Supplementary Data from An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging
Authors: Weber Judith , Bollepalli Laura , Belenguer Ana M. , Antonio Marco Di , De Mitri Nicola , Joseph James , Balasubramanian Shankar , Hunter Christopher A. , Bohndiek Sarah E. 
Published: April 2023

<p>Supporting Methods Supporting Figures 1-11 Supporting Tables 1-3 Supporting Figure 1. Synthesis route for the smart, targeted hydrogen peroxide probe. Reagents and conditions: (a) 4-Bromo-1-butyne, NaI, MeCN, reflux. (b) POCl3, DMF. (c) Aniline, EtOH. (d) NaOAc, EtOH, reflux. (e) Piperazine, Cs2CO3, MeCN. (f) 1-Azido-1-deoxy-Î²-D-glucopyranoside, CuSO4, Na-ascorbate, t-BuOH, H2O. (g) 4-Nitrophenylchloroformate, Pyridine, DCM. (h) 4, DIPEA, DMF. (i) 1-Azido-1-deoxy-Î²-D-glucopyranoside, Cu(CH3CN)4, TBTA, ACN. Supporting Figure 2. Calibration of the LC-MS/MS study. This study was performed in multiple reaction monitoring mode (MRM-MS). (A) Chemical structure of JW35mono which is used as internal standard (IS) for MRM-MS analysis. Standard calibration curves of JW41 and JW35 prepared in tumor or liver matrix were extracted for ex vivo quantification of JW35 and JW41total by MRM-MS. The signal ratio of the peak area of JW35 to the peak area of the IS versus the JW35 concentration (ï�­M) is plotted in (B); the peak area of JW41total versus its concentration (ï�­M) in (C). For each study a new calibration curve was acquired before and after running the samples. To validate the calibration line and the status of the analytical system, six quality control (QC) samples at three known concentrations were run before and after the biological samples. One representative calibration curve together with the QC samples are shown for JW35 and JW41total in (B) and (C) respectively. The colored symbols represent values obtained from the calibration samples, the crosses represent values obtained from the QC samples run before and after the biological samples, validating the calibration curve. The boronate groups of JW41 are partially hydrolyzed to give JW41hydro by the aqueous and acidic conditions required for the LC-MS/MS analysis. The linearity curves shown in (D) suggest that the loss of the concentration of JW41 is compensated by the gain in concentration of JW41hydro. Therefore, the peak area of the MRM transitions of JW41 and JW41hydro were added together to JW41total, which was used for the analysis. (E) Calculated total concentration of JW41total and JW35 24 h after injection of JW41 / JW41:JW35. The results of 4 tumors where the mice received an injection of JW41 alone and 3 tumors where mice received an injection of 2:1 JW41:JW35 solution are shown. Note these values cannot be interpreted as absolute due to the aforementioned hydrolysis challenges. Supporting Figure 3. LC-MS/MS chromatograms of JW41 (75 nM), JW35 (38 nM) and the internal standard IS (JW35Mono, 190 nM). Representative ion chromatogram of JW41, JW41hydro, JW35 and the IS are depicted in (A). Fragmentation of the parent compounds JW41 (m/z=1379.67), JW41hydro (m/z=1297.61), JW35 (m/z=560.30) and IS (m/z=914.48), are depicted in (B), (D), (F) and (H), respectively. Multiple reaction monitoring MRM-MS peaks for JW41 (monitoring m/z=1379.67 transition to m/z=913.48), JW41hydro (monitoring m/z=1297.60 transition to m/z=831.39), JW35 (monitoring m/z=560.30 transition to m/z=208.10) and IS (monitoring m/z=914.48 transition to m/z=161.11) are shown in (C), (E), (G) and (I), respectively. Supporting Figure 4. Linker evaluation. (A) Synthesis of the different dyes for linker evaluation studies (JW7/8/11/13/14/16/17). Reagents and conditions: (a) III, N-(4-hydroxyphenyl)acetamide, K2CO3, MeCN(anh). (b) tert-Butyl(4-hydroxyphenyl)carbamate, prepared from 4-aminophenol, di-tert-butyl-dicarbonate, THF(anh). (c) III, TEA, MeCN(anh) (d) JW11, TFA, DCM(anh). (e) III, piperazine, TEA, MeCN(anh). (f) JW8, imidazole, acetyl chloride, THF(anh). (g) JW8, di-tert-butyl dicarbonate, TEA, THF(anh). (h) JW8, TEA, trimethylacteyl chloride, THF(anh). To find an efficient linker unit able to elicit a shift in the optical properties of the dye upon reaction with H2O2 we explored benzene-1,4-diamine (B) and piperazine (C) as potential linker structures with different steric and electric nitrogen modifications. Representative, normalized absorbance spectra of the different dye products (solvent 15 Î¼M in MeOH) are shown. The results indicate that 4-aminophenol is not suitable as a linker for our purpose since no significant absorption shift is observed by derivatizing the nitrogen, whereas the piperazine-derivatives features promising characteristics with absorption spectra sensitive to sterically and electrical modifications on the nitrogen. Supporting Figure 5. Theoretical investigation of the effect of the different linker units towards the dye backbone. The minimum-energy conformations