General materials and instrumentationUnless otherwise noted, all reagents and solvents were ACS grade, purchased from commercial suppliers, and used without further purification. Deionized water was used for all experiments requiring the use of water. PSVue794 Reagent Kit was purchased from Cedarlane Labs (25101–1) and was used to prepare PSVue794 (2) from the Apo-PSS precursor (1) and a solution of 2 mM Zinc nitrate, 30 min before use. Lipopolysaccharide from Escherichia coli O55:B5, ready-made solution (1 mg/mL), 0.2 µm filtered, was purchased from Sigma-Aldrich (Cat#L5418-2ML).Absorbance and fluorescence scans were collected on a Tecan Infinite M1000 plate reader using Corning 96-well, clear bottom plates (non-treated surface) (Corning 3631), PerkinElmer film was used to seal any plates containing bacteria cells. Microwave reactions were performed using a Biotage Initiator 60 microwave reactor under standard settings. Radio-TLC was performed using a Bioscan AR-2000 imaging scanner on iTLC-SG glass microfiber chromatography paper (SG10001, Agilent Technologies) plates and Aluminum oxide plates using 1% HCl in methanol as the eluent. For each TLC performed, plates were spotted with ~ 3 µL (481 kBq). A background (represented as a blue underline) was integrated on each radio-TLC plate and subtracted from the integration of each peak. Sep-Pak C18 Plus Light Cartridge (55–105 µm) (WAT023501, Waters) was used for isolating radiolabeled products. High-performance liquid chromatography (HPLC) of the radiolabeled compounds was performed on a Waters 1525 Binary (Midford, MA), monitored simultaneously with 2998 photodiode array detector at 245/700 nm and in line radioactivity Bioscan gamma detector with NaI (TI) scintillator using the Empower software package. Analytical HPLC of each compound was performed on a Gemini 5 µm C18 110 Å (250 × 4.6 mm) Column (00G-443-E0, Phenomenex), operating at a flow rate of 1 mL/min. A PerkinElmer Wizard 1470 automatic gamma counter was used to measure the amount of radioactivity in samples from the bacteria binding assay and the biodistribution studies.Pertechnetate [99mTc][TcO4]− was obtained in 0.9% w/v saline from a 99Mo/99mTc generator supplied by Lantheus Medical Imaging. Caution: these materials are radioactive and should only be used in a properly licensed and equipped facility. Potassium boranocarbonate was prepared according to a literature method30, as was [99mTc(CO)3(OH2)3]+16.HPLC methodSolvent A: Water + 0.1% Trifluoroacetic acid (TFA), Solvent B: Acetonitrile + 0.1% TFA. Gradient: 0–2 min 90% A, 2–6 min 90–40% A, 6–8 min 40% A, 8–10 min 40–10% A, 10–11 min 10–90% A, 11–15 min 90% A.Photoacoustic phantom imagingPA phantom work was performed using a Vevo Phantom imaging chamber and the Vevo-3100/LAZR-X (FUJIFILM VisualSonics, Inc., Toronto, ON, Canada) imaging system equipped with a 680–970 nm laser. Samples were injected into Vevo Contrast Agent Phantom tubing (PN52807) that was threaded into the Vevo Phantom chamber. A 30 MHz, linear array US transducer equipped with integrated fibre optic cables (MX400 and LZ-400 [15–30 mJ(cm3)−1, 20 Hz repetition rate, 10 ns pulse width], FUJIFILM VisualSonics, Inc., Toronto, ON, Canada) was positioned overtop of the tubes.Spectral analysis was performed using ‘Spectro’ scans, where the PA signal of a 2D, x,y-cross-section of a sample is quantified at each wavelength (690–970 nm, step size of 5 nm, ~ 1 min/spectro scan, axial resolution of 50 µm) and is used to plot the spectral curve or ‘spectral signature’ from a defined region of interest (ROI). From this, the signal intensity of the chromophore of interest can be quantified by the signal unmixing algorithm utilized in the VevoLAB software (VisualSonics, Inc., Toronto, ON, Canada). This scan was also be used to demonstrate the merged spectra of multiple chromophores mixed. The 2D multispectral unmixing analysis was then performed on the spectrum, where the pre-programmed PA spectra of oxygenated and deoxygenated hemoglobin were unmixed from the spectra of 2, and were taken for each material in triplicate. Signals generated at 680, 692, 802, 924, and 942 nm were utilized in the signal unmixing algorithm. Images from the multiwavelength unmixing scans have been displayed using the