Table 1.
Changes of selection conditions in cell-SELEX process.
Figure 1.
Generation of aptamers by whole cell -SELEX process.
(A) DsDNA amplified by PCR during selection process was identified by 10% native polyacrylamide gel electrophoresis. Lane 1: pUC18DNA/Mspl/Marker. Lane 2, 3: dsDNA. (B) 7 M urea 8% denatured polyacrylamide gel electrophoresis was used to confirm obtained ssDNA at the end of each round. Lane 1: pUC18DNA/Mspl/Marker. Lane2: initial DNA pool GN. Lane 3: ssDNA. (C) Enrichment of selected pools was monitored by flow cytometry. FITC-labeled GN (red), the 3rd round ssDNA pools (black), the 5th round ssDNA pools (blue), the 11th round ssDNA pools (yellow) were incubated with U87-EGFRvIII cells respectively, then fluorescence intensity was detected. Unlabeled U87-EGFRvIII cells (green) were used as blank control. (D) Sequence homology analysis by MEME online software.
Table 2.
Kd value of the selected aptamers.
Figure 2.
Good specificity and high affinity of selected aptamers to U87-EGFRvIII cells.
(A) FITC-labeled aptamers U2 (red), U8 (black), U19 (green), U31 (yellow), GN (pink) were incubated with U87-EGFRvIII cells (right) and U87MG cells (left), respectively, then subjected to flow cytomety. Unlabeled cells (blue) were used as blank control. (B) Changes in cell number with detectable fluorescence reflected fluorescence intensity shift. Data represent mean ± SD of three independent experiments. * P<0.05, ** P<0.01. (C) Binding curve of U2, U8, U19 and U31 on U87-EGFRvIII cells. The mean absorbance from three independent experiments for each aptamer concentration was used to plot the binding curve.
Figure 3.
Aptamers U2 and U8 target EGFRvIII specifically.
(A) As protease K digestion time increasing from 3 min (black) to 10 min (blue), transition of the fluorescence intensity shift to the left illustrated a signal intensity decrease. The binding of FITC-labeled aptamers to intact U87-EGFRvIII cells (0 min) was used as positive control (red). (B) Changes in cell number with fluorescence as digestion time increasing. Data represent mean ± SD of three independent experiments. * P<0.05, ** P<0.01. #: There was no statistical difference at different time points in U31 group. (C) The specific interaction of U2 and U8 with EGFRvIII was determined by affinity purification on streptavidin beads of cell lysate treated with biotin-labeled aptamers followed by immunoblotting with anti-EGFR antibody. EGFRvIII has a molecular weight of 145 kDa compared with that of 170 kDa for EGFRwt. Obvious bands at 145 kDa indicated the spicific target of aptamers was EGFRvIII. Lanes (left to right): 1, GN with U87MG cell lysate; 2, GN with U87-EGFRvIII cell lysate; 3, U2 with U87-EGFRvIII cell lysate; 4, U8 with U87-EGFRvIII cell lysate; 5, U2 with U87MG cell lysate; 6, U8 with U87MG cell lysate. Three independent experiments were performed.
Figure 4.
188Re-labeled U2 imaged exnografts glioblastoma in mice successfully.
(A) Imaging of mice by SPECT 1 h after tail vein injection of free 188Re (left), 188Re-labeled GN (middle) and 188Re-labeled U2 (right). (B) Relative uptake values of the organs were counted and quantified using VG Acq data acquisition and processing system from SPECT. In (B), chart showed comparison of uptake values among different groups at 1 h after tail vein injection. (C) Up: Placement of real ex vivo organs. Down: Corresponding imaging of ex vivo organs by SPECT 3 h after tail vein injection of free 188Re (left), 188Re-labeled GN (middle) and188Re-labeled U2 (right). (D) Comparison of uptake values in ex vivo organs at 3 h after tail vein injection. (E–F) Imaging of mice 0.5 h (E) and 3 h (F) after intratumor injection of free 188Re (left), 188Re-labeled GN (right). (G) Changes of uptake values at 0.5 h and 3 h after intratumor injection. Asterisk indicated brain; pound = liver; triangle = bladder; arrow = tumor. Error bars depict means ± SD (n = 3). ** P<0.01.