Multimodal soft tissue markers for bridging high-resolution diagnostic imaging with therapeutic intervention

The liquid-based Carbo-gel technology enables real-time multimodal image guidance for improved therapeutic precision.

In the current synthesis, the NIR dye Cyanine 7.5 is chemically linked to the hydrophobic carbohydrate ester sucrose septaisobutyrate yielding the product Cy7.5-SSIB.

General experimental conditions:
All reactions were carried out under inert atmosphere (N2). Water sensitive liquids and solutions were transferred via syringe. Water used for washing of the isolated products was in all cases MilliQ water. Organic solutions were concentrated by rotary evaporation at 30-60°C at 200-0 mbar. Thin layer chromatography (TLC) was carried out using aluminum sheets pre-coated with silica 60F (Merck 5554). The TLC plates were inspected under UV light or developed using a cerium ammonium sulphate solution (1% cerium (IV) sulphate and 2.5% hexa-ammonium molybdate in a 10% sulfuric acid solution).
Reagents: Cyanine 7.5 NHS ester was purchased from Lumiprobe (Lumiprobe, Hannover, Germany), and dry solvents were purchased from Acros Organics (AcroSeal, extra dry over molecular sieves) (Thermo Fisher Scientific, Geel, Belgium). All other chemicals were purchased from Sigma Aldrich and were used as received.

Instrumentation: Nuclear Magnetic Resonance (NMR) of intermediates was acquired on a Bruker
Ascend (Bruker, Billerica, MA., USA) 400 MHz -operating at 401.3 MHz for 1 H and 100.62 MHz for 13 C -with a 5 mm H -Broadband Dual Channel z-gradient Prodigy cryoprobe at 298 K, using the residual non-deutorated solvent residue in the NMR solvents as internal standard. NMR of the final product was acquired with an 800 MHz Bruker Avance IIIHD spectrometer equipped with a TCI cryoprobe (Bruker, Billerica, MA., USA) in order to obtain optimal spectral resolution. All coupling constants (J) are expressed in Hz. The FID files were processed in Mnova Suite. MALDI-TOF MS was acquired on a Bruker Autoflex Speed mass spectrometer. The matrix used for MALDI-TOF was a mixture of 2,5 dihydroxy benzoic acid (DHB) spiked with sodium trifluoroacetate in ethanol (60 mg/mL). UPLC was conducted on a Waters Acquity Ultra performance LC system with Binary solvent manager and TUV detector. Preparative HPLC was conducted on a Waters 600 pump and controller with a Waters 2489 UV/Vis detector (Waters, Milford, MA., USA).

6'-OH-isobutyric sucrose (Sucrose septaisobutyrate) (3)
6'-TBDPS-isobutyric sucrose (2) (14.8 g, 13.8 mmol) was dissolved in dry THF (80mL). Acetic acid (12 mL, 0.21 mol) was carefully added dropwise. The reaction mixture was then cooled down, and 1.0M TBAF solution in THF (83 mL, 83 mmol) was added over 10-15 minutes through a syringe. The reaction was allowed to heat to room temperature over 30 minutes, hereafter it was warmed to 40 0 C and stirred at this temperature overnight. Then, TLC (Hexane:Ethyl acetate 3:1) showed completion of the reaction (rf product: 0.2). The reaction mixture was cooled to room temperature and first hexane (300 mL) then demineralized water (300 mL) was added. The mixture was stirred for 10 minutes, and hereafter poured into a separatory funnel. The organic phase was collected and the water phase was extracted with hexane (2 × 300 mL). The combined organic phases were washed with HCl (aq) (500 mL, pH= 2) and subsequently with phosphate buffer (3 × 300 mL, pH = 6.8). The organic phase was concentrated on celite and then purified by dry column purification (EtOAc in hexane with 2-4 % increments) to give the product.

