(CANCER RESEARCH SO.6371-6378. October I. 1990] Effect of Liposome Composition and Other Factors on the Targeting of Liposomes to Experimental Tumors: Biodistribution and Imaging Studies1
Alberto Gabizon,2 David C. Price, John Huberty, Robert S. Bresalier, and Demetrios Papahadjopoulos Cancer Research Institute ¡A.(j., I). P.] and Department of Radiology, [D. C. P., J. H.J, L'nirersity of California, San Francisco, California 9414}; (iastroinlestinal Research Laboratories, I eteram Administration Medical Center, and Department of Medicine, I 'nirersity of California, San Francisco, California 94121 [R. S. B.J; and Liposome Technology Inc., Mento Park, California 94025 ¡A.CiJ
ABSTRACT temperature, cholesterol, and careful size control result in in hibition of RES uptake with concomitant enhancement of We have examined the distribution of radiolabeled liposomes in tumor- tumor uptake (5). bearing mice after i.v. injection. Two mouse tumors (B16 melanoma, In this report, we describe tissue distribution and imaging J6456 lymphoma) and a human tumor (LS174T colon carcinoma) inoc ulated i.m., S.C.,or in the hind footpad were used in these studies. When studies with transplantable mouse and human tumor models various liposome compositions with a mean vesicle diameter of ~ 100 nm using 3 different radiolabels of liposomes. The findings here were compared using a radiolabel of gallium-67-deferoxamine, optimal indicate that the concentration of liposome-encapsulated radio- tumor localization was obtained with liposomes containing a phosphati- labels in tumors is well above that of most other tissues and dylcholine of high phase-transition temperature and a small molar frac approximates the values obtained in the liver. The evidence tion of monosialoganglioside or hydrogenated phosphatid)linositol (HPI). gathered strongly supports the validity of a direct tumor target At 24 h after injection, average values of tumor uptake higher than 10% ing approach with liposomes. of the injected dose per g and liver-to-tumor ratios close to 1 were reproducibly obtained. Increasing the molar fraction of HPI from 9% to 41% of the total phospholipid resulted in enhancement of liver uptake MATERIALS AND METHODS and decrease of tumor uptake. Methodological aspects that influence Preparation of Liposomes. The sources of materials used in this study- vesicle size appear to affect significantly liposome localization in the tumor. However, varying the phospholipid dose within a 10-fold range are the same as reported previously (5). The main fatty acid components of the hydrogenated phospholipids used were 66% stéarateand 33% caused only minor changes in the percent of injected dose recovered in palmitate for phosphatidylinositol, and 85% stéarateand15% palmitate the tumor. A high uptake by tumors was also observed using other radiolabels ¡|'II|imiliuand indium-111-labeled bleomycin ("'In-Bleo)| in for phosphatidylcholine. Liposomes were prepared by thin lipid film monosialoganglioside- and HPI-containing liposomes. In the case of '"In- hydration followed by repeated extrusions through polycarbonate mem Bleo, encapsulation in liposomes resulted in ~20- to 40-fold increase in branes of defined pore size (0.05 urn) as described previously (5). For hydration, we used isotonic solutions of saline (pH range, 6.0-7.0) with tumor accumulation of the radiolabel at 24 h after injection. The marked either DF (20-25 mivi), Bleo (5-15 units/ml), or ['Hjinulin (250 MCi/3 localization of liposomes in the mouse footpad inoculated with tumor as opposed to the contralateral mock-injected footpad was also documented ml). The mean vesicle size obtained with this methodology was in the by imaging experiments with gallium-67-deferoxamine and '"In-Bleo- range of 70 to 120 nm with a SD not larger than 30% of the Gaussian labeled liposomes. These results support the contention that some gly- mean as measured by dynamic laser scattering. In some