RAPID COMMUNICATION

Merocyanine 540-Sensitized Photoinactivation of Human Erythrocytes Parasitized by Plasmodium falciparum

By Orla M. Smith, Stephen A. Dolan, James A. Dvorak, Thomas E. Wellems, and Fritz Sieber

The purpose of this study was to evaluate the photosensitiz- ing dye merocyanine 540 (MC540) as a means for extracorpo- real purging of Plasmodium falciparum-infected erythro- cytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoi-

EROCYANINE 540 (MC540) is a photosensitizing M dye that has been used in preclinical models and a phase I clinical trial for the purging of leukemia and lymphoma cells from autologous bone marrow The virucidal properties of MC540 have prompted investiga- tions into its use as an agent for the inactivation of enveloped viruses in blood p r o d ~ c t s . ~

Plasmodium falciparum is the causative agent of the most serious form of malaria and is responsible for much morbidity and mortality in the tropics. Although normally transmitted by a mosquito vector, P fakiparum may also be transmitted through transfusion of infected blood from asymptomatic donor^.^-^ Here we report on the ability of MC540 to preferentially photosensitize red blood cells (RBCs) parasitized by either a chloroquine-resistant or a chloroquine-sensitive clone of P falcparum.

MATERIALS AND METHODS

Parasites. Chloroquine-resistant (Dd2) and chloroquine-sensi- tive (HB3) clones of P falcqpnmm* were maintained in vitro using modifications of the method of Trager and J e n ~ e n . ~ Parasitized RBCs were maintained as shallow layers in 75-cm2 tissue culture flasks at 37°C in an atmosphere of 90% N2, 5% C02, and 5% 0 2 .

Parasites were passaged by daily addition of fresh human RBCs (leukocyte-free, final cell density approximately 5 x 1O81mL) in RPMI 1640 growth medium (GIBCO, Grand Island, NY) supple- mented with N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES, 25 mmoliL), NaHC03 (28.6 mmol/L), hypoxanthine (360 KmoliL), gentamicin (10 pgimL), and pooled human serum (10% vol/vol). Leukocyte-free RBCs were prepared from a single donor using a Sephacell separation device (Baxter Healthcare/ Fenwall, Deerfield, IL).

Parasitized RBCs (3% to 5% para- sitemia, > 90% ring stage) were suspended in HEPES-buffered (10 mmol/L, pH 7.4) a-modified minimum essential medium (Sigma Chemical Co, St Louis, MO) supplemented with 12% of a selected lot of fetal bovine serum (Irvine Scientific, Santa Ana, CA) to yield hematocrits of 0.2%, 3%, or 15%, and placed into clear polystyrene tubes (16-mm diameter; Corning Glass Works, Corning, NY). MC540 (Kodak, Rochester, NY) was added from a 1 mg/mL stock solution in 50% ethanol to a final concentration of 15 wg/mL. Cell suspensions were then irradiated for up to 90 minutes at room temperature between two banks of five white fluorescent light bulbs (F20T12.CW; General Electric, Cleveland, OH) placed 28 cm apart and fitted with yellow acrylic filters to eliminate irradia- tion below 470 nm. The fluence rate at the sample site was approximately 26 W/m2 as determined by a United Detector Technology (Hawthorne, CA) power meter (model S351A equipped with detector model 262 and radiometric filter model 1158). Tubes were swirled manually every 10 minutes to prevent cell sedimenta-

MC54O-sensitized reactions.

etic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine- sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC54O-sensi- tized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood. o 1992 by The American Society of Hematology.

tion.’(’ Cell suspensions exposed to dye in the dark, to solvent (ethanol) and light for 90 minutes, or to neither dye nor light nor solvent served as controls.

Photosensitized and control cells were washed once with dye- free medium and aliquots containing a pretreatment concentration of 1 to 2 x IO7 parasitized cells were seeded into culture flasks containing 4 mL of growth medium supplemented with fresh erythrocytes (5 x lo8 cellslml). Cultures were monitored daily by microscopic examination of Giemsa-stained thin smears. Experi- ments were terminated when untreated control cultures reached 10% (ring stage) parasitemia.

