Yukihiro Namba

Photodynamic therapy targeted to tumor-induced angiogenic vessels

Cancer photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) may be effective not only by being directly cytotoxic to tumor cells, but also by being cytotoxic to the endothelium of tumor neovasculature. In the present study, we investigated the effect of PDT with an experimental liposomal formulation of BPD-MA on tumor-induced angiogenic vessels using a murine dorsal air sac model. First, hemostasis of neovasculature was examined by varying the regimen of PDT. Laser irradiation at 15 min after injection of 2 mg/kg liposomal BPD-MA (15 min PDT) caused complete blocking of blood flow in neovasculature. In contrast, PDT did not inhibit blood flow when the irradiation occurred 3 h after the injection of liposomal BPD-MA (3 h PDT). Next, the antitumor effect of PDT on Meth A sarcoma-bearing mice was investigated by using the hemostasis-inducing regimen. Tumor growth was strongly inhibited after the 15 min PDT with BPD-MA at a dose of 0.5-2 mg/kg. In contrast, 3 h PDT with BPD-MA at a dose of 2 mg/kg suppressed tumor growth only partially. The current study indicates that 15 min PDT causes strong suppression of tumor growth, perhaps through damaging endothelial cells in the tumor neovasculature rather than through a direct cytotoxic effect on tumor cells.

Tumor accumulation of novel RES-avoiding liposomes

For passive targeting of liposomes to tumor tissues, we earlier developed reticuloendothelial system (RES)-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA) (Namba, Y., Sakakibara, T., Masada, M., Ito, F. and Oku, N. (1990) Chem. Pharm. Bull. 38, 1663-1666). In this present study, we examined the blood clearance and biodistribution of PGlcUA-liposomes (dipalmitoylphosphatidylcholine/cholesterol/PGlcUA = 40:40:20 as a molar ratio) in normal and tumor-bearing mice. Liposomes containing dipalmitoylphosphatidylglycerol (DPPG) instead of PGlcUA was also examined as a control. When [3H]inulin-encapsulated PGlcUA-liposomes and DPPG-liposomes were intravenously injected into normal mice, approx. 50% of the 3H radioactivity was recovered from the liver, the bulk of RES, at 12 h after administration of DPPG-liposomes, while only approx. 20% of it was found there when PGlcUA-liposomes were administered. Radioactivity remaining in the plasma at 12 h after injection was 5-fold higher when PGlcUA-liposomes were injected than when DPPG-liposomes were used. Biodistribution of liposomes in tumor-bearing mice was also examined. Mice were inoculated with 10(7) S180 cells into the hind leg. After 1 week, liposomes were injected. Radioactivity of [3H]inulin originally encapsulated in the PGlcUA-liposomes accumulated in the tumor to an extent 3-4-fold higher than that of the marker in DPPG-liposomes. Liver/tumor ratio of the radioactivity was 12 for DPPG-liposomes and only 2 for PGlcUA-liposomes. This latter value is the lowest of various liposome formulations ever reported.

Effect of serum protein binding on real-time trafficking of liposomes with different charges analyzed by positron emission tomography

Liposomes have been used as carriers of various materials and as tools for gene transfer: for the latter purpose, positively charged liposomes are usually used. To evaluate the stability in the presence of serum and the in vivo behavior of such liposomes as well as those aspects of neutral and negatively charged liposomes, we investigated liposomal agglutinability in the presence of serum, serum protein binding to these liposomes, and real-time liposomal trafficking by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol without or with charged lipid were prepared in the presence of mannitol, and the turbidity change in the presence of serum was determined. Turbidity increase was not observed for so-called long-circulating liposomes, i.e., liposomes modified with glucuronic acid or with poly(ethylene glycol), or for negatively charged liposomes containing dicetyl phosphate (DCP), phosphatidylglycerol, or phosphatidylserine. On the contrary, a significant turbidity increase was observed when positively charged liposomes modified with stearylamine, stearyltrimethylammonium chloride or 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl bromide (DMRIE), which is known as a component of liposomes for gene transfer, were used. These liposomes were found to have bound a high amount of serum proteins after separation of unbound serum proteins by use of a spin column. The liposomal trafficking in vivo was determined for three kinds of liposomes, i.e., liposomes with DMRIE, those with DCP, and those without charged lipids. These liposomes were prepared in the presence of 2-[18F]fluoro-2-deoxy-D-glucose ([2-18F]FDG), and the [2-18F]FDG-labeled liposomes were administered to mice to perform PET scans. Positively charged liposomes containing DMRIE showed high accumulation in the liver compared with neutral and negatively charged liposomes. Since DMRIE-liposomes tended to aggregate in the presence of serum, and to be associated with serum protein, these characteristics may lead to the high uptake of DMRIE-liposomes by the liver.

