Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time

Abstract

Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools.

Azadipyrromethenes: from traditional dye chemistry to leading edge applications

“…Following t-butyl ester hydrolysis to 63 and conversion to the activated ester fluorochrome 64 by reaction with N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI), a terminal amine functionalized polyethylene glycol polymer was conjugated to make the final aqueous soluble bio-responsive NIR-fluorescent imaging agent 65 (Scheme 32). 83 This route is an encouraging Please do not adjust margins…”

“…In our own work we have utilized a multi-step synthesis to produce the bio-responsive NIR-fluorophore 65 which offers significant potential for use in live cellular and in vivo imaging. 83 Emission from the probe was shown to be highly selective for cellular lysosomes, capable of real-time continuous cellular imaging of fundamental cellular processes, and allowed for direct translation to in vivo tumour imaging. The starting point of the synthesis was the BF 2 -chelated bis-phenol azadipyrromethene 29h, which was monoalkylated with t-butyl bromoacetate to produce 61.…”

Development of a novel carboplatin like cytoplasmic trackable near infrared fluorophore conjugate via strain-promoted azide alkyne cycloaddition

“…[8][9][10] Significantly NIR probes have been successfully employed to image tumours in vitro and in vivo and as sensors for reactive oxygen species (ROS), reactive nitrogen species (RNS), thiols, ions, pH and enzyme activities. [8] The BF2-azadipyrromethene (NIR-AZA) fluorophore class, which have absorptions of c. 690 nm with emission at c. 725 nm in formulated aqueous solution, [11,12] offer excellent photophysical and stability properties and have attracted the attention of many research teams [10,13] including those interested in tracking metal-based drugs. [14] Specifically a number of Pt(II) anticancer type compounds have been reported where boron-fluorescent dipyrromethene (BODIPY) moieties have been tethered to Pt centres via prior direct covalent modification of the ammine carrier ligands.…”

BF 2 -azadipyrromethene NIR-emissive fluorophores with research and clinical potential

“…However, the atypical pharmacokinetics of ICG, which has a half-life of only four minutes and excretion that is exclusively via bile, severely limits any further expansion of its clinical uses [12,13]. We have recently embarked on the development of a new class of NIR-fluorophore, namely the BF 2 -azadipyrromethene or NIR-AZA class, which has excellent photostability and typical emission wavelengths in the range of 675 to 730 nm ( Figure 1) [14][15][16][17][18]. This has been successfully employed in real-time cellular microscopy and small animal imaging but, as yet, suitable longer wavelength derivatives have not been identified for clinical use.…”

“…This has been successfully employed in real-time cellular microscopy and small animal imaging but, as yet, suitable longer wavelength derivatives have not been identified for clinical use. To date, this class of fluorophore has shown no cellular or in vivo toxicity in the concentration ranges used M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 for imaging which is a positive indicator for clinical translation [14,16]. In contrast to the NIR-AZA class, ICG has longer wavelengths of absorption and emission at 787 and 818 nm which are preferred for clinical use as deeper light penetration is achievable with these longer wavelengths [19].…”