Supplementary Data from Evaluation of Novel Prostate-Specific Membrane Antigen-Targeted Near-Infrared Imaging Agent for Fluorescence-Guided Surgery of Prostate Cancer
Authors: Kularatne Sumith A. , Thomas Mini , Myers Carrie H. , Gagare Pravin , Kanduluru Ananda K. , Crian Christa J. , Cichocki Brandy N. 
Published: April 2023

<p>supplementary Figures & Legends, Tables, Materials & Methods, synthetic procedures, quantities and yield, and additional in vitro & in vivo experimental procedures SI Figure 1: (a) Chemical structures of S0456 and DUPA-FITC. (b) Excitation (Ex) & emission (Em) spectra of OTL78 (1 μM) andS0456 (1 μM) in 1 mL of PBS obtained using fluorometer. (c) Evaluation of PSMA expression levels in LNCaP, 22Rv1, PC3, and A549 using flow cytometry. (d) Dose dependent binding of DUPA-FITC and (e) competitive binding of OTL78 with respect to DUPA-FITC to 22Rv1 and PC3 cells in culture. Error bars represent SD (n = 2). (f)Binding and internalization of OTL78 to (i) 22Rv1 and (ii) LNCaP at 4 ºC by epifluorescence microscopy. Nuclear is stained with DAPI (a blue dye). SI Figure 2: NMRs of OTL78. (a) 1H - NMR and (b) 13C - NMR of OTL78 using Brooker 500 and 125 MHz spectrometer. SI Figure 3: Mass spectra of OTL78. (a) HPLC chromatogram using Waters UPLC system and (b) MS using UPLC coupled with MS. SI Figure 4. Tissue biodistribution analysis of OTL78: (a) IVIS images showing overlay of fluorescence images over white light images of selected tissues and (b) tumor-to-tissue ratio from tissue biodistribution data from mice bearing 22Rv1 tumor xenografts after administering increasing doses of OTL78. Error bars represents SD (n= 5). SI Figure 5: In vivo efficacy of OTL78. Tissue biodistribution analysis using fluorescence imaging of the mice with (a) PC3 and (b) A549, and (h) 22Rv1 orthotopic tumors at 2h post- injection. Representative fluorescence images from AMI imager showing mice bearing (c) 22Rv1 orthotopic (n=5 mice/group) and (d) tissue biodistribution analysis using fluorescence imaging of the same mice 2h after administering 10 nmol of OTL78. SI Figure 6. Quantitation of TBR of OTL78 using region of interest (ROI) and ImageJ analysis. (a) TBR calculated using ROI values obtained from IVIS or AMI imager after tissue biodistribution studies of 22Rv1 subcutaneous or orthotopic tumors bearing mice injected with 10 nmol of OTL78. Note: Since the primary tumor is in the prostate, tumor-to-prostate ratio is equal to one in orthotopic model. Error bars represents SD (n=5 mice/group). (b) Representative fluorescence image (in gray scale) of mouse bearing 22Rv1 subcutaneous tumor after injecting 10 nmol of OTL78. The plot of gray value versus distance (c) across the line and (d) within the box are shown in the Fig 4b. SI Figure 7. Comparison of surgeries performed under conventional and fluorescence-guided techniques. (a) Representative fluorescence images of mice before and after surgically removing 22Rv1 tumor xenografts by conventional (n=5 mice/group) or fluorescence-guided (n=5 mice/group) techniques till day 21. Mice were administered with OTL78 (10 nmol/mouse) 2 h before imaging using AMI image system. The cohort underwent on fluorescence-guided surgery were monitored over a 30 days. SI Figure 8: Time dependent tumor retention of OTL78. IVIS images showing time dependent whole-body fluorescence images over white light images of mice bearing 22Rv1 tumor after injecting 10 nmol of OTL78 and image at different time intervals (n=5 mice/group). SI Figure 9: Human serum binding studies of OTL78. Serum binding is defined as the ratio of area under the OTL78 peak in serum (sample): area under the OTL78 peak in PBS (negative control). n=3. SI Figure 10: Safety of OTL78. Histopathological analysis mice treated with OTL78 (10 µmol/mouse). a: Cerebellum, b: Cerebrum, c: Heart, d: Kidney, e: Large intestine, f: Liver, g: Lung, h: Skin, i: Muscle, j: Spleen, k: Stomach, l: Small intestine (n=5 mice/group). SI Table 1: Percent of basophile activation in healthy subjects SI Scheme 1: Reagents and conditions for synthesis of OTL78: (a) (i) 2: H2N-Tyr(OtBu)-OtBu.HCl, HATU, DIPEA, DMSO, RT, 2h; (b) H2/ Pd-C (10%), DCM/MeOH; (c) 5: Cbz-PEG2-CO2H, HATU, DIPEA, DMSO, RT, 2h; (d) H2/ Pd-C, DCM/MeOH; (e) 7, HATU, DIPEA, DMSO, RT, 2h; (f) TFA, RT, 1h; (g) (i) H2O/ NaOH (pH = 10), (ii) 11: S0456, H2O, 75 {degree sign}C, 3-4h.</p>