Hypersensitization of Multidrug Resistant Human Ovarian Carcinoma

Bioconjugate Chem. 1996, 7, 209−216

209

Hypersensitization of Multidrug Resistant Human Ovarian Carcinoma Cells by Pluronic P85 Block Copolymer Valery Yu. Alakhov,*,† Elizaveta Yu. Moskaleva, Elena V. Batrakova,‡ and Alexander V. Kabanov*,‡ Moscow Institute of Biotechnology, Inc., and Russian Research Center of Molecular Diagnostics and Therapy, Simpheropolskii Boulevard 8, Moscow 113149, Russia. Received September 11, 1995X

The chemosensitizing effects of Pluronic P85 block copolymer were studied using two human ovarian carcinoma sublines: the glycoprotein P (P-gp) multidrug resistant (MDR) SKVLB cells and non-MDR SKOV3 cells. The dramatic increase (up to 700 times) in the daunorubicin cytotoxic activity was observed in the presence of 0.01% (22 µM) to 1% (2.2 mM) copolymer in the case of SKVLB cells. By contrast, the copolymer induced a less than 3-fold increase in the drug activity in SKOV3 cells. As a result, the MDR subline demonstrated much higher response (“hypersensitivity”) to the daunorubicin/ Pluronic compared to that of the non-MDR cells. The copolymer increased the cytotoxic effects of other MDR type drugs (doxorubicin, epirubicin, vinblastine, and mitomycin C) by a factor of 20-1000 and non-MDR type drugs (methotrexate and cisplatin) by a factor of 2-5.5. The daunorubicin influx in the cytoplasm and nuclei of SKVLB cells was also increased in the presence of the copolymer, while in SKOV3 cells, it remained practically unchanged. However, the hypersensitization of the MDR cells by the copolymer could not be merely explained by the P-gp modulation. Therefore, the possible role of the copolymer in inhibition of non-P-gp drug resistance is hypothesized, which may also explain the sensitization of MDR cells with respect to non-MDR type drugs as well as sensitization of parental cells. The concentration dependence of the IC50 in MDR cells indicates that just the copolymer unimers are responsible for the hypersensitization effect. The results obtained suggest that Pluronic P85 can be used as a delivery system to enhance the activity of antineoplastic agents against MDR tumors.

Multidrug resistance (MDR) is often found in many types of human tumors that have relapsed after an initial favorable response to drug therapy (1, 2). The sensitivity of the MDR tumor cells to antineoplastic agents can decrease significantly (2), which hinders the efficacy of these drugs in tumor therapy. One mechanism for the appearance of MDR is a positive selection of cells with an amplified family of closely related genes encoding glycoprotein P (P-gp), a 170 kDa transmembrane protein that functions as an ATP-dependent drug efflux pump (3-7). In certain cases, up to 100-fold overexpression of P-gp in MDR cells has been observed (8). An increased expression of this protein leads to a lowered net accumulation of the drug in the cell and consequently to a decrease in drug effect toward MDR cells. One current approach used to overcome MDR in cancer cells is the administration of antineoplastic agents concurrent with agents that can modulate P-gp. Among the known MDR modulatory agents are P-gp inhibitors such as verapamil and SDZ PSC-833 that specifically bind with P-gp and inhibit its function (9). Another class of MDR inhibitors that has recently attracted attention is natural and synthetic surfactants. In particular, the ability of certain liposome formulations to decrease in vitro the resistance of MDR cells with respect to liposomebound drugs has recently been reported (9-15). The nonionic polyethoxylated surfactants, such as Cremophor,

Tween 80, and Solutol HS15, have also been used to modulate MDR (16-18). These reports suggest that various surfactants such as lipids and nonionic detergents have the ability to modulate MDR in cancer cells. We have previously proposed an approach to enhance the efficacy of certain types of drugs by using micelles of the polymeric surfactants, poly(oxyethylene-b-oxypropylene-b-oxyethylene) block copolymers (also known as “Pluronic copolymers”), as microcontainers for drug delivery (19). Pluronic micelles have first been used in vivo for the targeted delivery of a neuroleptic across the blood brain barrier to the brain (19, 20). Furthermore, Pluronic copolymers could reverse MDR in carcinoma cells in vitro (21-24) and were shown to substantially increase the activity of anthracycline antibiotics against drug sensitive and resistant tumors in vivo (24, 25). This paper studies the effects of Pluronic P85 block copolymer on the activity of antineoplastic drugs in MDR and sensitive human ovarian carcinoma cells (SKVLB and SKOV3, respectively). The data on the copolymer effect on daunorubicin cytotoxicity, drug-induced DNA damages, intracellular transport, and distribution in SKOV3 and SKVLB cell lines are presented. The copolymer effects on the cytotoxic activity of other MDR type drugs (doxorubicin, epirubicin, vinblastine, and mitomycin C) and non-MDR type drugs (methotrexate and cisplatin) are also explored.

