Stable Incorporation of a Lipophilic Daunorubicin Prodrug into Apolipoprotein E-Exposing Liposomes Induces Uptake of Prodrug via Low-Density Lipoprotein Receptor in Vivo

Experimental Procedures

Materials.

Human recombinant apoE₃, isolated from Escherichia coli, was a generous gift from Dr. T. Vogel (Biotechnology General Ltd., Rehovot, Israel) (Vogel et al., 1985). The apoE was dissolved in PBS (10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl) at a concentration of 2 mg/ml and was stored under argon at −80°C. [1α,2β(N)-³H]Cholesteryl oleate ([³H]CO), [1-¹⁴C]CO, and ¹²⁵I-labeled sodium salt were obtained from Amersham International (Amersham, Buckinghamshire, UK). [³H(G)]daunorubicin (DNR, daunomycin) was purchased from New England Nuclear Research Products (Boston, MA). Unlabeled and radiolabeled [³H]3α-O-(oleoyl)-5β-cholanic acid coupled to Ala-Leu-Ala-Leu-daunorubicin (further referred to as LAD; see chemical structure in Fig. 1) were synthesized as described previously (Versluis et al., 1998; purity >95%). The purity of the compounds was regularly checked by TLC on silica gel 60 F₂₅₄ (solvent: NH₄OH/CH₃OH/1,2 dichloromethane, 1:10:89). Egg yolk phosphatidylcholine (EYPC, 98%) was obtained from Fluka (Buchs, Switzerland). CO (97%) was from Janssen Chimica (Beerse, Belgium). Cholesterol oxidase, cholesterol esterase, peroxidase type II (200 U/mg), and PrecipathL were obtained from Boehringer Mannheim (Mannheim, Germany). 17αEE was purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade.

Preparation of LAD-Containing (apoE) Liposomes.

LAD-containing liposomes were prepared by sonication using a procedure described by Rensen et al. (1997a). EYPC (50 mg) and 1.9 mg CO were mixed in a 20-ml glass vial with 0 to 200 μg of LAD containing 0.2 μCi of [³H]LAD. For the in vivo experiments, 200 μg of LAD containing 1 μCi of [³H]LAD and 1.9 mg of CO containing 0.3 μCi of [¹⁴C]CO were added to 50 mg of EYPC. The solvent was evaporated under N₂ (1 h, room temperature), followed by vacuum desiccation overnight at 4°C. The lipids and prodrug were hydrated in 11.4 ml of 10 mM Tris·HCl buffer, pH 8.0, containing 0.1 M KCl. The mixture was subsequently sonicated (18 μm output) for 1 h under argon using a Soniprep 150 (MSE Scientific Instruments, Crawley, West Sussex, UK) equipped with a water bath to maintain the temperature at 54°C. After sonication, the liposomes were purified and concentrated by density-gradient ultracentrifugation at 285,000g for 18 h at 4°C, according to Redgrave et al. (1975). After ultracentrifugation, the liposomes, visible as a narrow opalescent layer at a density of 1.016 to 1.040 g/ml, were isolated by aspiration with glass capillary pipettes or by fractionation of the gradients. The isolated liposomes were stored under argon at room temperature. Liposomes prepared for the in vivo experiments were dialyzed against PBS and were used within 2 weeks after preparation. When indicated, the liposomes were provided with apoE by incubating the particles just before use for 30 min at 37°C with apoE at an apoE/phospholipid ratio of 0.1:1 (w/w).

Characterization of LAD-Containing (apoE) Liposomes.

The amounts of [³H]LAD and [¹⁴C]CO in the preparations were determined by measuring the radioactivity. The amounts of EYPC and unlabeled CO were determined using enzymatic kits for phospholipids and esterified cholesterol, respectively (Boehringer Mannheim, Mannheim, Germany). PrecipathL was used as standard in both assays. The size of the particles and the homogeneity of the population were determined by photon correlation spectroscopy at 27°C (90° angle) using a Malvern 4700c submicron particle analyzer (Malvern Instruments, Malvern, Worcs, UK). The net negative charge was determined by subjecting the (LAD-loaded) (apoE) liposomes to electrophoresis in a 0.75% (w/v) agarose gel at pH 8.8 (75 mM Tris·HCl-hippuric acid buffer, containing 0.65 mM EDTA). After electrophoresis, the gel was cut into small segments, which were dissolved in 0.75 ml of methyl-pyrrolidinone and subsequently counted for ³H and¹⁴C radioactivity.R_f values were determined relative to the front marker bromphenol blue. It was calculated that 1 mg of phospholipids represents 7.6 × 10¹³liposomes. For these calculations, it was assumed that the phospholipid bilayer length was 39 × 10⁻¹⁰ m and that the polar head of a EYPC occupies a surface of 4.2 × 10⁻¹⁹ m² (New, 1990).

