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To realize regenerative medicine, it is very important to eliminate the transformed stem cells selectively included in iPS cells, ES cells and adult stem cells derived from organs, because the transformed stem cells have a risk of tumorigenesis after the cell transplantation. Ueoka et al., have developed hybrid liposomes (HL) which selectively accumulated to membranes of tumor cells and have high inhibitory effects on the growth of tumor cells along with the induction of apoptosis. Therefore, we have investigated the application of HL23 (DMPC/10 mol%C(12)(EO)(23)) to the selective elimination of transformed stem cells using hepatoblast, which we could induce from human fetal hepatocytes by the treatment of 1 mM sodium butyrate for 8 days. During the induction process, the transformed cells appeared and produced abnormal prothrombin (PIVKA-II), which is a clinical marker for hepatoma, and also formed colonies in soft agar plate, which is a criteria for neoplastic cell transformation. On the other hand, by the treatment with 0.33 mM HL23 for 96 h during the induction process, PIVKA-II production rate of the cells and colonies formed in the soft agar plate also remarkably decreased less than those of the normal cells. Furthermore, the population of hepatoblasts in the remaining cells increased about four times. These results suggest that the transformed hepatic stem cells could be selectively eliminated by the treatment of HL23, and HL treatment of the stem cells would be a useful culture method for quality control of the stem cells to reduce a risk of tumorigenesis after the cell transplantation.

Accumulation of β amyloid (Aβ) peptides to nerve cells should be associated with the onset of Alzheimer's disease (AD). We prepared hybrid liposomes (HL) composed of 90 mol% phospholipids having various charged head groups (cationic L-α-dimyristoyltrimethyl ammonium propane (DMTAP), anionic L-α-dimyristoylphosphatidylserine (DMPS) or zwitterionic L-α-dimyristoylphosphatidylcholine (DMPC)) and 10 mol% polyoxyethylene(23) dodecyl ether (C(12)(EO)(23))), and investigated the inhibitory effects of HL on the accumulation of Aβ(1-40) peptides into human neuroblastoma (SH-SY5Y) cells in vitro. It is noteworthy that remarkable inhibitory effects on the accumulation of Aβ(1-40) peptides were observed for SH-SY5Y cells treated with anionic HL-DMPS, though the accumulation was not inhibited by cationic HL-DMTAP. On the other hand, the immediate fusion of HL-DMTAP into SH-SY5Y cells was confirmed using a confocal laser microscope. Interestingly, the specific interactions between anionic HL-DMPS and Aβ(1-40) peptides were observed using the thioflavin T (ThT) assay. In addition, the cytotoxicity of Aβ(1-42) peptides on the SH-SY5Y cells decreased after the treatment with HL-DMPS. These results suggest that anionic HL-DMPS could be used as a novel medicine for AD in the future.

In general, chemotherapeutic effects were low for non-small cell lung cancer (NSCLC) in the lung tumor. We examined the accumulation and antitumor effects of hybrid liposomes (HL-23) composed of phospholipid (L-α-dimyristoylphosphatidylcholine: DMPC) and PEG surfactant [polyoxyethylene(23)dodecyl ether: C₁₂(EO)₂₃] on NSCLC cells in vitro. Accumulation of HL-23 including a fluorescence probe [1-Palmitoyl-2-[12(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phosphocholine: NBDPC] was observed for NSCLC cells using a confocal laser microscope, but no accumulation of HL-23 in normal lung cells was observed. Furthermore, inhibitory effects of HL-23 on the growth of NSCLC cells were obtained on the basis of a WST-1 assay. It was also clarified that HL-23 induced apoptosis for NSCLC cells on the basis of Annexin-V binding and TUNEL assay. These results suggest that HL-23 could be applied in effective chemotherapies for NSCLC.

Hybrid liposomes (HLs) composed of vesicular and micellar surfactants have inhibitory effects on the growth of tumor cells in vitro and in vivo. Successful clinical chemotherapy with drug-free HLs to patient with lymphoma has been reported after approval by the Committe of Bioethics. However, the therapeutic effects of HLs on the metastasis of colon carcinoma cells have not yet been elucidated. In this study, the therapeutic effects of HLs composed of L-alpha-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (23) dodecyl ether [C(12)(EO)(23)] on the metastasis of colon carcinoma (Colon26) cells were examined in vivo. Marked high therapeutic effects were obtained in the hepatic metastasis mice model after the treatment with HLs. Furthermore, optical microscopic analysis indicated that HLs could induce the apoptosis of colon carcinoma cells in vivo. No toxicity was observed in the hepatic metastasis mice model after intravenously injecting HLs. Therapeutic effects along with the induction of apoptosis by HLs without any drugs on hepatic metastasis were revealed on the basis of optical microscopic analysis for the first time in vivo.

We established cell-line (CoRa 622 G6) of gastric carcinoma using cotton rats with spontaneous malignant gastric carcinoma with hypergastrinaemia. Inhibitory effects of hybrid liposomes (HL) composed of dimyristoylphosphatidylcoline (DMPC) and polyoxyethylene (n) dodecyl ether (C(12)(EO)(n): n=21, 23, 25) on the growth of CoRa 622 G6 cells were clarified on the basis of WST-1 assay. Fusion and accumulation of HL including fluorescence probe into CoRa 622 G6 cell membrane were clarified using confocal laser microscopy and total internal reflection fluorescence microscopy. Induction of apoptosis of CoRa 622 G6 cells after the treatment with HL was observed in fluorescence micrographs on the basis of Annexin-V binding assay and TUNEL method using confocal laser microscopy. The results in this study could contribute to the chemotherapy for patients with gastric carcinoma.