Target erythrocytes at a hematocrit of 3% were exposed to MC540 and light for 15, 30, 45, or 90 minutes, washed once, resuspended in growth medium, and cocultured at a density of 108/mL with HB3-parasitized erythrocytes (107/mL) that had been enriched to 250% parasitemia (schizont stage) using the gelatin selection technique.” After 18 hours at 3TC, Giemsa- stained thin smears were prepared, and 1,000 erythrocytes were examined microscopically to determine the percentage of invaded cells.

Flow cytometry was used to quantify the concentration of parasitized (DNA-containing) erythrocytes be- fore and after MC540-sensitized photoinactivation. Erythrocytes were washed once with phosphate-buffered saline (PBS), fixed with 0.1% glutaraldehyde in PBS for 16 hours at 4“C, washed free of fixative, stained with Hoechst 33258 (10 pg/mL; Polysciences, Warrington, PA), and analyzed with a Coulter Epics V Cell Sorter (Coulter Electronics, Hialeah, FL). Normal, nonparasitized RBCs were used to determine background fluorescence. To ensure that the Hoechst stain technique for DNA was detecting parasitized cells, Hoechst 33258-positive cells were sorted and cytocentrifuged onto microscope slides, and stained with Giemsa for microscopic analysis.

Invasion assay.

Flow cytometry.

From the Department of Pediatrics, Medical College of Wisconsin, Milwaukee; and the Laborato y of Malaria Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.

Submitted February 23, 1992; accepted April 14,1992. Supported by Grant CA 42734 fvom the National Cancer Institute

and the MACC Fund. Address reprint requests to Orla M. Smith, PhD, Department of

Pediatrics, Medical College of Wisconsin, MACC Fund Research Center, 8701 Watertown Plank Rd, Milwaukee, WI 53226.

The publication costs of this article were defvayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1992 by The American Society of Hematology. 0006-4971 192/8001-0040$3.0010

Blood, VOI 80, NO 1 (July I), 1992: PP 21-24 21

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22 SMITH ET AL

Fluorescence microscopy. Parasitized FU3Cs were exposed to MC540 and light for 15 minutes, washed, and resuspended in growth medium supplemented with 10 pg/mL Hoechst 33342 (Polysciences, Warrington, PA). Wet mounts were prepared using silicon oil as the mounting medium. Cells were visualized using an Axiophot microscope (Zeiss, Thornwood, NY) equipped for both transmitted differential interference contrast (DIC) and reflected fluorescence microscopy. DIC observations were performed at 590 nm. M a 4 0 was excited at 485 nm and its fluorescence visualized with a 515-565 nm barrier filter; Hoechst 33342-stained DNA was excited at 365 nm and visualized with a 397 nm barrier filter. Images were projected onto the faceplate of a Sony XC-77 CCD video camera (Sony Component Products Division, Cypress, CA). Video images #ere acquired with a DT2861 Arithmetic Frame Grabber (Data Translation, Marlboro, MA) operating in an IBM PC AT-compatible computer (ZEOS International, St Paul, MN). Image files were converted into tagged image file format for processing with Halo f/x (Media Cybernetics, Silver Spring, MD) and then converted to photographic prints with a LFR film recorder (Lasergraphics, Irvine, CA).

RESULTS

Exposure to MC540 and light inactivated the malaria parasite in a dose-dependent manner (Table 1). The chloroquine-resistant (Dd2) and the chloroquine-sensitive clones (HB3) of P falcipamm were equally susceptible to MC54O-sensitized photoinactivation (Table 1). The rate of dye-sensitized photoinactivation of the parasites was in- versely proportional to the density (hematocrit) of the cell suspension (Table 1).

Flow cytometric analyses of Hoechst 33258-stained eryth- rocytes confirmed that the frequency of parasitized (DNA- containing) cells was drastically reduced after exposure to MC540 and light (Table 2). When untreated, parasitized RBCs were sorted and analyzed by light microscopy, 85% (HB3) and 91% (Dd2), respectively, of all cells in the intensely staining fraction were positively identified as parasitized cells. Fluorescence microscopy also confirmed that the parasitized cells in a 7.2% parasitemic RBC preparation bound more dye than the nonparasitized cells (Fig 1). MC540 fluorescence was primarily associated with the parasite. The plasma membrane of parasitized host cells was dimly fluorescent whereas the plasma membrane of nonparasitized cells was virtually nonfluorescent (Fig 1).

