Folic acid, attached to polyethyleneglycol-derivatized,distearoyl-phosphatidylethanolamine, was used to target in vitro liposomes to folate receptor (FR)-overexpressing tumor cells. Confocal fluorescence microscopic observations demonstrated binding and subsequent internalization of rhodamine-labeled liposomes by a high FR-expressing, murine lung carcinoma line (M109-HiFR cells),with inhibition by free folic acid. Additional experiments tracking doxorubicin (DOX) fluorescence with DOX-loaded, folate-targeted liposomes (FTLs) indicate that liposomal DOX is rapidly internalized,released in the cytoplasmic compartment, and, shortly thereafter,detected in the nucleus, the entire process lasting 1–2 h. FR-mediated cell uptake of targeted liposomal DOX into a multidrug-resistant subline of M109-HiFR cells (M109R-HiFR) was unaffected by P-glycoprotein-mediated drug efflux, in sharp contrast to uptake of free DOX, based on verapamil-blockade experiments with quantitation of cell-associated DOX and flow cytometry analysis. Delivery of DOX by FTLs to M109R-HiFR cells increased continuously with time of exposure, reaching higher drug concentrations in whole cells and nuclei compared with exposure to free DOX. The in vitrocytotoxic activity obtained with DOX-loaded FTLs was 10-fold greater than that of the nontargeted liposome formulation, but was not improved over that of free DOX despite the higher cellular drug levels obtained with the targeted liposomes in M109R-HiFR cells. However,if M109R-HiFR cells were exposed to drugs in vitro and tested in an in vivo adoptive assay for tumor growth in syngeneic mice along a 5-week time span, FTL DOX was significantly more tumor inhibitory than free DOX. It is suggested that the biological activity of liposomal DOX released inside the cellular compartment is reduced in vitro due to the aggregated state of DOX, resulting from the liposome drug-loading process, and requires a long period of time and/or an in vivo environment for full expression.

FR3, a GPI membrane-anchored glycoprotein of 38 kDa (1), with extremely high affinity for folate (kDa ≈ 10−10m for α-isoform and kDa ≈ 10−9m for β-isoform; Ref. 2), is overexpressed in a wide variety of epithelial tumors (2, 3, 4, 5). The FR participates in the cellular accumulation of folates in a number of epithelial cells through a process of endocytosis (6). In this process, ligand-bound receptor is internalized and released from the receptor through intravesicular reduction in pH. Ligand-free receptor is then recycled to the cell surface (6, 7). The receptor-mediated uptake of folic acid has been proposed as a potentially useful target in cancer treatment (7, 8) and as a route to promote entry of attached macromolecules or liposomes into cells (9).

Liposomes with folate residues conjugated through a liposome-grafted PEG spacer are taken up avidly by KB cells (human nasopharyngeal cancer cell line; Refs. 10 and 11). The binding of these FTLs to KB cells is mediated by cell-surface FR, as demonstrated by competitive inhibition with excess free folate or with antiserum against the FR (10, 11). By providing a different pathway of tumor cell drug uptake, the use of targeted liposomes for chemotherapeutic delivery may conceivably circumvent the MDR drug efflux mechanism, leading to resistance (12, 13). The Pgp,located in the plasma membrane, is an active efflux pump of cytotoxic agents conferring multidrug resistance to cancer cells (12, 13). Intracellular entry of drug-loaded liposomes via endocytosis, followed by release of entrapped agent in cytoplasm (11), is an alternative route of drug entry that may enable bypassing Pgp-mediated efflux.

Here, we investigate the mechanism of drug delivery and activity of DOX-loaded FTLs as compared with free drug and drug encapsulated in nontargeted liposomes, and we report on drug uptake studies and tumor cell growth assays with DOX-sensitive and -resistant,high-FR-expressing murine tumor cells.

Liposome Preparation.

Preparation of liposomes and the sources of liposome components,including DSPE-PEG(3350)-Folate, were as described previously (14). DPPE-rhodamine was obtained from Avanti Polar Lipids(Birmingham, AL). The following formulations were prepared:(a) hydrogenated soybean phosphatidylcholine/cholesterol/DSPE- PEG-Folate/DPPE-rhodamine (molar ratio, 99.4:70:0.5:0.1); (b) same lipid composition (see Ref. 1), excluding DPPE-rhodamine, and loaded with DOX in the liposome water phase using an ammonium sulfate gradient (250 mm), as described previously (15).

