Novel Chondroitin Sulfate-binding Cationic Liposomes Loaded with Cisplatin Efficiently Suppress the Local Growth and Liver Metastasis of Tumor Cells in Vivo¹

GAGs³ exist in tissues mainly as proteoglycans (1). They include heparin, heparan sulfates, dermatan sulfates, keratan sulfates, hyaluronic acid, and CSs. GAG chains are highly anionic and hence have been implicated in numerous cellular functions, including cell adhesion (2, 3), cellular growth (4), tumor cell invasion (5), and viral infection (6).

Altered levels of production of and structural changes in GAGs have been reported in many neoplastic tissues (7, 8). In particular, an increased production of CSs has been found in transformed fibroblasts (9), mammary carcinoma cells (10, 11), and melanoma cells (12, 13). The selective and enhanced expression of CSs in certain types of malignant cells raises the possibility that the tumor-associated CSs may be a suitable molecular target for the selective delivery of anticancer drugs to malignant cells.

Liposomes have been used as carriers for the targeted delivery of anticancer drugs (14). Recent technical advances have established that liposomes containing PEG-conjugated lipids show reduced uptake by phagocytic cells and hence remain in the blood circulation for a long period of time (15). The sustained residence in the circulation enhances the liposomal accumulation in solid tumors (16, 17, 18) through tumor vessels where the permeability of endothelial barriers is much greater than it is in normal tissues. Experimentally, the unique tumoritropic accumulation of PEG liposomes has allowed the successful delivery of liposome-encapsulated drugs to solid tumors in vivo (19, 20). On the basis of these results, several liposomal formulations loaded with chemotherapeutic drugs are already being used clinically (21).

We recently developed a new formulation for long-circulating PEG liposomes that contain a new cationic lipid, TRX-20. We reported previously that TRX-20 liposomes preferentially bound to subendothelial cells and mesangial cells in vitro by recognizing CSs on the surface of these cells (22). When administered to mice with glomerulonephritis, the TRX-20 liposomes selectively accumulated in glomerular mesangial lesions where vascular permeability was increased and CSs were abundantly expressed (23). Furthermore, prednisolone encapsulated into TRX-20 liposomes showed an increased therapeutic efficacy, compared with the free drug (23). These results prompted us to investigate whether the TRX-20 liposomes could be used to selectively deliver anticancer drugs to tumor cells in vivo that have enhanced expression of CSs.

In this study, we examined in vitro and in vivo the applicability of TRX-20 liposomes to the targeted delivery of cisplatin to highly invasive LM8G5 and ACHN tumor cells expressing large amounts of CSs. We demonstrated that TRX-20 liposomes loaded with cisplatin successfully killed these tumor cells in vitro and effectively suppressed their local growth in s.c. sites, with a marked suppression of liver metastasis in vivo. Our results suggest that TRX-20 liposomes represent a novel strategy for the targeted delivery of anticancer drugs to CS-expressing tumor cells in vivo.

Female C3H/HeN mice and ICR nude mice, 10–14 weeks old, were obtained from Charles River Japan (Kanagawa, Japan) and kept in standard housing. All animal experiments were performed under the experimental protocol approved by the Ethics Review Committee for Animal Experimentation of Osaka University Graduate School of Medicine.

The ACHN human renal adenocarcinoma cell line was kindly provided by Dr. Tae Takeda (National Children’s Medical Research Center, Tokyo, Japan). The LM8G5 murine osteosarcoma cell line, which has a high potential for metastasis to the liver, was isolated from LM8 cells (Riken Cell Bank, Tsukuba, Japan) after five successive cycles of in vivo selection procedures (24). The HT-29 human colon adenocarcinoma cell line was obtained from American Type Culture Collection. The ACHN and HT-29 cells were maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum, 1% (v/v) 100× nonessential amino acids, 1 mm sodium pyruvate, 2 mm l-glutamine, 50 μm 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin. The LM8G5 cells were maintained in DMEM (Sigma-Aldrich) containing the same additives as above.