Radiolabeling of apoE.

The apoE was radioiodinated at pH 10.0 with carrier-free ¹²⁵I as described previously (Van Tol et al., 1978). Unbound ¹²⁵I was removed by gel filtration followed by extensive dialysis against PBS containing 1 mM EDTA. More than 98% of the radiolabel in the preparations was trichloroacetic acid precipitable. The specific activity of ¹²⁵I-labeled apoE was approximately 450 dpm/ng.

Determination of Plasma Clearance and Tissue Uptake of [³H]DNR- and [³H]LAD-Loaded (apoE) Liposomes in Rats.

Male Wistar rats (150–200 g) were maintained on normal chow and had free access to water. The animals were anesthetized by i.p. injection of sodium pentobarbital (60 mg/kg b.wt.), and the abdomen was opened. Subsequently, the rats were injected in the vena cava inferior with 0.1 nmol of [³H]DNR or with 5 nmol of [³H]LAD incorporated in [¹⁴C]CO-labeled (apoE) liposomes (4 mg of phospholipid). At the indicated times, blood samples of 0.3 ml were taken from the vena cava inferior, and 0.1-ml serum samples were assayed for radioactivity. The total amount of radioactivity in serum was calculated using the equation: plasma volume (ml) = [0.0219 × body weight (g)] + 2.66 (Bijsterbosch et al., 1989). At the indicated times, liver lobules were tied off and excised, and at the end of the experiment, the remainder of the liver was removed. The amount of liver tissue tied off successively did not exceed 15% of the total liver mass. The amount of radioactivity in the liver at each time point was calculated from the radioactivities and weights of the liver samples. Uptake by extrahepatic tissues was determined by removing the tissues at the end of the experiment and counting the radioactivity. Tissue radioactivity was corrected for radioactivity in plasma present in the tissue at the time of sampling (Bijsterbosch et al., 1989). When indicated, rats were injected s.c. for 3 consecutive days with 17α-EE (1 mg/ml in 1,2-propylene glycol) at a dose of 5 mg/kg b.wt. The effectiveness of the treatment was monitored by measuring loss of body weight and reduction of total serum cholesterol levels (Chao et al., 1979). On the fourth day, the animals were injected i.v. with [³H]LAD-containing [¹⁴C]CO-labeled (apoE) liposomes as described above.

Determination of Association of LAD to apoE Liposomes in Circulation.

Male Wistar rats (150–200 g) were injected with [³H]LAD-loaded [¹⁴C]CO-labeled apoE liposomes (5 nmol of [³H]LAD and 4 mg of phospholipid). After 30 min, the rats were sacrificed, and blood samples were collected. Then, 1-ml serum samples were subjected to density ultracentrifugation at 285,000g at 4°C for 18 h, according to Redgrave et al. (1975). After centrifugation, the gradients were fractionated into 0.5-ml aliquots, starting at the top of the centrifugation tube.³H and ¹⁴C radioactivities were measured, and the fractions were assayed for total cholesterol.

Serum Decay of LAD-Loaded apoE Liposomes in Wild-Type and LDL Receptor-Deficient Mice.

Male C57Bl/6J-129Sv LDL receptor (−/−) mice (Ishibashi et al., 1993; 22–26 g) and male wild-type C57Bl/6J mice (22–26 g) were maintained on normal chow and had free access to water. The mice were injected i.v. in the tail vein with [³H]LAD-containing [¹⁴C]CO-labeled apoE liposomes (0.5 nmol of [³H]-labeled LAD and 0.5 mg of phospholipid). At the indicated time points, blood samples were taken from the tail, and serum samples were assayed for radioactivity.