In addition, we also tested a nontargeted pegylated liposomal formulation of DOX (Doxil) provided by Sequus Pharmaceuticals (Menlo Park, CA), with a lipid composition as described previously (16). Unless otherwise indicated, the final DOX:phospholipid ratio was ∼150 μg/μmol. The mean vesicle size of all liposome formulations was in the range of 70–100 nm.

Cell Lines.

The high-FR M109 cell lines, including DOX-sensitive and -resistant variants, have been described previously (14). The cells were cultured in folate-depleted (∼2 nm, versus 2.26 μm folate under normal culture conditions) RPMI 1640 (Biological Industries, Ltd., Beyt Haemek, Israel) with 10% FCS (Life Technologies, Inc., Grand Island, NY). The two selected sublines adapted to grow under low folate conditions are referred to as M109-HiFR, for the sensitive line, and M109R-HiFR for the resistant line.

Confocal Microscopy.

Tumor cells were plated, 24 h before each experiment, on 22-mm coverslips inserted into 35-mm culture dishes. Exposure times of the cells to liposomes or DOX are indicated for each experiment. Phospholipid concentration and DOX concentration in these experiments were in the range of 30–150 μm and 10–40μ m, respectively. After medium removal, cells were washed with PBS and fixed with PBS-buffered (pH 7) 4% formaldehyde/1.5%methanol solution (Bio-Lab, Jerusalem, Israel) at 4°C for 15 min, then washed three times with PBS (Life Technologies, Inc.). Next,the coverslips were put on slides coated with buffered mounting medium consisting of 90%glycerol/10% PBS with 0.1%NaN3 and 3% DABCO (triethylenediamine;Sigma Chemical Co., St. Louis, MO), to prevent fading. To rule out any artifact caused by the processing of microscopic samples, such as liposomal drug leakage, the DOX tracking experiments were done with fixed cells and then repeated with live cells to confirm the observations. Microscopic visualization of live (nonfixed) cells was done in PBS containing 2 mm MgSO4/1 mm HEPES (pH 7.5; Sigma Chemical Co.). Examination was done with inverted Zeiss confocal laser scanning microscope (LSM410; Carl Zeiss, Jena, Germany). For rhodamine, maximum excitation was performed by a 543-nm line of the internal He-Neon laser, and fluorescence emission was observed above 570 nm with long-pass barrier filter LP-570. For DOX, maximum excitation was performed by a 488-nm line of internal Argon laser, and fluorescence emission was observed above 515 nm with long-pass barrier filter LP-515. A water immersion objective,C-Apochromat 63 × 1.2 W corr. (Zeiss), was used. Images were converted to TIFF format, and the contrast level and brightness of the images were adjusted by using the Zeiss LSM410 program.

Verapamil Blockade of Drug Efflux.

M109R-HiFR cells in monolayer were exposed to 5μ m DOX as free drug or in FTLs for 1 h, in the presence or absence of 10 mm verapamil (Teva,Netanya, Israel). Cells were washed to remove nonassociated drug and further incubated in the presence or absence of verapamil for 2 h. Cells were released from tissue culture dishes with 0.05%trypsin/0.02% EDTA (Life Technologies, Inc.), followed by PBS washing(centrifugation, 7 min, 500 × g), suspended and split into two fractions: one for cellular DOX determination and the other one for flow cytometry assay.

Measurement of Cell-associated DOX.

After cells were counted and pelleted by centrifugation, DOX was extracted by treatment with 0.075N HCl in 90% isopropyl alcohol at 4°C overnight. Following centrifugation, the supernatant was collected and used for fluorescent DOX determination at Ex 470 nm/Em 590 nm in a Kontron SFM25 spectrofluorimeter (Kontron, Zurich,Switzerland). Extracts from untreated cells were used as blank. DOX concentration was expressed as ng DOX-equivalents per 106 cells. In some experiments, cell-associated drug was also measured by high-performance liquid chromatography and fluorescence detection, as reported previously (17).

Flow Cytometry.

Suspended M109R-HiFR cells, as described above, were analyzed with a FACS-star plus (Becton Dickinson, Mountain View, CA)flow cytometer. Cells were passed at a rate of ∼1000 cells/s through a 70-μm nozzle, using saline as the sheath fluid. A 488-nm argon laser beam at 250 mW served as the light source for excitation. Fluorescence emission was measured using a 575-nm DF 26 band-pass filter.