The lipids used in this study were as follows: HSPC (Lipoid, Ludwigshafen, Germany), Chol (Solvay Pharmaceuticals B.V., Weesp, The Netherlands), PEG-PE (M_r = 5,000; Genzyme Pharmaceuticals, Liestal, Switzerland), and Lissamine rhodamine-PE (Molecular Probes, Inc., Eugene, OR). TRX-20 was synthesized as reported previously (22). CS A, CS B, CS C, CS D, CS E, hyaluronic acid, heparan sulfate, keratan sulfate, chondroitin, mAb CS-56 (mouse IgM, specific for CS; Refs. 25, 26), chondroitinase ABC, and heparitinase I were all obtained from Seikagaku Corporation (Tokyo, Japan). Heparin and mouse IgM were obtained from Sigma-Aldrich.

Liposomes were prepared as described (22) with some modifications. The lipid mixtures HSPC:Chol = 54:46 or HSPC:Chol:TRX-20 = 50:42:8, which include 0.2 mol % of rhodamine-PE, were dissolved in t-butyl alcohol and lyophilized. After hydration at 65°C in 0.9% NaCl for empty liposomes or in 7.1 mg/ml cisplatin (Heraeus, Hanau, Germany) in 0.9% NaCl for cisplatin-containing liposomes, the liposomes were prepared by vigorous vortexing, sonication, and extrusion through double-stacked polycarbonate membranes (Nucleopore Whatman, Inc., Clifton, NJ) to obtain sized liposomes (∼100 nm in diameter). Unentrapped, precipitated cisplatin was removed by successive filtering through a 0.45-μm pore filter and gel filtration (Sepharose 4FF; Amersham Pharmacia Biotech AB, Uppsala, Sweden). A PEG-PE solution was added to the liposomes and incubated at 60°C for 30 min to incorporate 0.75 mol % of PEG-PE into the liposomal outer membrane. The lipid concentration was determined using a phospholipid determination kit (Wako Pure Chemical Industries). The cisplatin concentration was determined by high-performance liquid chromatography.

GAGs (5 μg/well) were immobilized on Sumilon Amino-type 96-well plates (Sumitomo Bakelite, Tokyo, Japan) overnight at 4°C and treated with 50 milliunits/ml of chondroitinase ABC or 20 milliunits/ml of heparitinase I for 1 h at 37°C or left untreated. After the plates were washed with PBS, rhodamine-labeled TRX-20 liposomes (50 nmol of lipid) were added to them, and the plates were incubated for 1 h at 37°C. After being washed with PBS, the liposomes were solubilized by adding lysis buffer [20 mm Tris-HCl (pH 7.4), 0.1% SDS, 1% Triton-X100, and 1% sodium deoxycholate]. The fluorescence intensity was measured by a fluorescence plate reader, Fluoroskan II (Labsystems, Helsinki, Finland).

Tumor cells were seeded (3 × 10³ cells/well) into 96-well plates and incubated for 2 days. The cells were rinsed twice with PBS and fixed with 3% paraformaldehyde for 30 min. After the cells were washed with the washing buffer (PBS containing 0.1% BSA), they were blocked with 3% BSA in PBS and treated with or without 50 milliunits/ml of chondroitinase ABC or 20 milliunits/ml of heparitinase I for 1 h at 37°C. The cells were washed again in washing buffer, then incubated with diluted CS-56 (1:100 diluted ascites) mAb for 1 h, followed by peroxidase-conjugated goat antimouse IgG+M (Biosource International, Camarillo, CA; 1:1000). To quantify the reaction, o-phenylenediamine was used as a substrate.

The tumor cell monolayer was treated with chondroitinase ABC or heparitinase I as above or left untreated, then the rhodamine-labeled TRX-20 liposomes or PEG liposomes (50 nmol of lipid) were added, and the monolayers were incubated for 1 h at 37°C. The monolayers were washed with PBS, bound liposomes were solubilized by adding lysis buffer, and rhodamine fluorescence was measured as above.

