Shinya Suzuki

Modulation of doxorubicin resistance in a doxorubicin-resistant human leukaemia cell by an immunoliposome targeting transferring receptor

Using a doxorubicin-resistant subline (K562/ADM) of human leukaemia K562 cells (Tsuruo et al, 1986), the effect of immunoliposomes that targeted a cellular transferrin receptor (TFR) was examined by neutralization of doxorubicin (DOX) resistance. OKT9-CIL, prepared by conjugation of DOX-encapsulated liposome with an anti-TFR monoclonal antibody, OKT9 (Aisenberg and Wilkes, 1980), showed similar binding to both K562 and K562/ADM. Although an 80-fold higher sensitivity to free DOX on cell growth inhibition in K562 than in K562/ADM was found, the difference was clearly diminished after OKT9-CIL treatment through the increased sensitivity of K562/ADM. The cellular DOX level 30 min after the exposure of free DOX was 45-fold lower in K562/ADM than in K562, whereas nearly equivalent DOX levels were detected in K562 and K562/ADM after OKT9-CIL treatment. In addition, DOX in K562/ADM in the free DOX treatment was efficiently excreted by 54% within 120 min of incubation, whereas almost all DOX supplied by OKT9-CIL remained uncleared. Fluorescence microscopic observation showed that OKT9-CIL was internalized into juxtanuclear vesicles in K562/ADM cells. These results suggest that OKT9-CIL has a potency to accumulate DOX, resulting in augmentation of DOX cytotoxicity in DOX-resistant tumour cells.

Preparation of long-circulating immunoliposomes containing adriamycin by a novel method to coat immunoliposomes with poly(ethylene glycol).

Modifications of liposomes with poly (ethylene glycol) (PEG) have been reported to prolong blood circulation time of liposomes. In this report, an adriamycin-encapsulated immunoliposomes were modified with PEG by two different approaches: one is the pre-coating method using lipid derivative of PEG as described by Allen et al. The other is post-coating method which is presented here. The former pre-coating method did not allow coupling of antibody due to the steric hindrance of PEG which had been introduced on liposome surface. On the other hand, in the later post-coating method, PEG-succinylcysteine was synthesized and was successfully conjugated with immunoliposomes via maleimido linker. Resultant PEG-coated immunoliposomes containing adriamycin retained their binding activity and cytotoxicity to target cells, and also showed significantly prolonged blood circulating time as compared with conventional immunoliposomes. This is a novel method to coat immunoliposomes with PEG.

Cytotoxicity of anti-c-erbB-2 immunoliposomes containing doxorubicin on human cancer cells.

We have examined the selective cytotoxicity of immunoliposomes containing doxorubicin (chemoimmunoliposomes, CILs) targeting the c-erbB-2 gene product (gp185) or gp125. Anti-gp185 and anti-gp125 CILs were prepared by conjugation of doxorubicin-containing liposomes with monoclonal antibodies SER4 (IgG) and HBJ127 (IgG) respectively. Both CILs bound to human SKBr-3 breast cancer cells and MKN-7 human gastric cancer cells, which express both antigens in high density. The IC50 of anti-gp185 CILs on protein synthesis by SKBr-3 cells was respectively 2- and 25-fold lower than that of anti-gp125 CILs and unmodified liposomes. Furthermore, anti-gp185 CILs significantly inhibited neither the phytohaemagglutin response of normal lymphocytes nor protein synthesis of gp185-negative T24 bladder cancer. Quantitative analysis of cell-associated doxorubicin revealed that, compared with anti-gp125 CILs, anti-gp185 CILs required, respectively 4.5 and 4.3 times less doxorubicin association in SKBR-3 and MKN-7 cells, for 50% cytotoxicity. In addition, flow cytometric analysis showed that both SKBr-3 and MKN-7 internalised more anti-gp185 CILs and processed them more efficiently than anti-gp125 CILs. These results suggest that anti-gp185 CILs act selectively against gp185-expressing cancer cells and that gp185 is a more sensitive antigen for CIL cytotoxicity associated with endocytosis activity.