IN VIVO TRAFFICKING OF LONG‐CIRCULATING LIPOSOMES IN TUMOUR‐BEARING MICE DETERMINED BY POSITRON EMISSION TOMOGRAPHY

Various kinds of long-circulating liposome, such as ganglioside GM1-, polyethyleneglycol- (PEG-), and glucuronide-modified liposomes, have been developed for passive targeting of liposomal drugs to tumours. To evaluate the in vivo behaviour of such long-circulating liposomes, we investigated the liposomal trafficking, especially early trafficking just after injection of liposomes, by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and modifier, namely, GM1, distearoylphosphatidylethanolamine (DSPE)-PEG or palmityl-D-glucuronide (PGlcUA), were labelled with [2-18F]-2-fluoro-2-deoxy-D-glucose ([2-18F]FDG), and administered to mice bearing Meth A sarcoma after having been sized to 100 nm. A PET scan was started immediately after injection of liposomes and continued for 120 min. PET images and time-activity curves indicated that PEG liposomes and PGlcUA liposomes were efficiently accumulated in tumour tissues time dependently from immediately after injection. In contrast, GM1 liposomes accumulated less in the tumour as was also the case for control liposomes that contained dipalmitoylphosphatidylglycerol (DPPG) instead of a modifier. Long-circulating liposomes including GM1 liposomes, however, remained in the blood circulation and avoided liver trapping compared with control DPPG liposomes. These data suggest that PGlcUA and PEG liposomes start to accumulate in the tumour just after injection, whereas GM1 liposomes may accumulate in the tumour after a longer period of circulation.

Tumor imaging with technetium-99m-DTPA encapsulated in RES-avoiding liposomes

For passive targeting of liposomes to tumor tissues, we earlier developed reticuloendothelial system (RES)-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA). In the present study, we encapsulated technetium-99m (99mTc)-diethylenetriaminepentaacetic acid (DTPA) in PGlcUA-liposomes (dipalmitoylphosphatidylcholine:cholesterol:PGlcUA = 40:40:20 as a molar ratio) and studied the biodistribution of the liposomes in tumor-bearing mice. 99mTc-DTPA encapsulated in liposomes effectively accumulated in tumor tissues after intravenous administration. Corresponding to these results, tumor was strongly imaged by a gamma-camera when 99mTc-DTPA-encapsulated PGlcUA-liposomes were used.

Antitumor activity of vincristine encapsulated in glucuronide-modified long-circulating liposomes in mice bearing Meth A sarcoma

Liposomes modified with the uronic acid derivative palmityl-D-glucuronide (PGlcUA) have a long circulation time and tend to accumulate in the tumors of tumor-bearing mice. Taking advantage of this character, we investigated the therapeutic effect of vincristine (VCR) encapsulated in liposomes containing PGlcUA (dipalmitoylphosphatidylcholine/cholesterol/PGlcUA = 4:4:1 as a molar ratio) on tumor-bearing mice. VCR was loaded into liposomes by a remote loading method, and then free or liposomal VCR was injected intravenously into BALB/c mice bearing Meth A sarcoma implanted subcutaneously 5 days before hand. Single-dose administration of VCR (3.0 mg/kg) in PGlcUA-liposomes significantly suppressed tumor growth, and prolonged the survival time (T/C = 1.37). Furthermore, two-dose administration of the liposomes cured one third of the animals. The therapeutic effect of PGlcUA-liposomes was greater than that of control liposomes containing dipalmitoylphosphatidylglycerol instead of PGlcUA. PGlcUA-liposomes might thus be a useful tool for delivering antitumor agents to tumor tissues.

