Fig 1.
PSMA expression in the three cell lines (LNCaP, C4-2 and MKN45 cells) detected by Western blot.
LNCaP cells expressed a higher level of PSMA than C4-2 cells, whereas MKN45 cells exhibited no expression.
Fig 2.
Evaluation of binding affinity of biotinylated nanobody with PSMA using the competitive ELISA.
The curve with non-linear regression about the concentration of recombint PSMA ranging from 0.1 nM to 100 μM are obbtained.
Fig 3.
Particle size distributions and in vitro imaging of the Blank NBs and Targeted NBs.
A Malvern Zetasizer analyzer was used to examine the particle sizes. Blank NBs (without biotinylated nanobody) had an average diameter of 445.30 ± 32.96 nm (A), whereas Targeted NBs (with biotinylated nanobody) had an average diameter of 487.6 ± 33.55 nm (B). And there is no difference in their in vitro imaging at the same concentrations (C).
Fig 4.
Immunofluorescence validation of the Targeted NBs.
Microscopic observation of the Targeted NBs (A and B). The ring-like structures with thick membranes are NBs (B). The results demonstrated that Targeted NBs could specifically incorporate nanobody via biotin-avidin interactions.
Fig 5.
Binding of 3 types of cells to Blank NBs or Targeted NBs.
Under a light microscope, both LNCaP cells and C4-2 cells visibly bound to Targeted NBs (A and B), whereas MKN45 cells did not bind to Targeted NBs (C). None of the 3 types of cells displayed prominent binding to Blank NBs (D, E and F). Scale, 5 μm.
Fig 6.
Imaging results of the Targeted NBs in tumor-bearing nude mice at the peak nanobubble level.
Panels B, E and G show the classical cross-sections of 3 types of xenografts under B-mode ultrasonography. Panels A, D and H show the binding of Blank NBs to the xenografts at the peak intensity. Panels C, F and I show the binding of Targeted NBs to the xenografts at the peak intensity. The ultrasonographic images in both the LNCaP and C4-2 xenografts revealed that the imaging intensity was apparently higher than the imaging intensity achieved with the Blank NBs at the peak nanobubble level. In the MKN45 xenografts, the imaging results of the Targeted NBs and Blank NBs were comparable at the peak nanobubble level. Of all, blue areas represent the exnografts, while red and yellow areas respectively represent the different imaging areas in LNCaP and C4-2 exnografts.
Table 1.
Comparative ultrasonography indicator analysis of the Targeted NBs and Blank NBs in 3 types of xenografts.