A clinical trial of super-stable homogeneous lipiodol-nanoICG formulation-guided precise fluorescent laparoscopic hepatocellular carcinoma resection

Abstract

Background: Applying traditional fluorescence navigation technologies in hepatocellular carcinoma is severely restricted by high false-positive rates, variable tumor differentiation, and unstable fluorescence performance.

Results: In this study, a green, economical and safe nanomedicine formulation technology was developed to construct carrier-free indocyanine green nanoparticles (nanoICG) with a small uniform size and better fluorescent properties without any molecular structure changes compared to the ICG molecule. Subsequently, nanoICG dispersed into lipiodol via a super-stable homogeneous intermixed formulation technology (SHIFT&nanoICG) for transhepatic arterial embolization combined with fluorescent laparoscopic hepatectomy to eliminate the existing shortcomings. A 52-year-old liver cancer patient was recruited for the clinical trial of SHIFT&nanoICG. We demonstrate that SHIFT&nanoICG could accurately identify and mark the lesion with excellent stability, embolism, optical imaging performance, and higher tumor-to-normal tissue ratio, especially in the detection of the microsatellite lesions (0.4 × 0.3 cm), which could not be detected by preoperative imaging, to realize a complete resection of hepatocellular carcinoma under fluorescence laparoscopy in a shorter period (within 2 h) and with less intraoperative blood loss (50 mL).

Conclusions: This simple and effective strategy integrates the diagnosis and treatment of hepatocellular carcinoma, and thus, it has great potential in various clinical applications.

Keywords: Fluorescent laparoscope; Hepatectomy; Indocyanine green nanoparticles; Theranostics; Translational medicine.

Fig. 1

Schematic illustration of research process. A Supercritical anti-solvent process was employed to produce carrier-free nanoICG with enhanced imaging properties and anti-photobleaching capacity. B Superstable homogeneous intermixed formulation technology (SHIFT) was employed to produce SHIFT&nanoICG for theranostics. C Preoperative TAE adjuvant therapy with SHIFT&nanoICG as the embolic agent. D The patient received a precise laparoscopic hepatectomy under real-time fluorescence after TAE

Fig. 2

Preparation and characterization of nanoICG. A Schematic of nanoICG preparation via SPFT for clinical diagnosis and treatment. B, C mixture due to the pelleted Representative SEM images of nanoICG, scale bar: 1 μm (B) and 500 nm (C). D DLS of nanoICG. E The molecule structure of LC-MS of freeICG and nanoICG. F, G The fluorescence intensities of nanoICG in different pH conditions were detected by IVIS Lunima LT at the 745 nm excitation after 1 h incubation. H Fluorescence intensity of freeICG and nanoICG was detected at different times by a 745 nm excitation when pH is 6.5. Data represent mean ± SD, n = 3. * P < 0.05, **** P < 0.0001, Student’s t-test

Fig. 3

Preparation and characterization of SHIFT&nanoICG. A Schematic of SHIFT&nanoICG preparation via SHIFT for clinical diagnosis and treatment. B Clinical drug samples prepared for TAE treatment. C Photograph of the centrifuged formulation. D Photograph of SHIFT&nanoICG freshly prepared and stored for 30 days. E Viscosity of lipiodol, RL&nanoICG, and SHIFT&nanoICG. F CT capacities of lipiodol, RL&nanoICG, and SHIFT&nanoICG

Fig. 4

Cellular fluorescence properties and cytotoxicity. A, B The fluorescence study of the freeICG/nanoICG in LO2 cells. C, D The fluorescence study of the freeICG/nanoICG in Hepa1-6 cells. E, F The fluorescence study of the freeICG/nanoICG in HepG2 cells. G–I CCK-8 of the freeICG and nanoICG in LO2 (G), Hepa1-6 (H) and HepG2 (I) cells. Data represent mean ± SD, n = 3. *** p < 0.001, Student’s t-test

Fig. 5

Embolism and safety evaluation in clinical case of HCC. A–C MRI showed that a liver tumor at the right posterior lobe with a maximum diameter of 4.3 cm was observed (arrow): 4 × 2.4 × 4.3 cm³ (A: arterial phase, B: venous phase, C: coronal). D–F DSA angiography and SHIFT&ICG embolization (D: before embolization, E: during embolization, F: after embolization). G Blood AFP value (reference interval: 0.0 ~ 20.0 µg/L) before and 12 days after TAE. H–L Blood AST (reference interval: 15.0 ~ 40.0 U/L), ALT (reference interval: 9.0 ~ 50.0 U/L), T. Bil (reference interval: 1.7 ~ 26.0 µmol/L), Cre (reference interval: 57.0 ~ 97.0 µmol/L) and WBC value (reference interval: 3.5 ~ 9.5 10E9/L) before and 12 days after TAE

Fig. 6

The surgical navigation effect of SHIFT&nanoICG after TAE-assisted therapy. A The 3D reconstruction imaging before hepatectomy showed that the tumor was located in the right posterior lobe of the liver and was closely connected to the surrounding blood vessels. B The fluorescence imaging of primary lesion. C The fluorescence imaging of the incisal margin of the residual liver, with achieved R0 resection. D–F Whole resected tumor lesions and fluorescence imaging (white arrowhead = primary lesion, red arrowhead = microsatellite lesion). G–I The dissected tumor lesion and fluorescence imaging (white arrowhead = primary lesion, red arrowhead = microsatellite lesion). J–L The dissected tumor lesion by layer and fluorescence imaging. M–N The microsatellite lesion and fluorescence imaging. O The H&E staining of microsatellite lesion