Determination of Radioactivity

³H and¹⁴C radioactivities were determined in a Packard 1500 TriCarb liquid scintillation analyzer. Radioactivity (³H and ¹⁴C) in serum, liver, and other tissue samples was counted after combustion (recovery >97%) in a Packard Tri-Carb 306 Sample Oxidizer.¹⁴C radioactivity in the combusted samples was measured in a Carbosorb E/Permafluor mixture (2:3 v/v), and³H radioactivity was counted in Monophase S. Other ³H- and¹⁴C-containing samples were counted in Emulsifier Safe (aqueous solutions), Ultima Gold, or HIONIC Fluor (organic solutions) scintillation cocktails. All instruments and scintillation cocktails were from Packard Instrument Company Inc. (Downers Grove, IL).

Results

Preparation and Physicochemical Characterization of LAD-Loaded (apoE) Liposomes.

The incorporation of LAD into small liposomes was investigated by cosonication of EYPC and CO with a varying amount of [³H]LAD. The resulting particles were purified by density-gradient ultracentrifugation, and the amount of incorporated radiolabeled prodrug and the lipid composition were determined. Table 1 shows that LAD could be efficiently incorporated into the small liposomes. At the lower LAD concentrations (up to 15 μg of LAD/mg of CO), virtually all added LAD became incorporated. At higher concentrations, the efficiency was lower: 40 to 50%. Size, homogeneity, and lipid composition of the liposomes were not significantly changed by the incorporation of LAD, even when higher concentrations of LAD were used. The liposomes prepared with 100 μg of LAD/mg of CO formed a homogeneous (polydispersity <0.2) population of particles with a size of 29.3 ± 1.1 nm. It was calculated that these liposomes contained 11 ± 3 molecules of LAD per liposomal particle. All further experiments were performed with these liposomes.

To induce LDL receptor-mediated uptake, it is necessary to provide the liposomes with a specific LDL receptor-recognizing ligand. Therefore, LAD liposomes were incubated with recombinant apoE. Figure2 shows the association of apoE to the liposomes at different apoE/phospholipid ratios. The association of apoE to the liposomes was maximal at a ratio of 40 μg of apoE/mg of phospholipid. It can be calculated that at this ratio, approximately 9 molecules of apoE are associated per liposome. For all in vivo experiments, liposomes were incubated with 100 μg of apoE/mg of phospholipid to ensure that the liposomes were maximally provided with apoE. The excess of unbound apoE present in the preparations does not interfere with the LDL receptor recognition of the apoE liposomes because free apoE is not able to bind to the LDL receptor (Innerarity et al., 1979).

To investigate the effects of apoE association and LAD incorporation on the electric charge of the liposomes, [³H]LAD-loaded and/or [¹⁴C]CO-labeled liposomes were subjected to gel electrophoresis at pH 8.8. Table 2 shows that the association of apoE (R_f free apoE, 0.45) and/or the incorporation of LAD do not cause major changes in the electrophoretic mobility (i.e., the electric charge) of the particles. LAD-loaded apoE liposomes migrated in a slightly broadened band. However, the median R_f of the particles (0.14) was very similar to those of the free liposomes, the LAD-loaded particles, and apoE liposomes (R_f values 0.14, 0.09, and 0.14, respectively). Both labels of the LAD-loaded apoE liposomes migrated similarly, indicating that under these conditions the particles remain intact. The mobility of the liposomes was slightly lower than that of native LDL (R_f: 0.21 ± 0.01; mean ± S.D., n = 3). The combination of a low negative surface charge and the small size of the drug-carrier complex is beneficial with respect to avoiding non-LDL receptor-mediated uptake mechanisms by phagocytes of the reticuloendothelial system (Gabizon and Papahadjopoulos, 1988).

Serum Decay and Tissue Distribution of DNR and apoE Liposome-Associated LAD in Rats.

To investigate the effect of the lipophilic derivatization of daunorubicin and the subsequent incorporation of the prodrug into apoE liposomes on the biological fate of the drug, the serum decay and tissue distribution of both the prodrug LAD and free daunorubicin were determined (Figs.3 and 4). Incorporation into apoE liposomes considerably enhanced the half-life of the derivatized daunorubicin in the circulation. After 30 min, 50.3 ± 4.7% of the injected LAD was still circulating, whereas at that time, only 2.0 ± 0.9% of the injected amount of daunorubicin was recovered in the circulation. Daunorubicin was recovered in a wide variety of tissues, whereas the uptake of LAD in these tissues was much lower. The low disposition of LAD in the extrahepatic tissues, in particular in the heart, may reduce or even eliminate the side effects of free daunorubicin in these tissues.

