Gelatin beads as platforms for targeting molecule and anti-Fas antibody  Toshiya Yokozawa, Koichi Miyamura, Ryuichi Fujino, Shin Yonehara, Ryuzo Ueda, Mitsune Tanimoto, Hidehiko Saito  Experimental Hematology  Volume 28, Issue 10, Pages 1129-1136 (October 2000) DOI: 10.1016/S0301-472X(00)00528-2

Figure 1 Characterization of gelatin beads. (A) Scanning electron microscopic view of a gelatin bead as a spherical particle about 2.5 μm in diameter. (B) Amount of antibodies coated on gelatin beads detected by flow cytometry. Monoclonal antibodies (mAb)were fixed on the beads by coincubation with specific antibody (1–10 μg/mL) and 1.4 × 109 gelatin beads. Mean fluorescence intensity of gelatin beads bearing CH11 prepared with different amounts of CH11 is shown. Dose-dependent increase in surface mAbs was confirmed Experimental Hematology 2000 28, 1129-1136DOI: (10.1016/S0301-472X(00)00528-2)

Figure 2 Phase contrast microscopic view of the attachment of gelatin beads to different cell lines after a 1-hour incubation. Fp 0-10 beads attach to CD10+ cell lines NALM-6 (C) and MOLT-3 (F), but not to CD10- cell lines U-937 (D). A small number of beads are attached to the CD10± cell line K-562 (E). The efficiency of attachment correlates with the amount of NL-1 on gelatin beads in NALM-6 cells: Fp 0-0 (A), Fp 0-1 (B), Fp 0-10 (C) Experimental Hematology 2000 28, 1129-1136DOI: (10.1016/S0301-472X(00)00528-2)

Figure 3 Results of attachment scores as defined in the Materials and methods section. (A) Beads carrying NL-1 (Fp 0-1, Fp 0-10) are compared with Fp 0-0 using NALM-6, U-937, HL-60, K-562, and MOLT-3 at B/T ratio = 20. (B) Attachment scores of beads carrying both NL-1 and CH11 (Fp 1-0, Fp 1-1, Fp 1-10). The effect of additional NL-1 is demonstrated most clearly in CD10+ cell lines (NALM-6, MOLT-3) in both graphs Experimental Hematology 2000 28, 1129-1136DOI: (10.1016/S0301-472X(00)00528-2)

Figure 4 NALM-6 (A,B,C,F,G) and U-937 (D,E) were incubated with gelatin beads for 24 hours, after which apoptotic features of cells were observable. (A) Microscopic appearance at higher magnification showed that many gelatin beads attach to cells and that some display early apoptotic features. (B) Hoechst staining displayed in the right upper corner of (A) demonstrates condensation of the nucleus and cytoplasm. (C) Phase contrast microscopic view showing convolution of cellular outline and irregular cell surface. (F,G) May-Giemsa stain from the same slide shows condensation of the nuclear and cytoplasm (F) or destruction of features (G). (D,E) Late stages of apoptosis, showing nuclear fragmentation and marked convolution of nuclear and cellular outlines Experimental Hematology 2000 28, 1129-1136DOI: (10.1016/S0301-472X(00)00528-2)

Figure 5 Induction of apoptosis in U-937 (A,B) and NALM-6 (C) by gelatin beads. Cytotoxicity was measured by colorimetric DNA fragmentation assay and LDH release assay. (A) Cytotoxic effects with control beads (Fp 0-0), Fp 0-0 + free CH11, Fp 1-0, and Fp 10-0 were compared at different B/T ratios by DNA fragmentation assay. (B) Time course study (6, 12, 24, 48, 72 hours) using Fp 1-0 at B/T ratio = 20 demonstrated by DNA fragmentation assay (column) and LDH release assay (line). (C) Fp 1-0, Fp 1-1, and Fp 1-10 at different B/T ratios (10–80) were compared for their cytotoxic effects using DNA fragmentation assay. (D) Relative cytotoxicities of NALM-6, U-937, and HL-60 at B/T ratio = 20 (24 hours) measured by DNA fragmentaion assay. Cells were incubated with beads carrying small amounts of CH11 (Fp 1-0, Fp 1-1, and Fp 1-10) or those carrying large amounts of CH11 (Fp 10-0, Fp 10-1, Fp 10-10, indicated by asterisk) Experimental Hematology 2000 28, 1129-1136DOI: (10.1016/S0301-472X(00)00528-2)

Figure 6 Cytotoxic activities of beads (Fp 10-0) were examined in the peritoneal cavity of BALB/c nude mice. U-937 cells (1 × 107/mouse) were administered to mice followed by injection of Fp 10-0 (B/T ratio = 40/1), CH11 + Fp 0-0, or PBS. Cells were collected 24 hours later, and the total number of CD13+ cells was counted by flow cytometry. The number of CD13+ cells was remarkably reduced in comparison with those of controls (—-•—-). To detect the apoptotic CD13+ cells, CD13+/PI− and CD13+/PI+ populations were counted. Approximately half of the CD13+ cells from the mice treated with beads carrying CH11 were indicating apoptosis (black column). Those cells from control mice showed only background amounts of apoptotic change (white and shaded columns) Experimental Hematology 2000 28, 1129-1136DOI: (10.1016/S0301-472X(00)00528-2)