Effective delivery of an angiogenesis inhibitor by neovessel-targeted liposomes.

Angiogenesis is critical for tumor growth and metastasis, and several angiogenesis inhibitors have been developed for the treatment of cancer. Previously, we identified angiogenic vessel-homing peptide, Ala-Pro-Arg-Pro-Gly (APRPG), by use of a phage-displayed peptide library. APRPG peptide-modified liposomes have been revealed to be useful for the delivery of encapsulated drugs to angiogenic vasculature in tumor-bearing animals. In the present study, to assess the usefulness of APRPG-PEG-modified liposomes as a carrier of angiogenesis inhibitors in vitro and in vivo, we designed and validated APRPG-PEG-modified liposomal angiogenesis inhibitor. SU1498, an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, was successfully encapsulated into the liposomes. APRPG-PEG-modified liposomal SU1498 inhibited VEGF-stimulated endothelial cell proliferation in vitro. Moreover, APRPG-PEG-modified liposomal SU1498 significantly decreased tumor microvessel density in Colon26 NL-17 cell-bearing mice and prolonged the survival time of the mice. These findings suggest that APRPG-PEG-modified liposomes effectively deliver SU1498 to angiogenic endothelial cells in tumors and thus inhibit tumor-induced angiogenesis.

Enhanced desensitization efficacy by liposomal conjugation of a specific antigen.

Since liposomes are known as strong adjuvants, we attempted to use liposomes in immunotherapy as adjuvants, and to achieve desensitization in pre-sensitized mice. At first, we sensitized mice with intraperitoneal injection of model antigen, 100 microg ovalbumin (OVA), with Alum and treated them with liposome composed of distearoylphosphatidylcholine (DSPC) and cholesterol (2:1 as a molar ratio), which was coupled with a small amount of OVA (10 microg OVA in 400 nmol DSPC and 200 nmol cholesterol-liposome was injected into 20 g mouse). It is well known that antigen-specific immunotherapy increases IgG blocking antibodies and decreases in IgE antibodies. The treatment with i.v. injection of OVA-liposome at days 8, 10, and 12 after sensitization strongly suppressed OVA-specific IgE production without affecting IgG level after the boost (100 microg OVA with Alum). Moreover, the treatment with high-density OVA-liposome (10 microg OVA in 80 nmol DSPC and 40 nmol cholesterol-liposome/20 g mouse) not only strongly suppressed IgE levels but also reduced IgG production after the boost of OVA-sensitized mice suggesting the importance of liposomal characteristic in desensitization immunotherapy. Next we reduced the dose of OVA-liposome and the desensitization effect was also observed at the dose of as low as 1 microg OVA on OVA-liposome/mouse. On the contrary, free OVA did not affect the production of both IgG and IgE levels. Biodistribution study indicated that OVA-liposome was highly accumulated in spleen of OVA-sensitized mice compared to control liposome at 3 h after i.v. injection. These results suggest that the liposomal OVA effectively interacts with and desensitizes immune cells, therefore, liposomes coupling with a certain antigen may be effective in allergy immunotherapy.

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.

Selective release of non-electrolytes from liposomes upon perturbation of bilayers by temperature change or polyene antibiotics.

A new system for assaying the permeability characteristics of liposomes was established using Amicon cells equipped with a membrane filter (pore size, 0.3 micrometer). In this system, damage of liposomes during the assay procedure was negligible. Changes in permeability to non-electrolytes, such as glucose (Mr 180), sucrose (Mr 342), inulin (Mr 5000) and dextran (Mr 75000), induced by perturbation of the bilayers were examined with this system. The following results were obtained on the barrier properties of multilamellar liposomes modified by various treatments. 1. Amphotericin B and nystatin did not cause any change in permeability to glucose of egg yolk phosphatidylcholine liposomes prepared in physiological saline and containing trace amounts of radioactive markers in their aqueous compartments. Both antibiotics, however, induced nonspecific release of glucose, sucrose, inulin and dextran from liposomes that contained 0.3 M glucose in their aqueous compartments. These antibiotics first seem to form pores through which small ions can permeate; Na+ and Cl- can enter the liposomes through these pores, whereas glucose in the liposomes cannot pass out. As a result, the liposomes become swollen with consequent severe disruption of their membranes. 2. Filipin and digitonin disrupted the membrane structures, resulting in release of large molecules such as dextran even in the absence of an osmotic mechanism. 3. Perturbation of the phase equilibrium by temperature change resulted in formation of 'pores'. The penetration of cations and anions through these 'pores' was apparently much faster than that of glucose, since when liposomes swollen in 0.3 M glucose were incubated in salt solution they were disrupted by an osmotic mechanism releasing not only glucose but also dextran. Most of the 'pores' were not large enough to allow passage of large non-electrolytes, such as inulin and dextran, since no appreciable amounts of these markers were released from liposomes under conditions where there should be no osmotic gradient. 4. At a temperature well above the phase transition temperature, egg yolk phosphatidylcholine liposomes exhibited specific release of glucose. This process did not involve an osmotic gradient, indicating that it was mainly due to diffusion of the solutes through the bilayers.