To verify the stable association of LAD with the carrier during circulation, rats were injected with double-labeled ([³H]LAD and [¹⁴C]CO) LAD-loaded apoE liposomes, and a blood sample was collected at 30 min after injection. The serum was subjected to density ultracentrifugation, and the gradients were fractionated. The fractions were assayed for ³H and ¹⁴C radioactivity (LAD and liposomes, respectively) and for cholesterol (rat lipoproteins). Figure 5 shows that the distribution of liposomes and LAD over the fractions were identical, reaching a peak at a density of 1.085 g/ml, just between the rat LDL and high-density lipoprotein. The results suggest that after 30 min of circulation, LAD is still completely associated with the liposomes and that no redistribution of the prodrug to the rat lipoproteins had occurred.

LDL Receptor-Mediated Uptake of (apoE) LAD Liposomes in Control and 17αEE-Treated rats.

To allow evaluation of the role of LDL receptor-mediated uptake in the clearance of LAD-loaded apoE liposomes, rats were pretreated with 17αEE. This steroid induces in rats an enhanced expression of LDL receptors in the liver and therefore is widely used as a model to study the role of the LDL receptor in the clearance of circulating ligands (Chao et al., 1979; Harkes and Van Berkel, 1983). Both the behavior of the liposomes (labeled with [¹⁴C]CO) and [³H]LAD were studied. To examine the role of apoE, both apoE-containing liposomes and liposomes devoid of apoE were used. The apoE liposomes were cleared faster from the circulation than liposomes lacking apoE (Fig. 6, A and B). Thirty minutes after injection, 59 ± 5% of the injected [¹⁴C]CO label of the apoE liposomes was recovered in the serum. After injection of liposomes lacking apoE, 90 ± 3% of the injected liposomal label was still present in the serum at that time. The LAD present in the liposomes was also cleared faster when the liposomes were provided with apoE. However, at all time points, approximately 15% less [³H]LAD was recovered in the serum than in liposomal label. The ratios of the recovered labels (³H/¹⁴C; ratio at time of injection set at 1) did not change in time (0.86 ± 0.02 and 0.85 ± 0.02 for apoE liposomes and liposomes lacking apoE, respectively). Apparently, after an initial loss of some LAD from the liposomes, the LAD is cleared simultaneously with the liposomes. A similar phenomenon was also observed after the injection of LAD-loaded (apoE) liposomes in control rats (results not shown).

The liver uptake of LAD-loaded (apoE) liposomes, measured at 10 min after injection into 17αEE-treated rats or untreated control rats, is presented in Fig. 7. In the control rats, the uptake of both the liposomal [¹⁴C]CO label and [³H]LAD was low, and the presence of apoE did not significantly affect liver uptake. In the 17αEE-pretreated rats, the uptake of both labels was very similar to the uptake in control rats when liposomes lacking apoE were injected (approximately 6% of the dose). However, the hepatic uptake of apoE-enriched LAD-loaded liposomes in these rats was much higher: 30 ± 4% of the injected [¹⁴C]CO label and 18 ± 1.4% of the injected [³H]LAD were recovered in the liver (mean ± S.E.M., n = 4). Thus, a substantial amount of LAD was taken up by the liver together with its liposomal carrier. These results further indicate that the addition of apoE is crucial for the recognition of the drug-carrier complex by the LDL receptor.

Serum Decay of LAD-Loaded apoE Liposomes in Wild-Type Mice and Mice Lacking LDL Receptor.