cases, where colipid-containing liposomes previously shown to have long circulating indicated, liposomes were prepared by solvent injection (6) in the following way: lipids were weighed and dissolved in a 7:3 mixture of half-lives accumulate significantly in a variety of tumors and are prom ethanohdimethyl sulfoxide: the lipid solution was warmed to 60°Cand ising tools for the delivery of anti-tumor agents. injected through a 21- to 23-gauge needle, at a rate of =10 ml/min, into the bulk water phase (consisting of a isotonic solution of NaCl and INTRODUCTION 20 mM DF), which was kept at 60°Cand constantly stirred up; the organic solvents were diluted to 15% of the total volume, thus enabling Liposomes have been used as carriers of cytotoxic drugs with liposome formation. Ethanol, dimethyl sulfoxide, and unencapsulated a strategy based on reduction of toxicity and/or passive delivery DF were removed by repeated cycles of cartride dialysis (Diaflo hollow- to liver-infiltrating tumors (1, 2). The fast and dominant uptake fiber cartridge: Amicon, Danvers, MA) against isotonic saline or glu of liposomes by the RES' (3, 4) has prevented so far the cose 5%. Liposomes were concentrated =5-fold during the last dialysis adoption of a more direct strategy based on selective homing step. Liposomes were used either without any further manipulations or of liposomes to tumors. Targeting of liposomes to tumors after additional extrusion through 0.05 ^m-pore polycarbonate mem requires, most importantly, a prolonged circulation half-life of branes and Sephadex G-75 gel filtration to remove any DF released by intact liposomes which, in turn, depends on a reduction of the the extrusion procedure. Phospholipid concentration was determined by a phosphate assay rate of clearance by the RES. In addition, there is a need to (7, 8). Liposomes were stored either under argon or in vacuum-sealed minimize the leakage of liposome contents during their pro tubes at 5°Cand tested within 1 month after preparation. longed stay in the blood stream. Recent developments have Radiolabeling of Liposomes. The method of labeling preformed li shown that the inclusion of some glycolipids in the liposome posomes containing DF with 67Ga citrate has been described in detail bilayer coupled with phospholipids of high phase-transition (9). Labeling preformed liposomes containing Bleo (Bristol-Myers, Syracuse, NY) with '"InCI (New England Nuclear, Boston. MA) was Received 3/5/90: accepted 6/15/90. done following a similar technique. Briefly, 20 n\ of a 1 mg/ml solution The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in of oxine in ethanol or oxine hemisulfate in saline was added to an accordance with 18 U.S.C. Section 1734 solely to indicate this fact. aliquot of 100 to 200 jiCi of '"InCI and incubated at 60T for 15 min. 1Work supported until November 1987 by a grant from the National Cancer Bleo-containing liposome suspensions (5-15 (
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. LIPOSOME TARGETING TO TUMORS to Bleo with an efficiency close to 100% as assessed by separation on same phospholipid head group (dipalmitoylphosphatidylgly- thin-layer chromatography. cerol-DSPC versus PG-PC). Liposomes containing phospho- Tritium-labeled inulin (New England Nuclear) was encapsulated in lipids of widely different phase transition temperature (PG and liposomes by adding it to the hydration buffer (physiological saline). DSPC) resulted in low tissue recoveries probably as a result of The rest of the procedure was as described above for the thin lipid film/ extrusion method (5); 2.8% of the initial amount of [3H]inulin was phase separation and leakage of the label (5). Of note, skin entrapped in GMi-DSPC-Ch liposomes. uptake was several-fold higher in mice given injections of lipo Animals and Tumors. Age-matched C57BL/6 and BALB/c inbred somes with extended circulation time and high tumor uptake. female mice from Simonsen Laboratories (Gilroy, CA) and NCR-NU No such increase was observed in kidneys, intestine, and car athymic nude outbred female mice from the National Cancer Institute cass. The highest uptake values per g tissue were seen in the (Frederick, Ml)) were used in these studies. The mouse B16 melanoma spleen. However, given its small weight, the absolute uptake of was inoculated either i.m. into the hind leg (IO5 cells) or into the hind spleen was still below that of liver and skin for most formula footpad (2.5 x 10" cells) of syngeneic C57BL/6 mice. The mouse J6456 lymphoma (10) was inoculated into the hind leg (10'' cells) of syngeneic tions tested. BALB/c mice. The human LS174T colon carcinoma (11) was trans Biodistribution of (J\I,-containing Liposomes in Nude Mice planted by s.c. injection of IO7cells into the flank of nude mice. Cells Bearing the LS174T Human Colon Carcinoma were injected in serum-free RPMI-1640 medium (GIBCO, New York, NY). It was important to examine whether a high tumor uptake Biodistribution Studies. Radiolabeled liposomes were injected i.v. would also be observed in a human tumor model. For this into the tail vein of tumor-bearing mice. Tumor weight was in the range purpose, we tested one of the optimal formulations (GM,- of 0.5 to 2.0 g for mice bearing i.m. or s.c. tumors, and 0.1 to 0.4 g for DSPC-Ch) in nude mice bearing a human colon carcinoma, mice bearing footpad tumors. Retro-orbital bleeding (=1 ml) under LS174T. The results shown in Table 2 point at marked tumor ether anesthesia followed by complete dissection were done in all mice given injections of "Ga- or "'In-labeled liposomes. Organs were and skin uptake of liposomes, with an average value 5.3-fold weighed and their radioactivity quantitated in a gamma counter using higher than carcass uptake, exceeded only by liver and spleen. integral counting above 10 keV. Blood volume and correction factors Biodistribution of |3H]Inulin-labeled GM|-containing Liposomes for the blood content of various tissues were determined in age- and sex-matched mice with '"In oxine-labeled syngeneic erythrocytes as in Tumor-bearing Mice described previously (5). The results are expressed as %ID/g. Statistical The results presented in Tables 1 and 2 were obtained with significance of the differences between the %ID/g means was analyzed the 67Ga-DF label. To confirm these findings with an alternative using the 2-sided Student's / test. In the case of ['H]inulin-labeled liposomes, blood, liver, and tumor labeling method, we injected GM^DSPC-Ch liposomes with were examined for radioactivity content in a beta counter after solubil- encapsulated [3H]inulin in C57BL/6 mice bearing the B16 ization with Protosol (New England Nuclear). Quenching correction melanoma and in nude mice bearing the LS174T tumor and factors were obtained by measuring the radioactivity of blank tissues examined the label distribution in blood, liver, and tumor. As from noninjected mice spiked with known amounts of | 'I Ijimilin. seen in Table 3, the values obtained are of similar magnitude Imaging Studies. Imaging was performed with a Pho-Gamma IV to those observed when the same liposomes were labeled with Siemens scintillation camera with pinhole collimator at 13-inch dis 67Ga-DF. Liver-to-tumor ratios as low as 2 to 3 were obtained. tance from the animal lying in anterior view. Mice were anesthesized by i.p. injection of ketamine before each picture. The picture exposure Tissue Distribution Studies with HPI-containing Liposomes in time was 15 min. Images were digitized and stored in a DEC 11/34 Mice Bearing the B16 Melanoma computer using Gamma-II software for region of interest quantitative analysis. To assess the contribution of HPI to the liposome distribu tion pattern in tumor-bearing mice, we tested 3 preparations containing increasing molar fractions HPI and 1 preparation RESULTS containing no HPI, while the size and the other liposome Effect of Liposome Composition