma-bearing mice; Cancer Res 1992; 52:5135–5143. 8. Szoka F, Olson F, Heath T, Vail W, Mayhew E, Papahadjopoulos. Preparation of unillamellar liposomes of intermediate size by a combination of reverse phase evaporation and extrusion through polycarbonate membranes. Biochim Biophys Acta 1980; 601:559 –571.
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