When normal RBCs were exposed to MC540 and light

Table 2. Frequency of Parasitized (DNA-containing) Erythrocytes Before and After Exposure to MC540 and Light

Fluorescent Cells/ 1 0 Cells Analyzed

Cells Treatment HB3 Dd2

Parasitized None 55,590 102,721 Parasitized Dye, light 30 min 990 1,283 Parasitized Dye, light 90 min 136 100 Nonparasitized None 0 0

~ ~ ~ ___ ~~

Data are from a single determination (representative of two experi- ments).

for 45 minutes and then cocultured with HB3-parasitized erythrocytes, the invasion of RBCs by the parasite was close to normal (Table 3). However, when treatment with dye and light was extended to 90 minutes, invasion was inhib- ited by 45% (Table 3), suggesting that photodynamic damage to the cell surface of target erythrocytes interfered with parasite invasion.

DISCUSSION

In industrialized countries, concerns about the safety of the blood supply focus on the transmission of pathogenic viruses. In Third World countries where malaria and other protozoan diseases are endemic, the transmission of para- sites presents an equally serious problem. We have recently shown that simultaneous exposure to MC540 and light inactivates a wide range of enveloped viruses including the human T-cell leukemia/lymphoma virus type I, the human immunodeficiency virus and cytomegalovirus, but is rela- tively well tolerated by RBCs, pluripotent hematopoietic stem cells, factor VIII, and von Willebrand f a ~ t o r . ~ In this communication, we report that exposure to the same doses of MC540 and light also inactivates chloroquine-sensitive and chloroquine-resistant clones of P falcipamm.

Parasite invasion is known to induce rather drastic changes in all major components (lipids, proteins, carbohy- drates) of the erythrocyte plasma membrane.12 These parasite-induced surface alterations were probably respon- sible for the increased binding of MC540 by parasitized RBCs. In our studies, MC540 fluorescence was associated with the parasite and, to a lesser extent, with the plasma membrane of the host cell. By contrast, Kara et all3 had

Table 1. MC540-Sensitized Photoinactivation of Clones HB3 and Dd2 of Pfalciparum ~~

Parasitemia ( O h )

Treatment HB3 Dd2

0.2% Hct 3% Hct 15% Hct 0.2% Hct 3% Hct 15% Hct

None 9.8 f 0.5 9.8 f 2.1 10.3 5 1.2 8.6 2 1.6 13.2 f 2.7 10.0 f 2.1 Ethanol, light 90 min 10.7 f 1.1 8.2 f 0.8 9.6 5 0.5 10.6 2 1.7 12.6 f 2.2 8.6 2 2.2 Dye, dark 10.4 f 1.6 9.3 f 1.6 10.5 5 1.6 8.5 2 1.1 11.1 f 1.3 8.8 f 0.9 Dye, light 15 min 6.2 f 4.5 6.1 f 1.9 9.9 f 2.1 5.0 2 1.9 9.0 f 2.1 8.1 f 0.9 Dye, light 30 min 0.3 f 0.4 3.2 f 1.3 8.8 f 1.8 0.8 f 1.3 7.5 f 2.2 7.2 f 1.1 Dye, light 45 min 0 1.5 f 0.5 6.5 -c 1.4 0 3.9 2 1.4 6.5 f 0.9 Dye, light 90 min 0 0.1 f 0.1 2.6 f 0.9 0 0.1 f 0.1 3.1 f 0.7

Erythrocytes parasitized by the HB3 or the Dd2 clone of Pfalciparum (3% to 5% parasitemia; >90% ring stage) were exposed to MC540 and light for 15, 30, 45, or 90 minutes at hematocrits (Hct) of 0.2%. 3%, or 15%. and cocultured with fresh uninfected erythrocytes. Forty-eight hours later (when control cultures had reached approximately 10% parasitemia), the degree of parasitemia was determined by examining Giemsa-stained thin smears. Data represent means f SD of three experiments.