on the Biodistribution of 67Ga- components were kept the same. The results, presented in Fig. labeled Liposomes in Mice Bearing B16 Melanoma 1, indicate that the most favorable pattern of distribution was obtained with liposomes containing a phospholipid fraction of The first step of this study was to examine in tumor-bearing HPI of 9 to 23%. Liposomes with a higher HPI content showed mice the tissue distribution of various liposome preparations, a remarkable enhancement of liver uptake and minimal uptake different in composition but similar in size. For this purpose, 5 by the tumor. The effect appeared to be liver-specific since types of liposomes were tested in mice bearing B16 melanoma uptake by the spleen as well as other tissues was markedly using the 67Ga-DF radiolabel. Mice were sacrificed 24 h after reduced. In the case of DSPC-Ch liposomes lacking HPI, liver injection on the basis of previous experience indicating that and spleen uptake were both similarly increased as compared maximal tumor accumulation occurs around this time point with low HPI-containing preparations, although tumor uptake (5). The optimal formulations for further studies on tumor was not significantly affected. Thus, there appears to be differ targeting were selected on the following bases: (a) highest ent mechanisms by which the lack or the excess of the negatively absolute values of tumor uptake as reflected in the %ID/g charged lipid component, HPI, alter the pattern of tissue dis tumor; (b) lowest liver/blood and liver/tumor uptake ratios; tribution. and (c) highest tumor/carcass uptake ratio. As seen in Table 1, The effect of method of preparation and particle size distri the most favorable results were obtained with HPI- and GM,- bution on the pharmacological behavior of liposomes was stud containing formulations when considering both the absolute ied using a preparation made by solvent injection with or tumor uptake and the tissue uptake ratios. Injection of the free without extrusion. Solvent injection resulted in unilamellar 67Ga-DF label resulted in only 0.1% ID/g tumor (see Table 1, vesicles of small diameter (<150 nm) (6) without any need for footnote c). As expected, liposomes with fully saturated fatty extrusion. Extrusion through 0.05 /im-pore membranes further acyl chains showed a markedly improved distribution pattern reduced the average diameter and SD from 138 ±56 to 97 ± over those with unsaturated fatty acyl chains despite having the 22 nm. Larger vesicles were not examined since prior work in 6372
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. LIPOSOME TARGETING TO TUMORS Table 1 Biodistribution of'Ga-labeled liposomes in C57BL/6 mice bearing the BI6 melanoma" Liposomecompositionc%ID/g
(SD)Blood''Liver*SpleenKidneysIntestineSkinCarcass'Tumor''Tissuetissue (2.0)14.1 (0.6)6.0(0.2)1.8(0.1)0.2 (2.4)23.2(2.5)3.2 (0.3)17.1 (0.8)37.8 (2.9)3.3 (3.6)2.9 (0.3)0.5 (0.3)0.8(0.1)1.8(0.4)0.9(0.2)4.5(0.6)1.1 (0.9)0.9(0.1)1.9(0.9)0.7(0.1)8.4 (0.0)0.3 (0.0)0.7 (0.1)2.4 (0.0)0.4(0.1)1.8(1.0)119.019.84.5PG-DSPC-Ch1.4(0.4)9.8(0.5)0.2 (0.2)0.5 (0.0)1.5(0.4)7.06.57.5DPPG*-DSPC-Ch10.8(1.5)20.3 (0.0)5.3 (0.6)1.94.55.0HPI-DSPC-Ch8.4(0.4)1.72.710.6GM,-DSPC-Ch7.8(1.7)22.0(0.3)2.82.612.0
uptakeratiosLiver/bloodLiver/tumorTumor/carcassPG-PC-Ch0.3(0.1)35.7(1.9)61.7(16.6)2.3
°Tumor cells inoculated i.m. in the hind leg; dose, 1 fimol phospholipid/mouse i.V.; mice sacrificed 24 h after injection; results are the mean of 3 to 6 animals per experimental group. * DPPG. dipalmitoylphosphatidylglycerol. ' Molar ratio, 1-10-5. When the liposome radiolabel, 67Ga-DF, was injected in free form, tissue uptake was <0.2%ID/g, except for kidneys, which showed an uptake of 1.7%ID/g. d Blood, liver, and tumor values of dipalmitoylphosphatidylglycerol, HPI, and GM]-containing liposomes significantly different from those of PG-containing liposomes (IP < 0.05). ' Carcass consists of all skeletal muscles, bones, and appendages.