Tumor cells (8 × 10³/well) were cultured for 48 h in 8-well Lab-Tek chambers (Nalge Nunc International), then rhodamine-labeled TRX-20 liposomes or control PEG liposomes (50 nmol of lipid) were added for 1 h at 37°C. After extensive washing with PBS, the tumor cells were treated with chondroitinase ABC (50 milliunits/ml) for 1 h at 37°C or left untreated to evaluate whether the TRX-20 liposomes were surface-associated or internalized by the tumor cells. In some experiments, the tumor cells were incubated for 24 h before the chondroitinase ABC treatment to allow intracellular uptake of surface-bound liposomes. Subsequently, the tumor cells were incubated with 10 μm Hoechst 33342 (Molecular Probes, Inc.) for nuclear staining, fixed in 5% phosphate-buffered formalin, and examined with a fluorescence microscope BX50 (Olympus, Tokyo, Japan).

Tumor cells (1.5 × 10³ cells/well) were first incubated for 24 h to make a monolayer, then treated with free cisplatin or cisplatin entrapped in TRX-20 liposomes or PEG liposomes for 24 h. After the drugs were removed by washing the cells with PBS, the cells were further incubated for 60 h at 37°C. Cell proliferation was determined by water-soluble tetrazolium salt assay as described previously (27).

C3H/HeN and ICR nude mice were inoculated s.c. with LM8G5 (2 × 10⁶) and HT-29 (2.5 × 10⁶) cells, respectively, on day 0. On day 21, rhodamine-labeled TRX-20 liposomes or control PEG liposomes (0.5 mol/kg) were given to mice by intracardiac injection. The mice were sacrificed 24 h after the treatment, and peripheral blood samples were collected. Various tissues, including s.c. tumors, were removed after whole-body perfusion with heparinized 0.9% NaCl solution. Rhodamine-PE was extracted from tissue homogenates using the M-Per mammalian protein extraction reagent (Pierce Chemical Co., Rockford, IL) or from blood samples (diluted five times with 0.9% NaCl solution) by methanol and chloroform as described previously (28). The biodistribution of the liposomes was determined by measuring the rhodamine fluorescence as described above.

For the s.c. tumor models, C3H/HeN and ICR nude mice were inoculated s.c. with LM8G5 (2 × 10⁶ cells) and HT-29 (2.5 × 10⁶ cells), respectively, on day 0. On days 7 and 14, the mice were given free cisplatin or Cis-TRX20L or Cis-PEGL (3.5 mg/kg) by intracardiac injection (100 μl). Control mice received injections of sterile 0.9% NaCl solution. The s.c. tumor cell growth was monitored by measuring the three diameters of the tumor nodules, and the tumor volume was calculated using the following formula: volume = 1/6 × π × d1 × d2 × d3. In the LM8G5 liver metastasis model, C3H/HeN mice received an intracardiac injection of LM8G5 cells (1 × 10⁶ cells in 200 μl of PBS) on day 0. On day 3, 5 mg/kg of free cisplatin or cisplatin entrapped in liposomes were given as above. For the evaluation of liver metastasis, mice were sacrificed on day 14, the number of tumor nodules was counted macroscopically, and the liver weight was measured. In separate experiments, mice were monitored for their survival after treatment. To study the acute toxicity of different cisplatin formulations, ICR nude mice were given free cisplatin or cisplatin entrapped in TRX-20 liposomes or PEG liposomes (10 mg/kg) by intracardiac injection and monitored for survival.

The statistical differences observed between different groups regarding s.c. tumor cell growth and liver tumor burden were determined using the standard Student’s t test. The significance of the differences between the groups in the survival experiment was determined using the Mantel-Cox log-rank test.

