Basic Approach to Application of Liposomes.pdf

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Tohoku J. Exp. Med., 1992, 168, 361-369

Basic Approach to Application of Liposomesfor Cancer Chemotherapy

YOSHIYUKIASHIMOTOnd SHINYA UZUKIDepartment of Hygienic Chemistry, PharmaceuticalInstitute, Tohoku University,Sendai 980

HAsmMoTo,Y. and SuzuKI, S. Basic Approach to Application of Liposomesfor Cancer Chemotherapy. Tohoku J. Exp. Med., 1992, 168 (2), 361-369 Themethod for augmentation of systemic in vivo anticancer effect of liposomes (Lip)containing adriamycin (ADM) and endocytosis activity of cancer cells to liposomalpreparations have been studied. Encapsulation of ADM in liposomes increases itsmaximal tolerated dose and pretreatment of animals bearing tumor with tumornecrosis factor a (TNF) resulted in effective targeting of ADM-Lip to tumor,leading to its augmented therapeutic effect, but only when TNF and ADM-Lipwere administered with an appropriate interval. All human tumor cell lines testedshowed endocytosis activity to liposomes but the activity was differed amongdifferent tumor cell lines. adriamycin ; tumor necrosis factor a (TNF-a) ;liposomes ; antitumor effect

Monoclonal antibodies (mAbs) have been used for cancer therapy by takingadvantage of their selective binding to cancer cells bearing the correspondingantigens. As anticancer effect of a mAb itself is generally small, it is conjugatedwith a toxin or anticancer drug to deliver the agent to cancer cells.mAb-modified liposomes can be used as a carrier of an anticancer drug,because anticancer drugs can be holded in liposomes without chemical bindingwhich often inactivates the drug activity. We prepared mAb-modified iposomescontaining an anticancer drug (chemoimmunoliposomes OIL) (Hashimoto et al.1983a) and examined their anticancer activity. OIL showed selective binding torelevant target cancer cells and exhibited stronger anticancer activity than thedrug alone (Hashimoto et al. 1983b). In these experiments, OIL showed stronganticancer activity by a local injection. While i.v. injection of OIL did not showclear anticancer effect to s.c. tumor except a mouse mammary cancer system.This is probably due to poor permeability of liposomes through blood vessels ofcancer tissue. Another factor controling the efficacy of mAb-drug conjugates aswell as liposomes containing an anticancer drug in the vesicle would be en-docytosis activity of their target cancer cells, since they express anticancer effectafter internalization of target cancer cells. In these aspects, we studied themethod for augmentation of systemic in vivo anticancer effect of liposomes

Address for reprints : Aramaki aza Aoba, Aoba-ku, Sendai 980, Japan.361


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362 Y. Hashimoto and S. Suzuki

containing adriamycin and endocytosis activity of cancer cellspreparations.

to liposomal

MATERIALS AND METHODDrugsRecombinant human TNF-a (TNF), specific activity, 2.2 X 106units/mg from themurine L-cell assay was donated by Asahikasei Chemical Industry Co., Tokyo. For animalexperiments, TNF was dissolved in PBS containing 0.1% gelatin (vehicle) at 23pg/ml andaliquots of the solution were injected into mice. Adriamycin hydrochrolide (ADM) wasdonated by Kyowa Hakko Co., Tokyo.

Tumor cellsCultured human tumor cell lines, KU-1 and T24 urinary bladder cancer, MKN-7 andMKN-45 gastic cancer, SKBr-3 breast cancer, Molt-4 leukemia and K562 erythroleukemia

cells were used.Monoclonal antibodies

HBJ127 (IgGI) (Masuko et al. 1984) and AL-6 (IgM) were produced from the corre-sponding hybridomas prepared in our laboratory. HBJ127 recognizes a peptide epitope ofgp125 which expressed on human cells in association of cell proliferation and AL-6 isreactive with MBPE-containing liposomes. FITC-conjugated AL-6 was prepared by cou-pling AL-6 with FITC at a molar ratio of 1: 50. The molar ratio in the product was about1:12.Liposome preparations

All liposome preparations were basically prepared from dipalmitoylphosphatidyl-choline (DPPC), cholesterol, and dipalmitoylphosphatidylethanolamine (DPPE). Forpreparation of fluoresceine isothiothianate-labelled liposomes (F-Lip), dipalmitoylphosphat-dic acid (DPPA) was added in addition and FITC-conjugated DPPE was used forunmodified DPPE. Liposomes containg adriamycin (ADM-Lip) was prepared by activeADM-loading as described by Mayer et al. (1985) with a partial modification (Suzuki et al.1990) and mAb-modified liposomes (IL) were prepared as described (Hashimoto et al.1983a). Carboxyfluorescein-containing IL (CF-IL) were made by addingcarboxyfluorescein solution to lipid film. All liposome preparations were first obtained asmultilamellar liposomes and were sonically disrupted in a N2 atmosphere into smallunilamallar liposomes. The size of these liposomes was about 70-80 nm in diameter asdetermined by a peak elution volume in Sephacryl S-1,000 chromatography using polys-tylene beads of known diameters as a standard (Reynolds et al. 1983).Assay for target cell-bound liposomes