Studies on the interaction of Sendai virus with liposomal membranes. Sendai virus-induced agglutination of liposomes containing glycophorin.

Liposomes constituted with the major sialoglycoprotein of human erythrocytes, glycophorin, were used as models for studies on cell-virus interactions. Liposomes composed of egg yolk phosphatidylcholine, cholesterol and glycophorin were found to interact with the paramyxovirus HVJ to form aggregates. The aggregation process was temperature dependent: it was maximal at 0 degrees C and decreased with increase of the incubation temperature. The activity of viral neuraminidase is also temperature dependent, and it increases with increase of the incubation temperature; release of N-acetylneuraminic acid was negligible at 0 degrees C. Shift-up of the incubation temperature immediately cancelled HVJ-induced agglutination of liposomes. Viruses attached to liposomes seemed to be released into the supernatant when the 'virus-liposome' complex formed at 0 degrees C was incubated at 37 degrees C, possibly as a result of breakdown of the 'binding' site by neuraminidase. The characteristics of the interaction of HVJ with liposomes containing glycophorin appeared to be phenomenologically similar to those of HVJ-cell interaction.

Real-time analysis of liposomal trafficking in tumor-bearing mice by use of positron emission tomography.

Long-circulating liposomes are known to accumulate passively in tumor tissues of tumor-bearing animals. To evaluate the in vivo behavior of such liposomes, we investigated the real-time liposomal trafficking by a non-invasive method using position emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and palmityl-D-glucuronide (PGlcUA) in a molar ratio of 4:4:1 were prepared in the presence of 2-[18F]fluoro-2-deoxyglucose ([2-18F]FDG). [2-18F]FDG-labeled liposomes sized by extrusion through a filter with various-sized pores were administered to mice bearing Meth A sarcoma, and a PET scan was performed for 120 min. Small-sized, long-circulating liposomes (100 nm in diameter) constructed with PGlcUA tended to accumulate in the tumor tissues. On the contrary, control liposomes (100 nm in diameter) containing dipalmitoylphosphatidylglycerol instead of PGlcUA accumulated in the liver. Large-sized PGlcUA-containing liposomes (> 300 nm) also accumulated in the liver, as well as in the spleen. Time-activity curves indicated that the small long-circulating liposomes (< 200 nm) transiently accumulated in the liver right after the injection but that the accumulation there decreased time-dependently. These data suggest that, although the majority of small long-circulating liposomes remain in the bloodstream, some extravasate once into the interstitial spaces in the liver re-enter the bloodstream again, and finally accumulate in the tumor tissues. This PET technique might be useful for studying real-time liposomal trafficking and for tumor imaging.

Preparation of giant liposomes.

A method is described for the preparation of giant unilamellar lipid vesicles that are stable in electrolyte solution. In general, it involves dialysis of lipid and indifferent solute in a water-miscible organic solvent against an aqueous buffer. During dialysis the concentration of organic solvent decreases so that vesicles form under conditions where their internal contents are continuously hyperosmotic. Interlamellar attractive forces are neutralized, even between bilayer membranes with no net charge, and giant vesicles are generated in large numbers. The population is heterogeneous but most large vesicles have diameters between 10 and 100 micron. The method is simple. One procedure involves dialysis for a day or more of a methanol solution of phosphatidylcholine, supersaturated with methylglucoside, against an aqueous phase containing up to 1 M univalent electrolyte. The procedure is effective over a wide range of temperature and pH.

Potential usage of thermosensitive liposomes for macromolecule delivery.

Since transient swelling during phase transition of liposomes with hyper-osmotic internal aqueous phase may cause permeation of large molecules through the lipid bilayer, this type of liposomes can be useful for delivering macromolecules. In fact, thermosensitive liposomes containing an internal solution with osmotic pressure 2-fold higher than the physiologic level and sized through 200-nm pore, released encapsulated macromolecules such as dextran (M(r) 144,000) effectively when they were incubated at 40-42 degrees C. These liposomes were stable in the presence of serum compared to the liposomes with internal osmotic pressure more than 3-fold higher than the physiologic level.

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.

Electron microscopic study on the interaction of Sendai virus with liposomes containing glycophorin.

