Tumor-targeting nanodelivery enhances the anticancer activity of a novel quinazolinone analogue

Microtubules are self-assembling polymers of α- and β-tubulin heterodimeric proteins and are important cytoskeletal components involved in many cellular events, including maintenance of cellular shape, intracellular transportation, and regulation of motility and mitosis (1, 2). Because of the key role of microtubules in cell mitosis, targeting the microtubule system of eukaryotic cells represents an attractive strategy for the development of anticancer agents. The first Food and Drug Administration–approved antimicrotubule agents were Vinca alkaloids, which interact with monomeric tubulin and prevent polymerization (tubulin destabilizer). In contrast, taxanes promote the assembly of microtubules and stabilize microtubule structures (tubulin stabilizer; ref. 3). Both tubulin destabilizers and stabilizers interrupt microtubule dynamics by binding to tubulin, resulting in mitotic arrest that triggers signals for the mitochondrial pathway of apoptosis (4). Although Vinca alkaloids (vincristine, vinblastine, and vinorelbine) and taxanes (paclitaxel and docetaxel) are effective anticancer treatments, there are limitations to their use, including toxic side effects and drug resistance through the P-glycoprotein efflux pump (2, 5, 6). In addition, the relatively large molecular weights of these drugs result in complicated synthesis or isolation procedures. Therefore, the search for, and development of, novel small-molecule antimicrotubule agents with improved anticancer effects continues (7, 8).

One approach in the development of improved agents is the modification of the structure of thalidomide. The current small-molecule investigated in this article is a third-generation analogue of thalidomide. Thalidomide was developed in the 1950s as a nontoxic hypnosedative and was widely used to prevent morning sickness in pregnant women until it was withdrawn from the world market because of growing evidence of its teratogenic effects (severe infant limb defects from maternal thalidomide usage; ref. 9). Despite this, thalidomide has shown therapeutic value due to its immunosuppressive and anti-inflammatory effects when used for treating erythema nodosum leprosum, graft-versus-host disease (10–12), HIV-associated cachexia (13), HIV-associated oral aphthous ulcers (9, 14), and a variety of mucocutaneous disorders. In addition, thalidomide has been reported as having antiangiogenic effects on corneal angiogenesis induced by vascular endothelial growth factor or basic fibroblast growth factor (15–17). Thalidomide also has been found to be effective in cancers such as relapsed or refractory multiple myeloma (18).

Several groups have been developing thalidomide analogues with more potent antiangiogenic, anti-inflammatory, and anticancer properties (19–25). Our first thalidomide analogue was obtained by replacing the glutaramide ring of thalidomide with phenyl to develop the first active compound showing antiangiogenic and anticancer effects in endothelial cells and prostate cancer cells, respectively (23). Hamel et al. (24) and Hour et al. (25) generated quinazolinones by modifying the phthalimide ring of thalidomide, both resulting in cell death due to inhibition of tubulin function in cells. We optimized the quinazolinone with various substitutions, which led to molecule GMC-5-193 (GMC; Fig. 1).³

Drug delivery systems, such as nanoparticles, microspheres, lipidols, emulsions, activated carbon particles, and liposomes, have been used as carriers of anticancer drugs to alter their pharmacokinetics and thereby reduce toxic side effects and increase antitumor potency (26, 27). Over the past three decades, significant advances have been made in injectable liposomal delivery of anticancer agents and some of them have been introduced into the pharmaceutical market, taking advantage of their biological compatibility. In particular, tumor-targeted immunoliposomes, with the ability to target their payload to tumor cells, are recognized as having an enormous advantage for cancer therapy (28, 29). We have developed a tumor-targeting liposomal delivery system that incorporates an anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting molecule for delivery of gene medicine and small interfering RNA (30–34). Transferrin receptor levels are known to be elevated in various types of cancer cells, including breast, prostate, and pancreatic, and their levels are also correlated with cell proliferation (35, 36). Taking advantage of this, the TfRscFv-liposome complex (scL) has shown preferential targeting of tumor cells. Enhanced expression of scL-delivered genes has been shown in primary tumors as well as metastatic tumors but not in normal tissues (31).