ENDOCYTOSIS DOES NOT NECESSARILY AUGMENT THE CYTOTOXICITY OF ADRIAMYCIN ENCAPSULATED IN IMMUNOLIPOSOMES

We studied the relationship between endocytosis and cytoxicity of adriamycin (ADM) encapsulated in antibody-coated liposomes (immunoliposomes, IL) which are called chemoimmunoliposomes (CIL), by using several human cancer cell lines. IL coated with a monoclonal antibody, HBJ127 (IgG), which recognizes human gp125 antigen, specifically bound to gp125-positive target cancer cell lines, KU-1, T24, MKN-7, SKBr-3 and LS174T. Flow cytometric analysis using IL encapsulating carboxyfluorescein (CF) revealed that efficiencies of endocytosis varied among different cancer cells. The rate of IL internalization was in the order KU-1 > T24 > MKN-7 > SKBr-3 > LS174T. In 1 h incubation at 37 degrees C, all the four cell lines other than LS174T internalized about 60% of IL which were bound on their cell surfaces. KU-1, T24 and MKN-7, but not SKBr-3, significantly processed IL in endosome or lysosome. On the contrary, 80% of CIL bound to LS174T remained on the cell surface even after 2 h incubation. Furthermore, we evaluated the cytotoxic activities of CIL against the same panels of cancer cells. CIL inhibited the growth of all cancer cells tested in antibody-dependent manner, but, contrary to our expectation, KU-1 and T24 cells, which showed significant endocytosis activity, required a 7-14-fold higher amount of ADM binding than LS174T cells with low endocytosis activity for 50% cell growth inhibition. The difference of sensitivity to free ADM was only within 2.3-fold among those cancer cells. These results showing that liposomal ADM endocytosed is less effective than that remaining on the cell surface suggest that endocytosis is not necessarily required for cytotoxicity of CIL.

In vitro Targeting and Cytotoxicity of Adriamycin in Liposomes Bearing Monoclonal Antibody against Rat or Human gp125 Cell Proliferation-associated Antigen

Chemoimmunoliposomes (CIL) were prepared by entrapping adriamycin in monoclonal antibody (mAb)‐coated liposomes and examined for their binding capacity and cytotoxicity to relevant target tumor cells. Sonicated unilamellar liposomes were coated with B3 and HBJ127 mouse, mAbs, which recognize a rat and a homologous human cell proliferation‐associated surface antigen, gp125, respectively, and then adriamycin was entrapped in the liposomes by means of transmembrane Na±/K±gradients using valinomycin. These CIL selectively bound with relevant target tumor cells bearing the corresponding gp125 antigen, such as BC47 rat bladder cancer, FTL‐13 rat thymic lymphoma, T24 human bladder cancer and Molt‐4 human leukemia cells, although the binding capacities of the CIL to bladder cancer cells were relatively larger than those to lymphoma cells in both rat and human systems. This difference in the target cell binding was found to be attributable to the amount of gp125 antigen expressed on each target tumor cell, as determined by a Scatchard plot analysis. In accordance with the target cell binding capacities of CIL preparations, the CIL displayed much higher cytotoxic activity to bladder cancers than to lymphomas in both rat and human systems. In conjuction with our previous finding that gp125 antigen is expressed on tumor cells but not on resting normal cells, these findings indicate that CIL composed of anti‐gp125 mAb will be useful for tumor therapy and that the antitumor efficacy is dependent upon the extent of the antigen expression on target tumor cells.

Coating of liposomes with subunits of monoclonal IgM antibody and targeting of the liposomes.

SH-bearing subunits of IgM antibody were coupled through the Fc portion to sonicated liposomes consisting of N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine and cholesterol. Liposomes coated with subunits of monoclonal IgM antibody against a mouse mammary tumor-associated antigen selectively bound to the antigenic target cells. Assays of the liposomes for complement dependent cytotoxicity and spleen cell binding revealed inactivation of the liposome-coupled antibodies in both assays.