The interaction of liposomes containing glycophorin, a major sialoglycoprotein of human erythrocytes, with Sendai virus was studied by freeze-fractures and negative staining electron-microscopy. Viral envelopes were absorbed on liposomal membranes at 0 degrees C. When the temperature was shifted up to 37 degrees C, the viral envelopes fused with the liposomal membranes (envelope fusion). Particles representing viral membrane components formed clusters on liposomal membranes after incubation for more than 1 h at 37 degrees C.

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.

Applicability of anti-neovascular therapy to drug-resistant tumor: suppression of drug-resistant P388 tumor growth with neovessel-targeted liposomal adriamycin.

Anti-neovascular therapy, one of the effective anti-angiogenic chemotherapy, damages new blood vessels by cytotoxic agents delivered to angiogenic endothelial cells and results in indirect eradication of tumor cells. We previously reported that liposomes-modified with a pentapeptide, Ala-Pro-Arg-Pro-Gly (APRPG-Lip) homing to angiogenic site, highly accumulated in tumor tissue, and APRPG-Lip encapsulating adriamycin (APRPG-LipADM) effectively suppressed tumor growth in tumor-bearing mice. In the present study, we examined the topological distribution of fluorescence-labeled APRPG-LipADM as well as TUNEL-stained cells in an actual tumor specimen obtained from Colon 26 NL-17 carcinoma-bearing mice. The fluorescence-labeled APRPG-Lip dominantly localized to vessel-like structure: a part of which was also stained with anti-CD31 antibody. Furthermore, TUNEL-stained cells were co-localized to the same structure. These data indicated that APRPG-LipADM bound to angiogenic endothelial cells and induced apoptosis of them. We also investigated the applicability of anti-neovascular therapy using APRPG-LipADM to ADM-resistant P388 solid tumor. As a result, APRPG-LipADM significantly suppressed tumor growth in mice bearing the ADM-resistant tumor. These data suggest that APRPG-LipADM is applicable to various kinds of tumor including drug-resistant tumor since it targets angiogenic endothelial cells instead of tumor cells, and eradicates tumor cells through damaging the neovessels.

A novel non-viral gene transfer system, polycation liposomes.

To develop a novel non-viral gene transfer system, liposome was modified with cetylated polyethylenimine (PEI). This polycation liposome (PCL) showed remarkable transfection efficiency to COS-1 cells in vitro, in comparison with conventional cationic liposome preparations. Cytotoxicity against COS-1 cells and hemolytic activity of PCL or PCL-DNA complex were quite low in comparison with conventional cationic liposomes. Most conventional cationic liposomes require phosphatidylethanolamine or cholesterol as a component, though PCL did not. Egg yolk phosphatidylcholine- and dipalmitoylphosphatidylcholine-based PCL were as effective as dioleoylphosphatidylethanolamine-based PCL for gene transfer. Furthermore, the transfection efficacy of PCL was enhanced, instead of being diminished, in the presence of serum. Effective gene transfer was observed in all eight malignant and two normal line cells tested as well as in COS-1 cells. The effect of the molecular weight of PEI on PCL-mediated gene transfer was examined, and observed that PEIs with a molecular weight (Mr. Wt.) of 600 and 1800 Da were quite effective but PEI of 25,000 was far less effective. Effectiveness of gene transfer by using PCL was also observed in vivo: GFP and Luciferase genes were effectively expressed in mouse. We also discussed the mechanism of gene transfer by PCL. Taken together, PCL represents a new system useful for transfection and gene therapy.

Cancer chemotherapy administered by activated carbon particles and liposomes.

In cancer chemotherapy, a specific drug delivery system is expected since many anticancer drugs show toxicity against not only cancer cells but also against normal tissues. The dosage form comprising anticancer drugs adsorbed on activated carbon particles or encapsulated in liposomes is developed as a drug-delivery system which enhances the therapeutic efficacy and reduces the adverse effects. The dosage forms are versatile in size and electric charge, so that large amounts of the drugs are distributed to the "targeted" organs or tissues and lesser amounts are distributed to the whole body. The dosage forms are designed to release the drugs slowly for a long time at local sites. Through this process, practical use of the dosage forms as an anticancer drug carrier results in an enhancement of anticancer efficacy on the local lesion and a decrease of systemic toxicity.

Suppression of GD1alpha ganglioside-mediated tumor metastasis by liposomalized WHW-peptide.

GD1alpha ganglioside-replica peptides were recently isolated from a phage-displayed random pentadecapeptide library by assaying for inhibition of adhesion of RAW117-H10 lymphosarcoma cells to hepatic sinusoidal microvessel endothelial (HSE) cells. We show here that the Trp-His-Trp (WHW) peptide was identified as a minimal sequence of the GD1alpha-replica peptide WHWRHRIPLQLAAGR. The addition of WHW peptide-attached liposomes displayed efficient inhibition of liver metastasis of RAW117-H10 cells as well as of GD1alpha-mediated adhesion of RAW117-H10 cells to HSE cells in vitro. These results suggest that engineered liposomes for peptide delivery are applicable to treatment for metastasis.

