Cytotoxic activity of daunomycin and adriamycin encapsulated in immunoliposomes against avian myeloblastosis virus-infected cells

Journal of Virological Methods, Elsevier

131

19 (1988) 131-140

JVM 00688

Cytotoxic activity of daunomycin and adriamycin encapsulated in immunoliposomes against avian myeloblastosis virus-infected cells K.V.R.

Dhananjaya’.2

and A. Antony

‘Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore, India; 2Tumour Biology Laboratory, Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A. (Accepted

30 November

1987)

Summary

Immunoliposomes were prepared using the antibody raised against the avian myeloblastosis virus envelope glycoprotein, gp80. Adriamycin was encapsulated into immunoliposomes. More drug was delivered into target cells when the drug encapsulated in immunoliposomes was incubated with the cells. The drug encapsulated in immunoliposomes was able to inhibit the RNA synthesis twice more than free drug in the virus-transformed myeloblasts. Pre-treatment of cells with ammonium chloride, reversed the effect of drug encapsulated in immunoliposomes. The drugs encapsulated in immunoliposomes had marginal effect on the RNA synthesis of non-target cells, the yolk sac cells. Colony formation by virus-transformed cells and focus formation by virus-infected yolk sac cells was inhibited significantly by the drug encapsulated in immunoliposomes. Daunomycin; Adriamycin; Virus-transformed cells

Immunoliposomes;

Drug targeting; Cytotoxic activity;

Introduction

Most of the known anticancer agents have been found to be toxic to normal cells. The anthracycline antibiotics daunomycin and adriamycin were found to inhibit the growth of severe tumour cells in vitro (Di Marco, 1975). However, these antiCorrespondence too: K.V.R. Dhananjaya, Tumour Biology Laboratory, search Center, 1124 Columbia Street, Seattle, WA 98104, U.S.A.

0166-0934/88/$03.50

0

1988 Elsevier

Science

Publishers

B.V. (Biomedical

Fred Hutchinson

Division)

Cancer

Re-

132

biotics were shown to cause acute ~ardiotoxicity in vivo (Von Hoff et al., 197X). Liposomes have been shown to be good carriers of drugs as they minimize the toxic effects of encapsulated drug (Gregoriadis and Allison, 1980). Free liposomes however, are not target specific. In order to deliver the drugs specifically into target cells, several methods have been developed to attach cell specific antibodies to liposomes for the preparation of ‘immunoliposomes’ or antibody targeted liposomes (Martin and Papahadjopoulos, 1982; Huang et al., 1982; Heath et al., 1984; Torchillin et al., 1985). The effects of drugs encapsulated in immunoliposomes on the target cells have been reported (Machy et al., 2982; Huang et al., 1983; Heath et al.. 1983 and Matthay et al.. 1984). However, all these studies have been carried out in chemically induced tumors or spontaneous tumors of animal and human origin. In the above studies with immunoliposomes, methotrexate was used as an antitumor agent. Although extensive work has been carried out on the effects of daunomycin and adriamycin on several tumour cells, very little is known about their effects on virus-transformed cells. In this paper, the uptake and intracellular effects of the drugs encapsulated in immunoliposomes are described. The effect of the targeted drugs on the focus formation of the virus-transformed cells is also described.

Materials and Methods

Medium 199, tryptose phosphate broth and trypsin were obtained from Hindustan Dehydrated Media, Bombay, India. Sterile culture dishes (60 mm) were from A/S Nunc, Roskilde, Denmark and Becton and Dickinson, Oxnard, U.S.A. [“H]U ri d ine (sp eci fic activity 9700 mCi/mmol) was purchased from Bhabha Atomic Research Centre, Bombay, India. All other chemicals used were of analytical grade. Virus

BAI-A strain of Avian laboratory, was provided ology, Hyderabad, India. (Karnat~ka State Poultry

myeloblastosis virus originally obtained from J .W. Beards by Dr. M.R. Das, Centre for Cellular and Molecuiar BiThe virus was grown in leukosis-free white leghorn chicks Farm, Bangalore, India).

Methods Preparation of immunoliposomes

and encapsulation of drugs

Immunoliposomes were prepared using phosphatidyl choline:cholesterol:stearylamine in a molar ratio of 51:21:15 and palmitoylated antibody rabbit anti AMV gp80 as described by Huang et al. (1982). Adriamy~in was encapsulated into immunoliposomes and separated as described by Shen et al. (1982).