Cellular and Nuclear DOX Quantitation.

M109R-HiFR cells were exposed to free DOX or FTL-DOX for 1 h and 4 h. M109R-HiFR cells, released by typsinization as described above, were suspended at a concentration of 5 × 106 cells/ml for 10 min at 4°C in a 100 mm NaCl solution with 1 mm EDTA, 1% Triton X-100 (Sigma Chemical Co.), and 10 mm Tris buffer (pH 7.4;Sigma Chemical Co.). The suspension was then centrifuged (15 min,800 × g), and the resulting precipitate of cell nuclei was separated from the supernatant cell cytosol. DOX extraction from both fractions followed, as described before.

Cytotoxicity Studies.

M109HiFR and M109R HiFR cells in folate-depleted RPMI 1640 were seeded in 96-well plates at a density of 103cells/well (six replicates). Twenty-four h later, cells were exposed for 1 h to free DOX, FTL-DOX, or Doxil. The cells were washed twice and incubated further for 120 h in drug-free medium. In some experiments, cells were exposed continuously to drugs for 72 h. Cell growth was assayed colorimetrically using 2.5% glutaraldehyde as fixative, followed by methylene blue staining and absorbance measurements at 620 nm on an automated plate reader (18). Growth rates were calculated as reported previously (18).

In Vivo Adoptive Tumor Growth Assay.

Female BALB/c mice, 10 weeks of age, were obtained from the Hebrew University Animal Breeding House (Jerusalem, Israel) and maintained in a specific pathogen-free facility at Hadassah Medical Center. The experiments were done under ethical approval from our Institutional Animal Care and Use Committee. M109R-HiFR cells in in vitro suspension (107cells/ml) were exposed to 10 μm DOX either as free drug, Doxil, or FTL for 2 h, washed with PBS to remove non-cell-associated drug, and resuspended at a concentration of 2 × 107 cells/ml. Healthy, syngeneic BALB/c mice received injections into the right hind footpad with 50 μl(106 cells) from the above cell suspension. The footpad thickness was measured with calipers once or twice a week for 5 weeks. After 35 days, mice were sacrificed, the final number of tumors was recorded, and the control and tumor-inoculated footpads were sectioned at the ankle level and weighed. Tumor weight was estimated as the difference between the weight of the normal and tumor-bearing footpad. The statistical significance of differences in the final incidence of tumors per group was analyzed by contingency tables and the Fisher’s exact test.

FTL Binding to M109-HiFR Cells Is Mediated by the FR.

Association of rhodamine-labeled FTLs to M109-HiFR cells in folate-free RPMI 1640 was observed within 30 min of exposure (Fig. 1,A). At 50 min, subsequent liposome internalization and accumulation in the cell cytosol was observed, as shown in Fig. 1,B. However, under competitive free folate concentrations (2 mm, equivalent to∼1000-fold the concentration of liposomal folate), liposome binding was prevented completely, indicating involvement of FR in the liposome-targeting process at both 30- and 50-min time points (Fig. 1, C and D). Further evidence of the involvement of the GPI-anchored FR in the interaction of FTLs with M109 cells comes from experiments with phosphatidylinositol-phospholipase C-treated cells: exposure of phosphatidylinositol-phospholipase C-pretreated M109-HiFR cells to rhodamine-labeled FTLs (1 h at 4°C), resulted in no detectable binding, whereas the same liposomes were effectively bound by nontreated cells with no apparent temperature interference(data not shown).

DOX-loaded FTLs Bind to and Are Internalized into M109R-HiFR Cells.