Table 2 Biodistribution of*'Ga-labeled CM,-containing liposomes in nude mice 50.OA bearing the LSI 74T human colon carcinoma0 %ID/g tissue (SD) 13.6 24.2(0.1)19.3(2.6)38.3 BloodLiverSpleenKidneysLungsHeartIntestineSkinCarcassTumorExperiment(0.4)12.5(1.6)25.9(3.3)3.6
(4.5)3.3 (0.6)1.1 (0.3)0.8 o Q (0.2)1.0(0.2)1.0(0.0)5.2 (0.6)1.3(0.2)1.1
(0.2)4.3 (0.6)0.9(0.1)5.1 (0.3)0.8 (0.0)4.4 (0.4)Experiment (0.5) " Tumor cells injected s.c. in the flank; each mouse received i.v. 1 fimol phospholipid of GM|-DSPC-Ch (1-10-5) liposomes; mice were sacrificed 24 h 10 20 30 40 after liposome injection; the results are the average of 3 mice/experiment; a different liposome batch was tested in each experiment. HPI Content (molar % of total phospholipid) Fig. 1. Effect of liposome HPI content on liposome biodistribution in tumor- Table 3 Biodistribution off'H/inulin-labeled GM'¡-containingliposomes in bearing mice. 67Ga-DF-labeled DSPC-Ch liposomes and HPI-DSPC-Ch lipo tumor-bearing mice" somes containing increasing molar fractions of HPI (9,23, and 41 %) »ereinjected i.v. in C57BL/6 mice bearing i.m. leg implants of the B16 melanoma. Dose, 1 B16 tumor LS174T tumor fimol phospholipid per mouse. Mice were sacrificed 24 h after liposome injection. %ID/g(SD)BloodLiverTumorTissue tissue Each liposome formulation was tested in 3 mice. Liver and spleen values for (0.8)17.9(2.7)6.3 DSPC-Ch (HPI = 0) were significantly higher (2P < 0.05) than those for HPI- DSPC-Ch (HPI, 9-23%). Blood, spleen, and tumor values for HPI-DSPC-Ch (2.9)2.02.8 (HPI = 41%) were lower (IP < 0.05) than those for other formulations. O—O, Blood; •—•,liver;A A, spleen; A A, tumor. uptakeratiosLiver/bloodLiver/tumor4.9(0.1)21.9(0.4)10.6(0.2)4.52.19.0
" Tumor cells inoculated i.m. in the leg (B16) or s.c. in the flank (LS174T); dose. 1 f/mol phospholipid/mouse of GMi-DSPC-Ch (1-10-5) i.v.; mice sacrificed Table 4 Biodistribution of"JGa-labeled HPl-HPC-Ch liposomes in mice bearing 24 h after injection; results are the mean of 5 mice. the BI6 melanoma: effect of size reduction" No extrusion Extrusion tumor-free mice had shown that the circulation time of HPI- Mean(SD)%ID/g size in nm (22)3.7(2.1)11.8(2.8)9.5(1.6)5.7(1.1)1.9(0.2)1.4(0.1)1.3(0.3)13.2(2.3)3.20.910.2 and GMi-containing liposomes was markedly shortened when (SD)Blood"Liver*SpleenKidneysIntestineSkinCarcassTumor*Tissuetissue vesicles of 200-nm size or larger were used (data not shown). As seen in Table 4, the highest tumor uptake (13.2% ID/g) and lowest liver-to-tumor ratio (0.9) were obtained with the ex (0.2)1.6(0.2)1.0(0.1)0.8(0.1)7.7 truded liposomes. These results suggest that size optimization is a critical factor even within a narrow range of discrimination. In order to study the effect of lipid dose, we used the liposome (2.3)4.62.59.697 uptakeratiosLiver/bloodLiver/tumorTumor/carcass138(56)4.2(1.9)19.3(1.7)18.3(0.0)4.3 preparation made by solvent injection and extrusion. As shown in Fig. 2, tumor uptake was not significantly affected by increas ing the lipid dose over a 10-fold range (0.3 pmo\ to 3 /¿mol " Tumor cells inoculated in the hind footpad; dose, 1 ,,nml phospholipid/ phospholipid) reaching values between 10 and 15% ID/g in mouse i.v.; liposome composition, HPI-HPC-Ch (1-10-5) prepared by solvent agreement with the results obtained in Table 4. Liver-to-tumor injection with or without extrusion; mice sacrificed 24 h after liposome injection; ratios were close to 1 (1.2 to 1.3) for all 3 dose levels tested. results are the average of 3 mice. Similar results were obtained when 67Ga-DF-labeled GM,- * Blood values of nonextruded versus extruded liposomes not significantly different. Liver and tumor values of nonextruded versus extruded liposomes DSPC-Ch liposomes were tested in mice bearing the J6456 significantly different (2P = 0.017 a¡nd0.043 respectively). 6373