render mode with the PA signal overlaid on US B-mode (greyscale) signal of a cross-section of tubing. All image processing was carried out using the VevoLAB software. A ROI was drawn around the US image of the tube and, applying ROIs of the same size and shape to each image, a spectrum of the PA average signal was generated from the overlaid PA data of each image.PA signal analysis of 2Samples of 2 at 20 µM in Diluent X and 2 at 1 mM in Diluent X diluted to 64 µM in whole mouse blood (collected from cardiac puncture using EDTA coated syringes and immediately supplemented with 10% EDTA in 0.9% w/v saline [0.15 g/mL]) were prepared alongside appropriate blank controls. The samples were injected into Vevo contrast agent phantom tubing and Spectro scans were taken of the material in triplicate. The data was analyzed as described in ‘Photoacoustic Phantom Imaging’.Preparation of bacterial overnight (O/N) cultureStaphylococcus aureus cells (ATCC; 25,923) were incubated in tryptic soy broth (TSB) liquid media at 37 °C (450 rpm) for 18 h. The cells were centrifuged (2 min at 10 000 xg) and the pellet was resuspended in Hank’s Balanced Salt Solution (HBSS) to an OD600 = 19.9 for in vitro assay, or to an OD600 = 8.43 for in vivo studies.In vitro bacteria binding of 2 using fluorescence and photoacoustic detection2 (10 µM final concentration) was incubated with 50 µL of S. aureus suspension (1 × 109 CFUs in 50 µL HBSS) or 50 µL of HBSS (no-cell control), for 10 min in triplicate. The samples were washed 3 × and the pellet was resuspended in 250 µL of HBSS. 200 µL sample and 200 µL stock solution were transferred to a black flat clear bottom 96-well plate. A fluorescence scan (Ex = 736 nm; Em = 746–850 nm) was performed. The fluorescence intensities (at 829 nm) of blank samples were subtracted from samples of 2 incubated with or without bacteria and the change in intensity was evaluated. Approximately 20 µL of each sample was then injected into Vevo contrast agent phantom tubing. The ends of each tube were sealed with heat and Spectro scans were acquired of each sample in triplicate. The data was analyzed as described in the ‘Photoacoustic Phantom Imaging’ section.Radiosynthesis of [99mTc]Tc-PSVue794 (6)400 µL of Apo-PSS794 (1) (0.5 mg in 400 µL of methanol [0.87 mM]), was combined with 400 µL of [99mTc(CO)3(OH2)3]+ (4) (Prepared using a procedure reported by Causey, et al.16) (370 MBq, dissolved in 0.9% w/v saline, pH = 6–6.5), and mixed at room temperature for 1 h. Radio-TLC (1% HCl in methanol eluant) and HPLC (method A, UV = 700 nm tr = 8 min; \(\gamma\) trace tr = 8.8 min) were used to determine the radiochemical conversion (~ 85–90%). The material was then diluted in 10 mL of water and loaded onto a C18 cartridge for purification. Water was used to elute any remaining 4 from the cartridge and ethanol was used to elute 5 in 99% purity based on Radio-TLC. The ethanol was evaporated, and the material was reconstituted in 171.5 µL mL of Diluent X. 171.5 µL of Zn(NO3)2 (0.015 M in Diluent X) was added to 5 and stirred at 37 °C for 30 min. The material was then adjusted to the correct concentration using 10% ethanol in 0.9% w/v saline for subsequent experiments. HPLC UV = 700 nm tr = 8 min; \(\gamma\) trace tr = 8.8 min.Stability testing of 6The stability of compound 6, incubated in formulation buffer (Diluent X) (room temperature) and in mouse serum (37 °C), was assessed at 0, 2 and 18 h. At each time point an aliquot of the material was analyzed by radio-TLC (eluting in 1% HCl in methanol). For serum samples, the aliquot was mixed with cold acetonitrile and pelleted by centrifugation to remove the serum proteins.In Vitro Bacteria Binding of 6In a clear, conical bottom 96-well plate, 200 µL of 6 (37 kBq) in 10% ethanol in 0.9% w/v saline was incubated with S. aureus suspension (1 × 109 CFUs in 50 µL of HBSS) or 50 µL of HBSS (no-cell control), for 10 min (n = 3 per condition). The sealed plate was centrifuged for 1 min at max speed. The supernatants were removed and 200 µL of HBSS was added to resuspend the pellet. This was repeated a total of 3 times to remove the unbound fraction of 6. The washed pellet was resuspended in 200 µL of HBSS, 150 µL was transferred into gamma counting tubes, and the amount of radioactivity