In this study, we employed the tumor-targeting liposomal complex (scL) for delivery of GMC to improve the anticancer effects of this small molecule. Two different human tumor cell lines were used to show the applicability of this approach to diverse types of cancer. Our data reveal that delivery of GMC by means of the tumor-targeting liposomal complex resulted in enhanced cellular uptake of GMC in these tumor cell lines in vitro and in vivo and increased the cellular response to standard chemotherapeutic agents when compared with free (noncomplexed) GMC.

The compound GMC was synthesized and its structure (Fig. 1) was confirmed.³ GMC has a molecular mass of 359.4 Da. A stock solution (2.5 mg/mL) of the compound was prepared in DMSO. 1,2-Dioleoyl-3-trimethylammonium propane and dioleolylphosphatidyl ethanolamine were purchased from Avanti Polar Lipids. Sodium 3′-[1-(phenylamino-carbonyl)-3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonate (XTT) was purchased from Polysciences. N-methyl dibenzopyrazine methylsulfate was purchased from Sigma-Aldrich.

The human prostate cancer cell line DU145 (ATCC HTB-81) and normal human lung fibroblasts IMR-90 (ATCC CCL-186) were obtained from the American Type Culture Collection. The human melanoma cell line MDA-MB-435 was provided by the Tissue Culture Core Facility at Lombardi Comprehensive Cancer Center, Georgetown University Medical Center. Cells were maintained at 37°C in a 5% CO₂ atmosphere in Eagle's MEM with Earl's salts (DU145 cells) or improved MEM (MDA-MB-435 cells) supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L l-glutamine, and 50 μg/mL each of antibiotics, that is, penicillin, streptomycin, and neomycin. IMR-90 was cultured in Eagle's MEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L l-glutamine, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and 50 μg/mL each of the antibiotics. MDA435/LCC6 cells (a gift from Dr. Robert Clarke, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center) were cultured in improved MEM supplemented with 5% heat-inactivated fetal bovine serum, 2 mmol/L l-glutamine, and 50 μg/mL each of penicillin, streptomycin, and neomycin. Eagle's MEM was purchased from MediaTech, and other cell culture media and ingredients were obtained from Biofluids.

Cationic liposomal formulation LipA (1,2-dioleoyl-3-trimethylammonium propane/dioleolylphosphatidyl ethanolamine at a 1:1 molar ratio) was prepared using the ethanol injection method as described previously (31). TfRscFv/LipA/GMC (scL/GMC) complex was prepared as described previously (32). The molar ratio of LipA to GMC was 1:1. For in vitro experiments, the complex was further diluted with serum-free medium. For animal injections, 50% dextrose was added to each sample to a final concentration of 5% dextrose. The size of the complex was determined by dynamic light scattering at 25°C with a Zetasizer Nano ZS System. The mean particle size of scL/GMC in water was 168.9 ± 6.1 nm.

For confocal imaging, 5.0 × 10⁴ MDA-MB-435 cells per well were seeded on glass coverslips in a 24-well plate. Twenty-four hours later, cells were washed with serum-free medium, treated with scL/GMC complex or free GMC, and incubated for 6 h. After treatment, cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, and washed again with PBS. For nuclear staining, blue fluorescent counterstain reagent 4′,6-diamidino-2-phenylindole (DAPI) in Select FX Nuclear Labeling Kit for fixed cells (Molecular Probes) was used according to the manufacturer's protocol. Cells on glass coverslips were then mounted on glass slides using Antifade mounting solution (ProLong Antifade Kit, Molecular Probes). For imaging, an Olympus FLUOVIEW-300 laser scanning confocal system with ×60 oil immersion objective was used. For the 4′,6-diamidino-2-phenylindole, a 405 nm blue diode laser was used to excite the sample. The emitted light from the sample was directed with a mirror through a 430 to 460 nm band-pass filter and captured by the PMT detector. For the GMC, the 488 nm blue line of an argon laser was used to excite the sample. The emitted light from the sample was directed with a mirror through a 505 to 525 nm band-pass filter and captured by the PMT detector.