To examine the binding ability of TRX-20 liposomes to GAGs, rhodamine-labeled TRX-20 liposomes were added to various GAG species that had been immobilized on plastic plates. As shown in Fig. 1, TRX-20 liposomes showed strong binding to CS E, moderate binding to CS B and CS D, and almost no binding to the other GAG chains tested. Control PEG liposomes did not show significant binding to GAGs. The TRX-20 liposome binding to the CSs was abolished by pretreatment of the GAGs with chondroitinase ABC but not heparitinase I. These results confirmed our previous observations (22) and extended them by showing that TRX-20 liposomes preferentially recognize certain CS chains such as CS E, CSB, and CS D.

To examine whether TRX-20 liposomes could selectively bind tumor cells expressing high levels of CS on the cell surface, three tumor cell lines expressing different levels of CS were used. As shown in Fig. 2,A, the murine osteosarcoma LM8G5 and human renal adenocarcinoma ACHN cell lines showed strong reactivity to the anti-CS mAb CS-56, indicating that these cell lines express large amounts of CS on the cell surface. In contrast, the human colon adenocarcinoma HT-29 cells showed only weak reactivity to the anti-CS mAb, indicating that they express little CS on the cell surface. Interestingly, the binding of TRX-20 liposomes to these cell lines almost completely reiterated the study with mAb CS-56 in that, as shown in Fig. 2 B, the TRX-20 liposomes bound strongly to both LM8G5 and ACHN but very little bound to HT-29. The TRX-20 liposome binding was completely abrogated by chondroitinase but not heparitinase treatment, indicating that the TRX-20 liposomes preferentially bind to tumor cells that express large amounts of CS.

To determine whether TRX-20 liposomes could be internalized by tumor cells, the rhodamine-labeled TRX-20 liposomes and control PEG liposomes were examined for uptake. After 1 h of incubation followed by washing, the LM8G5 cells showed strong fluorescence from rhodamine-labeled TRX-20 liposomes (Fig. 3,A), which, however, almost completely disappeared after the chondroitinase ABC treatment (Fig. 3,B), indicating that the cell-associated fluorescence was mainly attributable to surface-bound liposomes. When LM8G5 cells were incubated for 24 h before the chondroitinase ABC treatment, they showed strong fluorescence that localized mainly to the cytoplasm; the cell-associated fluorescence remained unaltered even after the enzyme treatment (Fig. 3, C and D), indicating that TRX-20 liposomes were actually internalized by the tumor cells. When the same cells were incubated with rhodamine-labeled plain PEG liposomes, they showed little fluorescence (Fig. 3,E). In addition, when HT-29 cells, which show low binding of TRX-20 liposomes, were incubated with the liposomes, they also showed almost no cytoplasmic fluorescence (Fig. 3 F). These results indicate that TRX-20 liposomes are efficiently internalized by LM8G5 cells upon binding to the CSs expressed on the cell surface. Avid uptake of TRX-20 liposomes was also observed in ACHN cells (data not shown).

To evaluate the efficacy of TRX-20 liposomes for drug delivery to tumor cells, the in vitro cytotoxicity of Cis-TRX20L was examined in comparison with the cytotoxicity of Cis-PEGL and free cisplatin. As shown in Fig. 4, Cis-TRX20L exhibited a dose-dependent cytotoxicity against LM8G5 and ACHN cells, which was comparable with that observed with free cisplatin. In contrast, Cis-TRX20L showed only a low-grade cytotoxicity against HT-29 cells, which express only marginal levels of CS. Because free cisplatin efficiently killed HT-29 cells, these results suggest that the low cytotoxicity of Cis-TRX20L against HT-29 cells was not because of insensitivity of the HT-29 cells to cisplatin but rather to low liposomal binding of the tumor cells. In addition, Cis-PEGL, which showed little tumor cell binding, exhibited almost no cytotoxicity against any of the tumor cells examined. Taken together, these results demonstrate that Cis-TRX20L has selective and potent cytotoxic activities against tumor cells expressing large amounts of CS.