of JW13 (A), JW11 (B), JW35 (C) and JW41 (D) were obtained through geometry optimization in the gas phase using density functional theory. To quantify the relative orientation of the two terminal polycyclic moieties, a dihedral angle was defined as shown in the picture (defined with the main axis across the central aliphatic chain and terminating in the two distal benzene rings) and measured on the optimized structures. Supporting Figure 5. Theoretical investigation of the effect of the different linker units towards the dye backbone. The minimum-energy conformations of JW13 (A), JW11 (B), JW35 (C) and JW41 (D) were obtained through geometry optimization in the gas phase using density functional theory. To quantify the relative orientation of the two terminal polycyclic moieties, a dihedral angle was defined as shown in the picture (defined with the main axis across the central aliphatic chain and terminating in the two distal benzene rings) and measured on the optimized structures. Supporting Figure 7. TPA assay to investigate GLUT specific uptake. Pharmacological activation of glucose transport in (A) MDA-MB-231 and (B) MCF7 cells was induced with TPA (13 Î¼M) 1 h prior dye addition and led to increased uptake of JW41 and the positive control IR800CW-2DG but not of the negative control IR800CW. Statistical significance was assessed by unpaired 2 tailed t-test; *p<0.05, **p<0.01,****p<0.0001 Supporting Figure 8. In vivo characterization of 2:1 JW41:JW35 mixture in subcutaneous MDA-MB-231 tumors in nude mice. A) Representative PAI slice through MDA-MB-231 tumors (top row) and kidney, spleen and liver (bottom row) before and 15-20 min after injection of the 2:1 JW41-JW35 mixture. The regions of interest (ROIs) are indicated with white borders (LT = left tumor, RT = right tumor, L = liver, S = spleen, K = kidney, C = control region). The signal of JW41 is shown in yellow, the signal of JW35 in magenta. (B) Changes in the PA spectra and an increase in the PA signal between 700 and 810 nm were detected in the ROIs upon injection of the mixture. Representative graphs for tumor (top row, n = 4) and liver (bottom row, n = 2) are presented. (C) The time course of the total probe signal at the isosbestic point of the capped and uncapped probe up to 24 h after injection. (D) Fluorescence images before, 1 h and 24 h post injection of the JW41-JW35 mixture. Fluorescence in vivo imaging results support the PA findings indicating selective in vivo accumulation of the new probe in subcutaneous MDA-MB-231 tumors and the liver. (E) The specific increase in radiant efficiency in the left tumor and liver upon injection of the dye mixture (values from the control region, C, were used for background correction). Only the signal of the left tumor is considered since the position of the right tumor was hard accessible for the fluorescence imaging and the signal was mixed with the liver signal. (F) PA signal change at 765nm over time in the control regions confirming that the PA signal kinetic data (illustrated in C) are specific to the organs of interest. Supporting Figure 9. In vivo PA signal from control regions following injection of JW41 alone. ROIs illustrated in main text Figure 5. Supporting Figure 10. Time course of the in vivo PA signals of JW41 and JW35 derived using spectral unmixing. (A) JW41 PA signal time course following injection of JW41 alone. (B) JW41 PA signal time course following injection of the 2:1 mixture of JW41:JW35; (C) JW35 PA signal time course following injection of JW41 alone. (D) JW35 PA signal time course following injection of the 2:1 mixture of JW41:JW35. Background subtraction was performed in both cases by averaging the values obtained before injection (first 3 time points) and subtracting the average. Signals for the injection of JW41 alone are from ROIs drawn in main text Figure 5; signals for the injection of the mixture of JW41:JW35 are from ROIs drawn in Supplementary Figure 8. Supporting Figure 11. Ex vivo characterization of in vivo studies with JW35 alone and 2:1 JW41-JW35 mixture. (A) Representative H&E stained section of frozen MDA-MB-231 tumor from mouse treated with JW35 alone and (B) 2:1 JW41-JW35 mixture. The necrotic area is indicated with the white line. Representative widefield fluorescence image of a consecutive tumor section after formaldehyde fixation and staining with DAPI (blue) are shown to the right in both cases. Magnification of the widefield image allowing the localization of the dye signal into the cytosols of the cells. Supporting Table 1. Table of photophysical properties of JW41 and JW35. These were obtained from bulk UV-vis absorption and fluorescence measurements of the dyes. Supporting Table 2. Spectral unmixing studies of JW41 and JW35 in tissue-mimicking phantoms. Supporting Table 3. Concentrations of standard and QC samples for LC-MS/MS analysis.</p>