In this experiment, we followed free and liposomal DOX access to M109R-HiFR cells by tracking the intense fluorescence of DOX molecules. M109R-HiFR cell exposure to DOX as free drug or loaded in FTLs revealed that in both cases the drug gains access to the cell nucleus, as shown in Figs. 2 and 3, but the kinetics and apparently the route of uptake differ. The influx of free DOX through cell membrane was very rapid, as indicated by the bright cytoplasmic staining within 7 min of exposure (Fig. 2,A). At 30 min, the free drug was already completely localized in the nucleus (Fig. 2,B). The kinetics of cellular interaction with DOX-loaded FTL was remarkably different. Liposome attachment to the cell membrane was observed within 30 min (Fig. 3,A). By 60 min, internalization has taken place and liposomal DOX was detected in the cytosol, and in a few cells, the drug began to appear in the nucleus (Fig. 3,B). After 90 min,liposome-delivered DOX has reached the nucleus in most of the cells,whereas the cytoplasmic drug fluorescence has mostly disappeared (Fig. 3,C). In contrast to FTL, a formulation of nontargeted liposomes coated with PEG (known commercially as Doxil) showed absolutely no association with M109R cells (Fig. 3 D), even after 4 h of incubation. In this case, consistent with previous observations, the drug has no access to the cells unless leakage into extracellular medium occurs (18). This type of experiment was repeated with fresh and fixed cells with essentially similar results.

To examine drug efflux, M109R-HiFR cells were exposed to FTL-DOX or free DOX for 1 h and then further incubated in drug-free medium for 24 h. DOX fluorescence in cells treated with free drug disappeared almost completely in contrast to a marked residual fluorescence in cells treated with FTL-DOX (data not shown).

Intracellular Delivery of DOX via FTLs Overcomes Pgp-mediated Drug Efflux.

The activity of the Pgp efflux pump in M109R HiFR cells was examined by flow cytometry in the rhodamine-123 efflux assay, an indicator of Pgp-mediated resistance (19, 20), and found to be highly effective and sensitive to verapamil blockade (data not shown). In the next series of experiments, we tested the efflux of free DOX and FTL DOX and its sensitivity to verapamil by flow cytometry. In agreement with the rhodamine studies, flow cytometry analysis showed a shift in the curve, indicating a clear increase in cell fluorescence in M109R-HiFR cells after 1 h exposure to free DOX in the presence of verapamil (Fig. 4,A). In contrast, the cellular level of fluorescence in M109R-HiFR cells after a 1-h exposure to drug in FTL appears identical in presence or absence of verapamil (Fig. 4 B).

The flow cytometry observations were confirmed by quantitative fluorometry of DOX from cell extracts, a method that is relatively unaffected by quenching artifacts. Cell retention of free DOX was∼4-fold higher in the presence of verapamil, whereas identical drug levels accumulated in cells exposed to liposome-targeted drug in the presence or absence of verapamil (Table 1). Furthermore, when the cellular DOX levels in the absence of verapamil are compared, a 4–6-fold advantage for targeted liposomal DOX over free DOX is noticeable (Table 1). These results indicate that free drug diffusing into the cells is pumped out by Pgp and/or other proteins sensitive to verapamil blockade, whereas drug entry via liposome internalization provides an alternative delivery pathway not controlled by the MDR efflux machinery.

Additional experiments quantifying cell-associated drug in drug-sensitive and drug- resistant M109 cells (Table 2) confirm the enhancement of drug delivery with targeted liposomes to the resistant cells at 1 and 4 h of incubation. In the case of drug-sensitive cells (M109-HiFR), there was no advantage of FTL over free DOX (Table 2). Drug uptake was negligible when a nontargeted, liposomal DOX formulation (Doxil) was used (Table 2). The dependence of drug uptake on the number of FR-bound and internalized liposomes is supported by an experiment presented in Fig. 5 examining two formulations of FTL that differ only in their drug:lipid ratio by a factor of ∼10. Cellular levels of DOX were consistently higher by a similar factor of∼10, in favor of the high drug-to-lipid formulation. The progressive accumulation of targeted liposomal drug by tumor cells along a 24-h incubation period, without evidence of efflux or plateau level, is also apparent from the curves in Fig. 5.

Nuclear Delivery of FTL DOX.

To investigate quantitatively the delivery of targeted liposomal DOX to the nucleus, cell fractionation experiments were done. As seen in Fig. 6, most of the drug is found in the nuclear fraction with both free DOX and FTL DOX already after 1 h of incubation. The nuclear drug concentration obtained with FTL DOX clearly surpasses the concentration obtained with free DOX, especially after 4 h of incubation. In fact, the drug concentration in cells treated with free DOX did not increase at all when incubation was prolonged from 1 h to 4 h. In contrast, in the case of cells treated with FTL-DOX, there was a 2-fold increase during the same period. No significant amounts of metabolites were detected after a 1–4-h exposure of tumor cells to either free DOX or FTL-DOX by high-performance liquid chromatography analysis of cell-associated drug(data not shown), indicating that FTL delivers intact drug to the nucleus.