For in vitro cell survival studies, 5.5 × 10³ DU145 or MDA-MB-435 cells per well in 100 μL of the appropriate growth medium were plated in a 96-well plate. After 24 h, the medium was replaced with serum-free medium, overlaid with 100 μL of increasing concentrations of either scL/GMC complex, unliganded complex (L/GMC), free GMC, or LipA only in serum-free medium, incubated for 5 h, and then supplemented with fetal bovine serum. After incubation for an additional 43 h at 37°C in a humidified atmosphere containing 5% CO₂, the wells were washed with improved MEM without phenol red and a cell viability XTT-based assay was done as described previously (37). Formazan absorbance, which correlates to cell viability, was measured at 450 nm using a microplate reader (Ultramark, Microplate Imaging System, Bio-Rad). The IC₅₀ value (the concentration yielding 50% growth inhibition) was interpolated from the graph of the log of drug concentration versus the fraction of surviving cells.

For the chemosensitization study, 4.5 × 10³ DU145, MDA-MB-435, or IMR-90 cells per well in 100 μL medium were seeded in a 96-well plate. After 24 h, the medium was replaced with serum-free medium and overlaid with 100 μL of either scL/GMC complex or free GMC, in each case the concentration of GMC being either 1 or 1.25 μmol/L. Cells were then incubated at 37°C for 5 h, after which fetal bovine serum was added to each well and incubated an additional 19 h. The appropriate supplemented medium was then added either with or without chemotherapeutics in increasing concentrations and incubation continued for ∼48 h. Chemotherapeutic drugs used were doxorubicin (Bedford Laboratories), docetaxel (Taxotere; Aventis Pharmaceuticals), and mitoxantrone (Osi Pharmaceuticals). XTT assays were done to assess the degree of sensitization to the chemotherapeutics, and IC₅₀ values were calculated. Fold sensitization was determined as follows: IC₅₀ chemotherapeutic agent only / IC₅₀ each combination treatment.

For in vivo studies, human melanoma MDA435/LCC6 cells (derived from MDA-MB-435 cells) were used to generate lung metastatic tumors. MDA435/LCC6 cells (8 × 10⁶ cells per mouse) suspended in PBS were injected i.v. into the tail vein of 5-week-old female athymic nude mice. Ten to 12 weeks after injection, MDA435/LCC6 xenograft lung metastases were established. The mice were injected i.v. with 9 mg/kg GMC as either free GMC or scL/GMC. Three hours after injection, the liver and lung were excised and examined under a fluorescence microscope (Nikon SMZ-1500 EPI-Fluorescence stereoscope system). Images were acquired with an Hg lamp source using a “FITC-like” set of filters [excitation (nm), 480/40; dichroic (nm), 505 nm; emission (nm), BA535/50]. The care and use of laboratory animals have been approved by the Georgetown University Animal Care and Use Committee.

Taking advantage of the inherent fluorescence of GMC, confocal imaging was used to compare the internalization of GMC when delivered as free GMC with that when complexed with the targeted liposome (scL/GMC). MDA-MB-435 cells were treated for 6 h with either free GMC or scL/GMC, washed with PBS, fixed with 4% paraformaldehyde in PBS, and visualized by confocal microscopy. At 6 h post-transfection, green fluorescence was greater in cells treated with scL/GMC than in cells treated with free GMC (Fig. 2), representing ∼3-fold increase in intensity (arbitrary values measured by Metamorph: 220 for scL/GMC and 72 for free GMC). Differential interference contrast (DIC) images revealed the morphology of the cells. GMC green fluorescence was distributed throughout cells treated with either free GMC or scL/GMC, including in the cytoplasm and the nucleus. These results indicate that GMC is more easily taken up by MDA-MB-435 cells when delivered by means of the targeted liposomal complex.

