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The second near-infrared window (NIR-II) beyond 1000 nm has attracted attention for optical contrast imaging in small animals. We sought to assess whether commercially available NIR-II dyes could be easily formulated for this purpose. 13 hydrophobic NIR-II dyes were purchased and screened by formulating them in simple solubilizing agents with established use in humans: propylene glycol, Cremaphor EL, Kolliphor HS15 (HS15), Tween 80, and cyclodextrin. Based on the absorption at 1064 nm (matching the Nd:YAG laser output commonly used in photoacoustic imaging), three of the dyes were further assessed at varying dye and surfactant concentrations. Of these, benzo indole butyl diphenylaminocyclopentene heptamethine (BIBDAH) tetrafluoroborate in HS15 generally showed the most favorable NIR-II character. 1 mg mL−1 BIBDAH in 25% HS15 exhibited a single absorption peak at 1030 nm with a calculated intensity greater than 100, which was relatively stable for weeks in storage. Following intravenous administration to mice, determination of BIBDAH pharmacokinetics was possible by absorption measurements of sampled plasma, revealing a circulating half-life of about one hour. Most of the dye was taken up by the liver. BIBDAH was used in vitro and in vivo as a photoacoustic contrast imaging agent and its accumulation could be detected in subcutaneous tumors in mice. BIBDAH was used for fluorescence imaging of blood vessels in mice, including in the brain (through intact skull), and dye clearance from blood to the liver was visualized. Taken together, this study confirms that accessible, strongly-absorbing dye can readily be formulated for injection by simply dissolving them in biocompatible surfactants and used for high-contrast preclinical optical imaging in the second NIR window.

The strategy of structure-inherent tumor targeting (SITT) with cyanine-based fluorophores is receiving more attention because no chemical conjugation of targeting moieties is required. However, the targeting mechanism behind SITT has not yet been well explained. Here, it is demonstrated that heptamethine-cyanine-based fluorophores possess not only targetability of tumor microenvironments without the need for additional targeting ligands but also second near-infrared spectral window (NIR-II) imaging capabilities, i.e., minimum scattering and ultralow autofluorescence. The new SITT mechanism suggests that bone-marrow-derived and/or tissue-resident/tumor-associated immune cells can be a principal target for cancer detection due to their abundance in tumoral tissues. Among the tested, SH1 provides ubiquitous tumor targetability and a high tumor-to-background ratio (TBR) ranging from 9.5 to 47 in pancreatic, breast, and lung cancer mouse models upon a single bolus intravenous injection. Furthermore, SH1 can be used to detect small cancerous tissues smaller than 2 mm in diameter in orthotopic lung cancer models. Thus, SH1 could be a promising cancer-targeting agent and have a bright future for intraoperative optical imaging and image-guided cancer surgery.