This study aimed to develop a new processing method for the effective use of rice shochu distillation remnants. We examined the inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with various fungi. Interestingly, high inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with Aspergillus oryzae were obtained, although no inhibitory effect was observed in the case of synthetic medium. We therefore fractionated the medium of rice shochu distillation remnants with A. oryzae using anion-exchange and reverse-phase chromatography. Furthermore, we attempted to determine the chemical structure of compounds that showed high inhibitory effects on the growth of tumor cells. The chemical structure of 1-hydroxy-6-(1-methylpropyl)-3-(2-methylproryl)-2(1H)-pyrazinone was revealed on the basis of liquid and gas mass spectroscopies. This compound should be completely safe based on toxic test results using model mice.

It is known that hybrid liposomes composed of dimyristoylphosphatidylcholine (DMPC) and micellar surfactant (Tween 20) are effective for encapsulating drugs and for the treatment of meningeal gliomatosis in vivo without toxicity. In this study, we attempted to use these hybrid liposomes as a drug carrier system for the oral administration of insulin using diabetic model rats. The noteworthy aspects were as follows: (a) The hybrid liposomes of 90 mol% DMPC/10 mol% Tween 20 were stable and uniform for more than 4 weeks. (b) The blood glucose level of diabetic model rats decreased after the oral administration of hybrid liposomes including insulin. These results suggest that the oral administration of hybrid liposomes including insulin should be effective for the treatment of diabetes.

Prolonged survival was seen in a carcinoma model in mice intraperitoneally inoculated with B-16 melanoma cells after the intraperitoneal treatment with hybrid liposomes composed of L-alpha-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenedodecyl ether (C12(EO)n, n = 10 and 23 respectivery) which had a uniform and stable structure. No drug was administered. The therapeutic effects of the single-component liposomes composed of lipids with a variety of hydrophilic head groups and different hydrophobic alkyl chains were investigated. Markedly prolonged survival (248%) of mice was achieved after treatment with DMPC liposomes. However, DMPC liposomes have the disadvantage of an unstable structure, requiring daily sonication. On the other hand, no life-prolonging effects or toxicity occurred with the administration of the other single-component liposome employed in this study. Next, we successfully prepared stable, uniform liposomes composed of 90 mol% DMPC and 10 mol% C12(EO)n (n = 10 and 23, respectively), which have diameters of 70 nm and 100 nm, respectively. Interestingly, prolonged survival (173-186%) of mice was achieved after treatment with hybrid liposomes of 90 mol% DMPC/10 mol% C12(EO)n (n = 10 and 23). Finally, we conducted toxicity tests using normal rats to determine hybrid liposome stability. There were no abnormal findings in blood chemistry or relative organ weights at autopsy of normal rats after hybrid liposome administration. In addition, hybrid liposomes were metabolized in the liver after intravenous administration to normal mice. These results suggest that hybrid liposomes could be used as a new single chemotherapeutic agent in the treatment of carcinoma with no side effects.

The inhibitory effects of hybrid liposomes composed of lipids having a variety of head groups (zwitterionic L-alpha-dimyristoylphosphatidylcholine (DMPC), anionic L-alpha-dimyristoylphosphatidylglycerol (DMPG), and cationic 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)) and polyoxyethylene (10) dodecyl ether (C12(EO)10) on the growth of tumor cells (human lung carcinoma (RERF-LC-OK), human hepatoma (Hep-G2), human stomach tumor (GT3TKB)) in vitro were examined. The hybrid liposomes of DMPC/10 mol% C12(EO)10 and DMPG/10 mol% C12(EO)10 were fairly more effective for inhibiting the growth of all tumor cells employed in this study as compared with liposomes (DMPC and DMPG) or micelles (C12(EO)10). Especially, it is attractive that the highly specific inhibitory effect of the hybrid liposomes of DMPG/10 mol% C12(EO)10 without any antitumor drugs on the growth of RERF-LC-OK and GT3TKB was obtained for the first time.

Remarkably high inhibitory effects of the hybrid liposomes composed of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) and polyoxyethylenealkyl ether (C14(EO)n, n = 6-8 and C12(EO)n, n = 8-12)) on the growth of human lymphoma-human B-lymphocyte hybridoma (HF) cells in vitro were obtained. The hybrid liposomes composed of 90 mol% DMPC/10 mol% C14(EO)n (n = 6-8) or C12(EO)n (n = 8-12) were more fluid as compared with 90 mol% DMPC/10 mol% C14(EO)4 or C12(EO)n (n = 4, 23) hybrid liposomes on the basis of fluorescence polarization measurements. These results suggest that the inhibitory effects of the hybrid liposomes on the growth of HF cells should be related to the membrane fluidity. No toxicity to normal rats in vivo was observed in the experiment using 90 mol% DMPC/10 mol% C14(EO)7 or 90 mol% DMPC/10 mol% C12(EO)12 hybrid liposomes.