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AMV-transformed myeloblast cell culture AMV-transformed myeloblasts derived from leukaemic described earlier by Dodge and Moscovici (1973). Yolk sac cell culture Yolk sac cells derived from 13-day-old embryonated as described by Moscovici and Moscovici (1973).

chicks were cultured

chicken

as

eggs were cultured

Uptake of daunomycin by virus-transformed cells To determine the uptake of daunomycin by the virus-transformed cells the following samples (i) daunomycin; (ii) daunomycin encapsulated in free liposomes and (iii) daunomycin encapsulated in immunoliposomes, were incubated separately with 1 x lo6 myeloblasts in 1 ml culture medium. The drug level in all the cases was 20 mgiml. Cells were pelleted down at various intervals, washed with PBS, lysed with 1% SDS and extracted with 0.3 N HCl containing 50% ehtanol (Bachur et al., 1970). Daunomycin thus extracted was excited at 470 nm and the emission was monitored at 570 nm in a Shimadzu RF 540 fluorescence emission spectrometer. The amount of daunomycin in the samples was determined from a standard emission plot. Effect of daunomycin and adriamycin on RNA synthesis of normal and virus-transformed cells Myeloblasts (2 x 10”iml) in culture medium were incubated separately with various concentrations of free drug, drug encapsulated in free liposomes and drug encapsulated in immunoliposomes for 3 h at 37°C. [3H]-uridine (S&i) was then added to each tube and incubated further at 37°C in a shaker water bath for 16 h. The medium was discarded after centrifugation, cells were washed with cold PBS, and lysed with 1 ml of TM buffer (100 mM Tris-HCl, pH 7.4, 1mM MgCl,) containing 1% SDS. The tubes were vortexed, chilled 10% TCA was added and kept at 4°C for 2 h. The TCA insoluble precipitate was collected on Whatman GFiC filters, washed extensively with 10% TCA, 5% TCA, water, ethanol and ether. The filters were dried and radioactivity was determined in a LKB rackbeta counter using toluene based scintillant. In a separate set of experiments, cells were pretreated with 5 mM ammonium chloride for 30 min at 37°C and then incubated with immunoliposomes carrying various amounts of drugs at 37°C for 3 h. The cells were processed further as described above. Identically treated triplicates were used in each experiment. The mean values were plotted in the graph after calculation of standard error. Focus assay Secondary yolk sac cells (0.5 x 10”) were seeded into culture dishes and incubated at 37°C for 3 h in a water-jacketed CO, incubator. Cells were infected with AMV (1 x lO1” particles) for 18 h at 37°C. The cells were incubated with various concentrations of drug samples as mentioned above for 3 h at 37°C and overlaid with 4 ml of medium containing agarose. The agarose medium (100 ml) consisted

134

of 55 ml of 2 X Medium 199, 10 ml of 6% tryptose phosphate

broth, 5 ml of calf serum, 5 ml of chicken serum, 25 ml of 2.5% agarose, 200 U/ml penicillin, 200 kg/ml streptomycin and 200 kg/ml gentamycin. The overlay was allowed to solidify at room temperature for 5 min and the culture dishes were incubated at 37°C. A second overlay with 4 ml of the same medium was made on the sixth day and foci were counted on the fourteenth day. Before scoring for foci, 1 ml culture medium was added to the dishes and incubated at 37°C for 30 min (Moscovici et al., 1975). Identically treated triplicates were used in each experiment. Mean values were plotted after calculation of standard error. Colony assay of AMV-transformed

myeloblasts

The method used was described by Dodge and Moscovici (1973). Myeloblasts (0.5 x 106) were incubated with various concentrations of drugs for 4 h at 37”C, suspended in 4 ml of soft agarose (0.36% in myeloblast culture medium) and added to a basal layer of hard agarose (0.72% in myeloblast culture medium) in 60 mm culture dishes. After allowing the medium to solidify at room temperature, the culture dishes were incubated at 37°C in a water-jacketed CO2 incubator. The colonies were counted after 14 days. Identically treated triplicates were used in each experiment.

Mean

values

were plotted

after calculation

of standard

error.

Results Uptake of targeted and non-targeted drugs by myeloblasts The time kinetics of uptake of daunomycin as free drug, encapsulated in free liposomes and of daunomycin encapsulated in immunoliposomes are shown in Fig. 1. The results indicate that immunoliposomes deliver more drug into the myeloblasts than free liposomes.

Time

(h)

Fig. 1. Uptake of daunomycin by virus-transformed myeloblasts. A = Free daunomycin; nomycin encapsulated in free liposomes: l = daunomycin encapsulated in immunoliposomes. values of triplicate samples were plotted.

n

= dauMean

135

0’

I

1

I

I

I

2 Drug

1

I

3

I

4

(j-q)

Fig. 2. Effect of free drug and drug encapsulated in immunoliposomes on the RNA synthesis of yolk sac cells. Mean values of triplicate samples were plotted. 0 = Free adriamycin; o = Adriamycin encapsulated in immunoliposomes; A = free daunomycin; LI = Daunomycin encapsulated in immunoliposomes. The value (100%) in control is 4595 cpm.

