Ultralow Background Near-Infrared Fluorophores with Dual-Channel Intraoperative Imaging Capability

Abstract

Two of the most pressing challenges facing bioimaging are nonspecific uptake of intravenously administered contrast agents and incomplete elimination of unbound targeted agents from the body. Designing a targeted contrast agent that shows fast clearance from background tissues and eventually the body after complete targeting is key to the success of image-guided interventions. Here, this work describes the development of renally clearable near-infrared contrast agents and their potential use for dual-channel image-guided tumor targeting. cRGD-ZW800-PEG (800 nm channel) and ZW700-PEG (700 nm channel) are able to visualize tumor margins and tumor vasculature simultaneously and respectively. These targeted agents show rapid elimination from the bloodstream, followed by renal clearance, which together significantly lower off-target background signals and potential toxicity. To demonstrate its applicability, this multispectral imaging is performed in various tumor-bearing animal models including lung cancer, pancreatic neuroendocrine tumors, breast, and ovarian cancer.

Keywords: NIR imaging; multispectral imaging; renal clearance; targeted fluorophores; tumor targeting.

Figure 1.. Targeted NIR fluorophores for dual-channel tumor-vasculature imaging.

(a) Synthetic routes for ZW700-PEG, ZW800-PEG, cRGD-ZW700-PEG, and cRGD-ZW800-PEG. (b) Physicochemical and optical properties of the synthesized NIR fluorophores. (c) Optical spectra of NIR fluorophores. Abs, absorbance; Fl, fluorescence. All optical properties were measured at a concentration of 5 μM in 5% BSA solution.

Figure 2.. Optophysical properties of the targeted fluorophores.

(a) Thermal stability in distilled water at −80, −20, 4, and 25 °C for 7 d. (b) pH stability in various pH buffers ranging from pH 1-11. (c) Serum stability in 10% FBS solution, pH 7.4 at 37 °C for 24 h. (d) Plasma protein binding assay compared with ICG in whole mouse serum at 37 °C for 8 h. e) Photostability patterns obtained in the 90º geometry using a 630 or a 760 nm NIR light source after incubating each fluorophore at 25 μM in 10% FBS for 3 h. Data were expressed as mean (n= 3) ± S.D.

Figure 3.

Cellular binding assay of 700 emitting fluorophores (a,c) and 800 emitting fluorophores (b,d) on LLC (a,b) and NIT-1 cell lines (c,d). After incubation with 5 μM of each molecule for 24 h at 37 °C, images were taken in the Cy5 or Cy7 channel with custom filter sets. After washing, the cells were imaged using BioTek Cytation 5 (n=6, mean ± S.D.). All NIR fluorescence images were normalized identically for each experimental condition in ImageJ version 1.52p. Scale bars = 100 μm. A P-value of less than 0.05 was considered significant: ****P <0.0001.

Figure 4.

Biodistribution and pharmacokinetic (PK) parameters of targeted NIR fluorophores in normal mice. 25-50 nmol of each compound was injected into 25 g CD-1 mice and imaged 4 h post-injection. Shown are representative images of the intraoperative abdominal cavity and resected organs. NIR fluorescence images for each condition have identical exposure times and normalization (n = 3). PK parameters including blood half-life (t_1/2), area under the curve (AUC), clearance (Cl), volume of distribution (V_d), and urinary excretion (% injected dose, %ID) were calculated by Prism 9 software. Abbreviations used are: Bl, bladder; Du, duodenum; He, Heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen.

Figure 5.. Tumor targeting of cRGD-ZW800-PEG in LLC-bearing animal models.

(a) Shown are representative intraoperative images of tumors with and without skin. For kinetics, 50 nmol of cRGD-ZW800-PEG was injected intravenously and observed up to 24 h post-injection. For dose dependency, 10-100 nmol of cRGD-ZW800-PEG was injected intravenously 4 h prior to imaging. (b) Resected organs and cross-sectioned tumor (Tu) were compared with Mu. Abbreviations used are: Bl, bladder; Du, duodenum; He, Heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen. Scale bars = 5 mm. (c) Simultaneous intraoperative dual-channel imaging of tumors (800 nm NIR) and vasculature (700 nm NIR). 50 nmol of ZW700-PEG (red) was injected intravenously 4 h post-injection of cRGD-ZW800-PEG (green). NIR fluorescence images for each condition have identical exposure times and normalization (n = 3).