The role of the LDL receptor in the clearance of LAD-loaded apoE liposomes was also studied by comparing the serum decay of ([¹⁴C]CO/[³H]LAD-labeled) LAD-loaded apoE liposomes in homozygote LDL receptor-deficient mice and in wild-type mice (Fig. 8). A remarkable difference in the clearance of the drug-carrier complex in wild-type and LDL receptor deficient-mice was observed. After 4 h, 22 ± 5% and 45 ± 6% of the injected amounts of liposomal label were recovered in the serum of wild-type and LDL receptor-deficient mice, respectively. At this time, the recoveries of [³H]LAD in the serum were 16 ± 3% and 31 ± 5%, respectively. Like in the rats, an instantaneous loss of LAD of approximately 15% from the liposomes was observed in both strains of mice. After 30 min, the³H/¹⁴C ratios in the serum of wild-type and LDL receptor-deficient mice were 0.80 ± 0.02 and 0.84 ± 0.05 (mean ± S.D., n = 4; ratios at time of injection set at 1), respectively. After 4 h of circulation, the ratios decreased to 0.73 ± 0.09 and 0.68 ± 0.07, respectively. This indicates that at 4 h after injection of the LAD-loaded apoE liposomes, the circulating liposomes still carry 70% of the initially incorporated drug. The very similar rate of clearance of LAD and apoE liposomes that was found after the rapid initial loss of LAD from the liposomes suggests that LAD and the apoE liposomes are simultaneously cleared by the same mechanism. The difference in the clearance of the LAD-carrier complex in the two mice strains indicates that the LDL receptor is involved in the clearance of LAD-loaded apoE liposomes.

Discussion

The feasibility of chemotherapy in which native LDL loaded with cytotoxic drugs is used to eradicate tumors expressing the LDL receptor is strongly hampered by the limited availability of native LDL and artifacts induced by incorporation of drugs. The apoE-enriched liposomes that we recently described (Rensen et al., 1997a) and mimic LDL in physiological behavior form a novel system that allows on a pharmaceutical scale preparation of drug-carrier complexes for LDL receptor-mediated targeting of antineoplastic drugs to tumors. In this study, we incorporated a lipophilic derivative of daunorubicin (LAD) into these liposomes and demonstrate that the prodrug remains in vivo associated with the liposomes and that the drug-carrier complex is cleared by LDL receptors.

Unmodified daunorubicin and doxorubicin can be incapsulated into liposomes. The rationale for using a lipophilic prodrug in the present study is that the small (29 nm) liposomal particles have a phospholipid bilayer that composes 60% of their total volume. Because of the large volume of liposomal phospholipid, a high drug load may be expected with lipophilic drugs that are incorporated into the phospholipid bilayer. The small size of our liposomes is dictated by the use of apoE as a specific LDL receptor-homing marker. Larger (>50 nm) apoE-exposing lipid particles lose their LDL receptor specificity and are mainly cleared by the apoE remnant receptor (Rensen et al. 1997b).

The incorporation of the lipophilic daunorubicin derivative into the liposomes was easily achieved by cosonication of the drug with the lipid components. Up to 11 molecules of drug could be incorporated per particle without changing the size and lipid composition of the liposomes. The stoichiometry of the incorporation suggests that incorporation of higher amounts of LAD may be feasible. The incorporation efficiency (44% at the highest drug load) is not optimal. However, the suboptimal incorporation efficiency does not necessarily constitute a problem for pharmaceutical application because nonincorporated LAD can easily be regained. Nevertheless, in future experiments we will develop procedures to optimize the incorporation efficiency of LAD. The presence of LAD in the liposomes (up to 44 μg of LAD/mg of CO) did not affect the ability of the liposomes to acquire apoE. The liposomes acquired maximally 9 molecules of apoE per liposome, which should be sufficient to accomplish optimal binding to the LDL receptor. Pitas et al. (1980) demonstrated that dimyristoyl-phosphatidylcholine vesicles exposing minimally 4 apoE molecules per vesicle displayed a maximal binding affinity for the LDL receptor. Agarose gel electrophoresis of the drug-carrier complex showed that LAD-loaded apoE liposomes have a low negative surface charge. This is important to circumvent in vivo uptake by other uptake mechanisms than the LDL receptor-mediated pathway, such as uptake by the reticuloendothelial system (Gabizon and Papahadjopoulos, 1988;Patel, 1992).

The lipophilic derivatization of daunorubicin and the subsequent incorporation of the prodrug into apoE liposomes dramatically altered the biological fate of the drug. Free daunorubicin was cleared from the circulation with a half-life of less than 1 min, whereas the liposome-associated prodrug displayed a much longer half-life. Furthermore, the disposition of free daunorubicin in the heart (related to the dose-limiting cardiotoxicity) and other organs was not seen with the lipophilic daunorubicin derivative. The substantially reduced disposition of LAD in the heart and other extrahepatic organs probably allows the administration of higher doses of LAD than daunorubicin, which may lead to an increased therapeutic effect.