Long-circulating liposomes.

Liposomes have been investigated for use as drug carriers, and many previous studies have demonstrated enhanced efficacy of encapsulated drugs and the reduction of the side effects of drugs so entrapped. In many cases liposomal drugs are administered via the bloodstream. The stability in the bloodstream, clearance, and biodistribution are dependent on the composition, size, and charge of the liposomes. Rigid, small-size (100-200 nm) liposomes tend to be retained in the blood without degradation. Since the conventional liposomes are trapped in the reticuloendothelial system (RES), RES targeting by means of liposomes is easily achieved. This tendency of liposomes, however, is the most serious limitation when their target is not the RES. Many attempts have been made to avoid the RES-trapping and to prolong the circulation time of liposomes with monosialoganglioside GM1, polyethyleneglycol, glucuronide derivatives, and so on. When the targets are tumor tissues, these RES-avoiding, long-circulating liposomes passively accumulate in such tissues due to extravasation through the leaky vasculature in the tumor tissues. Therefore, long-circulating liposomes are useful tools, especially for tumor imaging and therapy. In this review, we show examples and discuss the mechanism of RES avoidance by these modifiers of liposomes, with special focus on the glucuronide as a modifier.

Potential usage of thermosensitive liposomes for site-specific delivery of cytokines.

Long-circulating liposomes reside long in the bloodstream, and transient swelling during phase transition of liposomes with hyper-osmotic internal aqueous phase causes release of macromolecules. Here we examined the applicability of long-circulating thermosensitive liposomes for delivery of tumor necrosis factor (TNF) by the heating of a local tumor-growing site after injection of TNF-loaded liposomes into tumor-bearing mice. Glucuronate modified thermosensitive liposomes with internal solution of two-fold higher osmotic pressure and sized through 200 nm-pore, released encapsulated [(131)I] human serum albumin at 42 degrees C in vitro and showed long-circulating character in vivo. Cytotoxic action of TNF encapsulated in long-circulating thermosensitive-liposomes (LCTS-liposomes) against L929 fibrosarcoma cells was enhanced at 42 degrees C in vitro. Furthermore, the tumor growth tended to be inhibited more by hyperthermia of mice bearing Meth A sarcoma than without heating after injection of TNF encapsulated in LCTS-liposomes. These results suggest that the cytokine can be released at the tumor site from the circulating CLTS-liposomes.

Targeting and anti-tumor efficacy of liposomal 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine in mice lung bearing B16BL6 melanoma.

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a potent anti-cancer agent, and we previously observed that liposomal formulation of 5'-O-dipalmitoylphosphatidyl derivative of CNDAC (DPP-CNDAC) is desirable for targeting. For targeting to pulmonary cancer, we investigated the in vivo behavior of liposomes containing DPP-CNDAC by a non-invasive method using positron emission tomography. Liposomes composed of DPP-CNDAC and cholesterol (DPP-CNDAC/CH liposomes) were markedly accumulated in mice lung bearing B16BL6 melanoma. In metastatic pulmonary cancer model, DPP-CNDAC/CH liposomes significantly reduced the lung colonization in a dose-dependent manner. The activity was significantly superior to conventional liposomal formulation or soluble CNDAC. These results suggest that DPP-CNDAC/CH liposomes are useful for metastatic pulmonary cancer.

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.

Permeability change of liposomal membrane induced by interleukin-1 alpha.

Interleukin-1 (IL-1) is produced and released by various cells, including activated macrophages, and plays important roles in inflammation as well as immune responses. Since the precursor of IL-1 has no signal peptide, the mechanism of IL-1 release has been an enigma. To investigate the possibility of direct interaction of IL-1 with the lipid bilayer, the interleukin-1 alpha (IL-1 alpha)- or beta (IL-1 beta)-induced permeability change of the liposomal membrane was determined. IL-1 alpha, but not IL-1 beta, caused an increase in the permeability of liposomes composed of phosphatidylserine (PS), at neutral and acidic pHs, as demonstrated by measuring the efflux of calcein. On the other hand, liposomes composed of phosphatidylcholine (PC) showed no increase in permeability when incubated with IL-1 alpha, suggesting the importance of acidic phospholipids in the interaction of IL-1 alpha with the membrane. Furthermore, permeability of liposomal membrane was markedly increased by IL-1 alpha in the presence of 1 microM calcium ions, although a permeability change was observed even in the absence of calcium ions. IL-1 alpha also induced the efflux of fluorescent dextran (average M(r) of 39,600), raising the possibility of translocation of IL-1 alpha through the cell membrane.