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PHOTOINACTIVATION OF PLASMODIUM FALCIPARUM 23

Fig 1. Erythrocytes parasitized bvPf8/cip8rum (clone Dd2; 7.2% parasitemia). Cells were exposed to MC540 (15 pg/mL) and light (26 W/m2, 15 minutes), washed, and counterstained with Hoechst 33342 (10 pg/mL). (A) Differential interference contrast. (B) MC540 fluorescence, excited at 485 nm and visualized with a 515- to 565-nm barrier filter. (C) Hoechst 33342-induced DNA fluorescence, excited at 365 nm and visualized with a 397-nm barrier filter. Bar, 10 pm.

Table 3. Invasion of MCWO-Treated and Untreated Erythrocytes by HB3 Parasites

Treatment

None Ethanol, light 90 min Dye. dark Dye, light 15 min Dye, light 30 min Dye, light 45 min Dye, light 90 min

Invasion Rate ( O h of total population)

30 f 4.4 35 f 0.3 29 f 2.4 31 f 2.6 27 f 1.2 25 f 3.3 16 f 0.3

Invasion Rate ( O h control)

100 118 f 16.5 97 f 6.2 104 f 6.4 91 f 17.3 84 f 1.6 55 f 6.9

Target erythrocytes (3% hematocrit) were exposed to MC540 and light and then cocultured with HB34nfected erythrocytes (51% para- sitemia, >90% schizont stage) at a ratio of 1O:l. After 18 hours, Giemsa-stained smears were prepared, and 1,000 erythrocytes were enumerated to determine the degree of parasitemia. Data represent means f range of two experiments.

reported that MC540 preferentially binds to the parasitopho- rous vacuole membrane of intracellular P falcipamm while Sherman and Greenan14 found that MC540 was primarily associated with the plasma membrane of the host cell. However, the staining of cells by MC540 is known to be affected by seemingly minor differences in experimental condition^.^^ Variations in type, concentration, or lot of serum or the radiation dose could easily account for reported differences in the staining of P falcipamm-infected cells. As a cell's susceptibility to MC540-sensitized photoly- sis is primarily determined by the amount of dye it binds,

greater dye binding was probably the major reason for the enhanced photosensitivity of parasitized RBCs.IS

Erythrocytic stages of the malaria parasite are known to be vulnerable to oxidative stress,16 and the cytotoxic and antiviral activities of MC540 are at least in part mediated by singlet oxygen (lO2).l7-I9 It is reasonable to speculate that IO2 may also play a role in the inactivation of P falcipamm. The nearly 1,000-fold reduction in parasitized cells shown in Table 2 may underestimate the antiprotozoan activity of MC540-sensitized photoirradiation because samples were fixed immediately after the photoirradiation step whereas the MC540-mediated photolysis of cells is generally not completed until several hours post photoirradiation.2°

MC540 has a low acute systemic toxicity, is rapidly cleared from the bloodstream, does not sensitize the skin of the recipient, and has tested negative in two standard mutagenicity tests.21J2 Because of the significant overlap between the absorption spectrum of MC540 and the absorp- tion spectrum of hemoglobin, RBCs have to be treated either as dilute suspensions or as thin films to allow adequate light penetration. This limitation may be over- come by using one of the recently described structural analogues of MC540 that absorb at longer wavelengths and match or exceed the antiviral and cytotoxic potency of MC540 while retaining its se lec t i~ i ty .~~

ACKNOWLEDGMENT

We thank Patrick McFadden and David Stephaney for flow cytometric analyses and Louis Miller and Gary Ward for helpful discussions.

REFERENCES 1. Sieber F, Spivak JL, Sutcliffe AM: Selective killing of leuke-

mia cells by merocyanine 540-mediated photosensitization. Proc Natl Acad Sci USA 81:7584,1984

2. Sieber F, Stuart RK, Rowley SD, Sharkis SJ, Sensenbrenner L L Dye-mediated photolysis of normal and neoplastic hematopoi- etic cells. Leuk Res 11:43,1987

3. Sieber F: Extracorporeal purging of bone marrow grafts by dye-sensitized photoirradiation, in Gee AP (ed): Bone Marrow Processing and Purging: A Practical Guide. Boca Raton, FL, CRC, 1991, p 263