After treatment with various concentrations of CF-IL, cells were analized for theirfluorescence intensity using FACScan. Cells treated with IL were stained with FITC-AL6IgM or FITC-coupled rabbit anti-moused immunoglobuline antibody. Fluorescence inten-sity on cell surface was analyzed as described above.For the assay of liposomes internalized into cells, cells were treated with CF-IL, weresolubilized, and incubated for 20 min at 3TC. The cell lysate was measured for itsfluorescence intensity.Assay for tissue distribution of liposomes to tumor tissue

BALB/c mice were transplanted intradermally (i)d.) with 2 X 106Meth-A cells in theleft back. Seven days after the tumor inoculation, when the tumor nodules grew to 7 to 9


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Application of Liposomes for Cancer Chemotherapy 363mm in diameter, the mice were injected i.v. with TNF at 2.3 ,ug/mouse from a tail vein, andat certain times after the TNF injection, mice were injected i.v. with ADM-Lip (80,ug ofliposomal ADM/mouse). The mice were sacrificed 30 min after the injection of ADM-Lip,and tumor tissue was immediately removed by surgical operation. The tumor tissue wasstored at -20C under dark until analysis. ADM in tumor tissue was measured accordingto the method of Gabizon et al. (1982). Distribution of liposomes to normal organs or tissuewas similarly determined (Suzuki et al. 1990).Histological analysis for tissue distribution of F-Lip

To visualize the localization of liposomes, mice bearing Meth-A tumor were injectedwith F-Lip in a dose of 4.5,umol of total lipid/mouse 1 hr after preinjection of TNF (2.3,ug/mouse) or vehicle in a volume of 0.1 ml. One, 3 and 6 hr after the F-Lip injection, frozensections of tumor tissue were prepared from individual mouse tumor and were observed forthe F-Lip distribution under a fluorescence microscope.In vivo assay for antitumor effect of drugs

Groups of mice were i.v. treated twice (2 cycles) with ADM (50,ug/mouse), ADM-Lip(50 jug of ADM), TNF (2.3,ug), or combined regimens of TNF and ADM or ADM-Lip, ondays 7 and 11 after tumor inoculation and the antitumor effect of these regimens wasdetermined in terms of tumor growth inhibition and cure rate or prolongation of the survivaltime of the mice. Control mice received vehicle solution or saline.

RESULTSANDDICUSSIONAugmentation by TNF pretreatment for intratumoral accumulation and antitumoractivity of ADM in liposomes

Encapsulation of certain drugs in liposomes results in the reduction of theirside effects in experimental animals without losing their antitumor activity asrevealed from the case of adriamycin (ADM) entrapped in liposomes (ADM-Lip),which shows the same degree of therapeutic effect as free ADM but with reducedcardiotoxicity (Gabizon et al. 1982; Van Hoesel et al. 1984; Balazsovits et al.1989). However, the vascular permeability of liposomes is generally too poor to

Fig. 1. Effect of TNF pretreatment on transfer rate of ADM-Lip to Meth-A tumorin BALB/c mice. Thirty min after TNF injection (i.v.), ADM-Lip wereinjected i.v. and the amount of ADM in tumor was assayed periodically.(Cited from Ref. of Suzuki et al. 1990).


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allow efficient targeting of liposomal drugs to remote tumor tissue. Therefore, toevoke more efficient tumor targeting of liposomal preparations containingantitumor drug, a combined use of a drug capable of enhancing the extravasationof liposomas will be helpful. As a such drug, we selected TNF which induceshemorrhagic necrosis of transplanted animal tumors by acting on tumor vessels.In this section, we described that pretreatment of tumor-bearing mice with TNFwhich enhanced the accumulation of ADM-Lip n tumor tissue and led to augmen-tation of the antitumor activity of ADM-Lip (Suzuki et al. 1990).As shown in Fig. 1, a pretreatment with TNF increased the content of ADMin tumor tissue which reached a plateau at 2 hr (about 2 fold of the TNF-untreatedcontrol, p


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Application of Liposomes for Cancer Chemotherapy 365ADM. Relatively large quantity of ADM was incorporated into the liver andspleen by administration of either free ADM or ADM-Lip and small amount in thekidney and heart. ADM-Lip accumulated in spleen in higher extent than freeADM. Pretreatment with TNF did not affect the accumulation of either freeADM or ADM-Lip in all these organs.