In Vitro Cytotoxic Activity of DOX Delivered by FTL,Nontargeted Liposomes, or as Free Drug.

We explored whether delivery of DOX via FTL would increase the drug cytotoxicity against FR-overexpressing cells. M109-HiFR cells were exposed to free or liposomal DOX for 1 h, then washed and further incubated for 120 h in fresh medium. As seen in Fig. 7 A, the growth inhibition curve of DOX in FTL was similar to that of free DOX, but clearly superior (10-fold drop in IC50) to that of DOX in nontargeted liposomes, stressing the key role of liposome binding and internalization in enhancement of cytotoxic activity.

A similar cytotoxicity assay was done using the MDR subline M109R-HiFR (Fig. 7,B). Once more, a clear enhancement of cytotoxicity was obtained when the folate-targeted formulation is compared with the nontargeted formulation. However,despite higher drug levels accumulating in cells exposed to the targeted liposomal drug (Tables 1 and 2), this type of assay did not reveal increased cytotoxic activity of the targeted preparation compared with free DOX. Cytotoxicity assays using continuous exposure to drug (72 h) pointed to similar results in terms of the relative activity of the different forms (targeted, nontargeted, free) of DOX delivery.

Superior Tumor-inhibitory Activity of DOX Delivered by FTL in an in Vivo Adoptive Tumor Growth Assay.

To examine the biological activity of drug delivered by FTL in another model, we exposed M109R-HiFR cells in vitro to the test drug and thereafter inoculated them into the mouse footpad. In this way, the growth of cells is tracked along a much longer time span than in in vitro experiments, and the influence of in vivo micro-environmental factors is brought into play. However,unlike therapeutic experiments, this type of in vivoadoptive assay is unaffected by pharmacokinetic and biodistribution factors that would have complicated the interpretation of results. The results (Table 3) point to a statistically significant decrease of the number of tumor takes in mice injected with tumor cells exposed to FTL, as compared with free DOX,Doxil, and control, after 5 weeks follow-up. Tumor weights were also smaller for the FTL group (Table 3). The kinetics of tumor growth, as estimated by the mean footpad tumor thickness, shows a clearly slower growth rate for the FTL group from the second week after inoculation over the other treatment groups (Fig. 8).

Recent reports have indicated the feasibility of using folate-conjugated liposomes to target drugs to cancer cells and to augment their in vitro efficacy (10, 11). Folate-targeted systems possess tumor cell specificity due to the frequent overexpression of the FR in human carcinomas. Folates and its conjugates enter cancer cells by FR-mediated endocytosis (6, 7). The normal permeability barriers that limit drug entry into cells are bypassed, allowing even macromolecules or liposomes to enter FR-bearing cells readily (7). Folate conjugates such as proteins and nucleic acids have been observed to remain intact for hours following uptake by cancer cells (9, 21). Apparently, vitamin-mediated delivery may constitute a more protected pathway for intracellular delivery.

We have examined the likelihood that targeted liposomes will be taken up by carcinoma cells with overexpressed FR in an in vitrosystem. In this study and a previous study (14), the folate residue, anchored to the liposome bilayer by its γ-carboxyl group through a PEG-DSPE linker, was found to function as a specific and efficient targeting agent. The involvement of cell surface FR in the binding and internalization of FTL into M109-HiFR was validated by inhibition of association and uptake of these liposomes in the presence of excess free folate and, by the loss of liposome binding due to enzymatic cleavage of GPI, the FR cell membrane anchor (1). Although soluble PEG-folate seems to have less affinity than free folate for the FR, liposomal PEG-folate actually has an increased binding affinity due to the multivalency of liposomal binding (14).

In contrast to rhodamine-PE, a liposome bilayer component that localizes in the cytoplasm, FTL DOX readily gains access to the cell nucleus after internalization. The small size of the pores of the nuclear membrane makes this compartment inaccessible to liposomes. Therefore, this observation suggests that during the process of vesicular docking on cell surface and/or subsequent internalization path, destabilization of the liposomal carrier occurs, resulting in release of entrapped DOX and accumulation of the drug in the nucleus. It is unclear what triggers drug release from endocytosed liposomes. The acidic milieu of the lysosomal or endosomal vesicles (6, 22) is unlikely to have an effect because the liposomes used here and the gradient retaining the drug are stable at pH 4–5 within the relevant time frame (23). However, loss of cholesterol or bilayer damage by enzymatic activity of phospholipases will destroy the proton gradient of liposomes and lead to DOX leakage.

