Intraoperative Assessment and Photothermal Ablation of the Tumor Margins Using Gold Nanoparticles

Abstract Surgical resection is commonly used for therapeutic management of different solid tumors and is regarded as a primary standard of care procedure, but precise localization of tumor margins is a major intraoperative challenge. Herein, a generalized method by optimizing gold nanoparticles for intraoperative detection and photothermal ablation of tumor margins is introduced. These nanoparticles are detectable by highly sensitive surface‐enhanced Raman scattering imaging. This non‐invasive technique assists in delineating the two surgically challenged tumors in live mice with orthotopic colon or ovarian tumors. Any remaining residual tumors are also ablated by using post‐surgical adjuvant photothermaltherapy (aPTT), which results in microscale heat generation due to interaction of these nanoparticles with near‐infrared laser. Ablation of these post‐operative residual micro‐tumors prolongs the survival of mice significantly and delays tumor recurrence by 15 days. To validate clinical translatability of this method, the pharmacokinetics, biodistribution, Raman contrast, aPTT efficiency, and toxicity of these nanoparticles are also investigated. The nanoparticles have long blood circulation time (≈24 h), high tumor accumulation (4.87 ± 1.73%ID g−1) and no toxicity. This high‐resolution and sensitive intraoperative approach is versatile and can be potentially used for targeted ablation of residual tumor after resection within different organs.

SKOV3 and CT26 cells were seeded in the 96-well plates at a density of 5 x10 3 cells per well in complete medium (DMEM containing 10% FBS and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin)) at 37 °C and under a humidified atmosphere containing 5% CO 2 . Cells without treatment were used as control. After 48 h, 10 uL of MTT solution (5 mg/mL) was added to each well and incubated for an additional 4 h at 37 °C for MTT formazan formation. Then, the media were completely removed, and 200 μL DMSO was added to each well to dissolve MTT formazan at 37 °C. The plates were gently shaken for 5 min to ensure the dissolution of formazan.
The absorbance values were measured at 570 nm wavelength, using a microplate reader (Infinite F200, TECAN, Austria). Triplicates were used for each condition.

In vivo photoacoustic imaging (PAI)
Mice bearing SKOV3 tumors (n = 3) were i.v. injected with Au-Ur@DTTC NPs (2 OD) and imaged using a PA system with an 808-nm laser as the excitation source. For in vivo photoacoustic imaging, BALB/c nude female mice (6-8 weeks, purchased from SLAC, Shanghai, China) with subcutaneous tumor xenografts (n = 3) were anesthetized with 2.5% isoflurane delivered via a nose cone, and then Au-Ur@DTTC NPs (200 μL) was injected to each mouse via the tail vein. After this injection, mice were scanned at different time post-injection times (1 h, 5 h, and 24 h, MSOT inVision 128, iThera medical, Germany) to record the PA images under 808 nm laser excitation, consuming 5 mins for each time point. Anesthetized animals were placed in supine position and PA images were acquired with an in-plane resolution of approximately 150 μm. Linear-mode-based reconstruction and linear regression spectral were applied using ViewMSOT (iThera Medical).

Radiolabeling of [ 64 Cu]Au-Ur@DTTC NPs
For conjugation of radiometal chelator to nanoparticles, DOTA-LA (1 mg/mL, 10 µL) was mixed with 1 mL aqueous solution of Au-Ur@DTTC NPs for 4 h at room temperature. For radiolabeling, aliquots of Au-Ur@DTTC NPs (DOTA) (0.5 mL) in 0.1 mol/L sodium acetate solution (pH 5.5) were mixed with an aqueous solution of 64 CuCl 2 (~1 mCi) at 37 °C for 30 min. The radiolabeled nanoparticles were then purified by centrifugation at 3800 g for 5 min and re-dispersion in PBS (3 times). The radiolabeling efficiency and the stability of labeled nanoparticles were analyzed using a Bioscan IAR-2000 TLC scanner (Washington, DC, USA).

In vivo pharmacokinetics
Five Swiss female mice (8 weeks) were injected with [ 64 Cu] Au-Ur@DTTC NPs (0.074 MBq per mouse) via tail veins, and blood samples (10 μL) were collected from their retinal vein before and after intravenous injection at different time points (0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, and 24 h). The radioactivity of the blood samples was measured by a gamma counter (Packard Cobra, Ramsey, MD, USA). The pharmacokinetic parameters of the blood were analyzed by WinNonlin 5.0.1 software (Pharsight Corporation, Palo Alto, CA), and the decay curves of the copper content in the blood samples were generated and fitted with a two-compartmental model to determine the blood half-life of the nanoparticles.

In vivo biodistribution
[ 64 Cu] Au-Ur@DTTC NPs (0.74 MBq per mouse) were injected into female nude mice (8 weeks old, n = 5) with subcutaneous SKOV3 tumors (the volume of the tumors was nanoparticles. Heart, liver, spleen, lung, kidneys, stomach, intestine, muscle, bone, brain, blood, and tumors were collected and weighted, and radioactivity was measured using a gamma counter (Packard Cobra, Ramsey, MD, USA).

Figure S1. Synthesis and characterization of gold nano-urchins (Au-Ur). (A-C)
TEM images of Au-Ur synthesized by addition of 100, 200, and 500 mM sodium hydroxide solutions to gold precursor solution. Insets: photographs of the corresponding nanoparticle solutions. By adjusting the sodium hydroxide concentration, the mean particle diameters were tuned to ~55 nm, ~72 nm, and ~92 nm, respectively. In addition, the color of the three solutions was distinguishable and changed from purple-red to blue, and then to lilac, due to the red-shift characteristic surface plasmon resonance of Au-Ur.