It is desirable to have non-invasive imaging methods able not only to render the expression and distribution of receptors, enzymes and other targets relevant to
Methods
Dextran targeting the prostate-specific membrane antigen (PSMA) (Fig. 1b) was synthesized according to Fig. 2a. The binding affinity of urea-Dex10 to PSMA was measured using a standard, fluorescence-based assay2,3. For in vivo studies, both PSMA(+) PC3-PIP cells and PSMA(-) PC3-flu cells were inoculated in the opposite flanks of male SCID mice (n = 5). The in vitro CEST contrast of dextran was assessed using a vertical bore Bruker 11.7 T MRI scanner as described previously.4 In vivo MR studies were carried out on a Biospec11.7 T horizontal MRI scanner as described previously.5 CEST MR images were acquired before and within the first hour after the tail vein injection of dextran (100 mL, 375 mg/ml). For the dynamic study, CEST images were repetitively acquired at the offsets of ± 0.6, ± 0.8, ± 1.0, and ± 1.2 ppm using a modified fat-suppressed RARE sequence (CW saturation pulse, B1=1.8 µT and 3 seconds, TR/TE=5000/5 ms, RARE factor=10). To correct the B0 inhomogeneity, WASSR scans were acquired before and after the CEST acquisitions. The in vivo CEST contrast was quantified by averaging the MTRasym=(S-Δω – S+Δω)/S0 at 1.0 ppm. The change in CEST MRI was quantified by ∆MTRasym(t)= MTRasym (t)- MTRasym (t0).
PSMA-targeted CEST MRI probes were synthesized by conjugating 10 kD dextran (Dex10, size ~ 4-6 nm) with a low-molecular-weight urea-based ligand (MW ~ 400). A high binding affinity to PSMA was confirmed in vitro, i.e. Ki = 2 nM (IC50 = 10 nM). The CEST signal of urea-Dex10 was found to be almost identical to that of the non-conjugated Dex10 (Fig. 2), attributed to only a small portion (i.e., 2.5 mmol/mole) of hydroxyl groups was functionalized in the conjugate. The CEST MRI detectability (1% MTRasym) was estimated to be approximately 20 µM.
We then tested the urea-Dex10 in a well-validated prostate cancer xenograft model2. Our results showed that CEST MRI was able to detect the dynamic tumor uptake of urea-Dex10 (Figs. 3a, b). Initially (< 15 min), both tumors showed a substantial increase in CEST signal, i.e., 1.28 ± 0.16% and 1.23 ± 0.11% (∆MTRasym). Later (>15 min), CEST signal in PSMA(-) PC3-flu tumors decreased while that in PSMA(+) PC3-PIP tumors remained, indicating specific binding of urea-Dex10. At the final time point (60 min), the CEST enhancement (∆MTRasym) in PSMA(+) PC3-PIP tumors was approximately two times higher than that in PSMA(-) PC3-flu tumors, i.e., 1.33 ± 0.16% vs 0.53 ± 0.11%. A significant difference was found between the PSMA(+) and PSMA(-) tumors (P = 0.018, Student’s t-test, two-tailed and unpaired, n= 5, Fig. 3c), showing the ability to use urea-Dex10 to detect PSMA-expressing prostate tumors. Fluorescence microscopy (Fig. 3d) confirmed higher uptake of labeled urea-Dex10 in PSMA(+) PC3-PIP tumors than that in PSMA(-) PC3-flu tumors.
R21EB015609, R03EB021573, R01EB019934 and R01EB015032
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(5) Xu, X., et al. Magn. Reson. Med. 2015, 74, 1556-63.
(6) Chandran, S. S., et al. Cancer Biol. Ther. 2008, 7, 974-82.