Biomimetic Approach to Promote Cellular Uptake and Enhance Photoacoustic Properties of Tumor-Seeking Dyes.

The attachment of glucose to drugs and imaging agents enables cancer cell targeting via interactions with GLUT1 overexpressed on the cell surface. While an added benefit of this modification is the solubilizing effect of carbohydrates, in the context of imaging agents, aqueous solubility does not guarantee decreased π-stacking or aggregation. The resulting broadening of the absorbance spectrum is a detriment to photoacoustic (PA) imaging since the signal intensity, accuracy, and image quality all rely on reliable spectral unmixing. To address this major limitation and further enhance the tumor-targeting ability of imaging agents, we have taken a biomimetic approach to design a multivalent glucose moiety (mvGlu). We showcase the utility of this new group by developing aza-BODIPY-based contrast agents boasting a significant PA signal enhancement greater than 11-fold after spectral unmixing. Moreover, when applied to targeting cancer cells, effective staining could be achieved with ultra-low dye concentrations (50 nM) and compared to a non-targeted analogue, the signal intensity was >1000-fold higher. Lastly, we employed the mvGlu technology to develop a logic-gated acoustogenic probe to detect intratumoral copper (i.e., Cu(I)), which is an emerging cancer biomarker, in a murine model of breast cancer. This exciting application was not possible using other acoustogenic probes previously developed for copper sensing.

Xanthene-Based Nitric Oxide-Responsive Nanosensor for Photoacoustic Imaging in the SWIR Window.

Shortwave infrared (SWIR) dyes are characterized by their ability to absorb light from 900 to 1400 nm, which is ideal for deep tissue imaging owing to minimized light scattering and interference from endogenous pigments. An approach to access such molecules is to tune the photophysical properties of known near-infrared dyes. Herein, we report the development of a series of easily accessible (three steps) SWIR xanthene dyes based on a dibenzazepine donor conjugated to thiophene (SCR-1), thienothiophene (SCR-2), or bithiophene (SCR-3). We leverage the fact that SCR-1 undergoes a bathochromic shift when aggregated for in vivo studies by developing a ratiometric nanoparticle for NO (rNP-NO), which we employed to successfully visualize pathological levels of nitric oxide in a drug-induced liver injury model via deep tissue SWIR photoacoustic (PA) imaging. Our work demonstrates how easily this dye series can be utilized as a component in nanosensor designs for imaging studies.

Development of NIR-II Photoacoustic Probes Tailored for Deep-Tissue Sensing of Nitric Oxide.

Photoacoustic (PA) imaging has emerged as a reliable in vivo technique for diverse biomedical applications ranging from disease screening to analyte sensing. Most contemporary PA imaging agents employ NIR-I light (650-900 nm) to generate an ultrasound signal; however, there is significant interference from endogenous biomolecules such as hemoglobin that are PA active in this window. Transitioning to longer excitation wavelengths (i.e., NIR-II) reduces the background and facilitates the detection of low abundance targets (e.g., nitric oxide, NO). In this study, we employed a two-phase tuning approach to develop APNO-1080, a NIR-II NO-responsive probe for deep-tissue PA imaging. First, we performed Hammett and Brønsted analyses to identify a highly reactive and selective aniline-based trigger that reacts with NO via N-nitrosation chemistry. Next, we screened a panel of NIR-II platforms to identify chemical structures that have a low propensity to aggregate since this can diminish the PA signal. In a head-to-head comparison with a NIR-I analogue, APNO-1080 was 17.7-fold more sensitive in an in vitro tissue phantom assay. To evaluate the deep-tissue imaging capabilities of APNO-1080 in vivo, we performed PA imaging in an orthotopic breast cancer model and a heterotopic lung cancer model. Relative to control mice not bearing tumors, the normalized turn-on response was 1.3 ± 0.12 and 1.65 ± 0.07, respectively.

Activity-Based Nitric Oxide-Responsive Porphyrin for Site-Selective and Nascent Cancer Ablation.

Nitric oxide (NO) generated within the tumor microenvironment is an established driver of cancer progression and metastasis. Recent efforts have focused on leveraging this feature to target cancer through the development of diagnostic imaging agents and activatable chemotherapeutics. In this context, porphyrins represent an extraordinarily promising class of molecules, owing to their demonstrated use within both modalities. However, the remodeling of a standard porphyrin to afford a responsive chemical that can distinguish elevated NO from physiological levels has remained a significant research challenge. In this study, we employed a photoinduced electron transfer strategy to develop a panel of NO-activatable porphyrin photosensitizers (NOxPorfins) augmented with real-time fluorescence monitoring capabilities. The lead compound, NOxPorfin-1, features an o-phenylenediamine trigger that can effectively capture NO (via N2O3) to yield a triazole product that exhibits a 7.5-fold enhancement and a 70-fold turn-on response in the singlet oxygen quantum yield and fluorescence signal, respectively. Beyond demonstrating excellent in vitro responsiveness and selectivity toward NO, we showcase the potent photodynamic therapy (PDT) effect of NOxPorfin-1 in murine breast cancer and human non-small cellular lung cancer cells. Further, to highlight the in vivo efficacy, two key studies were executed. First, we utilized NOxPorfin-1 to ablate murine breast tumors in a site-selective manner without causing substantial collateral damage to healthy tissue. Second, we established a nascent human lung cancer model to demonstrate the unprecedented ability of NOxPorfin-1 to halt tumor growth and progression completely. The results of the latter study have tremendous implications for applying PDT to target metastatic lesions.

Synthesis of Silicon-Substituted Hemicyanines for Multimodal SWIR Imaging.

SWIR dyes offer many advantages over their more common NIR congeners; however, the available options are limited. New SWIR imaging agents can be accessed by remodeling existing NIR molecules (i.e., hemicyanines (HDs)). In this study, we synthesized SWIR-HD, a modified HD featuring dimethylsilicon and benzo[cd]indolium groups that are designed to red-shift the absorbance and emission to 988 and 1126 nm, respectively. SWIR-HD was employed to visualize the liver and tumors via multimodal SWIR imaging.

Near-infrared II photoacoustic probes for nitric oxide sensing.

In this chapter, we introduce a two-phase tuning approach for developing highly sensitive photoacoustic probes for imaging nitric oxide (NO) in the near-infrared (NIR)-II window. Due to the synthetically challenging nature of current NIR-II dye platforms, our two-phase tuning approach circumvents this issue by first allowing one to tune the reactivity using a synthetically accessible dye. We have used a physical organic workflow to understand the reaction kinetics and identify the most reactive sensing component. The selected reactive trigger is then introduced to phase two where it is appended to a range of well-established NIR-II dyes. This strategy is used to select the ideal photoacoustic probe for NIR-II imaging in vivo. Here, we have detailed procedures for synthesis, in vitro studies, and in vivo imaging.

New directions of activity-based sensing for in vivo NIR imaging.

In vivo imaging is a powerful approach to study biological processes. Beyond cellular methods, in vivo studies allow for biological stimuli (small molecules or proteins) to be studied in their native environment. This has the potential to aid in the discovery of new biology and guide the development of diagnostics and therapies for diseases. To ensure selectivity and an observable readout, the probe development field is shifting towards activity-based sensing (ABS) approaches and near-infrared (NIR) imaging modalities. This perspective will highlight recent in vivo ABS applications that utilize NIR imaging platforms.