Article Figures & Data
Figures
- Fig. 1.
Structure and internalization of G8:3DNA. (A) Schematic representation of the G8 IgM mAb coupled to a two-layer 3DNA nanocarrier intercalated with doxorubicin. (B) Proposed mechanism of targeted cytotoxicity in which doxorubicin diffuses from 3DNA following antibody binding and internalization of the conjugate into acidic compartments of the cell. (C–E) Anterior lens tissue was incubated for 2 hours with G8:3DNA:Cy3 (red) and LysoSensor dye that fluoresces green at acidic pH (C–E) or 3DNA:Cy3 and LysoSensor dye (F–H). Nuclei were labeled with Hoechst dye (blue). Colocalization of red and green (arrows in C and D) appears yellow in the merged image in E. Minimal incorporation of the nontargeting 3DNA:Cy3 conjugate was visible in the tissue (arrows in F-H). Bar, 9 μm in F-H, 5.67 in C and D, and 2.25 μm in E.
- Fig. 2.
G8:3DNA:Dox kills Myo/Nog cells in human lens tissue. Anterior human lens tissue was incubated with G8:3DNA (A–C), 3DNA:Dox (D–F), or G8:3DNA:Dox (G–I) for 24 hours, beginning on the second day in culture. Lenses were fixed and labeled with antibodies to G8, α-SMA, or αB-crystalline and fluorescent secondary antibodies (green) and TUNEL reagents (red). Nuclei were stained with Hoechst dye (blue). Cells labeled with α-SMA also were targeted with G8:3DNA:Dox (J-L). Lens epithelial cells labeled with αB-crystalline were unaffected by G8:3DNA:Dox (M-O). Bar, 9 μm. Overlap of red and green appears yellow in merged images. G8:3DNA:Dox (G–I), but not the control conjugates (A–F), specifically killed Myo/Nog cells in lens explants. Bar, 9 μm.
- Fig. 3.
Expression of Myo/Nog and muscle cell markers in long-term cultures of human lens tissue. Anterior lens tissue was cultured for 30 days and labeled with antibodies to G8, noggin, MyoD, sarcomeric myosin, α-SMA, and fluorescent secondary antibodies. Nuclei were stained with Hoechst dye (blue). Overlap of red and green appears yellow in merged images. G8+ cells contained noggin (A–C), MyoD (D–F), sarcomeric myosin (G–I), and α-SMA (J–L). Bar, 9 μm.
- Fig. 4.
Effect of G8:3DNA:Dox on myofibroblast accumulation in long-term cultures of human lens tissue challenged by wounding. Anterior lens tissue was incubated with DMEM/F12 medium alone or medium containing 3DNA:Dox or G8:3DNA:DOX for 24 hours on day 1 only or days 1 and 13. The tissue was wounded on day 29 and stained for G8, fluorescent secondary antibodies (red), and α-SMA (green), or G8 and αB-crystalline the following day. Nuclei were stained with Hoechst dye (blue). Overlap of red and green appears yellow in merged images. G8+/α-SMA+ cells were present around the wound in cultures lacking drug (A–C) and those treated with 3DNA:Dox (G–I). Myo/Nog cells extended processes onto the capsule (I). G8+/α-SMA+ cells were rare in cultures treated with a single dose of G8:3DNA:Dox (n = 4) (D–F). Two doses of G8:3DNA:Dox completely eliminated G8+/α-SMA+ cells (J–L). Lens epithelial cells stained for αB-crystalline were unaffected by two doses of G8:3DNA:Dox [inset in (L)]. Bar, 9 μm.
Tables
- TABLE 1
Depletion of Myo/Nog cells with G8:3DNA:Dox in human lens cultures
Anterior human lens explants were incubated with medium alone (untreated), G8:3DNA, 3DNA:Dox, or G8:3DNA:Dox for 24 hours. Cells were double-labeled with the G8 mAb and fluorescent secondary antibodies, and TUNEL reagents. The results are the mean ± standard deviation. Untreated, G8:3DNA and 3DNA:Dox, n = 5; G8:3DNA:Dox: n = 8. Significant differences were found between the percentages of TUNEL+ cells in all three control groups compared with G8:3DNA:Dox (P < 0.0001), untreated and 3DNA:Dox (P = 0.0005), and G8:3DNA and 3DNA:Dox (P = 0.02). % G8+ or TUNEL+ = (number of fluorescent cells ÷ total number of cells in 20 fields) × 100. % G8+ with TUNEL = (number of G8+ cells colabeled with the second primary antibody ÷ total G8+ cells) × 100. Reciprocal pairs are also presented. G8:3DNA:Dox, but not control conjugates, specifically killed G8+ cells.
Treatment % G8+ % TUNEL+ % G8+ with TUNEL % TUNEL+ with G8 Untreated 4 ± 1 0.7 ± 0.2 0 0 G8:3DNA 4 ± 1 0.5 ± 0.3 0 0 3DNA:Dox 4 ± 2 0.1 ± 0.1 0 0 G8:3DNA:Dox 5 ± 1 5 ± 1 99 ± 2 99 ± 2 - TABLE 2
Expression of Myo/Nog and muscle cell markers in 30-day human lens cultures
Anterior human lens explants were cultured for 30 days and doubled-labeled with antibodies to G8 and noggin, MyoD, α-SMA, and sarcomeric myosin and fluorescent secondary antibodies. The results are the mean ± standard deviation. The number of subject is indicated in parentheses.
Markers Percent Positive G8 6 ± 4 (18) Noggin 5 ± 3 (7) MyoD 1 ± 2 (4) Myosin 4 ± 3 (4) α-SMA 9 ± 5 (9) G8+ cells with Noggin 100 (4) Noggin+ cells with G8 99 ± 3 (4) G8+ cells with MyoD 24 ± 24 (4) MyoD+ cells with G8 100 (4) G8+ with myosin 53 ± 29 (4) Myosin+ cells with G8 100 (4) G8+ cells with α-SMA 82 ± 22 (6) α-SMA+ cells with G8 67 ± 32 (6) % positive = (number of fluorescent cells ÷ total number of cells in 20 fields) × 100. % G8+ with second primary antibody = (number of G8+ cells co-labeled with the second primary antibody ÷ total G8+ cells) × 100. Reciprocal pairs are also presented. All G8+ cells contained Noggin. Subpopulations of Myo/Nog cells contain different muscle proteins.
- TABLE 3
Effects of G8:3DNA:Dox on the accumulation of G8+ and α-SMA+ cells in 30-day, wounded human lens explants
Anterior human lens explants were incubated with medium alone, 3DNA:Dox or G8:3DNA:Dox for 24 hours on the second and thirteenth day in culture. Wounds were created in the epithelium on day 29. Cells were double-labeled with antibodies to G8, fluorescent secondary antibodies, and α-SMA on day 30. The results are the mean ± standard deviation. The number of cultures is indicated in parentheses.
Marker % Positive No drug % Positive G8:3DNA % Positive 3DNA:Dox % Positive G8:3DNA:Dox G8 9 ± 2 (4) 9 ± 1 (4) 8 ± 4 (n = 8) ***0 (n = 7) α-SMA 9 ± 2 (4) 10 ± 1 (4) 7 ± 3 (n = 6) ***0 (n = 7) % Positive = (number of fluorescent cells ÷ total number of cells in 20 fields) × 100. The number of cultures scored is indicated in parentheses. Two doses of G8:3DNA:Dox completely eliminated G8+/α-SMA+ cells in long-term lens cultures.
↵*** P < 0.0001.
