Development of a Squaraine-Based Molecular Probe for Dual-Modal in Vivo Fluorescence and Photoacoustic Imaging

Abstract

Dual-modular imaging approaches combining near-infrared (NIR) fluorescence (FLI) and photoacoustic imaging (PAI) require suitable contrast agents to produce dual-modular signals. Although nanoparticles have been used to develop PAI agents, small molecule-based imaging agents have not been extensively studied, highlighting the need to design new fluorophores with an enhanced multifunctional ability. Thus, in this study, we designed a novel squaraine (SQ)-based dye and reported its rational preparation and conjugation with a cancer targeting peptide. Specifically, benzoindole-derived SQ (BSQ) showed strong absorption and fluorescence properties at above 650 nm under aqueous conditions, with a maximum absorption and emission at 665 and 680 nm, respectively. Moreover, PA signal scanning experiments revealed a maximum signal intensity in the range 680−700 nm. BSQ was also conjugated with cyclic arginine−glycine−aspartic acid (cRGD) to improve its active targeting ability for the α v β 3 integrin, which is overexpressed in various cancer and angiogenic cells. A series of in vitro, in vivo, and ex vivo FLI studies showed that the cRGD conjugated BSQ (BSQ−RGD 2 ) successfully stained and targeted α v β 3 integrin-overexpressing tumor cells and xenografts, which were clearly visualized by FLI and PAI. Therefore, BSQ−RGD 2 can successfully be applied to dual-modular imaging of the specific biomarker in living animals.

Targeted contrast agents and activatable probes for photoacoustic imaging of cancer

“…112 Relative to antibody targeting, RGD-based peptides are readily accessible, exhibit low immunogenicity, and can be conveniently conjugated to various molecular structures via conventional conjugation chemistry. Although a vast number of RGD-based TCA for PA imaging have been developed, 20,113–143 we highlight several notable examples below.…”

The Chemistry of Organic Contrast Agents in the NIR‐II Window

“…Squaraine dyes are akind of zwitterionic fluorophore that contain the four-membered-ring system of squaric acid and display high absorption and emission properties from the visible to the NIR-I region. [104] Yaoe tal. constructed the small-molecule fluorophore SQ1 (9r)b yu sing the ethylgrafted 1,8-naphtholactam as the donor and malonitrilesubstituted squaric acid as the acceptor based on aD -A-D structure.…”

“…Squaraine dyes are a kind of zwitterionic fluorophore that contain the four‐membered‐ring system of squaric acid and display high absorption and emission properties from the visible to the NIR‐I region [104] . Yao et al.…”

NIR-I Dye-Based Probe: A New Window for Bimodal Tumor Theranostics

“…In the recent 5 years, NIR-I fluorophores in the reported bimodal FI/PAI probes are often served as the CAs of PAI as well. These fluorophores, including natural origin ( Zhou et al, 2017 ; Zheng et al, 2018 ; Siwawannapong et al, 2020 ), cyanine derivatives ( Baart et al, 2021 ; Doan et al, 2021 ; Mu et al, 2021 ; Nishio et al, 2021 ), and other types of organic dyes ( Park et al, 2020 ; Shin et al, 2021 ; Wang et al, 2022 ), had strong and broad optical absorption in the NIR-I region to provide PA signals. Apart from the broad applications of the traditional cyanine dyes, researchers have devoted much attention to developing new structures to obtain better absorption and emission properties in this field.…”