Near-Infrared Illumination of Native Tissues for Image-Guided Surgery

Abstract

Our initial efforts to prepare tissue-specific near-infrared (NIR) fluorescent compounds generated successful correlation between physicochemical properties and global uptake in major organs after systemic circulation and biodistribution. Herein, we focus on the effects on biodistribution based on modulating electronic influencing moieties from donating to withdrawing moieties at both the heterocyclic site and through meso-substitution of pentamethine cyanine fluorophores. These selected modifications harnessed innate biodistribution pathways through the structure-inherent targeting, resulting in effective imaging of the adrenal glands, pituitary gland, lymph nodes, pancreas, and thyroid and salivary glands. These native-tissue contrast agents will arm surgeons with a powerful and versatile arsenal for intraoperative NIR imaging in real time.

Figure 1

Photodegradation profile in light and dark conditions of a select subset of NIR fluorescent contrast agents. The absorbance values for the individual fluorophores were measured (at the wavelength of maximum absorption) at different intervals to determine the percent absorption remaining.

Figure 2

Compounds 20–22, targeting to salivary glands and thyroid glands, and biodistribution of resected organs in CD-1 mice. Ten nmol of each fluorophore was injected intravenously into 25 g CD-1 mice 4 h prior to imaging and resection. Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; SG, salivary gland, Sp, spleen. Arrows indicate the targeted gland.

Figure 3

Compounds 23–25, targeting to salivary glands and thyroid glands, and biodistribution of resected organs in CD-1 mice. Ten nmol of each fluorophore was injected intravenously into 25 g CD-1 mice 4 h prior to imaging and resection. Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; SG, salivary gland, Sp, spleen. Arrowheads indicate the targeted gland.

Figure 4

Compounds 26–28, targeting to thyroid glands and pituitary gland, and biodistribution of resected organs in CD-1 mice. Ten nmol of each fluorophore was injected intravenously into 25 g CD-1 mice 4 h prior to imaging and resection. Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; LN, lymph node; Lu, lungs; Mu, muscle; Pa, pancreas; SG, salivary gland, Sp, spleen. Arrows indicate the targeted gland. For pituitary glands, arrowheads indicate the anterior portion and arrows for posterior pituitary.

Figure 5

Compounds 31–33, targeting to inguinal and lumbar lymph nodes, and biodistribution of resected organs in CD-1 mice. Ten nmol of each fluorophore was injected intravenously into 25 g CD-1 mice 4 h prior to imaging and resection. Abbreviations used are Bl, bladder; Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; LN, lymph node; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen. Arrows indicate the targeted gland.

Figure 6

Compounds 36–38, targeting to adrenal glands and pituitary gland, and biodistribution of resected organs in CD-1 mice. Ten nmol of each fluorophore was injected intravenously into 25 g CD-1 mice 4 h prior to imaging and resection. Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; SG, salivary gland, Sp, spleen. Arrows indicate the targeted gland. For pituitary glands, arrowheads indicate the anterior portion and arrows for posterior pituitary.

Figure 7

Cell viability assay of selected NIR fluorophores using NIH/3T3 fibroblasts. Cell viability was plotted 1 day post-treatment of compounds at a concentration of 2 μM (left) or 10 μM (right), compared to untreated control as measured by the alamarBlue assay. Data are representative of N = 6 independent experiments per condition (mean ± SD).

Scheme 1

Synthesis of 700 nm Emitting Pentamethine Cyanines Featuring Neutral (X = H) and Electron-Withdrawing (X = F, Cl, Br, CF₃) Moieties

Scheme 2

Synthesis of 700 nm Emitting Pentamethine Cyanines Featuring Electron-Donating Methoxy Groups

Scheme 3

Synthesis of 700 nm Emitting Pentamethine Cyanines Featuring Electron-Donating Methylene Dioxy Substitution on the Phenyl Rings