* Corresponding authors. † Present address: Supratek Pharma Inc. and Immunology Research Center, Institute Armand-Frappier, University of Quebec, 513 Boulevard des Prairies, Case Postale 100, Laval, Quebec, Canada H7N 4Z3. ‡ Present address: Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6025. X Abstract published in Advance ACS Abstracts, February 1, 1996.

MATERIALS AND METHODS

1043-1802/96/2907-0209$12.00/0

Cells. Human ovarian carcinoma cell lines (SKVO3 and SKVLB) were cultivated in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. SKVLB cells were obtained from SKVO3 cells by long-time cultivation in the presence of vinblastine (26). To maintain the resistance of SKVLB cells, during each fourth passage, these cells were cultivated in the presence of 400 ng/mL daunorubicin. © 1996 American Chemical Society

210 Bioconjugate Chem., Vol. 7, No. 2, 1996

Anticancer Drugs. Daunorubicin, doxorubicin, vinblastine, mitomycin C, methotrexate, and cisplatin were purchased from Sigma (St. Louis, MO). Epirubicin (fapmarubicin) was purchased from Farmitalia C. Erba (Milan, Italy). Preparation of the Micellar Form of the Drugs. Pluronic P85 [poly(oxyethylene-b-oxypropylene-b-oxyethylene) block copolymer] characterized by the general structure formula

Alakhov et al.

each set designated as T, P, and B. Cells in all tubes were lysed with 4.5 M urea. DNA in P tubes was submitted to alkali treatment (29) for 30 min at 0 °C and then 60 min at 15 °C. DNA in B tubes was submitted to alkali treatment for 30 min at 20 °C. DNA in T tubes was not exposed to alkali. After neutralization of the solution in tubes P and B, ethidium bromide was added to each tube and the relative fluorescence values of the samples (λex ) 520 nm, λem ) 590 nm) were determined. The percentage of the double-stranded DNA after alkali treatment (D) was determined as

D ) (P - B)/(T - B) × 100% was purchased from Serva (FRG) and used without further purification. The copolymer was dissolved at various concentrations (0.01-5%) in RPMI-1640 at 4 °C, and the solutions obtained were sterilized by filtration through a 0.2 µm filter. The drugs were then diluted in these solutions (3 nM to 100 µM) and incubated for 30 min at 37 °C prior to cell experiments. Characteristics of Pluronic P85 Micelles and Drug Partitioning. The cmc,1 the micelle hydrodynamic radius, and the surfactant aggregation number in the absence and presence of daunorubicin were determined using surface tension measurements, quasielastic light scattering, and ultracentrifugation as previously described in ref 27. The partitioning of daunorubicin in the Pluronic P85 micelles was studied by fluorescence spectroscopy as previously described (27). Briefly, the daunorubicin was dissolved in Pluronic solutions of various concentrations, and the drug fluorescence spectra (λex ) 471 nm) were recorded using a Hitachi F4000 spectrofluorimeter at 20 °C. The fluorescence intensity data at various Pluronic concentrations were used to determine the drug partitioning coefficients (27). Cytotoxicity Test. The cells were cultured in drugfree medium for a minimum of four passages prior to experimental use. They were subsequently harvested by trypsinization, suspended in fresh medium, plated at 2000-3000 cells/well in 96-well plates (Costar, Cambridge, MA), and cultured over 2 days to allow reattachment. The medium was then replaced with fresh medium (100 µL per well). Drug solutions with or without the copolymer were added to the cells (100 µL per well). Cells were incubated with drugs for various time intervals at 37 °C and 5% CO2, then washed three times with fresh medium, and cultured for 4 days. After 4 days, the drug cytotoxic activity was evaluated using the XTT assay (28). Briefly, 50 mL/well of sterile 1 mg/mL XTT (Sigma, St. Louis, MO) in RPMI-1640 containing 5 µL/ mL of 1.54 mg/mL phenazine metasulfate (Sigma, St. Louis, MO) in PBS was added to the cells and incubated with the cells for 16 h. The absorbance at 450 nm was determined on a microplate reader. All experimental points were carried out in triplicate. Values shown are mean. The standard error of the mean did not exceed 10% of the mean values (p < 0.05). Drug-Induced DNA Strand Breaks. The DNA damage in drug-treated cells was determined using a direct fluorescence assay (29) modified as reported in ref 30. Briefly, control and drug-treated cells were distributed in three sets of samples each containing four tubes, 1 Abbreviations: BSA, bovine serum albumin; cmc, critical micelle concentration; HLB, hydrophilic-lipophilic balance; SDS, sodium dodecylsulfate; PBS, phosphate-buffered saline; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium 5-carboxanilide inner salt.