Cryo-SEM imaging of X-mark and XPV-mark
Cryo SEM of hydrated X-mark and XPV-mark formulations revealed an internal pore structure in each gel with pores of different sizes. Pore size decreased in diameter the further from the surface of the gel the pore was located. Larger pores were often surrounded by much smaller pores in both formulations. The XPV-mark formulation exhibited pores that were consistently larger than those observed in the X-mark formulation as shown in Supplementary figure S2 where the XPV-mark formulation had pores up to ~250 µm in diameter whereas the X-mark formulation only had pores with a diameter up to ~125 µm.
Materials and methods: X-mark and XPV-mark formulations were injected into buffer in a 12 well plate and aged for 1 week at 37 ºC to ensure adequate ethanol diffusion out of the gel. Gel samples were then adhered to SEM stubs with a 50:50 mixture of colloidal graphite powder (agar scientific) and Tissue-Tek OCT compound (Ted Pella) and plunge frozen in liquid nitrogen. Frozen samples were then loaded onto a Leica EM VCT 100 Cryo Transfer Shuttle and transferred to a Leica EM MED020 freeze fracture and high vacuum coating system. Samples were then fractured, sublimated for 1 minute at -90ºC and sputter coated with 6 nm of carbon/platinum. After coating, the samples were transferred via the VCT 100 Cryo Transfer Shuttle under vacuum and at -140ºC to the Thermo Scientific Quanta 3D FEG FIB/SEM for subsequent SEM imaging. Imaging was performed at high vacuum at -140ºC with an accelerating voltage of 2 kV.  0, 2.5, 5.0, 10, 20, 30, 40 and 50 %w/w xSAIB were CT imaged (C) and the corresponding CT contrast was quantified and presented as function of the xSAIB content (D).

X-ray Visibility of XPV-mark -CT contrast dependency on xSAIB content
Liquid marker formulations with 0, 2.5, 5.0, 10, 20, 30 ,40 or 50w/w% xSAIB in LOIB and fixed amount of EtOH (18.0 w/w%) and DC2 (0.10 w/w%) were prepared using the general procedure. The liquid marker formulations (200 µL) were added to separate wells in a 96 well microtiter plate. Evaluation of radiopacity of the liquid marker formulations as a function of xSAIB concentration was performed on a Siemens SOMATOM Definition AS + CT scanner (Siemens Health Care, Erlangen, Germany). The CT settings were a tube voltage of 120 kVp, 300mAs, 200mm FOV and extended CT-scale. Slices were reconstructed in a coronal plane with a B30s reconstruction kernel. HU contrast was quantified using Eclipse v 13.7 software package. The CT contrast was shown to depend linearly on the xSAIB content up to 40% w/w for XPV-mark formulations, where after the CT contrast increased further non-linearly. The CT contrast followed the regression line:
Analytical HPLC analysis was conducted in order to detect the formation of TIPA caused by chemical hydrolysis of xSAIB in the X-mark markers in the degradation buffer medium as illustrated in Fig. S3. TIPA was chosen as the preferred analyte to monitor over time due to its UVabsorbance at 256nm. 200 µL aliquots from the aqueous buffer phase above all markers were removed at designated intervals after 10, 20 and 30 days and replaced with fresh buffer to mimic the sink effect in vivo. After 30 days, aliquots and markers dissolved in MeCN were analyzed by analytical HPLC to monitor the pH dependent degradation of the markers based on the AUC from 2-(2,4,6triiodophenoxy)acetic acid (TIPA) formed by hydrolysis of xSAIB. Fig. S3. X-mark components after complete efflux of EtOH and degradation (% of xSAIB) of X-mark. SAIB and xSAIB and the primary degradation products formed by chemical hydrolysis of the multiply ester bonds of SAIB and xSAIB (A). Markers in buffer at pH 4.00-8.00 inside glass vials at Day 0, ~Day 30, ~Day 60 and ~Day 90 following incubation at 37°C. Reported values represents mean ± SEM, n = 3 (B).
Analytical HPLC analysis was conducted using a Shimadzu LC-2010 analytical HPLC (Shimadzu Corp., Kyoto, Japan) by employing a Waters XTerra ® C8 5 µm (4.6 x 150 mm) column (Waters Corporation, MA, USA). Analysis was conducted using a linear gradient from 0-100% Eluent B over 15min followed by 100% B for 5 minutes (Eluent A: 5% MeCN in MQ-H2O + 0.1% TFA; Eluent B: MeCN + 0.1% TFA). Flow rate: 1.00mL/minutes, injection volume: 25µL and UVdetection at 220 nm and 256 nm. Minimal X-mark marker degradation (< 0.15%) was observed after incubation of markers at pH 4.00, 5.00, 6.00, 7.00 and 8.00 at 37°C for >90 days as monitored by the amount of TIPA released from the markers. The pH of the degradation medium had little effect on the hydrolysis rate of the markers and was found to affect the partition of TIPA between the hydrophobic markers and the hydrophilic buffer phase.