To further manifest the effect of TNF on the targeting of ADM-Lip to tumor,mice bearing Meth-A tumor were treated with TNF or vehicle (control) followedby an i.v. injection of F-Lip 1 hr later. At 1 hr, F-Lip were observed only inintravascular space of tumor vessels in both TNF-treated and control mice.Fluorescein profiles in the tumor tissue were similar at 3 and 6 hr. In the tumortissue of control mice, tumor cells were tightly contacted with little interstitialarea, and the weak fluorescence was observed in localized areas of the tissue. Incontrast, tumor tissue of the the TNF-treated mice showed a loose orientation oftumor cells with wide interstitial spaces, and nuclei of the tumor cells showedpyknotic changes. In the lesions of tumor tissue, dense accumulation of afluorescent material (F-Lip) was observed, especially in intracellular space ofpyknotic cells.As TNF pretreatment resulted in the selective accumulation of liposomes intumor tissue, we examined whether TNF pretreatment really augment in in vivoantitumor effect of ADM-Lip or not. The effect of the drugs on tumor growth isshown in Fig. 3. In the control mice received vehicle or saline, tumor grewprogressively and all mice died within 41 days after the tumor transplantation.The treatment with ADM alone or ADM-Lip alone caused significant retardationof tumor growth, but it did not prolong the survival times of the mice as comparedto controls. The tumor nodules in mice receiving TNF alone blackened at thecentral area by 24 hr, and black scabs were formed at a part of, or all, area of the

Fig. 3. Effect of TNF pretreatment on the growth inhibitory effect of ADM-Lip.The treatment with TNF and ADM-LIP was performed in 2 cycles at daysshowing by arrows. (Cited from Ref, of Suzuki et al. 1990).


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tumor surface by 48 to 72 hr after the initial treatment. In these mice, tumor cellsthat remained in a rim of the necrotic tumor regrew to form a fresh tumor nodule.By contrast, the combination therapy with TNF and ADM-Lip with 1-hr interval(1 hr pretreatment with TNF) resulted in remarkable retardation of the tumorgrowth and led to 5 tumor-free mice out of 13 mice tested (p


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Application of Liposomes for Cancer Chemotherapy 367

fusion with lysosomes, leading to the release of CF from the vesicle showingdequenched fluorescence. Therefore, we assessed the endocytosis activity ofvarious tumor cell lines to HBJ127-IL by measuring dequenching of encapsulatedCF. In both KU-1, MKN-7 and T24 cells, CF-dequenching was manifested after20-30 min and it reached maximal level after 2 to 4 hr. Maximal fluorescenceintensity in these cells was about six fold higher than the initial level, whereas inMKN-45, SKBr-3, Molt-4 and K562 cells, it was about two fold of initial level.

The endocytosis and fluorescence dequenching activities of tumor cells wascompletely blocked by addition of colchicin. A well known lysosome inhibitor,NH4C1(Weinstain et al. 1984) allowed the internalization of CF-IL in part (60%as compared with control), but it strongly inhibited the CF-dequenching process.NaN3 and chloroquin also inhibited the dequenching at concentrations to inhibitendocytosis of cells (Djikstra et al. 1984).

Finally we visually observed the fate of CF-IL in tumor cells (Fig. 4). KU-1cells coated with HBJ127-CF-IL at 4C showed ring shaped weak fluorescence onthe cell-surface (Fig. 4a). Ten min after the incubation at 37C, scattering smallfluorecent particules was observed in peripheral part of cytoplasma (Fig. 4b).After 30 min incubation, this vesicular fluorescence became brighter and largerand to show perinuclear localization (Fig. 4c). Intensity of the cytosolic fluores-cence also increased at the time. These observation indicated that CF inliposomes was leaked by exposure of CF-IL to the acidic lysosomal environmentand distributed to cytosol. When cells were incubated in the presence of 10 mMNH4C1 for 1 hr, only perinuclear fluorescence were observed. By contrast, cellstreated with colchicin showed only cell-surface fluorescence. These observationswere accordance with the quantitative evalation of CF-dequenching bycytofluorometry. The internalization and the following processing of HBJ127-ILby KU-1 cells were as rapid as those in well-known ligand-receptor systems (Presset al. 1986).

Fig. 4. Internalization of HBJ127-CF-IL into KU-1 cells. a, Liposomes bound ontumor cell surface at 4C; b, after incubation at for 10 min 37C ; c, after 30min.


