Extended‐release PEG–luciferin allows for long‐term imaging of firefly luciferase activity in vivo

Abstract

Bioluminescence has gained favour in the last decade as an approach for observing tumours in vivo in a nondestructive manner. This very sensitive technique is based on light emission by the reaction of luciferin with the enzyme luciferase, as measured by a photodetector. Ever since the development of recombinant tumour cell lines that have been engineered to produce luciferase, a vast number of experiments have been carried out examining tumour growth, tumour metastasis and the effect of therapeutic regimens in such cases. A primary stumbling block, however, is the relatively short circulatory half-life of luciferin. In this paper, we propose the PEGylation of 6-amino-D-luciferin to extend its in vivo circulatory half-life, thus making the possibility of long-term observations in animals possible. The covalent attachment was through a carbamate linker that is known to hydrolyse in vivo, releasing the parent compound. Based on our studies, longer emission of the PEGylated luciferin was observed, as compared to free luciferin in mice bearing PC3 prostate tumours expressing luciferase. This result suggests that this reagent can be used in applications requiring extended monitoring of luciferase activation in vivo.

A Biocompatible in Vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice

“…74,75 This rapid change in the intensity of the signal observed from D-luciferin injection in animals has been recognized as a real problem in BLI field for application of this important modality for reliable imaging and quantification of various biological processes. 73,74,76 For example, BLI has been widely used to quantify the size of the tumor burden in xenograft models, in which animals are injected with luciferase transfected human cancer cells. 77,78 This animal model, in combination with BLI, has been one of the major tools in drug discovery for screening of anticancer drugs in animals.…”

“…77,78 This animal model, in combination with BLI, has been one of the major tools in drug discovery for screening of anticancer drugs in animals. 79,80 However, since the signal from injected luciferin changes significantly over time, 73,75,76 it can be difficult to assess the exact size of the tumor, as the timing of injection and duration of the imaging session can introduce significant errors. Presently, stabilization of the signal from D-luciferin is even more urgent as new 3D imaging BLI technologies become routine and require longer imaging times.…”

“…75 Similarly, the Denmeade group reported the synthesis of PEGylated luciferin derivatives that result in more stable and prolonged light production from D-luciferin, albeit with a significant reduction in overall light output (Table 1). 76 …”

Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions

“…For bioluminescence imaging in mammalian cells using firefly luciferase as a reporter gene, membrane-permeable luciferin esters were reported [79,91,92], and 6 0 -amino-D-luciferin amides including glycine [93] and the transporter conjugate [94] were synthesized and applied in mice [95]. To improve the circulatory half-life time of luciferin for in vivo tumor imaging, 6 0 -amino-D-luciferin modified with polyethylene glycol (PEG-luciferin) was prepared [96].…”

D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminescent activity

“…A solution to all above problems has been proposed by preparing the PEGylated D-6′-aminoluciferin (PEG-6-ADL, compound 46, Figure 8) by reaction of a PEG-carbonyl chloride and D-6′-aminoluciferin (20a). 69 PEGylation of D-6′-aminoluciferin (20a) was expected to improve tumour uptake via the enhanced permeability and retention (EPR) effect 70 and by the linker-dependent hydrolysis that occurs at higher rates at slightly acidic pH, as compared to normal physiological pH 71 When PC3-Luc-bearing animals were treated with PEG-6-ADL an initial photon flux was observed in the first 30 min and this is consistent with data from experiments using D-luciferin (1). However, following the initial burst effect, luciferin is both cleared from circulation and consumed within the tumour, leading to a rapid decrease in the bioluminescent signal over ~30-60 min.…”