UDP-GaNAc : polypeptide N-acetylgalactosarninyltransferases(GaNAc trapsferases) catalyze the initial reaction in O-linked (mucin type) oligosaccharide biosynthesis. To attempt to inhibit the synthesis of O-glycan, we transfected antisense cDNA of GalNAc transferase-type 1 (GalN Ac-T1) into a human gastric cancer cell line, JRST. A decreased expression of GalNAc-T1 in the level of both mRNA and protein was observed in the resultant transfectants. They demonstrated a significantly increased sensitivity to NK and LAK cells in vitro compared with parental cells and mock transfectants. Although there was no significant difference in in vitro cell proliferation among parental cells, mock transfectants or antisense transfectants, in vivo growth rate of antisense transfectants using SCID mice was clearly lower than that of parental cells and mock transfectants. From these results, we conclude that antisense suppression of GalNAc-T1 could increase the sensitivity of tumor cells to NK and LAK cells, suggesting that this strategy may be of use as a new immunogene therapy.

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To evaluate the usefulness of liposomes as oral vaccines, the stability of liposomes, and the serum and intestinal IgA antibody responses to antigen associated with liposomes after oral administration were examined. In the liposomes tested, liposomes composed of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), and cholesterol (Chol) (1 : 1 : 2, molar ratio), distearoylphosphatidylcholine (DSPC) and Chol (7 : 2, molar ratio), and DSPC, DPPS, and Chol (7 : 3 : 2 or 1 : 1 : 2, molar ratio) were stable in acidic solution (pH 2.0), bile, and pancreatin solution. After the oral immunization of antigen (ganglioside GM1)-containing liposomes composed of DPPC, DPPS, and Choi (1 : 1 : 2, molar ratio) to mice, the serum and intestinal IgA antibody responses against ganglioside GM1 were found. Furthermore, when monophosphoryl lipid A was incorporated into liposomes containing ganglioside GM1, further augmentation of serum IgA responses to ganglioside GM1 was observed. On the other hand, the oral administration with liposomes composed of DPPC, Chol, and ganglioside GM1 (unstable liposomes), ganglioside GM1 mixed with liposomes composed of DPPC, DPPS and Chol, and ganglioside GM1 alone was unable to induce any detectable anti-ganglioside GM1 IgA antibody responses. These results suggest that liposomes which showed the stability to acidic solution, bile, and pancreatin solution would serve effectively as an oral delivery vehicle for inducing mucosal immune responses.

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Previously we reported the preparation and characterization of fusogenic liposomes (FL) which have highly immunogenic envelope glycoprotcins derived from Sendai virus on their surface. In this report, we investigated the ability of antigen-encapsulated FL to stimulate antigen-specific humoral immunity in mice. Anti-OVA antibody production was markedly increased in serum from mice subcutaneously immunized with ovalbumin (OVA) in FL compared to OVA alone or OVA in simple liposomes. However, these enhancements were not observed in mice immunized with OVA simply mixed with empty-FL. Moreover, consistent with mitogenic effects of Sendai virus' envelope glycoprotein, empty-FL were mitogenic for cultured murine splenocytes and B cells were especially proliferated. And this stimulation depended on protein concentration derived from Sendai virus. These results indicate that fusogenic liposome functions as an efficient immunoadjuvants in inducing antigen-specific antibody production and that for efficient adjuvancy it is necessary to encapulate antigens into FL. Moreover it was speculated that B cell stimulation which was given by glycoprotein derived from Sendai virus participated in adjuvancy of FL.

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We previously reported that fusogenic liposomes (FL) prepared by fusing simple liposomes with Sendai virus perticles, could introduce their contents directly and efficiently into the cytoplasm. In this report, we investigated that exogenous antigen was processed and presented by class I major histocompatibility complex (MHC) molecules after delivery into cells by using FL. FL could introduce fragment A of diphtheria toxin, maintaining its cytotoxicity, into the cytoplasm of cells efficiently in vitro. Consistent with this, cells treated with ovalbumin (OVA) in FL were as susceptible to lysis by class I MHC-restricted, OVA specific cytotoxic T lymphocytes (CTL) as OVA-transfected clones. However, cells treated with naive OVA or OVA in simple liposomes were failed to lysis by OVA-specific CTL. These results indicated that FL could direct a exogenous antigen into class I MHC presentation pathway as a result of introduction of them into the cytoplasm. Thus, FL may facilitate induction of CD8⁺ CTL responses in vaccinations with exogenous soluble antigens, and they may serve as readily avaiable tools for dissecting class I MHC immunity to viruses and other intracellular pathogens.