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AcknowledgmentsThis work was supported in part by a Grant for Special Project Research onBiosciences from the Ministry of Education, Science and Culture, Japan. Cancer

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R.S. & Falk, RE. (1989) Analysis of the effect of liposome encapsulation on thevesicant properties, acute and cardiac toxicities, and antitumor efficacy of doxor-ubicin. Cancer Chemother.Pharmacol., 23, 81-86.2) Brett, J., Gerlach, H., Nawroth, P., Steinberg, S., Godman, G. & Stern, D. (1989)Tumor necrosis factor/cachectin increases permeability of endothelial cellmonolayers by a mechanism involving regulatory G proteins. J. Exp. Med., 169,1977-1991.3) Dijkstra, J., Van Galen, M. & Scherphof, G.L. (1984) Effects of ammoniumchloride and chloroquine on endocytic uptake of lipsomes by kupffer cells in vitro.Biochim. Biophys. Acta, 804, 58-67.4) Gabizon, A., Dagan, A., Goren, D., Barenholz, Y. & Fuks, Z. (1982) Liposomes asin vivo carriers of adriamycin : Reduced cardiac uptake and preserved antitumoractivity in mice. Cancer Res., 42, 4734-4739.5) Goldblum, SE., Hennig, B., Jay, M., Yoneda, K. & McClain, C.J. (1989) Tumornecrosis factor a-induced pulmonary vascular endothelial injury. Infect. Immun.,57, 1218-1226.6) Hashimoto, Y., Sugawara, M., Masuko, T. & Hojo, H. (1983a) Antitumor effect ofactinomycin D entrapped in liposomes bearing subunits of tumor-specific mono-clonal immunoglobulin M antibody. Cancer Res., 43, 5328-5334.

7) Hashimoto, Y., Sugawara, M. & Endoh, H. (1983b) Coating of liposomes withsubunits of monoclonal IgM antibody and targeting of the liposomes. J. Immunol.Methods, 62, 155-162.8) Horvath, C.J., Ferro, T.J., Jesmok, G. & Malik, A.B. (1988) Recombinant tumornecrosis factor increases pulmonary vascular permeability independent of neutro-phils. Proc. Natl. Acad. Sci. USA, 85, 9219-9223.9) Masuko, T., Yagita, H. & Hashimoto, Y. (1984) Monoclonal antibodies against cellsurface antigens present on human urinary bladder cancer cells. J. Natl. CancerInst., 72, 523-530.10) Mayer, L.D., Bally, M.B. & Cullis, P.R. (1985) Uptake of antineoplastic agentsinto large unilamellar vesicles in response to a memarane potential. Biochim.Biophys. Acta, 816, 294-302.11) Press, OW., Vitetta, E.S., Farr, A.G., Hansen, J.A. & Martin, P.J. (1986) Evalua-tion of ricin A-chain immunotoxins directed against human T cells. CellularImmunol., 102, 10-20.12) Reynolds, J.A., Nozaki, Y. & Tanford, C. (1983) Gel-exclusion chromatograpgy on51000 Sephacryl : Application to phospholipid vesicles. Anal. Biochem.,130, 471-474.13) Suzuki, S., Ohta, S., Takashio, K., Nitanai, H. & Hashimoto, Y. (1990) Augmenta-tion for intratumoral accumulation and anti-tumor activity of liposome-encapsulated adriamycin by tumor necrosis factor-a in mice. Int. J. Cancer, 46,1095-1100.14) Tanaka, T., Suzuki, S., Masuko, T. & Hashimoto, Y. (1989) In vitro targeting andcytotoxicity of adriamycin in liposomes bearing monoclonal antibody against rat orhuman gpl25 cell proliferation-associated antigen. Jpn. J. Cancer Res., 80, 380-386.


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Application of Liposomes for Cancer Chemotherapy 36915) Van Hoesel, Q.G.C.M.,Steerenberg, P.A., Crommelin, D.J.A., van Dijk, A., van Oort,

W., Klein, S., Douze, J.M.C., de Wildt, D.J. & Hillen, F.C. (1984) Reducedcadiotoxicity and nephrotoxicity with preservation of antitumor activity of doxor-ubicin entrapped in stable liposomes in the LOU/M Wsl rat. Cancer Res., 44, 3698-3705.16) Weinstein, J.N., Ralston, E., Leserman, L.D., Klausner, R.D., Dragsten, P., Henkart,P. & Blumenthal, R. (1984) Self-quenching of carboxyfluorescein fluorescence :uses in studying liposome stability and liposome-cell interaction. In : LiposomeTechnologyIII, edited by G. Gregoriads CRC Press, Roca Baton, FL, p. 183.

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Basic Approach to Application of Liposomes.pdf