Pgp is known to reduce cellular drug accumulation by acting as an ATP-dependent efflux pump of a great many structurally distinct hydrophobic compounds (12). The manner in which Pgp recognizes these different substrates is unknown. Pgp has been found in the membrane, Golgi apparatus, and nucleus (24, 25). In the M109R-HiFR cell line, confocal microscopy studies,together with fluorometric measurements of cell-associated drug,indicate that DOX readily enters the cell and concentrates in the nucleus, but is subsequently excreted very effectively with no observable nuclear fluorescence left at 24 h. The efflux of drug is blocked by verapamil, indicating that it is Pgp mediated (19). Removal of DOX from the nucleus is presumably mediated by nuclear Pgp (24) or other proteins involved in the nuclear-cytoplasmic trafficking and compartmentalization of drugs (26). A notable finding of this study is the failure of the efflux mechanism to pump out and reduce the rate of accumulation of DOX when delivered by FTLs. The mechanism for this is unclear because the liposome-targeted drug is not retained in endosomal vesicles, but rather concentrates effectively in the nucleus, albeit with a slower kinetics than free drug. It is likely that part of the liposomal drug is in a different physical form than free drug. There is evidence of intraliposomal precipitation of DOX in liposomes loaded via an ammonium sulfate-generated gradient (27, 28). Aggregation or molecular stacking due to self-association of the intracellular liposomal drug is a possibility, because DOX dimerization has been observed for concentrations greater than 10 μm(28, 29, 30). This would explain the inability of cells to pump out liposomal DOX, and, at the same time, the lack of a cytotoxic advantage for folate-targeted liposomal DOX over free DOX in the in vitro tests, despite higher drug levels accumulating in cells exposed to the former. Aggregation and precipitation of DOX after liposome encapsulation using the ammonium sulfate method is a fully reversible process, with complete resolubilization and restoration of drug activity on collapse of the gradient in extracellular medium (18, 28). However, the high intracellular concentration of targeted liposomal DOX and short time scale of in vitrocytotoxicity experiments may not enable this process to be completed for full biological expression of the drug activity. In contrast, the results of the in vivo adoptive assay of tumor growth give a clear indication that targeted liposomal DOX has a greater tumor-inhibitory potential than free DOX in line with the levels of drug delivered to tumor cells. One factor that may account for the relatively higher biological activity of FTL in vivo is the lag period of growth commonly observed after in vivo tumor implantation and the long time span of the experiment (>30 days),which may enable disaggregation and full bioavailability of intracellular drug before rapid cell growth occurs.

In conclusion, our results support the proposition that FTL offers an attractive means of delivering DOX into tumor cells, which is insensitive to Pgp-mediated drug efflux and more effective than free DOX and nontargeted liposomal DOX. FR-mediated drug delivery has the potential to circumvent multidrug resistance and may be especially useful if it can be coupled with the in vivo pharmacological advantages of long-circulating liposomal delivery systems, such as stable drug retention and tumor accumulation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Supported by the Israel Science Foundation(Jerusalem, Israel) and by ALZA Corporation (Mountain View, CA).

                
3

The abbreviations used are: FR, folate receptor;GPI, glycosyl-phosphatidylinositol; PEG, polyethyleneglycol; FTL,folate-targeted liposomes; Pgp, P-170 glycoprotein; MDR,multidrug-resistance; DOX, doxorubicin; DSPE,distearoyl-phosphatidylethanolamine; DPPE, dipalmitoyl-PE; HiFR, high expression of FR in M109 cells.

Fig. 1.

Confocal microscopy after 30 min(a and c) and 50 min (band d) of incubation of rhodamine-labeled FTL with M109-HiFR cells at 37°C, in the absence (a and b) or presence of 2 mm free folic acid, a 1000-fold excess over FTL folate (c and d).

Fig. 1.

Confocal microscopy after 30 min(a and c) and 50 min (band d) of incubation of rhodamine-labeled FTL with M109-HiFR cells at 37°C, in the absence (a and b) or presence of 2 mm free folic acid, a 1000-fold excess over FTL folate (c and d).