(1)

where P, B, and T represent fluorescence measured in P, B, and T samples, respectively. Daunorubicin Transport and Distribution in the Cells. The transport and subcellular distribution of free daunorubicin and daunorubicin in the copolymer solution were studied by fluorescent spectroscopy. The cells were cultured in 50 mL flasks until the formation of monolayers. The medium was then replaced with fresh medium, and 10 µg/mL daunorubicin or 10 µg/mL daunorubicin in 1% Pluronic P85 solution was added at 37 °C for various time intervals of 5-120 min. The cells were then transferred to 4 °C, washed three times with cold PBS, and detached by scraper. Daunorubicin accumulation in the cells was assayed by measuring cell fluorescence at λex ) 471 nm and λem ) 556 nm, corresponding to the maxima in daunorubicin excitation and emission spectra. Under these conditions, the cell fluorescence was mainly due to the portion of the drug localized in the cytoplasm and plasma membrane, while the fluorescence of the nuclear portion of the drug was quenched (31). To analyze daunorubicin binding to the DNA, the cells were treated with 0.1% SDS for 30 min at room temperature. The treatment led to an increase in the fluorescence by a factor of appropximately 2-10, which was due to the destruction of the daunorubicin-DNA complex [the model experiment on the complexation of daunorubicin with DNA from salmon sperm demonstrated 95% quenching of the fluorescence in the complex, while after destruction of the complex with 0.1% SDS, fluorescence was restored to the level observed in the absence of DNA; in the absence of DNA, SDS only marginally (by a factor 1.1) increased the fluorescence]. The portions of the drug localized in the cytoplasm and plasma membrane and drug bound to the nuclear DNA were determined from the fluorescence data using the following equations:

Ic ) I1 - γIn and In ) I2 - Ic

(2)

where I1 and I2 are the fluorescence intensities measured in the daunorubicin-loaded cells before and after SDS treatment, respectively, Ic and In are the portions of the drug fluorescence corresponding to the drug in the cytoplasm and plasma membrane (Ic) and DNA (In), γ is the coefficient accounting for the daunorubicin quenching in the DNA ()0.05), and is the coefficient accounting for the change of the daunorubicin quantum yield in the 0.1% SDS solution ()0.9). Efflux Studies. Cell monolayers were incubated for 1 h at 37 °C with 10 µg/mL daunorubicin or 10 µg/mL daunorubicin in 1% Pluronic P85 solution to obtain substantial levels of drug accumulation in the cell. The cells were then transferred to 4 °C to inhibit P-gpmediated efflux in SKVLB cells, washed three times with cold PBS, and incubated again at 37 °C for various time intervals in drug-free medium. After the incubation, the

Bioconjugate Chem., Vol. 7, No. 2, 1996 211

Hypersensitization of Human Ovarian Carcinoma Cells

Figure 1. Cytotoxicity of (1 and 2) free daunorubicin and (3 and 4) daunorubicin/Pluronic with respect to (1 and 4) SKVLB and (2 and 3) SKOV3 cells. Cells were incubated with the drugs for 120 min. Pluronic P85 concentration equals 1%. Daunorubicin concentration is varied.

cells were again transferred to 4 °C, washed three times with cold PBS, and detached by scraper. The amount of drug in cytoplasm and plasma membrane and DNA was determined as described above.

Figure 2. (a) Effect of the duration of the cell exposure to the drug on the cytotoxic effect of (1 and 3) free daunorubicin and (2 and 4) daunorubicin/Pluronic in (1 and 4) SKVLB and (2 and 3) SKOV3 cells. (b) Dependence of the resistance factor (IC50 ratio in SKVLB and SKOV3 cells) on the duration of the cell exposure to the free daunorubicin and daunorubicin/Pluronic. Pluronic P85 concentration equals 1%. Table 1. Effect of Dilution of Pluronic P85 on the Cytotoxicity of the Daunorubicin/Pluronic with Respect to Resistant SKVLB Cellsa Pluronic concentration % wt