Glucuronate-Modified, Long-Circulating Liposomes for the Delivery of Anticancer Agents

Liposomes are useful as drug carriers in drug delivery systems, especially for drugs with severe side effects such as antitumor agents. The conventional formulations of liposomes are opsonized by plasma proteins in the bloodstream and trapped in the reticuloendothelial system (RES). Therefore, liposomes with reduced opsonization are expected to have prolonged circulation and to accumulate in tumor tissue due to the leaky endothelium of the tissue. To avoid RES trapping of liposomes, two approaches have been considered. Liposomes may mimic cells circulating in the blood to escape host recognition as foreign substances, or liposomes may be covered with a hydrophilic barrier to escape recognition. For the latter purpose, poly(ethylene glycol) is widely used. For the former purpose, here we focus on the characteristics, in vivo trafficking, and usage in cancer therapy of glucuronate-modified liposomes. Glucuronate-modified liposomes bind to a lower extent to macrophage-like cells in vitro and passively accumulate in tumor tissue evaluated by a technique using positron emission tomography. Glucuronate-modified liposomes with extended circulation are useful for delivering anticancer agents to tumors and reducing the toxic side effects of the agents.

Liposomes modified with a synthetic Arg-Gly-Asp mimetic inhibit lung metastasis of B16BL6 melanoma cells

Administration of large amounts of synthetic peptides based on the Arg-Gly-Asp (RGD) sequence has been shown to suppress tumor metastasis. To overcome the rapid degradation of peptides in the circulation, an RGD mimetic, L-arginyl-6-aminohexanoic acid (NOK), was synthesized and conjugated with phosphatidylethanolamine (PE) (NOK-PE) for liposomalization. Cell adhesion assays revealed that B16BL6 murine melanoma cells adhered to immobilized NOK-PE. This adhesion was inhibited by addition of either soluble RGDS or NOK at similar concentration in a dose-dependent manner. Administration of NOK-PE liposomes (equivalent to ca. 500 microg RGD peptides) via the tail vein completely inhibited lung colonization of B 16BL6 cells. The same dose of soluble NOK was not effective in inhibition of the tumor metastasis. In addition, injection of NOK-PE liposomes via the tail vein inhibited spontaneous lung metastasis of B16BL6 cells from the primary tumor site in the hind footpad. These results suggest that NOK, a structural mimetic of RGD, is capable of suppressing metastasis by blockade of the binding of the integrins present on tumor cells to the RGD-containing extracellular matrix.

Liposomal Applications to Cancer Therapy

are relatively non-toxic to most cell types. Liposomes have been used successfully as carriers for drugs and biological components in various in vitro [1-4], in vivo [5,6], and clinical [7-9] investigations. These lipid vesicles have been shown to enhance the dose efficacy of pharmaceuticals by prolonging the circulatory transit time, isolating the agents from the systemic background, increasing the concentrations of the agent at target sites, and producing a sustained release mode of action [10-13]. In addition, drug delivery via liposomes can markedly reduce the toxicity of a variety of antitumor and antimicrobial agents, and yet preserve or enhance their therapeutic effects. In this review, we focused on two aspects of liposomal applications besides the efficacy cf anticancer chemotherapy with drugs encapsulated in liposomes. When liposomes are administered into the bloodstream, they tend to accumulate in the reticuloendothelial system (RES), also called the mononuclear phagocyte system. Thus liposomes encapsulating immunomodulators are effectively delivered to macrophages and can potentially activate the host defense mechanisms.

Liposomal modification with uronate, which endows liposomes with long circulation in vivo, reduces the uptake of liposomes by J774 cells in vitro

For overcoming rapid removal of liposomes from the bloodstream, we developed reticuloendothelial system (RES)-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA). In this current study, we examined the in vitro interaction of PGlcUA-liposomes with J774 cells derived from mouse macrophages. Liposomal association with J774 cells at 37 degrees C did not increase compared with the binding at 4 degrees C when liposomes were modified with PGlcUA. RES-avoiding ability was not specifically endowed by glucuronate but by uronates in general, since palmityl-D-galacturonide showed a similar effect on liposomal clearance in vivo and liposomal uptake in vitro. These facts indicate that modification of the liposomal surface with uronic acid derivatives endows liposomes with a long circulation time in the bloodstream by reducing their uptake by macrophages.