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Fig. 2.

Confocal microscopy of free DOX uptake by M109R-HiFR cells cultured in folate-depleted RPMI 1640 at 37°C. Incubation times: 7 min (a) and 30 min(b).

Fig. 2.

Confocal microscopy of free DOX uptake by M109R-HiFR cells cultured in folate-depleted RPMI 1640 at 37°C. Incubation times: 7 min (a) and 30 min(b).

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Fig. 3.

Confocal microscopy of liposomal DOX uptake by M109R-HiFR cells cultured in folate-depleted RPMI 1640 at 37°C. a, b, and c, binding,internalization, and nuclear localization of DOX delivered by FTL after a 30-min, 60-min, and 90-min incubation, respectively. d,cells exposed for 4 h to nontargeted PEG-coated liposomes encapsulating DOX (Doxil).

Fig. 3.

Confocal microscopy of liposomal DOX uptake by M109R-HiFR cells cultured in folate-depleted RPMI 1640 at 37°C. a, b, and c, binding,internalization, and nuclear localization of DOX delivered by FTL after a 30-min, 60-min, and 90-min incubation, respectively. d,cells exposed for 4 h to nontargeted PEG-coated liposomes encapsulating DOX (Doxil).

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Fig. 4.

Flow cytometry of M109R-HiFR cells exposed for 1 h to 10 μm DOX as free drug(a) or encapsulated in FTL (b) in the presence or absence of 10 mm verapamil. X axis, fluorescence intensity; Y axis, cell number; black line, no verapamil; gray line, verapamil added. In the case of free DOX(a), the curve shifts to the right, pointing to an increase in the amount of drug retained in the cells in the presence of verapamil, whereas in the case of FTL-DOX (b) no such shift occurs.

Fig. 4.

Flow cytometry of M109R-HiFR cells exposed for 1 h to 10 μm DOX as free drug(a) or encapsulated in FTL (b) in the presence or absence of 10 mm verapamil. X axis, fluorescence intensity; Y axis, cell number; black line, no verapamil; gray line, verapamil added. In the case of free DOX(a), the curve shifts to the right, pointing to an increase in the amount of drug retained in the cells in the presence of verapamil, whereas in the case of FTL-DOX (b) no such shift occurs.

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Fig. 5.

Drug accumulation by M109R-HiFR cells exposed to high and low drug:lipid ratio FTL. DOX concentration, 10μ m. The DOX:phospholipid ratios of the high and low ratio preparations were, respectively, 137.6 μg/μmol and 11.3μg/μmol.

Fig. 5.

Drug accumulation by M109R-HiFR cells exposed to high and low drug:lipid ratio FTL. DOX concentration, 10μ m. The DOX:phospholipid ratios of the high and low ratio preparations were, respectively, 137.6 μg/μmol and 11.3μg/μmol.

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Fig. 6.

Accumulation of DOX in nuclei and cytosol of M109R-HiFR cells after exposure to free and FTL DOX. M109R-HiFR cells were exposed to 10 μm DOX for 1 and 4 h.

Fig. 6.

Accumulation of DOX in nuclei and cytosol of M109R-HiFR cells after exposure to free and FTL DOX. M109R-HiFR cells were exposed to 10 μm DOX for 1 and 4 h.

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Fig. 7.

Cytotoxicity of DOX delivered as free drug, in nontargeted liposomes (Doxil) or in FTL against M109-HiFR cells (A) and M109R-HiFR cells (b)exposed for 1 h to the drug, washed, and further incubated for 120 h in drug-free medium. ○, free DOX-treated; □,FTL-DOX-treated; ▴, Doxil-treated. Note the significant shift to the right (i.e., decreased toxicity) of all of the growth curves in b as compared with a, as a result of the greater resistance of M109R-HiFR cells to DOX. Each point consists of six replicates; SDs do not exceed ± 15%.

Fig. 7.

Cytotoxicity of DOX delivered as free drug, in nontargeted liposomes (Doxil) or in FTL against M109-HiFR cells (A) and M109R-HiFR cells (b)exposed for 1 h to the drug, washed, and further incubated for 120 h in drug-free medium. ○, free DOX-treated; □,FTL-DOX-treated; ▴, Doxil-treated. Note the significant shift to the right (i.e., decreased toxicity) of all of the growth curves in b as compared with a, as a result of the greater resistance of M109R-HiFR cells to DOX. Each point consists of six replicates; SDs do not exceed ± 15%.