Effect of adriamycin and daunomycin on the RNA synthesis in yolk sac virus-transformed myeloblasts Adriamycin and daunomycin at 4 kg concentration inhibited the RNA in yolk sac cells to 83 and 78%, respectively. When drugs encapsulated noliposomes were used at the same concentration only 10% of the RNA

Adriamycin

cells and synthesis in immusynthesis

(pg)

Fig. 3. Effect of adriamycin on the RNA synthesis in virus-transformed myeloblasts. Mean values of triplicate samples were plotted. n = Free adriamycin; l = Adriamycin encapsulated in free liposomes; A = Adriamycin encapsulated in immunoliposomes. The value (100%) in control is 3923 cpm.

136

100

80 P 2

0 ”

60

‘ii %. 8 40 E 2 x

20

Drug +g) Fig. 3. Effect of ammonium chloride on the inhibition of RNA synthesis in myeloblasts by adriamycin Mean values of triplicates were (0. 0) and daunomycin (~3. A) encapsulated in immunoliposomes. chloride treatment. plotted, 0. L% = with ammonium chloride treatment. 0. A = without ammonium The value (100%) in control is 3680 cpm.

was inhibited (Fig. 2). In virus-transformed myeloblasts, the inhibition of RNA synthesis by adriamycin encapsulated in immunoliposomes was found to be more significant than that observed in the case of free drug or the drug encapsulated in free liposomes (Fig. 3). When encapsulated in immunoliposomes, as low as 1 kg of drug was able to inhibit 50% RNA synthesis in myeloblasts. Similar results were obtained when the experiment was carried out using daunomycin (data not shown).

2.5

5.0

7.5

10.0

Adriamycincpg) Fig. 5. Effect of adriamycin on the colony formation by virus-transformed myeloblasts. Mean values l = Adriamycin encapsulated in free liposomes; n of triplicates were plotted. A = Free adriamycin; = Adriamycin encapsulated in immunoliposomes. In the control 85 colonies were observed.

137

2

4

Incubation

6

8

time(h)

Fig. 6. Time kinetics of inhibition of colony formation. Mean values of triplicates were plotted. w = Free adriamycin; l = Adriamycin encapsulated in free liposomes; A = Adriamycin encapsulated in immunoliposomes. In the control 82 colonies were observed.

Effect of ammonium chloride on the inhibition of RNA synthesis in formed myeloblasts by adriamycin and daunomycin When virus-transformed myeloblasts were pre-treated with 5mM chloride, the inhibition of RNA synthesis by the drugs encapsulated liposomes was arrested to the extent of 90% (Fig. 4). In the untreated RNA synthesis was inhibited to 90%.

Adriamycin

virus-transammonium in immunocontrol the

(pg)

Fig. 7. Inhibition of focus formation of AMV-infected yolk sac cells. Mean values of triplicates were plotted. n = Free adriamycin; l = Adriamycin encapsulated in free liposomes; A = Adriamycin encapsulated in immunoliposomes.

138

Effect of daunomycin and adriamycin on colony formation of virus-trunsformed myeloblasts At 5 kg concentration the drug encapsulated in immunoliposomes inhibited 70% of colony formation by virus-transformed cells. On the other hand, the free drug inhibited 40% of colony formation (Fig. 5). In a time course experiment, drugs encapsulated in immunoliposomes exhibited significant cytotoxicity at all the time points tested (Fig. 6). Drugs encapsulated in free liposome had less effect. Similar results were also obtained with daunomycin (data not shown). Effect of daunomycin and adriamycin on the focus formation by virus in yolk sac cells Adriamycin at 5 kg inhibited 80% of the focus formation by yolk sac cells transformed by AMV (Fig. 7) whereas free drug inhibited 35% of focus formation at the same concentration. Drug encapsulated in free liposomes had a little effect on focus formation by virus as compared to immunoliposomes. Similar results were also obtained with daunomycin (data not shown).