Molar ratios of GMC to targeted liposome (scL) were determined for optimal efficient delivery to cells. The dose response of scL/GMC on MDA-MB-435 cells was determined. As shown in Fig. 3A, IC₅₀ values of GMC-treated MDA-MB-435 cells were 12 μmol/L for free GMC and 3 μmol/L for scL/GMC, indicating a 4-fold increase in cell killing after treatment with the targeted liposome-delivered GMC (scL/GMC). Treatment of cells with liposome alone (LipA), in concentrations up to 6 μmol/L, did not result in any cytotoxicity. Only at the highest concentration of LipA (LipA concentration equivalent to 50 μmol/L scL/GMC) was some growth inhibition observed, that is, 50% growth inhibition. Similar results were obtained with DU145 cells (Fig. 3B), showing a 3-fold increase in cell death after treatment with scL/GMC compared with free GMC (IC₅₀, 4 and 12 μmol/L, respectively; Fig. 3B). Moreover, the importance of the tumor-targeting ligand is shown in Fig. 3C with MDA-MB-435 cells. Whereas there was very little difference observed between the free GMC (IC₅₀, 12 μmol/L) and the unliganded complex (L/GMC; IC₅₀, 9 μmol/L), there is a significant difference in IC₅₀ values between the complex with and the complex without the TfRscFv targeting moiety. The IC₅₀ value with the unliganded complex (L/GMC) is 9 μmol/L, whereas that of the full complex is 1.9 μmol/L, a >4-fold increase in cell killing when the ligand is included in the complex.

We have shown previously that GMC disrupts and destabilizes microtubules.³ In view of this, we hypothesized that the combination of GMC with conventional chemotherapeutics would enhance the anticancer effects of conventional chemotherapies. First, the ability of scL/GMC to sensitize MDA-MB-435 cells to docetaxel was explored (Fig. 4A). Docetaxel is a microtubule-targeted, tubulin-polymerizing agent that has shown a high level of clinical activity. Cells treated with a combination of liposome (LipA) and docetaxel showed an IC₅₀ value (9 nmol/L) similar to that of cells treated with docetaxel only, revealing a lack of toxicity of the liposomes themselves. Whereas treatment with 1.25 μmol/L free GMC resulted in an ∼21-fold increase in sensitization of MDA-MB-435 cells to docetaxel-induced cell death (IC₅₀, 0.43 nmol/L), encapsulation of the same concentration of GMC (1.25 μmol/L) within the targeted liposome nanocomplex resulted in an 80% reduction in cell survival even without treatment with docetaxel; therefore, the IC₅₀ was unable to be determined. In view of this, another experiment was done using a lower concentration of GMC (1 μmol/L; Fig. 4B). As in the previous experiment, administration of docetaxel alone and LipA plus docetaxel resulted in similar IC₅₀ values (12 and 9 nmol/L, respectively). GMC (1 μmol/L) delivered by the targeted liposome (scL/GMC) resulted in a dramatic sensitization of MDA-MB-435 cells to docetaxel (Fig. 4B), exhibiting an IC₅₀ value of 0.4 nmol/L. This represents a 30-fold increase in sensitization when compared with cells treated with docetaxel only and a 3-fold increase in sensitization over cells treated with free GMC and docetaxel (IC₅₀, 1.2 nmol/L).