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Fig. 8.

In vivo adoptive assay of tumor growth. M109R-HiFR cells (106/mouse)were inoculated into the right hind mouse footpad after prior in vitro exposure to 10 μm free DOX, Doxil,or FTL-DOX for 2 h. The mean size of tumor-inoculated footpad along time of observation is shown.

Fig. 8.

In vivo adoptive assay of tumor growth. M109R-HiFR cells (106/mouse)were inoculated into the right hind mouse footpad after prior in vitro exposure to 10 μm free DOX, Doxil,or FTL-DOX for 2 h. The mean size of tumor-inoculated footpad along time of observation is shown.

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Table 1

DOX levels in M109R-HiFR cells(ng/106 cells) in the presence or absence of verapamila

Exposure time (min) 30 60 
Free-DOX 16 ± 1 28 ± 3 
Free-DOX+ verapamil 60 ± 4 112 ± 3 
FTL-DOX 104 ± 4 133 ± 12 
FTL-DOX+ verapamil 101 ± 10 124 ± 6 
Exposure time (min) 30 60 
Free-DOX 16 ± 1 28 ± 3 
Free-DOX+ verapamil 60 ± 4 112 ± 3 
FTL-DOX 104 ± 4 133 ± 12 
FTL-DOX+ verapamil 101 ± 10 124 ± 6 
a

M109R-HiFR cells in monolayer (24-multiwell plates) were exposed for 30 min or 1 h at 37°C to 5 μm (2.9 μg/ml) free DOX or FTL-DOX in the presence or absence of 10 mm verapamil. Thereafter, cells were washed and incubated further for 2 h in the presence or absence of 10 mm verapamil.

Table 2

DOX accumulation in cells (ng/106cells) exposed to free DOX, nontargeted liposomal DOX (Doxil), and targeted liposomal DOX (FTL-DOX)a

Cell testedM109-HiFRM109R-HiFRM109R-HiFR
Exposure time 1 h 1 h 4 h 
Free DOX 153 ± 3 81 ± 8 110 ± 11 
Doxil 5.2 ± 0.3 1.3 ± 0.1 Not done 
FTL-DOX 142 ± 2 180 ± 10 285 ± 8 
Cell testedM109-HiFRM109R-HiFRM109R-HiFR
Exposure time 1 h 1 h 4 h 
Free DOX 153 ± 3 81 ± 8 110 ± 11 
Doxil 5.2 ± 0.3 1.3 ± 0.1 Not done 
FTL-DOX 142 ± 2 180 ± 10 285 ± 8 
a

M109R-HiFR and M109-HiFR cells in monolayer (24-multiwell plates) were exposed to 10μ m DOX at 37°C. The drug concentration in M109R-HiFR cells treated with FTL-DOX was significantly greater than with free DOX (P < 0.0001, ttest).

Table 3

In vivo adoptive tumor growth assay in mice inoculated with M109R-HiFR tumor cells exposed in vitro to free DOX, FTL-DOX, and Doxila

Cell treatmentFinal tumor incidenceb (%)Tumor weight (mg) median (range)
Untreated 13 /20 (65%) 381 (48–825) 
Free DOX 8 /19 (42%) 239 (32–683) 
Doxil 10 /19 (53%) 397 (13–512) 
FTL-DOX 2 /20 (10%) 57 (27–87) 
Cell treatmentFinal tumor incidenceb (%)Tumor weight (mg) median (range)
Untreated 13 /20 (65%) 381 (48–825) 
Free DOX 8 /19 (42%) 239 (32–683) 
Doxil 10 /19 (53%) 397 (13–512) 
FTL-DOX 2 /20 (10%) 57 (27–87) 
a

Results of two experiments. DOX concentration in vitro, 10 μm.

b

Fisher’s exact test: FTL-DOX versus untreated, P = 0.0008; FTL-DOX versus Doxil, P = 0.0057; FTL-DOX versus free DOX, P = 0.0310. All other comparisons, not significant.

We thank D. Rund, A. Taraboulos, and Y. Barenholz for fruitful discussion, and O. Drize for technical assistance.

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