Discussion The anthracycline antibiotics daunomycin and adriamycin were found to be very effective anti-cancer agents, especially against leukemia (Di Marco, 1975; Von Hoff et al., 1978). In addition to their anti-cancer properties, these antibiotics caused acute cardiotoxicity and cardiac failure when injected into animals or human beings (reviewed by Von Hoff et al., 1978). Hence there is a necessity to develop a specific drug delivery system which can kill the target cells without appreciably damaging normal cells. Though the antiviral activity of daunomycin against herpes simplex (Di Marco et al., 1968), Vaccinia and Polio viruses (Cohen et al., 1969) is known. not much is known on the effect of these drugs against retroviruses and virus-transformed cells, except the inhibition of focus formation by murine sarcoma virus in mouse embryonal cells (Cassaza et al., 1972). We have prepared immunoliposomes using rabbit anti AMV gp80 antibody, characterized these liposomes and their interaction with virus-transformed cells (manuscript under preparation). Since no specific drug delivery system is available for antiviral drugs at present, we explored the possibility of using immunoliposomes (Huang et al.. 1982; Heath et al., 1984) for their use in the antiviral chemotherapy. As a first step towards these studies we studied the effect of drugs encapsulated in immunoliposomes against virus-infected cells in culture. In the present studies, we observed that more drug was delivered into target cells when drugs were encapsulated in immunoliposomes. The increased cytotoxicity of drug correlated with the amount of drug delivered into cells. Ammonium chloride which increases the pH of lysosomes (Ohkuma and Poole, 1978) reversed the effect of drug encapsulated in immunoliposomes on the RNA synthesis of virustransformed cells. The lysosomal enzyme system, after treatment with ammonium

139

chloride, may not be active to degrade the liposomes due to the increased internal pH. Alternatively, increased pH of lysosome may not allow the weakly acidic drug molecule to escape from lysosomes (De Duve et al., 1974). Similar effects of ammonium chloride on the inhibition of nucleic acid synthesis by methotrexate encapsulated in immunoliposomes were reported (Heath et al., 1983 and Matthay et al., 1984). The drugs encapsulated in immunoliposomes had marginal effect on the RNA synthesis of normal cells. Recently, Onuma et al. (1986) reported that adriamycin exhibited increased cytotoxicity against bovine leukemia cells when encapsulated in immunoliposomes as compared to free drug. The present studies have revealed that immunoliposomes are better vehicles for drug delivery than free liposomes. The targeted drugs inhibited the colony formation by virus-transformed cells and focus formation by AMV in yolk sac cell cultures suggesting their possible use in chemotherapy in vivo.

Acknowledgements

This work was supported by a grant from the Department of Science and Technology, Government of India. KVRD is thankful to the Indian Institute of Science, Bangalore for a fellowship. References Bachur, N.R., Leon Moore, A.. Bernstein, J.G. and Liu, A. (1970) Tissue distribution and disposition of daunomycin in mice: fluorometric and isotopic methods. Cancer Chemother. Rep. part 1, 54, 89-94. Cassazza. A.M., Silvestrini, R. and Gambarucci, R. (1972) Activity of daunomycin and adriamycin and some other daunomycin derivatives on murine sarcoma virus (Moloney) MSV. Eur. J. Clin. Biol. Res. 17, 622-630. Cohen, A., Hartley, E.M. and Ress, K.E. (1969) Antiviral effect of daunomycin. Nature 222, 36-38. De Duve, C., De Barsy, T., Poole, B., Trouet, A., Tulkens, P. and Van Hoof, F. (1974) Lysosomotropic agents. Biochem. Pharmacol. 23, 2495-2531. Di Marco, A. (1975) Adriamycin (NSC-123127): Mode and mechanism of action. Cancer Chemother. Rep. Part 3, 6, 91-106. Di Marco, A.. Terni, M., Silvestrini, R., Scarpinato, B., Boglioli, E. and Antonelli, A. (1968) Effect of daunomycin on Herpes Virus hominis in human cells. Giorn Microbial. 16, 25-35. Dodge, W.H. and Moscovici, C. (1973) Colony formation by chicken hematopoietic cells and virus induced myeloblasts. J. Cell. Physiol. 81, 371-386. Gregoriadis, G. and Allison, A.C. (Eds) (1980) Liposomes in Biological Systems. John Wiley and Sons Ltd.. Chichester. Heath, T.D., Montgomery, J.A., Piper, J.R. and Papahadjopoulos, D. (1983) Antibody-targeted liposomes: increase in specific toxicity of methotrexate-y-asparate. Proc. Nat]. Acad. Sci. U.S.A. 80, 1377-1381. Heath, T.D., Bragman, K.S., Matthay, K.K., Lopez-straubinger, G. and Papahadjopoulos, D. (1984) Antibody targeted liposomes: the development of cell specific cytotoxic agent. B&hem. Sot. Trans. 12, 34@342. Huang, A., Tsao, Y.S.. Kennel, S.J. and Huang, L. (1982) Characterization of antibody covalently coupled to liposomes. Biochim. Biophys. Acta 716, 140-150. Huang, A., Kennel, S.J. and Huang, L. (1983) Intractions of immunoliposomes with target cells. J. Biol. Chem. 258, 14034-14040.

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