The firm association of a drug with its carrier is a prerequisite for the successful targeting of drugs by a drug-carrier system in vivo. Data from our recent study suggest that LAD can form a stable drug-carrier complex (Versluis et al., 1998). In this study, LAD and liposomes were recovered in the same fraction after density-gradient centrifugation of a serum sample taken 30 min after injection of LAD-loaded liposomes into a rat. This indicates that LAD does not significantly redistribute to serum (lipo)proteins. However, we also observed in the present study in both mice and rats a loss of approximately 15% of LAD from the liposomes shortly after injection. This probably represents a nonsufficiently tight associated prodrug. The remaining part was cleared at a rate similar to that of the liposomes, which points to a stable anchoring of LAD into the liposomes.

The role of the LDL receptor in the clearance of LAD-loaded apoE liposomes was clearly demonstrated in 17αEE-pretreated rats. These rats have an elevated expression of LDL receptor in the liver and are widely used as a model to investigate LDL receptor-mediated uptake of circulating ligands (Chao et al., 1979; Harkes and van Berkel, 1983). The LDL receptor expression in control rats is very low (Nagelkerke et al., 1986). In the pretreated rats, the LAD-loaded apoE liposomes were more rapidly cleared from the circulation than in control rats. The hepatic uptake of the liposomal label was enhanced 5-fold, and 30 min after injection, the liver contained approximately 30% of the injected dose. The liver uptake of these apoE liposomes therefore was comparable to that reported for [³H]CO-labeled LDL in 17αEE-pretreated rats (approximately 25% of the dose at 30 min after injection; Pieters et al., 1991). These results clearly indicate the involvement of the LDL receptor. The uptake of the liposomes lacking apoE in the 17αEE-pretreated rats was very similar to the uptake of (apoE) liposomes in untreated rats. This finding indicates that the presence of apoE on the liposomes is essential for the LDL receptor recognition. Furthermore, the relatively rapid tissue uptake of the apoE liposomes indicates that the 29-nm-diameter particles can readily extravasate and become available for receptor-mediated uptake. The hepatic uptake of apo E liposomes in the control rats was low, which indicates that the presence of apoE on the particles does not induce liver uptake by other mechanisms (e.g., the remnant receptor).

Further evidence for the involvement of the LDL receptor in the clearance of LAD-loaded apoE liposomes was obtained by comparing the clearance of the particles in LDL receptor-deficient mice with that in wild-type mice. In LDL receptor-deficient mice, the half-lives of both the prodrug and the liposomal label were approximately 2 times higher than that in wild-type mice. This finding agrees well with the earlier findings of Ishibashi et al. (1993), who reported that compared with wild-type mice, the half-life of LDL in LDL receptor-deficient mice was increased 2.5-fold. The difference in clearance between the two mice strains is specifically LDL receptor mediated. An initial rapid loss of approximately 15% of the liposome-associated LAD was also observed in mice injected with the LAD-loaded apoE liposomes. Subsequently, the drug and the carrier were cleared very similarly. At 4 h after injection of the LAD-loaded apoE liposomes, the circulating particles still carried 70% of the initially incorporated drug.

In conclusion, in the present study, we show that the lipophilic daunorubicin prodrug LAD can be stably incorporated into apoE-enriched liposomes. Compared with free daunorubicin, the liposome-associated LAD showed a remarkably increased circulation half-life and a substantially reduced tissue disposition, which may reduce the dose-limiting side effects of daunorubicin. LAD was firmly anchored into the liposomes, and the biological fate of the drug was largely determined by its carrier. The LDL receptor plays an important role in the clearance of the LAD-loaded apoE liposomes, as was clearly demonstrated in 17αEE-pretreated rats and in LDL receptor-deficient mice. The presence of apoE appeared to be essential for the LDL receptor-mediated processing of the drug-carrier complex. Because the affinity of apoE liposomes for the LDL receptor is 15-fold higher than that of LDL (Rensen et al., 1997a), the drug-loaded apoE liposomes probably have a competitive advantage over circulating endogenous LDL for binding the LDL receptor. The present encouraging data indicate that a novel therapy based on LDL receptor-mediated selective delivery of antineoplastic drugs to tumor cells is feasible.