Carbohydrate based biomarkers enable hybrid near infrared fluorescence and 64Cu based radio-guidance for improved surgical precision

Increasing numbers of lung tumors are identified at early disease stages by diagnostic imaging in screening programs, but difficulties in locating these during surgical intervention has prevented an improved treatment outcome. Surgical biomarkers that are visible on diagnostic images, and that provide the surgeon with real-time image guidance during the intervention are thus highly warranted to bridge diagnostic precision into enhanced therapeutic outcome. In this paper, a liquid soft tissue marker for near infrared fluorescence and radio-guidance is presented. The biocompatible marker is based on the carbohydrate ester, sucrose acetate isobutyrate, ethanol, and a multifunctional naphthalocyanine dye, which enable near infrared fluorescence image-guided resection at short, medium and long tissue depths. Naphthalocyanine dyes have high quantum yields and may further act as chelators of radionuclides. Upon injection of the liquid marker, a gel-like depot is formed in situ at the site of injection, wherein the fluorescent dye and radionuclide is retained. The radiolabeled markers were optimized for minimal fluorescence quenching and high retention of the positron emission tomography radionuclide 64Cu. The performance of the radiolabeled marker was tested in vivo in mice, where it displayed high photostability over a period of 4 weeks, and high retention of 64Cu for 48 hours. The retention and biodistribution of 64Cu was quantified via PET/CT, and the fluorescence emission by an in vivo imaging system. The presented data demonstrate proof-of-concept for naphthalocyanine markers as multimodal imaging agents that can bridge the precision of diagnostic imaging into surgical interventions.

: Mass spectra of the formed Cu-NC reference complex. The peak of 1000 presents the mass of Cu-NC complex. The peak of 1002 presents the mass of Cu-NC complex + 2H.

Method and results:
The release of NC dye from the NC-mark was investigated in vitro, by injecting 300 µL of NC-mark (0.1% w/w NC) into 5 mL of phosphate buffer saline (PBS, 5 mM, 150 mM NaCl, pH 7.0). The sample was following stored in the dark at 37°C, and dye release was monitored by UV-vis spectroscopy after 1, 3, 6 hour and 1, 2, 4, 6 days. UV-vis spectra of 0.5 ml PBS release buffer sample was recorded in quartz cuvettes from 200 nm to 850 nm using a Nanodrop 2000c (Thermoscientific, US) spectrophotometer. A PBS solution (release standard) corresponding to 10% release of NC was prepared from a NC solution in acetonitrile (0.05 mg/mL). At preparation, NC partially precipitated after dilution into PBS. The supernatant of the 10% release standard solution was taken after storage at room temperature overnight. The concentration of the supernatant thus contains less NC than intended, and therefore represents a lower estimate of the 10% release standard.
The UVvis absorption of the release samples (6D-1, 6D-2, 6D-3) was found to be negligible and nearly non-detectable compared to the 10% release standard solution (Fig. S2), which indicate minimal release of NC over the period of 6 days. Figure S2: In vitro release of NC dye from the marker. UVvis spectra of NC in the PBS release media on day 6 after injection into buffer (conducted in triplicate). A standard corresponding to 10% release was included for reference.

Preparation of NC-2 and PC-3 markers
NC-2 marker formulation: SAIB was heated to 70℃, and SAIB was poured into a glass vial. SAIB A solution of PC-3 dissolved in chloroform (50 µL, 1 mg/mL) was pipetted into a glass vial, and the chloroform was evaporated at room temperature under nitrogen flow. Subsequently, marker formulation (SAIB:xSAIB:ethanol 70:10:20, 1.0 g) was added into the vial to achieve a PC-3 concentration of 0.005% w/w for fluorescence emission measurement. The resulting mixture was sonicated at 70℃ for 15 minutes and followed by vortexing. The PC-3 marker formulation was further diluted using SAIB:xSAIB:ethanol 70:10:20 to a PC-3 concentration of 0.001% for absorbance measurement.

UV-vis absorbance measurements
Each marker solution (0.2 mL) was pipetted into a 96-well plate, and the UV-vis spectrum (400 -1000 nm) was recorded by a multimode microplate reader (Spark®, Tecan) with bandwidth of 3.5 nm.

Fluorescence emission measurements
Each marker formulation (1.0 mL) was transferred to a quartz cuvette (Helma, 10mm light path), and the fluorescence spectrum was collected by a fluorescence spectrometer (OLIS DM 45) with excitation/emission bandwidth of 26 nm and integration time of 0.2 seconds. An excitation wavelength of 650 nm was used for the PC-3 marker formulation. An excitation wavelength of 750 nm was utilized for NC-2 marker formulation. Figure S3. Absorbance and fluorescence spectra of NC-2 and PC-3 dissolved in marker formulations. (A)