in each tube was determined using a gamma counter. A 100 µL aliquot of 6 (from the 0.37 MBq stock) was measured and corrected to represent a 100% signal control for each concentration. The counts per minute (CPM) from each sample were corrected based on the dispensed volume measured and specific binding was calculated by subtracting the non-specific binding (CPM from the samples without bacteria cells) from the total binding (CPM from the samples with bacteria cells). The percent binding of each sample was calculated using Equation SI-2.Animal studiesAll procedures were conducted according to the guidelines of the Committee for Research and Ethics Issues of the International Association for the Study of Pain, and guidelines established by the Canadian Council on Animal Care and the McMaster University Animal Research Ethics Board. All experimental protocols were approved by the McMaster University Animal Research Ethics Board. All experimental protocols and results are reported in accordance with ARRIVE guidelines. In each animal study 3–4 female BALB/c mice (7–10 weeks old, Charles River Laboratories, Raleigh, NC) from the same litter, weighing around 20 g, were used. The mice were sterile housed and maintained at 21 °C with a 12-h light/dark cycle and were provided autoclaved food and water ad libitum.
Staphylococcus aureus-induced myositis modelAn aliquot of S. aureus (1 × 108 CFUs in 50 µL of HBSS) was innoculated intramuscularly (I.M.) on the right hindlimb of mice (n = 4). 50 µL of the vehicle control, HBSS, was injected IM into the contralateral hindlimb of the same mice. The mice were monitored for 18 h post-inoculation of the bacteria, tissues were then collected and analyzed as described in the section titled ‘Histological Analysis’. Confounders were not controlled in the study design. No data points were excluded from the experimental analysis.Biodistribution of 6Four healthy control mice and 4 mice with established S. aureus-induced myositis were injected in the lateral tail vein with 200 µL (0.74 MBq) of 6 formulated in 10% ethanol in 0.9% w/v saline. 12 h post-injection, animals were anesthetized and euthanized by cardiac puncture followed by cervical dislocation. Fluids, bone (knee), and select tissues were collected, weighed, and quantified on a gamma counter. Decay correction was used to normalize organ activity measurements to the time of dose preparation for data calculations. Data are expressed as percent injected dose per gram of tissue/fluid (%ID/g). Confounders were not controlled in the study design. No data points were excluded from the experimental analysis.Lipopolysaccharide-induced myositis modelLipopolysaccharide (LPS) (1 mg/mL, 100 µL/mouse) was injected intramuscularly (IM) on the right hindlimb of mice (n = 4). 50 µL of the vehicle control, HBSS, was injected IM into the contralateral hindlimb of the same mice. The mice were monitored for 15 h post-injection of LPS, tissues were then collected and analyzed as described in the section titled ‘Histological Analysis’. Confounders were not controlled in the study design. No data points were excluded from the experimental analysis.Histological analysisMice were euthanized with sodium pentobarbitol and perfused with lactate Ringer’s solution and tissues were fixed with 10% formalin. The right and left hind limb, and the spleen were collected and decalcified in a solution of 10% formalin + 8% EDTA. The trimmed tissues were embedded into paraffin, sliced, and stained with hematoxylin–eosin (H&E). A veterinary research pathologist, blinded to the treatments, examined the slides under a Nikon Eclipse 50i light microscope and morphological features of the tissues were described. One slice of each tissue (the treated hindlimb, the contralateral hindlimb, and the spleen) from each mouse in the LPS- and bacteria-induced myositis groups and 1 naïve mouse were analyzed and contributed to the main diagnoses of each condition. The images selected for the figure are representative for each condition and taken from one mouse per group.The bone marrow was present within the slices of the hindlimbs due to the decalcification of bone. The microphotographs were only provided for the tissues where pathological morphology was observed. This is true for