4. O'Brien JM, Montgomery RR, Burns WH, GalTney DK, Sieber F Evaluation of merocyanine 540-sensitized photoirradia- tion as a means to inactivate enveloped viruses in blood products. J Lab Clin Med 116:439,1990

5. Grant DB, Perinpanayagam MS, Shute PG, Zeitlin RA: A case of malignant tertian malaria after blood-transfusion. Lancet 2469,1960

6. Chojnacki RE, Brazinsky JH, Barrett 0: Transfusion- introduced fulcipancm malaria. N Engl J Med 279:984,1968

For personal use only.on January 9, 2019. by guest www.bloodjournal.orgFrom

24 SMITH ET AL

7. Espinal CA, DeMorales P: Indirect ELISA test for malaria in blood donors. Trans R SOC Trop Med Hyg 78:645,1984

8. Wellems TE, Panton U, Gluzman IY, do Rosario VE, Gwadz RW, Walker-Jonah A, Krogstad D J Chloroquine resistance not linked to mdr-like genes in a Plasmodium f a l c i p m cross. Nature 345253,1990

9. Trager W, Jensen JB: Human malaria parasites in continuous culture. Science 193:673,1976

10. Smith OM, Traul DL, McOlash L, Sieber F Evaluation of merocyanine 540-sensitized photoirradiation as a method for purging malarially infected red cells from blood. J Infect Dis 163:1312,1991

11. Jensen JB: Concentration from continuous culture of eryth- rocytes infected with trophozoites and schizonts of Plasmodium falcipamm. Am J Trop Med Hyg 27:1274,1978

12. Howard RJ: Alterations in the surface membrane of red blood cells during malaria. Immunol Rev 61:67,1982

13. Kara UAK, Stenzel DJ, Ingram LT, Kidson C The parasito- phorous vacuole membrane of Plasmodium falcipamm: Demonstra- tion of vesicle formation using an immunoprobe. Eur J Cell Biol 46:9,1988

14. Sherman IW, Greenan JRT: Altered red cell membrane fluidity during schizogenic development of malarial parasites (Plasmodium falcipamm and P. lophurae). Trans R SOC Trop Med Hyg 78:641,1984

15. Sieber F Merocyanine 540 (yearly review). Photochem Photobiol46:1035,1987

16. Clark IA, Chaudhri G, Cowden WB: Some roles of free radicals in malaria. Free Rad Bioi Med 6:315,1989

17. Kalyanaraman B, Feix JB, Sieber F, Thomas JP, Girotti AW Photodynamic action of merocyanine 540 on artificial and natural cell membranes: involvement of singlet molecular oxygen. Proc Natl Acad Sci USA 84:2999,1987

18. O'Brien JM, Gaffney DK, Wang TP, Sieber F Merocyanine 540-sensitized photoinactivation of enveloped viruses in blood products: Site and mechanism of phototoxicity. Blood 80:277,1992

19. O'Brien JM, Singh RJ, Feix JB, Kalyanaraman B, Sieber F Action spectra of the antileukemic and antiviral activities of merocyanine 540. Photochem Photobiol54851,1991

20. GafTney DK, Schober SL, Sieber F Merocyanine 540- sensitized photoinactivation of leukemia cells: Role of oxygen and effects on plasma membrane integrity and mitochondrial respira- tion. Exp Hematol18:23,1990

21. Sieber F Dye-mediated photolysis of tumor cells: Implica- tions for autologous bone marrow transplantation, in Gale RP, Champlin R (eds): Recent Advances in Bone Marrow Transplanta- tion. New York, NY, Liss, 1987, p 865

22. O'Brien JM, Sieber F Mutagenicity of merocyanine 540- mediated photosensitization. Exp Hematol17:166,1989

23. Giinther WHH, Searle R, Sieber F Structure-activity rela- tionships in the antiviral and antileukemic photoproperties of merocyanine dyes. Semin Hematol2988,1992

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1992 80: 21-24  

OM Smith, SA Dolan, JA Dvorak, TE Wellems and F Sieber parasitized by Plasmodium falciparumMerocyanine 540-sensitized photoinactivation of human erythrocytes 

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