IC50, ng/mL

resistance reversion index, IC50,(-)P85/IC50,(+)P85

1 0.5 0.1 0.05 0.01 0

7 10 12 16 20 5000

714 500 417 312 250 -

RESULTS

Cytotoxicity of Pluronic Copolymers. At concentrations less than 5% and incubation periods less than 120 min, Pluronic P85 alone had no cytotoxic effects in either SKOV3 or SKVLB cells. Under these conditions, cytotoxicity of the copolymer, as determined by the XTT assay, did not exceed 10%. Since higher concentrations of the copolymer or longer incubation times produced some cytotoxicity, we studied below the copolymer concentration and incubation times equal or less than 1% and 120 min, respectively. Reversion of MDR by Pluronic P85. The cytotoxicity of free daunorubicin and daunorubicin in Pluronic P85 solution (daunorubicin/Pluronic) with respect to SKOV3 and SKVLB cell lines was examined using XTT assay (Figure 1). An about 700-fold decrease in the IC50 of daunorubicin in the presence of Pluronic P85 was observed in SKVLB cells. By contrast, the copolymer had a much less significant effect on drug efficacy in the case of SKOV3 cells. The daunorubicin/Pluronic cytotoxicity curves observed in this case became more steep compared to the free drug curves, which was indicative of the amplification of the drug effect. However, the IC50 values of daunorubicin/Pluronic observed in a series of repeated measurments (data not presented) were decreased less than 3-fold compared to the free drug IC50 values. As a result, the resistant subline demonstrated considerably higher response to the cytotoxic action of the daunorubicin/Pluronic compared to the response in the parental line. Effect of Incubation Time on Drug Cytotoxicity. The dependence of cytotoxic effects on the time of exposure with SKOV3 and SKVLB cells is shown in Figure 2. The time dependencies of IC50 values observed for SKOV3 and SKVLB cells treated with the free drug were parallel (Figure 2a). The IC50 values decreased 5-9-fold with an increase in the time of cell exposure to drugs from 5 to 120 min. Therefore, the cell resistance factor did not change appreciably within the time interval studied and approximated a value of 10 (Figure 2b). A dramatically different picture was observed with the cells treated with the daunorubicin/Pluronic. During the time interval, the IC50 in SKVLB decreased approximately 400-fold, while in SKOV3 cells, it changed less signifi-

a

Cells treated with the drug for 120 min.

cantly (Figure 2a). The effects on SKVLB and SKOV3 cells were equivalent after 30-40 min. At longer incubation times, the resistance of the cells with respect to the daunorubicin/Pluronic was inverted. As a result, the index of cell resistance decreased from 1.7 (5 min) to 0.014 (120 min) (Figure 2b). Effect of Pluronic Concentration. The dependence of the cytotoxic effects on the concentration of the copolymer was examined for SKVLB cells (Table 1). The decrease in Pluronic P85 concentration from 1% (2.2 mM) to 0.01% (22 µM) was accompanied by only a 3-4-fold increase in IC50, corresponding to a decrease in the resistance reversion index from about 700 to 250. Therefore, even after dilution by a factor of 100, the copolymer preserved the ability to significantly reverse drug resistance in SKVLB cells. Drug-Induced DNA Strand Breaks in SKOV3 and SKVLB Cells. The free drug significantly increased the level of strand breaks in SKOV3 cells compared to the level of breaks in untreated cells (Figure 3). Approximately the same increase in strand breaks was observed after treatment of these cells with daunorubicin/Pluronic. By contrast, while free daunorubicin did not produce excessive strand breaks in the SKVLB cells, significant DNA damage was observed in SKVLB cells when the drug was administered in the copolymer solution. Influx and Intracellular Distribution of Free Daunorubicin. The data on the daunorubicin influx were plotted as the kinetic curves of Ic and In vs time of cell incubation with the drug for SKOV3 (Figure 4) and SKVLB cells (Figure 5). These curves characterize the kinetics of daunorubicin accumulation in the cytosol and cell membrane (Figures 4a and 5a) and drug binding with the DNA (Figures 4b and 5b). The influx of daunorubicin into the cytoplasm of SKOV3 cells was described by a curve with a maximum; during first 30 min, the Ic value increased, while subsequent incubation of the cells with


Figure 3. Daunorubicin-induced DNA strand breaks in (1 and 2) SKVLB and (3 and 4) SKOV3 cells treated with (1 and 3) free daunorubicin and (2 and 4) daunorubicin/Pluronic. The ratio of D in drug-treated and untreated cells (Dt/Dc × 100%) is shown. Pluronic P85 concentration equals 1%.

Figure 4. Kinetics of daunorubicin influx in SKOV3 cells in the absence and presence of 1% Pluronic P85: (a) accumulation in cytoplasm and plasma membrane (Ic) and (b) binding with DNA (In).

