Fig. 1. The profiles of CsCl isopycnic ultracentrifugation. The fluorescence of 100-nm beads (a) was observed in a plate reader. Liposomes (b) were detected by the absorbance (300 nm). BNC (c) and the BNC–liposome complex containing 100-nm beads (d) were measured by ELISA.

Fig. 2. Electron-micrographs of liposomes, BNC and the BNC–liposome complex. Liposomes (a), BNC (b), and the BNC–liposome complex (c) were observed using TEM, following negative staining. Scale bar, 100 nm. The sizes of BNC, 100-nm beads, liposomes, and the BNC–liposome complex were detected by dynamic light scattering method (5 batches, 12 measurements for each batch) (d).

Fig. 3. Optimization of BNC–liposome complex for delivering 100-nm beads. BNC–liposome complex (black circles, 5 µg as a protein) or liposomes (white circles) containing 100-nm yellow-green beads was used to transfect about 5 × 10⁴ cells of HepG2 (a) and WiDr (b). The amount of liposome (as a lipid) was expressed as the ratio to that of BNC (as a protein). The 100 nm beads-derived fluorescence was measured by a plate reader (4 batches, 6 measurements for each batch; mean ± s.d.).

Fig. 4. Ex vivo delivery of 100-nm beads to human hepatocytes using the BNC–liposome complex. 100-nm yellow-green beads were transfected in HepG2 cells by electroporated BNC (EP, white square) and the BNC–liposome complex (BNC-Lipo, black square) (6 batches, 3 measurements for each batch; mean ± s.d.) (a). BNCs containing 100-nm red beads were applied to HepG2, A431, and WiDr cells for 6 h. The fluorescence of cells to which beads were added with BNC (black square) or without BNC (gray square) was measured by a plate reader (6 batches, 3 measurements for each batch; mean ± s.d.) (b) and observed under a confocal microscope (c). Scale bar, 50 µm. The cytotoxicity of BNC–liposome complex was determined by MTT assay using HepG2 cells (6 batches, 3 measurements for each batch; mean ± s.d.) Dashed line indicates 70% of viability (d).

Fig. 5. In vivo delivery of 100-nm beads to human liver-derived tumor using the BNC–liposome complex. BNCs containing 100-nm beads were injected (i.v.) in the mouse xenograft model bearing a NuE cell-derived tumor and an A431 cell-derived tumor. At 16 h after the injection, lectin was injected (i.v.) and sacrificed. Fluorescence was observed in sections from the NuE-derived tumor under a confocal microscope (3 batches, 5 mice for each batch). Scale bar, 100 µm.

Fig. 6. BNC–liposome complex containing GFP expression plasmid. The sizes of the liposomes containing DNA, and the BNC–liposome complex containing DNA were detected by a dynamic light scattering method (5 batches, 12 measurements for each batch) (a). At 48 h after the transfection, GFP-derived fluorescence was observed under a confocal microscope (6 batches, 3 measurements for each batch). Scale bar, 100 µm (b).

Fig. 7. In vivo delivery of GFP expression plasmid to human hepatocytes using the BNC–liposome complex. The BNC–liposome complexes containing the plasmid were injected (i.v.) in the mouse xenograft model bearing a NuE-derived tumor and an A431-derived tumor. At 1 week after the injection, GFP-derived fluorescence was observed in sections from the NuE- and A431-derived tumor (a) and the regular tissues (liver and kidney) (b) under a confocal microscope (3 batches, 5 mice for each batch). Scale bar, 100 µm.

Fig. 8. Ex vivo delivery of large GFP expression plasmid to human hepatocytes using the BNC–liposome complex. The BNC–liposome complex containing the 35-kbp plasmid was applied to HepG2 and A431 cells. At 2 day after the transfection, GFP-derived fluorescence was observed under a confocal microscope (6 batches, 3 measurements for each batch). Scale bar, 100 µm.