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20.0 tissue distribution experiments. C57BL/6 mice bearing B16 melanoma implants in the right hind footpad were used as the £ 10.0- animal tumor model. The left hind footpad was mock-injected with cell-free medium. In the first experiment, HPI-HPC-Ch (mean size, 100 ±39 nm) and PG-PC-Ch (mean size, 74 ±15 5.0 nm) liposomes labeled with 67Ga-DF were injected into 3 mice each, and free ft7Ga-DF was injected into 1 mouse. Images were
O Q obtained within l h (blood pool image), and at 24, 42, and 70 2.0 h after injection. Figs. 4 (A to />), 5 (A to D), and 6 (A and B) u show successive images obtained with HPI-HPC-Ch, PG-PC- Z 1.CH Ch, and free label, respectively. The label was more rapidly excreted from animals receiving PG-PC-Ch than from animals 0.5 receiving HPI-HPC-Ch. The average number of counts ob 0.0 0.5 1.0 1.5 2.0 2.5 3.0 tained at 70 h was 20.3% of the initial blood pool image counts for the former, and 30.3% for the latter. Free 67Ga-DF was Phospholipid dose (umoles / mouse) rapidly excreted into the urine, and only 10% of the blood pool Fig. 2. Effect of phospholipid dose on the biodistribution of liposomes in tumor-bearing mice. "Ga-DF labeled HPI-HPC-Ch liposomes were injected i.v. image counts were recovered at 42 h injection. With both types in C57BL/6 mice bearing footpad implants of the B16 melanoma. Mice were of liposomes, there was marked label concentration in the upper sacrificed 24 h after liposome injection. Each dose level was tested in 3 mice. O, Blood; •,liver; A. carcass; A. tumor. abdomen (liver and spleen). In addition, label concentration in the tumor area was clearly enhanced in mice receiving HPI- HPC-Ch as compared with mice receiving PG-PC-Ch. When lymphoma in the dose range of 0.25 to 5 /jmol phospholipid the mice were sacrificed and the dissected tissues checked in a per mouse, namely no significant change in the relative tumor -y-counter at 70 h post-injection, the %ID/g tumor and liver- uptake (data not shown). to-tumor ratios were 1.8 (SD = 0.3) and 3.8 for PG-PC-Ch, Tissue Distribution Studies with '"In-Bleo-labeled Liposomes and 7.2 (SD = 1.0) and 1.9 for HPI-HPC-Ch. An additional imaging experiment was carried out with '"In- To evaluate the potential significance of liposome uptake by Bleo-labeled HPI-HPC-Ch liposomes and free '"In-Bleo. Two tumors with regard to a cytotoxic drug, we examined the biodistribution of Bleo labeled with ' "In, either as free complex mice given injections of liposomes and 1 mouse given an or encapsulated in GMi- and HPI-containing liposomes. These injection of the free drug were imaged within l h (blood pool experiments were done with 2 mouse tumor models: the B16 image), and at 18, 44, and 67 h after injection. When free '"In- melanoma and the J6456 lymphoma. The results are summa Bleo was injected, the label was concentrated in the urinary rized in Table 5 and Fig. 3. Liposome encapsulation increased bladder, and only 10% of the blood pool image counts were Bleo retention in all body tissues, probably by diminishing renal recorded at 18 h, thus pointing at the fast renal excretion rate. clearance. The tumor uptake of liposome-encapsulated Bleo No label uptake was detected in the tumor-injected footpad. In was relatively high as compared with other