GMC-induced sensitization of cancer cells to the chemotherapeutic agents doxorubicin and mitoxantrone was also evaluated. Doxorubicin is used to treat a variety of cancers including breast, ovarian, lung, thyroid, gastric, transitional cell bladder cancer, soft tissue and osteogenic sarcomas, neuroblastoma, Wilms' tumor, Hodgkin's and non-Hodgkin's lymphoma, acute myeloblastic leukemia, acute lymphoblastic leukemia, and even Kaposi's sarcoma related to AIDS. In vitro studies show doxorubicin to be potent in inducing cell death in various cancer cell lines including MDA-MB-435. Mitoxantrone, a synthetic antineoplastic anthracenedione, was also used in these studies due to its use in combination with corticosteroids as a first-step chemotherapy for the treatment of patients with advanced hormone-refractory prostate cancer. As shown in Fig. 4C, MDA-MB-435 cells treated with doxorubicin only exhibited an IC₅₀ of 700 nmol/L, whereas GMC (1 μmol/L), either in free form (free GMC) or complexed with the targeted liposome (scL/GMC), sensitized cells to doxorubicin-induced death, with IC₅₀ values of 620 and 73 nmol/L, respectively. Thus, GMC delivered by the targeted liposome resulted in a 9.6-fold increased sensitization to doxorubicin, whereas free GMC resulted in a 1.1-fold increase in sensitization to doxorubicin (Fig. 4C). Figure 4D shows that DU145 cells treated with either free GMC or scL/GMC (1.25 μmol/L GMC) were also sensitized to mitoxantrone-induced cell death, exhibiting IC₅₀ values of 21 and 11 nmol/L, respectively, and representing a greater increase in sensitization by scL/GMC (3.2-fold) than by free GMC (1.7-fold) when compared with mitoxantrone treatment only (IC₅₀, 35 nmol/L; Fig. 4D).

To confirm that the increases in sensitization of cells to chemotherapeutic drugs were not due to nonspecific cytotoxicity from GMC or scL/GMC, the normal human lung fibroblast cell line, IMR-90, was used (Fig. 5A and B). IMR-90 cells, when treated with docetaxel or mitoxantrone, showed no appreciable sensitization by LipA, free GMC, or scL/GMC when compared with the cells treated with docetaxel or mitoxantrone only.

These in vitro data support the observation that GMC delivered to cells by the targeted liposome sensitizes cancer cells to conventional chemotherapeutics more effectively than when delivered as free GMC.

We have shown previously that our TfRscFv-liposome can deliver DNA preferentially to tumor cells after systemic administration (31). The following studies were done to determine whether the TfRscFv-liposome could deliver a different class of payload, that is, a small molecule, also preferentially to tumor cells after systemic administration. Athymic nude mice carrying MDA435/LCC6 xenograft metastatic tumors, primarily in the lung, were injected i.v. with free GMC or scL/GMC at a concentration of GMC of 9 mg/kg/mouse. Three hours after injection, liver and lung were excised and examined under a fluorescence microscope. Figure 6 shows the same field photographed in bright-field (left) or with fluorescence (right). In the bright-field image of the mouse treated with free GMC, large tumors can be observed throughout the lung, but the fluorescent image shows only weak fluorescence in all the tissues. In the bright-field image of the mouse injected with scL/GMC complex, metastases are distinguishable as being denser than the lighter bubbly appearance of the normal lung tissue, and the fluorescent image shows the clearly discernible fluorescence of GMC in these lung metastases. Thus, GMC fluorescence in the lung metastases is ∼2-fold stronger in this mouse than in the mouse treated with free GMC when measured by Metamorph, representing a greater amount of GMC present in these metastases. In contrast to these images, the normal liver shows only weak fluorescence. These results show that tumor-specific uptake of GMC after systemic administration is enhanced when GMC is delivered by the targeted liposome complex.

Several studies using thalidomide analogues suggest that their antiproliferative effect in several types of cancer cells may be due to inhibition of tubulin polymerization (25, 26). These analogues initiate formation of abnormal mitotic spindles in cancer cells. Our previous study clearly showed that GMC, a thalidomide analogue, inhibits human cancer cell proliferation.³ The potency of this molecule is similar to other well-known antimitotic agents such as colchicine, combretastatin, and Vinca alkaloids. These compounds are tubulin destabilizers and have been found to inhibit the function of the mitotic spindle. Most, however, have been toxic with limited ability to track their intracellular mechanism. GMC is a fluorescent tubulin destabilizer that is advantageous for intracellular and intratumoral detection.