Serum cytokine levels and tolerability of X-mark in mice over a 98-day study period
Please refer to main text materials and methods section for study details. Blood sampling was performed by facial vein puncture and direct collection of blood into Eppendorf tubes. The blood samples are allowed to reach room temperature. The blood samples are subsequently centrifuged (10 minutes, 4°C, 1500 rpm.) and the serum collected and stored at -80°C until further processing.  EtOH (50:30:20) or SAIB:EtOH (80:20) and again 3d, 8d, 26d, 53d, 76d and 98d from both groups for assessment of cytokine response. Serum levels of IFN-g (B), TNF-a (C) and interleukin 6 (IL-6) (D) were analyzed using a bead-based sandwich immunoassay and a commercially available Mouse Cytokine/Chemokine Magnetic Bead Panel kit (Cat#MCYTOMAG-70K-03) on the Luminex LX100 (Millipore Corporation, Burlington, MA., USA). (E) EtOH efflux from X-mark (SAIB:xSAIB:EtOH (50:30:20)) injections of 50 µl and 200 µl, respectively, evaluated by reduction in segmented volume of the injected markers on CT scans performed at multiple time points (0,30,60,90, 120 minutes and 3, 6 and 24 hours ) up to 24 hours after injection. Comparison by t-test demonstrated a significant difference in mean time to complete ethanol efflux between subcutaneous 50 µL markers (165 ± 45 minutes) and 200 µL markers (330 ± 133 minutes) (p=0.009). (F) EtOH efflux from 50 µL X-mark (SAIB:xSAIB:EtOH,50:30:20) in adipose tissue and sub cutaneous space, respectively, evaluated by reduction in segmented volume of the injected markers on CT scan performed at multiple time points (0, 30, 60, 90, 120 minutes and 3, 6 and 24 hours) up to 24 hours after injection. Comparison by t-test demonstrated no significant difference in mean time to complete ethanol efflux between subcutaneous markers (165 ± 45 minutes) and markers in adipose tissue (138 ± 45 minutes) (p = 0.675). (G) Representative slice of 50 µL Xmark (SAIB:xSAIB:EtOH,50:30:20) in adipose tissue (arrow) and sub cutaneous space (arrow head) of rat on 24 hours pi. CT-scan. The black line surrounding the markers, highlighted in yellow, is the marker contouring delineation used for image analysis.
EtOH solvent efflux of X-mark SAIB:xSAIB:EtOH (50:30:20) ethanol efflux kinetics as function of injection volume and injected tissue composition during the initial 24 hours after injection.

Fig. S5. Evaluation of injection practicability and short-term performance of X-mark in pigs.
Practicability and imaging characteristics were investigated by injection of SAIB:xSAIB:EtOH (50:30:20) into liver using EUS (22G. endoscopic injection needle), into lung by percutaneous fluoroscopy guided injection (22G. 63 mm needle) and thymus using EBUS (22G. endoscopic injection needle). Imaging performance was evaluated by CT imaging (top row) and fluoroscopy (middle row) and the marker in thymus was imaged post mortem in one pig imaged using MRI and CT (bottom row, MRI T1 and T2 weighted images and CT). For the CT-evaluation all measurements of HU and contouring were performed in Matlab 2013a or Osirix. A maximum intensity projection (MIP) was used for three directions to select the liquid marker for evaluation. Three-dimensional ROIs of the liquid marker was then used for evaluation with lower window setting of 400 HU. (A) EBUS assisted injection of two 200 µl marker depots using a 22G. endoscopic injection needle. Please refer to video S1 for recorded EBUS assisted injection of marker (B) EUS assisted injection of one 200 µl marker depot using a 22G. endoscopic injection needle. (C) Ultrasound guided percutaneous injection of one 200 µl marker depot using a 22G. 63 mm needle. Images from injection sites includes Day 0,2,9,15,27,37,45, CT scans and the maximum intensity projection (MIP) from CT scan day 2.
Practicability and performance of XPV-mark in pigs  A and B). Injection volumes were either 600µl (A) or 300µl (B). Please refer to video S2 for real time fluoroscopy of injection of 600µl XPV-mark (DV: dorsoventral). (C) CT scanning of lungs after removal from the thoracic cavity allow for the determination of the center of marker to lung-surface distance at which marker was palpable, maximal center of marker to lung-surface distance was estimated to 17 mm.

Spectroscopic characterization of the Cy7.5-SSIB dye
The NIR dye Cy7.5-SSIB was formulated in markers based on SAIB or LOIB and characterized by fluorescence or absorbance. The fluorescence emission was furthermore investigated as function of dye concentration in SAIB based markers.