Figure 5. Kinetics of daunorubicin influx in SKVLB cells in the absence and presence of 1% Pluronic P85: (a) accumulation in cytoplasm and plasma membrane (Ic) and (b) binding with DNA (In).

daunorubicin led to a decrease in Ic (Figure 4a). The In value increased over 120 min and at all times studied was significantly (from 4 to 100-fold) higher than Ic (Figure 4b). This type of kinetic behavior is characteristic of a two-stage process:

drug in external medium S drug in cytoplasm and membrane S fluorescence emitted stage one drug in nucleus fluorescence quenched stage two The first stage consists of drug transport from the extracellular medium to the cytoplasm and membrane, while the second stage is transport to the DNA, leading

Alakhov et al.

Figure 6. Binding of (1 and 2) free daunorubicin and (3 and 4) daunorubicin/Pluronic with DNA in (1 and 4) SKVLB and (2 and 3) SKOV3 cells. Cells were incubated with the drugs for 120 min. Pluronic P85 concentration equals 1%. Daunorubicin concentration is varied.

to fluorescence quenching. A different picture was observed in the SKVLB cells treated with free daunorubicin. In this case, both Ic and In were significantly lower than those in SKOV3 cells. The decreased ability of these cells to accumulate the drug can be explained by the P-gpmediated efflux of the drug from the cell. No maximum in Ic time dependence was observed in this case over the time period studied. This is consistent with the decreased capacity of the nuclear DNA to bind the drug. In contrast to the case of SKOV3 cells, the drug binding with DNA in SKVLB cells was characterized by a curve indicating saturation (Figure 5b). Furthermore, the concentration dependence of daunorubicin binding with DNA suggests that the binding in SKVLB cells is significantly lower than that in the parental subline (Figure 6). First, the maximal binding of daunorubicin observed in SKVLB cells was about 15 times less than the binding in SKOV3 cells. Second, the apparent binding constant in SKVLB cells was 10 times lower than that in SKOV3 cells. Effect of Pluronic P85 on the Influx and Intracellular Distribution. No significant changes in Ic and In vs time dependence were observed when daunorubicin/ Pluronic was added to SKOV3 cells instead of the free drug (Figure 4). However, copolymer caused dramatic changes in the response of SKVLB cells to daunorubicin, resulting in a substantial elevation in the total influx of the drug. In this case, after a 40 min incubation, the Ic values approximated the maximal value observed with the SKOV3 cells (Figure 5a), suggesting that the drug in the presence of Pluronic P85 is efficiently transported into the cytoplasm and membrane. In contrast to the results with SKOV3 cells, the daunorubicin concentration in the cytoplasm of SKVLB cells remained constant after reaching the maximal value (40 min) which is consistent with the lower capacity of the DNA of SKVLB cells to bind daunorubicin compared to that in SKOV3 cells. However, while the binding of the daunorubicin/Pluronic with DNA in SKVLB cells was several-fold lower than the binding observed in SKOV3 cells, it was increased compared to the binding of the free drug in the SKVLB cells (Figure 5b). The concentration dependencies of the drug binding suggest that, although the maximal binding of daunorubicin/Pluronic observed in SKVLB cells was essentially unaffected, the apparent binding constant was decreased at least 10 times compared to the binding of free drug and was equal to that observed for the parental subline (Figure 6). Efflux of the Free and Micelle-Incorporated Drug. A rapid decline in both Ic and In (85 and 95%, respectively,



Table 3. Molecular Characteristics of Pluronic P85 and Parameters Characterizing the Multimolecular Micelles Formed by This Copolymer in Aqueous Solution at 37 °Ca characteristics of the copolymerb block lengths n

m

molecular mass

HLB

38

51

4500

12-18

a

Figure 7. Efflux of (1 and 2) free daunorubicin and (3 and 4) daunorubicin/Pluronic from (a) the cytoplasm and plasma membranes and (b) nuclei of (1 and 4) SKVLB and (2 and 3) SKOV3 cells. The daunorubicin levels in the cytoplasm and membranes and nuclei are expressed as the percentage of the initial levels (Ic/Ico × 100 and In/Ino × 100, respectively). Table 2. Effect of Pluronic P85 on Reversion of Resistance of SKVLB Ovarian Carcinoma Cells with Respect to Various Drugs IC50, ng/mL drug

free drug

drug in 1% pluronic P85

doxorubicin epirubicina vinblastine mitomycin C methotrexate cisplatin

6000 60000 1200 800 500 400

300 2000 1.1 10 90 200

a Cells were incubated with drug for 60 min. In all other cases, incubation time was 120 min.