In vitro, in both human melanoma (MDA-MB-435) and prostate (DU145) cell lines, dose-response studies revealed IC₅₀ values of free GMC of 12 μmol/L after treatment for 2 days. Delivery of GMC by the tumor-targeting liposome nanodelivery system (scL/GMC) reduced the IC₅₀ values in MDA-MB-435 and DU145 cells by 4- and 3-fold, respectively (Fig. 3), revealing an increase in sensitivity of cells to GMC. At these IC₅₀ values, the liposome itself did not show any toxic effects on both cell lines. Figure 3C shows that the increase in the effectiveness of GMC in inducing cell death results from enhanced cellular uptake of GMC when delivered by the targeted liposome. Transferrin receptors are elevated in DU145 and MDA-MB-435 cells (35, 36) and have been shown to play a key role in receptor-mediated endocytosis of TfRscFv-liposomes (30–34), also resulting in increased cellular uptake. Thus, in this study, confocal images of MDA-MB-435 cells treated with the small molecule revealed that GMC can be more efficiently taken up by the cells when delivered by the targeted liposome than when delivered as free GMC (Fig. 2), as shown by the ∼3-fold increase in green fluorescence in cells treated with scL/GMC than in cells treated with free GMC. These data are similar to our previous reports showing that incorporation of the tumor-targeting moiety, TfRscFv, in the complex enhanced tumor-specific cellular uptake, systemic delivery of payload to both primary tumor and metastases, and efficacy (30–34).

Combination therapy, in which cancer patients are routinely treated with a combination of cancer therapies, has been shown as having many advantages over single-agent therapies. A combination of antimicrotubule agents docetaxel and estramustine has been evaluated in the treatment of metastatic, androgen-independent prostate cancer with positive results (38). Combination therapy comprising antimicrotubule agents plus doxorubicin or cisplatin is proposed as an effective regimen for breast cancers (39, 40). In addition, thalidomide, the parent compound of GMC, is being used more extensively in the treatment of various cancers such as multiple myeloma (41), colon cancer (42), and prostate cancer (43) in combination with cyclophosphamide, dexamethasone, capecitabine, irinotecan, or docetaxel. Combining dacarbazine and thalidomide has been reported as a promising strategy for the treatment of melanoma (44). Similarly, our current studies have shown increased sensitization of cells to conventional chemotherapeutics by combining treatment with the thalidomide analogue GMC.

GMC was shown to increase sensitization of MDA-MB-435 and DU145 cells to docetaxel/doxorubicin and mitoxantrone, respectively. However, MDA-MB-435 cells exhibited greater sensitivity to docetaxel than to doxorubicin (IC₅₀, 9-12 and 700 nmol/L, respectively), and a similar greater sensitivity to combination treatment with both GMC and docetaxel, than with both GMC and doxorubicin. It is not clear why the two chemotherapeutic drugs exhibited such differences in inducing cell death in the MDA-MB-435 cells, although this could be a cell-specific phenomenon. It is interesting to note that synergistic combinations of taxanes and antimicrotubule agents have been explored in several studies. Although expression of the mdr1 gene has been known shown to be the main cause of drug resistance, several studies have shown that cancer cells resistant to microtubule-targeting agents include alteration in microtubule structure due to changes in β-tubulin isotype content and tubulin content (2). An increase in β-tubulin III content has been repeatedly related to taxane resistance in vitro (2, 45) and in clinical settings. Recent clinical evidence supports β-tubulin III content as a marker of poor response to taxanes in different cancers, including non-small cell lung cancer (46), breast cancer (47), and ovarian cancer (48). Moreover, when paclitaxel-resistant lung cancer cells, A549-T24, were treated with antisense oligonucleotides to β-tubulin III, a 39% increase in sensitivity to paclitaxel was shown (49). Similarly, a synergistic effect between the tubulin stabilizer paclitaxel and the tubulin destabilizer vinorelbine against human melanoma lines was reported (50). Unfortunately, a combination of these chemotherapeutics is not currently in clinical use due to severe neurotoxic effects (51). In view of the above, the distinguishable synergistic effect of the scL/GMC complex in combination with docetaxel in MDA-MB-435 cells may be due to the fact that GMC targets β-tubulin III, thereby increasing sensitivity to docetaxel.

