Role of Albumin in Accumulation and Persistence of Tumor-Seeking Cyanine Dyes

Abstract

Some heptamethine cyanine dyes accumulate in solid tumors in vivo and persist there for several days. The reasons why they accumulate and persist in tumors were incompletely defined, but explanations based on uptake into cancer cells via organic anion transporting polypeptides (OATPs) have been widely discussed. All cyanine-based "tumor-seeking dyes" have a chloride centrally placed on the heptamethine bridge (a "meso-chloride"). We were intrigued and perplexed by the correlation between this particular functional group and tumor uptake, so the following study was designed. It features four dyes (1-Cl, 1-Ph, 5-Cl, and 5-Ph) with complementary properties. Dye 1-Cl is otherwise known as MHI-148, and 1-Ph is a close analog wherein the meso-chloride has been replaced by a phenyl group. Data presented here shows that both 1-Cl and 1-Ph form noncovalent adducts with albumin, but only 1-Cl can form a covalent one. Both dyes 5-Cl and 5-Ph have a methylene (CH₂) unit replaced by a dimethylammonium functionality (N⁺Me₂). Data presented here shows that both these dyes 5 do not form tight noncovalent adducts with albumin, and only 5-Cl can form a covalent one (though much more slowly than 1-Cl). In tissue culture experiments, uptake of dyes 1 is more impacted by the albumin in the media than by the pan-OATP uptake inhibitor (BSP) that has been used to connect uptake of tumor-seeking dyes in vivo with the OATPs. Uptake of 1-Cl in media containing fluorescein-labeled albumin gave a high degree of colocalization of intracellular fluorescence. No evidence was found for the involvement of OATPs in uptake of the dyes into cells in media containing albumin. In an in vivo tumor model, only the two dyes that can form albumin adducts (1-Cl and 5-Cl) gave intratumor fluorescence that persisted long enough to be clearly discerned over the background (∼4 h); this fluorescence was still observed at 48 h. Tumors could be imaged with a higher contrast if 5-Cl is used instead of 1-Cl, because 5-Cl is cleared more rapidly from healthy tissues. Overall, the evidence is consistent with in vitro and in vivo results and indicates that the two dyes in the test series that accumulate in tumors and persist there (1-Cl and 5-Cl, true tumor-seeking dyes) do so as covalent albumin adducts trapped in tumor tissue via uptake by some cancer cells and via the enhanced permeability and retention (EPR) effect.

Figure 1.

a, MDA-MB-231 cells incubated with 1-Ph (20 μM) SFM; b, same experiment but preblocked by incubating with 250 μM BSP for 5 min; c same as a, but cells pretreated with 1 mM DMOG to induce hypoxia. Figure S1 shows data from identical experiments but using 1-Cl. Throughout, images were taken by Leica Confocal Microscope at 20×/0.75 NA.

Figure 2.

1-Cl (20 μM) in a, serum free media; b, media with 10% FBS; c, 250 μM BSP serum free; and d, 250 μMBSP with media containing 10%FBS; and e, quantification of the data in a−d by FACS.

Figure 3.

Interaction of a, 1-Cl; and b, 1-Ph (1 μM) with HSA (50 μM) at pH 7.4 in PBS buffer at 37 °C observed via UV−vis spectroscopy. c, Summary of key UV experiments for albumin interactions with 1, 5, and BSP.

Figure 4.

MDA-MB-231 cells incubated with the following. a, 1-Cl (10 μM throughout) and ~1 equiv BSA-FITC (mixed then used as a noncovalent adduct, i.e., before a covalent adduct could form). b, 1-Cl preincubated with ~1 equiv of BSA-FITC for 24 h to form covalent adduct. c, 1-Cl with BSA-FITC preincubated with 6-maleimidohexanoic acid for 24 h to block Cys. d, 1-Ph (10 μM) and ~1 equiv BSA-FITC. Throughout the incubation conditions were at 37 °C and 5% CO₂ in 20 min unless otherwise indicated. Images at 20× magnification.

Figure 5.

a, Tumor localization of 1-Cl, 1-Ph, 5-Cl, and 5-Ph. b, Time course of tumor to background ratios (TBRs). Arrows indicate tumors. Scale bars = 1 cm. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6.

Intraoperative imaging and biodistribution after 48 h for 25 nmol: a, 1-Cl; b, 1-Ph; c, 5-Cl; and, d, 5-Ph, in C57Bl/6 mice with subcutaneous triple negative breast tumor (E0771). Scale bars = 1 cm. He, heart; Lu, lung; Li, liver; Pa, pancreas; Sp, spleen; K_i, kidney, Du;duodenum; In, intestine; Mu, muscle; and Tu, tumor.