Effect of Functional Groups Introduced into Radioiodinated Asymmetric Cyanine Dyes on Their In Vivo Behavior

Abstract

After intravenous administration, some heptamethine cyanine dyes bind to serum albumin and accumulate in tumors via the enhanced permeability and retention effect. Additionally, asymmetric cyanine dyes have two indole moieties which allow conjugation of a low-molecular-weight anticancer agent to one of the N-substituent side chains. Therefore, asymmetric heptamethine cyanine dyes have been studied as possible platforms for tumor-targeted drug delivery. However, the effects of functional groups on the opposite side chains on their in vivo behavior have not been explored in detail. Therefore, in this study, we aimed to clarify these effects by designing three model asymmetric cyanine dyes. For each dye, the side chain opposite the drug-conjugation site was modified with either an alkyl chain, a carboxyl group, or a sulfonyl group. A 125I atom was introduced on the other side of the molecule to enable quantitative biodistribution analysis. The resulting compounds are referred to as [125I]Bu, [125I]COOH, and [125I]SO3H. The albumin binding affinities (Kb values) for the corresponding nonradioactive compounds, Bu, COOH, and SO3H, were 0.82, 1.17, and 1.18 × 106 L/mol, respectively. In biodistribution studies, [125I]COOH showed higher radioactivity in the blood and tumors than [125I]Bu, whereas [125I]SO3H showed lower radioactivity levels. Notably, these differences did not correlate with their Kb values. Fluorescence analyses of the compounds incubated in serum-containing medium demonstrated that the fluorescence of COOH and Bu plateaued after approximately 1 and 2 h, respectively, whereas that of SO3H continued to change beyond 3 h, consistent with a slower time-dependent change in the protein-associated state of SO3H. Moreover, longer preincubation times of the radiolabeled asymmetric cyanine dyes in serum-containing medium decreased their transfer from the medium to the cellular side, with the greatest decrease observed for [125I]COOH. These results suggest that functional groups affect albumin-binding affinity, serum protein-associated states, and subsequent transfer to the cellular side, thereby influencing blood retention and tumor accumulation. Our findings provide a basis for the rational optimization of asymmetric cyanine dyes by modulating albumin interactions that influence their in vivo behavior.