during the first 60 min) was observed after transfer of the drug-treated SKVLB cells in a drug-free medium (Figure 7). This decline was consistent with the P-gpmediated efflux of daunorubicin from the cells. In contrast, only a 33% decrease in Ic and no decrease in In were observed in the SKOV3 cells treated with the free drug. Both the Ic and In components of the efflux were significantly reduced in SKVLB cells treated with the daunorubicin/Pluronic. In this case, the Ic is only decreased by 47% of the initial value and In is even slightly increased ()15%) over a 60 min incubation period. This demonstrates that the copolymer provides for an increase in the daunorubicin retention time in the SKVLB cells. Some increase of daunorubicin efflux in the presence of copolymer was observed in the SKOV3 cells. Interestingly, the efflux of daunorubicin/Pluronic from the cytoplasm was exactly the same ()47%) in both SKOV3 and SKVLB cell lines (Figure 7a). It is probable that this efflux component accounts for the passive diffusion of the drug from the cells, facilitated in the presence of the copolymer. Effect of Pluronic P-85 on the Activity of Other Cytotoxic Drugs. Table 2 compares the cytotoxic effects of several types of anticancer drugs in Pluronic P85 solutions with the effects of the free drugs on SKVLB cells. The cytotoxicity of MDR type drugs, doxorubicin, epirubicin, vinblastine, and mitomycin C, increased 201000 times in the presence of 1% Pluronic P85. Less significant changes (only 2-5.5 times) were observed in the IC50 of non-MDR type drugs, cisplatin and methotrexate. Pluronic Micellar System and Drug Partitioning. To characterize daunorubicin and Pluronic P85 interactions, the micelle formation and daunorubicin partitioning in the micelles have been studied. The Pluronic P85 solutions used in the current study were optically transparent over a wide range of temperatures (18-40 °C) and surfactant concentrations (0.001-1%). A multimolecular

characteristics of the micellesc micelle radius, aggregation cmc, % nm number 0.005

7.3 ( 0.3

57 ( 16

b

From ref 27. The hydrophilic-lipophilic balance (HLB) was calculated from Glc relative retention ratios (32). c The Critical micelle concentration (cmc), micelle hydrodynamic radius, and aggregation number (i.e. number of copolymer molecules constituting one micelle) were determined using the fluorescent probe technique, quasi-elastic light scattering, and ultracentrifugation (27).

micellar species of the copolymer was formed at copolymer concentrations exceeding the cmc as measured by surface tension, light scattering, and ultracentrifugation. The cmc, the micelle hydrodynamic radius, and the Pluronic P85 aggregation number (i.e. the number of the copolymer molecules in one micelle) are presented in Table 3. Under the conditions studied, these parameters were not affected by the pH (pH 3-9), the ionic strength (0.2 M), or the presence of anticancer drugs (5 mg/mL) or serum proteins (3% BSA) in the system. At concentrations exceeding the cmc, daunorubicin was partitioned between the bulk aqueous phase and the hydrophobic micelle core formed by the oxypropylene chain blocks. Under these conditions, the dynamic exchange of the drug molecules between the bulk phase and micelle microphase takes place. A partitioning model for a drug noncovalently incorporated into the micellar microcontainer has been reported previously (27). According to this model, the fraction of the drug that is incorporated in the micelles is expressed by

R)

Cmic P([Pluronic] - cmc) ) Co 119 + (P - 1)([Pluronic] - cmc)

(3)

where Cmic and Co are the bulk concentration of the drug in the copolymer solution and the total bulk concentration of the drug in the system, respectively, [Pluronic] is the copolymer concentration (% wt), and P is the partitioning coefficient that is expressed by

P ) [drug]m/[drug]w

(4)

where [drug]m and [drug]w are the local drug concentrations in the micellar and aqueous phases, respectively. The change in the polarity of the microenviroment after daunorubicin incorporation in the micelle resulted in an increase in the fluorescence of the drug. The daunorubicin partitioning coefficients were determined as previously described for other probes (27) from the dependencies of daunorubicin fluorescence at λex ) 471 nm and λem ) 556 nm on Pluronic P85 concentration using the linear plots (Figure 8),

Imax - I 119 1 ) I - Io P([Pluronic] - cmc) P

(5)

where Io is the fluorescence intensity in the absence of the copolymer, I is the fluorescence at the given copolymer concentration, Imax is the fluorescence at the “saturating” concentration of the copolymer (when fluorescence reaches the maximal value), and 119 is the coefficient that depends on the partial specific volume of the copolymer (27). The value of P approximated 185. Using


Alakhov et al.