tissues, with the the case of "'In-Bleo encapsulated in HPI-HPC-Ch liposomes exception of liver and spleen (Table 5). These factors resulted (Fig. 7, A to D), 50% of the blood pool image counts were still in 2 favorable parameters of tissue distribution pattern: (a) a recovered at 67 h. The label was concentrated in the upper substantial increase of the label concentration in tumors of mice abdomen (liver and spleen) and in the tumor area. Computer treated with liposome-encapsulated Bleo as opposed to free ized quantitation of regions of interest showed a 5- to 11-fold Bleo (=20- to 40-fold); and (b) an increased uptake by tumor increased uptake by the tumor as compared with the normal as opposed to carcass in mice treated with liposome-encapsu lated Bleo (~7- to 16-fold). As found previously using a 67Ga- mock-injected contralateral leg. Upon sacrifice and 7-counting of the dissected organs, the %ID/g tumor and carcass were 7.9 DF liposome label (5), peak increases in tumor concentration ±0.3 and 1.2 ±0.0, respectively (tumorcarcass ratio, 6.6). were observed at 24 h after injection (Fig. 3). Imaging Studies with Free and Liposome-encapsulated 67Ga-DF and '"In-Bleo DISCUSSION The use of -y-ray-emitting radiolabels enabled us to carry The results of this study provide strong evidence on the ability imaging studies to complement the information obtained from of at least 2 liposome formulations to accumulate in transplant-
Table 5 Biodistribution offree '"In-Bleo and "'In-Bleo encapsulated in GM¡-and HPI-containing liposomes in tumor-bearing mice" (SD)*BloodLiverSpleenKidneysLungsHeartIntestineSkinCarcassTumorFreeO.I%ID/g tissue
tumorGMl-DSPC-Ch7.4 tumorGMl-DSPCCh2.5
(0.0)0.4 (0.9)23.0(1.1)37.8 (0.3)26.7 (0.3)0.5 (0.9)44.8 (0.0)43.2(1.1)45.4 (0.0)0.3 (2.4)54.7 (0.4)0.8 (0.6)80.9(2.1)3.5 (0.0)3.0(1.0)0.2(0.1)0.4 (2.7)4.5(1.2)1.5(0.4)1.2(0.1)1.2(5.0)12.1 (0.1)1.9(0.6)1.0(0.3)0.8 (0.6)6.6 (1.1)2.6(1.0)2.0 (0.2)1.9(0.6)2.2(1.1)1.8(0.1)1.4(0.1)0.6(0.6)2.8 (0.4)3.3 (0.0)0.1 (0.2)1.9(0.1)1.9(0.2)1.0(0.1)6.8(0.3)0.2(0.1)0.3 (0.5)3.4(0.1)2.9(0.1)1.1 (0.0)0.2 (0.6)2.9 (0.0)0.1 (0.9)1.1 (0.2)0.2(0.1)0.4 (0.0)0.3 (0.2)9.2(1.9)HPI-HPC-Ch2.2 (0.0)9.6(1.6)HPI-DSPC-Ch3.8(0.2)17.3(7.7) (0.0)B16 (2.3)Free0.6 (0.3)J6456 * Tumor cells inoculated i.m in the hind leg; phospholipid dose. 1 to 1.4 ^mol/mouse; Bleo dose. 5 mU/mouse. except for the B16 tumor HPI-HPC-Ch group, which received 50 mU/mouse; mice sacrificed 24 h after injection: results are the average of 3 mice. * Except for kidneys, lungs, and heart, all other tissue values were significantly higher for "'In-Bleo in liposomes as compared with free '"In-Bleo (2P < 0.05). 6374
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50.0 most normal tissues. This is exemplified by the high tumor-to- «j D carcass ratios. In most cases, tumor uptake was still below the
'+J« 20.0 uptake by liver and spleen, organs with fenestrated capillaries § 10-°- and rich in cells of the RES (12). Nevertheless, we were able to obtain very low liver-to-tumor ratios in the range of 1 to 2. This -T 5.0 substantial improvement in the pattern of liposome biodistri
Fig. 4. Imaging of tumor-bearing mouse given an injection of "Ga-DF-labeled HPI- HPC-Ch liposomes. C57BL/6 mice bearing right hind footpad implants of the B16 mel anoma received i.v. 1.3 /.mol phospholipid and 29.5 nCi of the label in liposome-en- trapped form. Anterior views were obtained 1 (A), 24 (B), 42 (C), and 70 (D) h after injection. Note the higher uptake of label in the tumor-injected footpad as compared with the normal mock-injected footpad.