In our experiments, the effect of GMC on tumor cells was dramatically different from the effect of GMC on normal human lung fibroblasts IMR-90 under the same conditions, in that the normal lung fibroblasts showed no significant sensitization to chemotherapeutics when treated with either free GMC or scL/GMC. These data indicate that TfRscFv-targeted liposomal delivery targets the GMC complex to cancer cells but not to normal cells, thereby increasing its therapeutic index. The current in vitro experiments show that although GMC sensitizes cancer cells to conventional chemotherapeutic drugs the effect of GMC is greatly enhanced when delivered by the targeted liposome. Therefore, GMC nanoimmunocomplexes (scL/GMC) in combination with conventional chemotherapeutics may likely result in distinct advantages over treatment with free GMC or with conventional chemotherapeutics only and may permit a reduction in the effective dose of chemotherapeutic with a subsequent reduction in the incidence of adverse side effects.

The primary focus of this study was to deliver GMC specifically to tumor cells in vivo after systemic administration. scL/GMC administered i.v. to athymic nude mice carrying MDA435/LCC6 xenograft metastatic tumors primarily in the lung resulted in GMC delivery specifically to the metastatic tumor cells, to a greater extent than free GMC administered i.v., as determined by observation of intensity of green fluorescence in the lung metastases. Tumor-specific uptake of GMC after systemic administration of scL/GMC was clearly observed, correlating with our previous reports showing tumor-targeting delivery of gene medicine (30–34).

When it is established that scL/GMC could be delivered to metastatic tumors in vivo, the next step will be to determine antitumor efficacy. As discussed previously, GMC exhibits antimicrotubule activity, and it is also possible that the effectiveness of the small molecule as a cancer treatment may be due to antiangiogenic activity. Thalidomide is known to have antiangiogenic activity (16) and several thalidomide analogues have also been reported as exhibiting antiangiogenic activity (19, 23). As a thalidomide analogue, it is possible that GMC may also exhibit antiangiogenic activity, which may work cooperatively with antimicrotubule activity for cancer treatment, although further study on its mechanism of action is required.

These studies have shown efficient and tumor-specific targeted delivery of the novel anticancer small-molecule GMC. GMC can be delivered to tumors specifically and more efficiently when incorporated into a tumor-targeting liposome complex than as a free small molecule. As a result, a significantly higher level of in vitro chemosensitization in human melanoma and prostate cancer cells was evident when GMC was delivered by the targeted liposome nanodelivery system than when delivered as free GMC. Similarly, treatment of cancer cells with scL/GMC more efficiently sensitized cells to the chemotherapeutics docetaxel, doxorubicin, or mitoxantrone. In contrast, there was no increase in toxicity in normal lung fibroblasts. These data also showed a synergistic effect between the nanoimmunoliposome complexed small molecule and docetaxel in a melanoma lung metastases animal model. In humans, a substantial advantage to such a synergistic combination treatment would be the possibility of reducing the dose of conventional chemotherapeutic agents while maintaining or even increasing their effectiveness, with a concomitant decrease in toxic side effects, thereby increasing the quality of life for cancer patients.

We thank Dr. Leanne Sleer for advice and assistance in preparation of this article and Guillermo Palchik and Dr. Thane Govindan for assistance with confocal and fluorescence microscopy.