FAP-α-Instructed Coumarin Excimer Formation for High Contrast Fluorescence Imaging of Tumor

Abstract

Emissive excimers, which are formed by planar polycyclic aromatic fluorophores (e.g., coumarin), enable high contrast tumor imaging. However, it is still challenging to "turn on" excimer fluorescence in physiological dilute solutions. The biocompatible CBT-Cys click condensation reaction enables both intra-and intermolecular aggregations of the as-loaded fluorophores on the probe molecules, which may promote the generation of emissive excimers in a synergistic manner. As a proof-of-concept, we herein design a fluorescence probe Cbz-Gly-Pro-Cys(StBu)-Lys(coumarin)-CBT (Cbz-GPC(StBu)K(Cou)-CBT), which can be activated by FAP-α under tumor-inherent reduction conditions, undergo a CBT-Cys click reaction, and selfassemble into coumarin nanoparticle Cou-CBT-NP to "turn on" the excimer fluorescence. In vitro and in vivo studies validate that this "smart" probe realizes efficient excimer fluorescence imaging of FAP-α-overexpressed tumor cells with high contrast and enhanced accumulation, respectively. We anticipate that this probe can be applied for diagnosis of FAP-α-related diseases in the clinic in near future.

CBT‐Cys click reaction for optical bioimaging in vivo

“…As a proof‐of‐concerpt, Gao et al recently designed a smart CBT‐Cys FLI probe Cbz‐Gly‐Pro‐Cys(StBu)‐Lys(coumarin)‐CBT ( 1 ) (Figure 2B). 19 1 can be activated by fibroblast activation protein alpha (FAP‐α) 59 to yield C(StBu)K(Cou)‐CBT, which underwent CBT‐Cys click reaction under reduction conditions to form a cyclic dimer Cou‐CBT‐Dimer, subsequently self‐assembling into nanoparticle Cou‐CBT‐NP to turn coumarin excimer fluorescence “on.” This probe enabled in vivo FLI of intratumoral FAP‐α activity with enhanced tumor‐to‐background contrast, biosafety, and tumor accumulation.…”

“…58 As a proofof-concerpt, Gao et al recently designed a smart CBT-Cys FLI probe Cbz-Gly-Pro-Cys(StBu)-Lys(coumarin)-CBT (1) (Figure 2B). 19 1 can be activated by fibroblast activation Abovementioned fluorophores need visible/UV light for excitation, thus suffer from severe light attenuation, tissue absorption, and autofluorescence interference. 60 In contrast, near-infrared (NIR) dyes confer significantly improved tissue penetration depth and signal-tobackground contrasts, thus they are more suitable for in vivo bioimaging applications.…”

“…These cyclic dimers/oligomers could further self‐assemble into nanostructures (e.g., particles) through π–π stacking or hydrophobic interaction 16 . Of note, various physiological stimuli, such as pH, 17 reduction, 18 or enzyme, 19 could be employed to activate CBT‐Cys click reaction. This facilitates on‐demond fabrication of activatable molecular probes/prodrugs with controllable assembly of nanostructures in living cells 15 …”

Enzyme-Instructed CBT-Cys-like Click Cyclization Reactions for Bioimaging

“…(d) Time course average fluorescence intensities of tumors (dotted circles) of the mice in (c) (mean ± SD, n = 5, *** P < 0.001). Reproduced with permission from ref . Copyright 2022 American Chemical Society.…”

“…54 Upon enzymatic cleavage of the DEVD peptide by caspase-3, the solubility of the obtained probe significantly decreased, which 4a). 55 The Cbz-GP peptide sequence within the probe can be enzymatically cleaved by fibroblast activation protein α (FAP-α), 56 which results in the generation of C(StBu)K(Cou)-CBT. Subsequently, the reduction of disulfide bonds facilitated by GSH triggers a CBT-Cys click reaction, thereby leading to the formation of cyclic Cou-CBT-dimers.…”