High and selective accumulation of drug carriers at the tumor site is essential for the success of drug targeting in vivo. To examine whether TRX-20 liposomes would selectively accumulate in solid tumors expressing CS in vivo, rhodamine-labeled TRX-20 liposomes, or rhodamine-labeled control PEG liposomes were administered by intracardiac injection to mice bearing a s.c. LM8G5 or HT-29 tumor, and liposome distribution was determined 24 h after the injection. As shown in Fig. 5, in animals bearing an LM8G5 tumor, TRX-20 liposomes accumulated in s.c. LM8G5 tumors at twice the levels of the PEG liposomes, whereas both liposomal preparations were retained in the blood circulation and accumulated in the liver equally well. Interestingly, in mice bearing the HT-29 tumor, both the TRX-20 liposomes and PEG liposomes showed low accumulation in the tumor but comparably high accumulation in the blood and liver. In both groups of tumor-bearing mice, TRX-20 and PEG liposomes accumulated in the lung and kidney only marginally. Collectively, these results demonstrate that TRX-20 liposomes but not PEG liposomes can selectively accumulate in a s.c. tumor expressing large amounts of CS, whereas both TRX-20 and PEG liposomes have long circulation times and accumulate preferentially in the liver.

We then examined the therapeutic efficacy of TRX-20 liposomes loaded with chemotherapeutic drugs. C3H/He and ICR nude mice were given a s.c. inoculation of LM8G5 cells and HT-29 cells, respectively, and were then treated with Cis-TRX20L, Cis-PEGL, or free cisplatin on days 7 and 14 (3.5 mg/kg). In mice inoculated with LM8G5 cells, Cis-TRX20L was quite effective in suppressing the s.c. tumor growth compared with free cisplatin and Cis-PEGL (Fig. 6,A). By contrast, in mice given HT-29 cells, Cis-TRX20L, Cis-PEGL, and free cisplatin were all only marginally effective with no significant differences in their efficacies of growth suppression (Fig. 6,B). It is interesting to note that although Cis-PEGL was much inferior to free cisplatin in in vitro cytotoxicity (Fig. 4), its in vivo effect was equivalent to that of free cisplatin, which may be attributable to a long half-life of Cis-PEGL in the blood circulation. As shown in Fig. 6 C, acute toxicity experiments with a large-dose bolus administration of various cisplatin preparations indicated that Cis-TRX20L and Cis-PEGL had much reduced toxicity compared with free cisplatin. Collectively, these data show that Cis-TRX20L has selective cytotoxicity against tumor cells that express large amounts of CS in vivo and may provide an efficient and safe means to deliver anticancer drugs to tumor tissues.

Another remarkable property of Cis-TRX20L was shown in its potent activity of inhibiting experimental metastasis to the liver. Mice given an intracardiac injection of LM8G5 cells develop numerous metastatic nodules in the liver within 14 days. When Cis-TRX20L was given to the tumor-bearing mice on day 3 after the tumor inoculation, it markedly reduced the number and size of metastatic nodules in the liver (Fig. 7, A and B). In contrast, Cis-PEGL was much less effective than Cis-TRX20L in reducing the number of metastatic nodules, although it decreased the size of individual tumor nodules moderately (Fig. 7, A and B). The free form of cisplatin was almost completely ineffective (Fig. 7, A–C). Measurement of the liver weight, which reflects tumor load, gave similar results, indicating that Cis-TRX20L almost completely suppressed the increase in liver weight, whereas Cis-PEGL was moderately effective, and free cisplatin was nearly ineffective (Fig. 7,C). Consistently, Cis-TRX20L was most effective in increasing the mean survival time of the tumor-bearing mice compared with Cis-PEGL and free cisplatin (P < 0.005; Fig. 7 D). These results clearly show that Cis-TRX20L is much more potent in inhibiting liver metastasis than either Cis-PEGL or free cisplatin.

In this study, we have demonstrated that TRX-20 liposomes preferentially recognize CS E, CS B, and CS D, that they selectively accumulate in s.c. solid tumors that express high levels of CS, and that they significantly suppress tumor growth when cisplatin is encapsulated within them. We also showed that Cis-TRX20L was far superior to Cis-PEGL or free cisplatin in suppressing the liver metastasis of a high CS-expressing tumor, LM8G5.