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We previously reported that some modulators, which enhanced the antitumor activity of doxorubicin, increased the doxorubicin concentration specifically in the tumor. In an attempt to elucidate a part of the mechanism, we compared the membrane transport and the antitumor activity of pirarubicin with those of doxorubicin on M5076 ovarian sarcoma which is known to have low sensitivity to doxorubicin. Pirarubicin uptake by the M5076 showed 2.0 fold of doxorubicin uptake. The 50% cell growth inhibitory concentration and the 50% lethal concentration of pirarubicin were less than those of doxorubicin in vitro. Thus, the cytotoxicity of pirarubicin on M5076 cells was much stronger than that of doxorubicin. It has been suggested that the difference in these cytotoxicities depended on the uptake amounts of antitumor agents by M5076 cells. On M5076 sarcoma bearing mice, doxorubicin did not affect the tumor weight, whereas pirarubicin significantly reduced the tumor weight compared to the control level. Furthermore, pirarubicin prolonged the survival time of M5076 transplanted (iv) mice to 1.5-fold. These in vivo experiments indicated that pirarubicin was more effective on M5076 sarcoma compared to doxorubicin. These results suggested that the increase in the uptake amount of anthracycline in tumor cells might provide the enhancement of antitumor activity.

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Nimustine (ACNU), one of water-soluble nitrosoureas, is used for treatment of some types of tumors. Since this drug can penetrate through the blood-brain barrier, it has been used especially for the treatment of primary brain tumors. ACNU is, however, decomposed rapidly in the blood or cerebrospinal fluid (CSF). In order to overcome this short-coming, we attempted to encapsulate ACNU in liposomes. Liposomes composed of egg phosphatidylcholine were prepared according to the procedure of the reverse-phase evaporation vesicle (REV) method. ACNU could be encapsulated efficiently into liposomes using a transmembrane pH gradient. Encapsulation efficiencies of ACNU with this method were shown to be more than 80%. The mean diameter of liposomes encapsulating ACNU (ACNU liposome) was about 300 nm. These liposomes were stable for more than 7 days at 4°C in storage. When 1 ml of ACNU solution(1 mg/ml) was added to 15 ml of artificial CSF at 37°C, concentrations of ACNU in the solution were 0.28 μg/ml at 3 hr after addition, and no ACNU was detected at 6 hr after addition. On the other hand, when 1 ml of ACNU liposome(1 mg/ml) was added to artificial CSF, concentrations of ACNU in the solution and liposomes were 6.71 and 2.62 μg/ml, respectively at 3 hr after addition. ACNU was detected at 9 hr in artificial CSF and liposomes. These results suggest that the pH gradient method is useful for encapsulation of ACNU in liposomes and the liposome formulation is useful for stabilization of ACNU in artificial CSF.

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We studied the effect of chitosan film containing bFGF on wound healing in genetically diabetic mice. Full thickness wounds of skin (2 cm²) were created on the back of each mouse. The chitosan film was prepared as follows : hydroxypropyl chitosan acetate buffer solution (6%w/v) was casted in a Teflon cast (1 × 1 cm) and dried. The wounds were treated with chitosan film, with chitosan film containing bFGF (0.6 μg/cm² and 2.0 μg/cm²), and without any film (control group). On the 7 day after wounding, wound area was reduced significantly in the groups treated with chitosan film and both of chitosan films containing bFGF compared with control group. On the 14 day after wounding, in chitosan film containing bFGF (2.0 μg/cm²) group, wound area was significantly reduced compared with the other groups. Histological examination revealed thickening of granulation tissue and an increase in the number of capillaries in chitosan film containing bFGF (2.0 ²g/cm²) group. These results suggested that chitosan film containing bFGF is effective on wound healing in genetically diabetic mice in a dose-dependent fasion.

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In this study we analyzed the binding ability of complement component 3 (C3) associated with liposomes after incubation in rat serum in order to determine the effect of cholesterol content of liposomes on the complement system liposomes interactions and the fate of liposomes in vivo. Liposomes having different cholesterol content showed no difference in clearances from blood circulation, while in both hepatic and renal clearances the liposomes showed marked difference. The hepatic clearance increased with increasing cholesterol content and the renal clearance decreased with increasing cholesterol content. Further, it appeared that those liposomes had different C3 binding potentials : cholesterol-rich (44 mol%) liposomes had higher affinity for C3 than cholesterol-poor (22 mol%) liposomes. Interestingly, there was a good correlation between the C3 binding ability and hepatic clearance of liposomes, while there was an inverse correlation between the C3 binding ability and renal clearance of liposomes. These results suggested that the extent of interaction of liposomes with complement system directly related to the distribution of liposomes in liver, leading to a possibility that the liposomes having high affinity for C3 are phagocytosed by Kuppfer cells via complement receptors. Release of encapsulated carboxy fluorescein from liposomes was inhibited in heat-treated rat serum, less done in EDTA-treated one. This finding indicated that degradation of liposomes in in vivo or in vitro was attributed to a heat-labile serum component, but not complement system. We clarified in this study that the complement-liposome interactions play a critical role on the hepatic uptake of liposomes, but not on the degradation of liposomes in blood circulation.

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