Anisotropy in lithium ion conduction in laminated thin films of montmorillonite nanosheets

Laminated thin films of lithium-ion-exchanged montmorillonite nanosheets were prepared, and their anisotropy in lithium ion conduction was investigated. For the films with 0.70 µm thickness, the lithium ion conductivities at 30 °C in the directions parallel and perpendicular to the film plane (σ//, σ⊥) were determined by alternative current impedance measurements and found to be 4.3 × 10−2 and 5.7 × 10−6 S cm−1, respectively. The dependence of σ⊥ on the nanosheet size for the films composed of nanosheets with different lateral sizes was also investigated. σ⊥ decreased as the lateral size of the nanosheets increased. A model for the lithium ion conduction path in the nanosheet-laminated films is proposed, and the dependence of conduction on the nanosheet size is discussed.

Augmentation by Tumor Necrosis Factor α of the Systemic Therapeutic Effect of Lymphokine-activated Killer Cells in Adoptive Immunotherapy of Murine Tumor

The therapeutic effect of a combined modality of lymphokine‐activated killer (LAK) cells and tumor necrosis factor α (TNFα) on MBL‐2 tumor in C57BL/6 mice was studied. Murine LAK cells induced from splenocytes by interleukin 2 (IL2) could lyse MBL‐2 target cells in vitro, but no enhancement of the LAK activity was found by the treatment of LAK cells with TNFαin vitro. However, the treatment of MBL‐2 with TNFα enhanced the sensitivity to LAK cells. Moreover, administration of TNFα to mice bearing solid MBL‐2 tumor led to increased tumor vascular permeability within 1 h, and resulted in the enhanced accumulation of systemically transferred LAK cells in tumor tissue. Based on these results, we treated MBL‐2‐bearing mice with TNFα and then with LAK cells 1 h later, No therapeutic effect was observed when tumor‐bearing mice were treated with TNFα alone or LAK cells plus IL2. However, adoptive immunotherapy using LAK cells and TNFα had therapeutic effects, i.e. growth inhibition of tumor nodules and prolongation of survival. These results indicated that appropriately timed pretreatment of tumor‐bearing mice with TNFα augmented the anti‐tumor efficacy of LAK cells.

Electrode Properties of Ba0.5Sr0.5CoyFe1-yO3-δCathode Materials for Solid Oxide Fuel Cell

The electrode properties of Ba0.5Sr0.5CoyFe1-yO3-δ {BSCF(5/5/10y/10(1-y))} on a Ce0.8Sm0.2O1.9 electrolyte were investigated. BSCF powders were synthesized by a solid-state reaction. The electronic conductivities of BSCFs increased with increasing Co content and BSCF(5/5/8/2) showed the highest conductivity of 38 S cm-1 at 600oC. The electrode resistance was measured on symmetric cells, BSCF/Ce0.8Sm0.2O1.9/BSCF, over the temperature range from 600oC to 800oC. The electrode resistances decreased with increasing Co content and BSCF(5/5/8/2) showed the lowest electrode resistance in the whole temperature range, having an electrode resistance of 0.62 cm2 at 600oC.

High-Efficiency Mirror Grinding of AlN by Ultra-Precision Plane Honing

Plane honing machining of aluminum nitride (AlN) was investigated in this study. AlN has advantageous thermal and dielectric characteristics and is in great demand as a semiconductor mounting board or packaging parts material. However, as AlN is a sintered material, the AlN grains readily detach during machining making it difficult to obtain fine surface roughness at high efficiency. In previous studies, we have developed a new plane honing method that makes it possible to grind hard and brittle materials. In this paper, plane honing experiments of AlN were carried out using a resinoid or vitrified bond wheel. Fine surface roughness could be obtained using the resinoid bond wheel but there was low-machining efficiency due to burying and detachment of abrasives. The vitrified bond wheel, however, enabled high efficiency grinding but resulted in traces of detached AlN grains forming at the machined surface. Highly efficient mirror grinding of AlN could be achieved by using a vitrified bond wheel for semi-finishing and a resinoid bond wheel for finishing.