Molecular Targeted NIR-II Probe for Image-Guided Brain Tumor Surgery

Abstract

Optical imaging strategies for improving delineation of glioblastoma (GBM) is highly desired for guiding surgeons to distinguish cancerous tissue from healthy and precious brain tissue. Fluorescence imaging (FLI) in the second near-infrared window (NIR-II) outperforms traditional NIR-I imaging with better tissue penetration, higher spatial and temporal resolution, and less auto fluorescence and scattering. Because of high expression in GBM and many other tumors, urokinase Plasminogen Activator Receptor (uPAR) is an attractive and well proven target for FLI. Herein we aim to combine the benefit of a NIR-II fluorophore with a high affinity uPAR targeting small peptide. A targeted NIR-II fluorescent probe was developed by conjugating an in-house synthesized NIR-II fluorophore, CH1055, and a uPAR targeting peptide, AE105. To characterize the in vivo distribution and targeting properties, a dynamic imaging was performed in orthotopic GBM bearing nude mice ( n = 8). Additionally, fluorescence guided surgery of orthotopic GBM was performed in living animals. CH1055-4Glu-AE105 was easily synthesized with >75% yield and >98% HPLC evaluated purity. The retention time of the probe on analytical HPLC was 15.9 min and the product was verified by mass spectrometry. Dynamic imaging demonstrated that the uPAR targeting probe visualized orthotopic GBM through the intact skull with a tumor-to-background ratio (TBR) of 2.7 peaking at 96 h. Further, the orthotopic GBM was successfully resected in small animals guided by the NIR-II FLI. By using a small uPAR targeting NIR-II probe, FLI allows us to specifically image and detect GBM. A real-time imaging setup further renders FLI guided tumor resection, and the probe developed in this work is a promising candidate for clinical translation.

Figure 1

(a) Peptide sequence and schematic molecular structure of CH1055-4Glu-AE105; (b) analytical HPLC of purified probe; (c) MALDI-TOF mass spectrum of CH1055-4Glu-AE105; (d) affinity to the uPAR receptor for the NIR-II labeled peptide (CH1055-4Glu-AE105) and the nonlabeled peptide (AE105); (e) absorbance and fluorescent emission of CH1055, demonstrating an absorbance peak at ∼750 nm and an emission peak at ∼1055 nm. The fluorescent emission spectrum was obtained with an 808 nm excitation laser.

Figure 2

(a) Dynamic imaging of CH1055-4Glu-AE105 in orthotopic GBM models in the unblocked (top images) and blocked (10 times 4Glu-AE105, bottom images) group (n = 4 per group); (b) corresponding MRI with clear tumors (yellow arrows) in both unblocked and blocked mice; (c) quantification analysis of NIR-II images. Mean tumor-to-background ratio over time between blocked (green bars) and unblocked (blue bars) animals; (d) side-to-side comparison between a NIR-I-AE105 probe and the NIR-II probe (CH1055-4Glu-AE105). NIR-I TBR = 1.4 vs NIR-II TBR = 2.7.

Figure 3

For both Complete resection (a) and Incomplete resection (b), left-hand side pictures are taken prior to tumor resection and right-hand side pictures are after resection. Complete resection: no signal left around the tumor at the postoperative NIR-II picture and it is considered a complete resection. Yellow circle indicates the margin (M = 36,000 Gray Value) and the blue circles indicates the tumor free background signal (B = 35,000 Gray Value) resulting in a margin-to background ratio = 1.0. Incomplete resection: clear signal at the tumor margin at the postoperative picture indicating positive resection margins. Yellow circle indicates the positive margin (M = 52,500 Gray Value) and the blue circles indicates the tumor free background signal (B = 35,000 Gray Value) resulting in a margin-to-background ratio = 1.5. On ICH, blue arrows are pointing at uPAR positive stained tumor cells.

Figure 4

Biodistribution of CH1055-4Glu-AE105. (a) Visual distribution of signal intensity (normalized) for major organs; (b) normalized quantification of the signal (n = 2); (c) plasma stability curve and table with normalized area under curve (AUC) (0 h as reference).

Figure 5

Special stained liver. (a) portal triad, HE staining; (b) liver parenchyma with apoptotic hepatocytes (arrows), HE staining; (c) central vein, HE staining; (d) central vein, modified Sirius connective tissue staining.