the spleen as well. While the spleen tissue was analyzed from each mouse within each group, the microphotographs were only provided from tissues that displayed morphological changes relative to healthy tissue.In vivo photoacoustic imaging studiesPAI was performed using the Vevo-3100/LAZR-X imaging system. Mice were anesthetized, and the fur of their right hindlimb was thoroughly removed before being placed in a prone position on a platform that enabled the monitoring of their respiration and heart rate. A generous amount of US gel was applied before imaging with the transducer (MX400/LX400) positioned over the leg. At each imaging time point, Spectro scans and 3D multispectral unmixing scans were taken of each hindlimb. The transducer was positioned laterally overtop of the hindlimb, corresponding with the inoculation site. Scan distances averaged 20 mm and scans were taken every 0.33 mm (~ 7 min/3D scan).3D multiwavelength unmixing scans were performed, where the pre-programmed PA spectra of oxygenated and deoxygenated hemoglobin are unmixed from the spectra of 2, across a defined z,x axis to construct images depicting dye-specific PA signal present within the hindlimb. Designated wavelengths (680, 692, 802, 924, and 942 nm), based on notable signals generated within individual chromophore spectra (oxygenated and deoxygenated hemoglobin, or 2), were used by the Vevo-3100/LAZR-X unmixing algorithm. The series of 2D cross-sectional scans collected were then reconstructed by the instrument into a 3D image, where the signals from each chromophore are assigned a colour and overlaid onto a b-mode ultrasound scan. The scans are displayed as either render mode or texture mapping images where the PA signal is overlaid on US B-mode (greyscale) signal for sections of the hindlimb. Signals from oxygenated and deoxygenated hemoglobin were subtracted from the images to reduce visual complexity. The spectrally unmixed signal of 2, in the 3D multiwavelength unmixing scans, was quantified using 3D ROI drawn around the inoculation sites. All signal unmixing algorithms and image processing were carried out using the VevoLAB software. Image processing consisted of signal optimization, with parameters set the same for each image within the study. 3D regions of interest were drawn around the inoculation site.Spectral analysis was performed using ‘Spectro’ scans (as described in the “Photoacoustic Phantom Imaging’ section) and was used to verify whether the spectral signature of the in vivo signal matched that of the exogenous dye. The ROI used for each spectral curve was displayed in a multiwavelength unmixing scan (oxygenated and deoxygenated hemoglobin unmixed from 3) derived from the Spectro scan.Photoacoustic imaging of LPS-induced sterile inflammation using 2Three hours after treating mice with LPS (n = 3), as described previously, the mice were injected intravenously with 75 µL of 2 (1 mM) and were imaged, as described in the section titled ‘In vivo photoacoustic imaging studies’, on the Vevo LAZR X, pre-injection, and 12 h post-injection. Confounders were not controlled in the study design. No data points were excluded from the experimental analysis.Photoacoustic imaging of S. aureus-induced bacterial infection using 2Six hours after inoculating mice (n = 3), as described previously, the mice were injected intravenously with 75 µL of 2 (1 mM) and were imaged, as described in the section titled ‘In vivo Photoacoustic Imaging Studies’, on the Vevo LAZR X, pre-injection, and 12 h post-injection. Confounders were not controlled in the study design. No data points were excluded from the experimental analysis.Statistical analysesData are reported as mean ± standard error of the mean (SEM). Concentration and bacteria-binding data were analyzed by one-way analysis of variance (ANOVA). Differences between test groups and control were assessed using Dunnett’s Multiple Comparison Test on GraphPad Prism version 9 (GraphPad Software, San Diego, CA). In vivo PAI data were analyzed using a two-way ANOVA. Differences between tested groups and control were assessed using the Tukey Test on GraphPad Prism (Threshold *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).
Photoacoustic imaging of a cyanine dye targeting bacterial infection