Figure 8. Linear plot for determination of the daunorubicin partitioning coefficient. The data were obtained using two concentrations of doxorubicin (0.4 and 0.08 µg/mL) and varying concentrations of Pluronic P85. P value was determined from the slope of the linear regression line using Microsoft Excel 5.0 program. Table 4. Fractions of Daunorubicin Incorporated into the Micelles at Various Pluronic P85 Concentrations at 37 °C Pluronic P85 concentration, %

R × 100, %

1 0.5 0.1 0.05 0.01

61.0 44.0 12.3 6.5 0.4

this partitioning coefficient, the fractions of the drug that is incorporated in the micelles were determined from eq 3. The values obtained for various Pluronic P85 concentrations are presented in Table 4. DISCUSSION

Two sublines of human ovarian carcinoma cells, SKVLB and SKOV3, have been chosen as a model for this study. The SKVLB subline, which has been selected from the parental SKOV3 cell, is known to be an MDR type due to P-gp overexpression (26). The major result of this study is that Pluronic P85 dramatically increased the cytotoxicity of daunorubicin with respect to the MDR cells and much less significantly changed the activity of the drug with respect to the non-MDR subline (Figure 1). The copolymer effect in MDR cells approximated 700-fold (Table 1) and significantly surpassed the MDR-reversing effects of liposomes and polyoxyethylated surfactants that have been previously reported (9-18). Further, the study on daunorubicin transport into the cells suggested that the copolymer enhanced the drug influx into the cytoplasm and its binding with the DNA in the MDR cells (Figures 4-6). Once taken up by the resistant cells, the daunorubicin/Pluronic was retained much longer in the nuclei and cytoplasm of the MDR cells than the free drug which was rapidly eliminated from the cells due to the P-gp-mediated efflux (Figure 7). The drug uptake in the sensitive cells was not significantly altered in the presence of the copolymer, providing further evidence to support an effect of the copolymer on P-gp. The data on the DNA strand breaks suggest that the mechanism of drug action in the SKVLB cells was not changed by the copolymer. The enhanced cytotoxic effect of daunorubicin in the presence of Pluronic P85 correlated with the increase in the drug-induced DNA damage that is presently considered to be a major mechanism of anthracycline cytotoxicity (Figure 3). Furthermore, no significant changes in DNA damage were observed in the presence of the copolymer in the sensitive subline which is also

consistent with the cytotoxicity and transport data. Our data also suggest that the sensitization of MDR cells in the presence of the copolymer is a general phenomenon characteristic for a broad range of MDR type and nonMDR antineoplastic agents (Table 2). Another important result of this study is that in the presence of Pluronic P85 daunorubicin became significantly more active (“hypersensitive”) with respect to SKVLB cells compared to the parental subline (Figure 2). This result cannot be explained merely by increased drug transport into the MDR subline. Most currently known modulators of MDR (i.e. inhibitors of P-gp) render MDR cells sensitive to MDR type drugs so that the IC50 values approach those of sensitive parental cells. It is known that anthracyclines are easily transported across the plasma membrane of the non-MDR cells (31). By contrast, in the absence of the copolymer, free daunorubicin was poorly transported into the SKVLB cells due to the P-gp-mediated efflux. Since the cytoplasm concentration of daunorubicin in SKVLB cells in the presence of the copolymer approximated the drug concentration in the SKOV3 cells (Figures 4a and 5a), we conclude that, once the efflux system for the drug in SKVLB cells is overcome, these copolymer-treated cells become more sensitive to the drug’s cytotoxic action compared with the parental subline. The reason for the hypersensitivity of SKVLB cells with respect to daunorubicin/Pluronic is not yet clear. It cannot be explained by the cytotoxic effect of the copolymer, since under the conditions studied the copolymer did not affect cell viability. Interestingly, data on the daunorubicin influx and intracellular distribution suggest that the DNA of SKVLB cells have a significantly lower capacity with respect to the drug binding compared with the DNA of SKOV3 cells (Figure 6). This result gives independent support of a recent observation that MDR is associated with the changes in intracellular distribution of anthracyclines, specifically with a decreased accumulation of these drugs in the nucleus (33). Furthermore, our binding and cytotoxicity data also suggest that in the presence of the copolymer the lower amounts of the drug that intercalated in the DNA of the MDR cells caused substantially higher cytotoxic effects compared to the non-MDR subline. We hypothesize that in addition to P-gp inhibition the copolymer affects some non-P-gp mechanisms of drug resistance that are yet to be identified in ovarian carcinoma cells. This hypothesis may also give grounds for reconciliation of the sensitization of MDR cells with respect to non-MDR type drugs as well as sensitization of parental cells with respect to daunorubicin. Extensive studies on non-P-gp mechanisms of drug resistance are currently ongoing (34-36). It is worth mentioning in this respect that we have recently observed (data in preparation) several-fold sensitization of parental cells and hypersensitization of MDR cells by Pluronic copolymers on MDR and non-MDR Chinese hamster ovary (CHr C5 and AUX-B1) and human breast carcinoma (MCF-7 ADR and MCF-7) cell lines. The results obtained also raise the question about the mechanism of the P-gp-modulatory effects of Pluronic P85. One possible explanation for increased drug influx is an inhibitory effect of Pluronic P85 on the P-gp function in SKVLB cells. Such inhibition may result from a direct interaction of the copolymer molecules with the P-gp or from some copolymer-induced changes in the structure of the plasma membrane that affect P-gp function. However, other mechanisms for the copolymer effect that account for the bypass of the efflux pump, rather than its inhibition, must also be considered. One possible mechanism consists of a copolymer-induced increase in