Previous in vitro and in vivo studies demonstrated that long-circulating liposomes, such as those sterically stabilized by conjugating PEG to the lipid bilayers, significantly increase the therapeutic efficacy of antitumor drugs (19, 20). In addition, coupling-specific ligands such as mAbs (17, 29, 30) or surface-bound, site-specific molecules (31) to the PEG terminus are even more beneficial in improving the therapeutic efficacy of these drugs. In this study, we used a new cationic lipid TRX-20 (22) as a targeting device for such PEG-coated liposomes. TRX-20 liposomes can selectively recognize certain types of CS chains, including CS E, CS B, and CS D, but not other CSs, although all CSs are composed of disulfated disaccharides and have highly anionic properties. It should be stressed in this regard that the TRX-20 liposomes interacted only poorly with non-CS GAGs, including common extracellular matrix components such as heparan sulfate and hyaluronic acid, which may at least partly account for their low uptake by the lung and kidney, which abundantly express these extracellular matrix components (7, 32). Currently, the precise structural requirement for TRX-20 liposomes to bind specific CS oligosaccharide chains remains unclear.

CSs exist on the cell surface or in the extracellular space as CSPGs (1). Cell surface CSPGs have been implicated in cell adhesion and motility (33). In particular, CD44-related CSPG (34) and melanoma CSPG (5) have been reported to regulate melanoma cell motility and invasive behavior on extracellular matrix components such as collagen I (5, 34) and fibrinogen (2). Although we do not know what kind of CSPGs are expressed on the surface of the LM8G5 or ACHN cells that were used in this study, CSs are likely to provide a suitable target for liposome binding and internalization by tumor cells. Further study to identify the CSPGs involved in the TRX-20 liposome binding will help increase the accuracy of drug targeting to tumor tissues.

Although the present results show a significantly increased efficacy of Cis-TRX20L to suppress the growth and metastasis of tumor cells in vivo compared with other cisplatin preparations, it should be pointed out that the treatment of mice with Cis-TRX20L did not result in the complete eradication of the tumor cells with the dose and schedules used in this study. Although Cis-TRX20L significantly suppressed liver metastasis and prolonged the mean survival time of the mice, all of the animals died within 25 days after inoculation with the LM8G5 cells in our model; death was mainly because of metastasis to tissues other than liver. This may be partly because Cis-TRX20L was given only once on day 3, partly to a limited penetration of these long-circulating liposomes into the interior of established solid tumors in vivo (35) and partly to their low accumulation in tissues other than liver. Clearly, additional development of these liposomes should be pursued to rectify these deficiencies. Detailed pharmacokinetic and pharmacodynamic studies are in progress in our laboratory to improve efficacy and safety of Cis-TRX20L.

In humans, significant increases in CS content have been reported in a variety of epithelial and mesenchymal neoplasms, including pancreatic carcinoma (36), colon carcinoma (37), rectum carcinoma (38), hepatocellular carcinoma (39), and prostate carcinoma (40). In addition, the enhanced expression of CS often correlates with the metastatic ability of tumor cells (41, 42). Hence, these observations point to the usefulness of tumor-associated CSs for the targeted delivery of anticancer drugs by CS-tropic vehicles such as TRX-20 liposomes.

In summary, we have demonstrated that TRX-20 liposomes that preferentially bind to certain CSs can be successfully used for the delivery of anticancer drugs to tumors that express large amounts of CS in vivo. TRX-20 liposomes are particularly effective in suppressing tumor metastasis to the liver. Although much additional study of relevant tumor models is required to improve the efficacy of TRX-20 liposomes, the results in this study warrant additional development of these liposomes for potential investigation into their use in clinical tumor chemotherapy.

We thank Dr. Tae Takeda for the ACHN cells. We also thank Dr. Takako Hirata for critical reading of the manuscript, and Shinobu Yamashita and Miyuki Komine for secretarial assistance.