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Fig. 5. Imaging of tumor-bearing mouse given an injection of "Ga-DF labeled PG-PC- Ch liposomes. C57BL/6 mice bearing right hind footpad implants of the B16 melanoma received i.V. 1.3 ¿imuÃphospholipid and 46.5 ßC'iofthe label in liposome-entrapped form. Anterior views were obtained 1 (A), 24 (fi), 42 (C), and 70 (O) h after injection. In contrast to Fig. 4, the difference in uptake between the tumor- injected footpad and the normal mock-injected footpad is barely noticeable. Note also that the urinary bladder is visualized in the l h blood pool image.
Fig. 6. Imaging of tumor-bearing mouse given an injection of free 67Ga-DF. C57BL/6 mice bearing right hind footpad implants of the B16 melanoma received i.v. 33 nC\ of the free label. Anterior views were obtained 1 (A) and 42 (B) h after injection. In contrast to Figs. 4 and 5, the label is rapidly excreted, thus resulting in accumulation of the label in the urinary bladder. No label uptake occurs in the tumor-injected footpad.
hydrogenated phosphatidylinositol, and a relatively small vesi splenic uptake was relatively high, thus resulting in an overall cle diameter (=100 nm). The differences in tumor uptake be suboptimal biodistribution pattern when compared with lipo tween unextruded and extruded solvent injection-prepared li some preparations containing a low molar ratio of HPI (Fig. posomes (Table 4) suggest that even small changes in size have 1). As we proposed previously (5), the effect of HPI and CM, an important pharmacological impact. may be related to prevention of vesicle aggregation in addition The observations of Proffitt et al. (18, 19) and Ogihara et al. to decrease of the recognition of the liposome surface by plasma (20, 21) in tumor-bearing mice and subsequent reports on opsonins. Interestingly, a high molar ratio of HPI overrides the imaging studies in cancer patients (22, 23) with small DSPC- favorable effect of a low molar ratio by shifting liposome Ch neutral vesicles would appear to indicate that significant distribution to the liver and concomitantly reducing the amount tumor targeting is achievable without any need for negatively reaching other organs. The increased liver uptake of these charged glycolipids. These observations (18-23) were made highly negatively charged vesicles is likely to be the result of with weak chelation complexes ('"In-nitriloacetic acid, 67Ga- opsonization and receptor-mediated endocytosis (24). nitriloacetic acid), which result in high tissue backgrounds as Accumulation of liposomes in tumors may result from ex opposed to 67Ga-DF (9). Using such neutral vesicles, we also travasation into the interstitial tumor space or endocytosis by found a high uptake by the B16 melanoma, although hepato- capillary endothelial cells of the tumor microvasculature. Al- 6376
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Fig. 7. Imaging of tumor-bearing mouse given an injection of "'In-Bleo-labeled HPI- HPC-Ch liposomes. C57BL/6 mice bearing right hind footpad implants of the B16 mela noma received i.v. 5 /*mol phospholipid and 23.5 fiCi of the label (=0.25 units Bleo) in liposome-entrapped form. Anterior views were obtained I (A), 18 (A), 44 (C), and 67 (D) h after injection. Note the high uptake of label in the tumor-injected footpad as compared with the normal footpad.
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Alberto Gabizon, David C. Price, John Huberty, et al.
Cancer Res 1990;50:6371-6378.
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