Figure 9. Schematic representation of the dynamic equilibrium in the Pluronic P85 and drug solution. The copolymer exchange between multimolecular micelles and bulk solution is characterized by the cmc. The drug exchange between the micelles and bulk solution is characterized by the partitioning coefficient.

the permeability of the cell plasma membrane. The increase in membrane permeability with respect to the drug can result in increased drug influx that compensates for the efflux pump component and leads to a net increase in total uptake. There are previous reports that Pluronic copolymers enhance passive diffusion of low-molecular mass compounds across plasma membranes into cells (37). Our previous work (38) has demonstrated that Pluronic P85-based micellar systems provide for enhanced transport of negatively charged ATP molecules into the cytoplasm that otherwise cannot be transported into a cell. Finally, data obtained in our laboratory (in preparation) also suggest that Pluronic P85 increases the ion permeability of liposome membranes while not affecting the liposome integrity. Interestingly, the increased ion transport in liposomes was observed over the same range of copolymer concentrations that is studied in this work. Another important problem that is raised by this work is the evaluation of the roles of the multimolecular micellization of the copolymer and drug partitioning in the micellar system in the MDR modulation phenomenon. Originally, the drug delivery concept advanced by us implied that the drug molecules noncovalently incorporate in the core of the Pluronic micelles that serve as carriers for the drug delivery and targeting (19, 20). The validity of this consideration has previously been demonstrated in the experiments on the endocytic uptake of fluorescein incorporated in the Pluronic P85 micelles (39). However, this consideration is not necessarily true for daunorubicin transport in MDR cells reported in this work. Generally, in the daunorubicin/Pluronic systems, both the free and micelle incorporated fractions of the drug and copolymer are present (Figure 9). First, the drug molecules are partitioned and dynamically exchanged between the bulk aqueous solution and the micellar microphase. Therefore, the question is, in what form, micellar or free, are the drug molecules transported into the MDR cells? The answer to this question is obtained in part from the partitioning study performed in this work. Significant MDR reversion is observed at the low copolymer concentration (0.01%) (Table 1) when according to the data of the partitioning study (Table 4) only 0.4% of the total drug is incorporated into the micelles. This result suggests that the effects observed with the MDR cells involve interaction of the free drug form with the cells. We hypothesize therefore that the copolymer interacts with the plasma membrane and enhances the influx of the free drug form presented in


the extracellular media. The study of the validity of this hypothesis is in progress. Second, the copolymer molecules are also partitioned and dynamically exchanged between the multimolecular micelles and the bulk solution (Pluronic unimers). Therefore, the question is, what form of the copolymer, micellar or unimer, is responsible for the effects observed in the MDR cells? The answer to this question can be obtained after the study of MDR modulatory effects of the drug/copolymer system and drug transport in the MDR cells at various concentrations of the copolymer. Some preliminary conclusions can be made on the basis of the cytotoxicity vs concentration dependence presented in this paper (Table 1). The hypersensitization of MDR cells was observed at as low a concentration of the copolymer as 0.01% (22 µM), which is very close to the cmc (27). The increase of the copolymer concentration up to 1% (2.2 mM) leads to a proportional elevation in the micelle concentration, while the unimer concentration remains practically unchanged. Since only 3-fold change in IC50 is observed in this concentration range, we hypothesize that just the copolymer unimers are responsible for the hypersensitization effect. The major practical outcome of this work is that Pluronic P85 can possibly be used as a delivery system for enhancing the activity of antineoplastic agents against MDR tumors. Furthermore, since MDR cells are more susceptible to the cytotoxic effects of daunorubicin/ Pluronic than the parental cells, one can assume that it is possible to reduce the development of MDR during chemotherapy by using the copolymer-based drug forms. ACKNOWLEDGMENT

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