Figure 1. In Vitro Bioluminescence Assay. 1. Substrate dose-response analysis: (1) Total flux(p/s) of six different luciferases at different substrate concentrations (μM). (2) The light intensity of six luciferases at different substrate concentrations(μM); 2. ATP dose-response analysis: (1) Total flux(p/s) of six different luciferases at different ATP concentrations(μM). (2) The light intensity of six luciferases at different ATP concentrations(μM).

Table 1.The bioluminescence properties of 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin

Substrates	λmax(nm)	Vmax (s-1)	Km (mol/L)
dLuc	560	9.68×1010	10.9
aLuc	588	1.89×1010	6.63
5-cyL	598	4.67×109	0.344
6-cyL	610	3.69×109	0.274
7-cyL	605	5.90×109	0.281
8-cyL	613	4.367×109	0.313

[Cell bioluminescence imaging]

In order to measure the bioluminescence intensity of these analogues, our team measured bioluminescence imaging in living cells. The luciferase-expressing in U87 cells were cultured in a conditioned system. After the incubation period, the medium was removed and we add different concentration of 5-cyL, 6-cyL, 7-cyL and 8-cyL. In the same condition of the substrate concentration, the bioluminescence intense has an obvious positive correlation with the concentration of U87-Luc cells and the three novel bioluminescence substrates has high sensitivity, good cell permeability and bioluminescence activity (Figure 2).

Figure 2. Cell Bioluminescence imaging in U87-Luc cells.(1) Total flux(p/s) of five different luciferases at different Cell-concentrations(μM). (2) The light intensity of five luciferases at different Cell-concentrations(μM).

[Tumor Xenografts Mice Imaging]

In a cell-based assay, our compounds exhibited good biological activity. Subsequently, we carried on further bioluminescence imaging of well-established mice xenograft tumor models. The 6-weeks-old female nude mice were inoculated subcutaneously with luciferase-expressing U87-Luc cells. After tumor xenografts in mice generated, the nude mice with tumor xenografts were injected i.p. with 100 μL of 5-cyL, 6-cyL, 7-cyL, D-luciferin and aminoluciferin (1 µM, 100 µM, 1 mM and 10 mM). The bioluminescence signals were measured by a Xenogen IVIS Spectrum imaging system (Figure 3A). The cyL compounds all yielded a >10-fold higher bioluminescent signal than D-luciferin at equivalent doses. Especially, 7-cyL showed 25-fold higher bioluminescence intensity than D-luciferin (Figure 3B). More interestingly, low to 1 µM dose of aminoluciferin analogues still could show high and detectable bioluminescent signal, while D-luciferin at equivalent concentrations provided either weak or no signal. These results fully demonstrated that the three aminoluciferin analogues have higher sensitivity for bioluminescence imaging in live mice. Meanwhile, up to 7 h light emission time of aminoluciferin analogues were significantly greater than 10 min of D-luciferin and aminoluciferin, beneficial to real-time dynamic monitoring imaging (Figure 3C). In addition, 5-cyL, 6-cyL and 7-cyL have higher Cy5.5 filter (695–770 nm bandpass) transmittance ratio, contributing to the imaging of deep tissue in live organism.

Figure 3. Bioluminescence imaging of mouse xenograft tumor models with aminoluciferin analogues. a) Maximum total photon flux from with nude mice inoculated ES-2-Luc tumors. Mice were injected i.p. with 5-cyL, 6-cyL, 7-cyL, D-luciferin and aminoluciferin (1 µM, 100 µM, 1 mM and 10 mM) and imaged 10 min after injection. b) Quantification of the images from (a); error bars, s.e.m. (n = 3 experiments). c) Time–response analysis of BLI in mouse xenograft tumor models with 1 mM cyL.

[FVB-Tg Transgenic Mice Imaging]

In light of in vivo imaging activity, we chose 7-cyL that performed excellent in the nude mice with tumor xenografts assay for further bioluminescence imaging in transgenic mouse ubiquitously expressing firefly luciferase (FVB-Tg mice). As expected, when 100 μL 7-cyL (1 mM) injected i.v to FVB-Tg mice, its bioluminescence intensity presented up to 10-fold higher than that of D-luciferin and aminoluciferin (Figure 4A). Moreover, in the measurement process, our aminoluciferin analogues have not only higher bioluminescence intensity but also longer in vivo bioluminescence time (Figure 4B). Interestingly, when computing the ratio of brain BLI intensity relative to the whole-body bioluminescence signal in FVB-Tg mice, we found that 7-cyL has higher brain bioluminescence ratio than D-luciferin and aminoluciferin (Figure 4C). As a result, we believed that 7-cyL has an excellent ability to penetrate the blood-brain barrier.

Figure 4.Bioluminescence imaging comparison of 7-cyL, D-luciferin and aminoluciferin (1 mM) in FVB-Tg mice. a) Photon flux from FVB-Tg mice injected i.v. with 100 µL of 1 mM 7-cyL、D-luciferin and aminoluciferin. b) Time–response analysis of BLI in FVB-Tg mice with 1 mM 7-cyL、D-luciferin and aminoluciferin. c) The ratio of brain BLI intensity in FVB-Tg mice with 7-cyL, D-luciferin and aminoluciferin (1 mM) (n = 3). Error bars, s.e.m. ****P < 0.0001 (t-test).

[Brain tumor bioluminescence imaging]

As we know, the blood-brain barrier as a protective barrier limits the access of many small molecules to this tissue. So, the bioluminescence imaging in the brain is particularly challenging. In view of the real biological activity of 7-cyL in living mice, we asked whether 7-cyL could improve bioluminescent signals in the brain. To evaluate the ability of 7-cyL to access the blood-brain barrier, we imaged nude mouse that had been inoculating with luciferase-expressing U87-Luc cells in brain hippocampus. After two weeks, these nude mice with tumor xenografts in the brain were injected i.p. with 100 μL of 7-cyL, D-luciferin and aminoluciferin (1 mM). Significantly, 7-cyL showed 30-fold higher bioluminescence intensity than D-luciferin (Figure 5). It is not difficult to see that 7-cyL can readily cross the blood-brain barrier and access deep brain regions in living mice. Overall, 7-cyL has distinctive advantages in brain bioluminescence imaging.

Figure 5.Bioluminescence imaging comparison of 7-cyL, D-luciferin and aminoluciferin (1 mM) in brain. a): Photon flux from nude mouse inoculated with U87-Luc cells in brain hippo-campus after i.v. injection with 100 μL of 1 mM 7-cyL, D-luciferin and aminoluciferin; b): Maximum bioluminescence emission for mice with 7-cyL, D-luciferin and aminoluciferin (1 mM) (n = 3). Error bars, s.e.m. ****P < 0.0001 (t-test); c): Time–response analysis of BLI in nude mouse brain with 1 mM 7-cyL, D-luciferin and aminoluciferin.