MRI Bimodal Probes for in vivo Imaging

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Activatable NIR Fluorescence/MRI Bimodal Probes for in vivo Imaging by Enzyme-Mediated Fluorogenic Reaction and Self-Assembly Runqi Yan, Yuxuan Hu, Fei Liu, Shixuan Wei, Daqing Fang, Adam J. Shuhendler, Hong Liu, Hong-Yuan Chen, and Deju Ye J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b03649 • Publication Date (Web): 07 Jun 2019 Downloaded from http://pubs.acs.org on June 7, 2019

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Journal of the American Chemical Society

Activatable NIR Fluorescence/MRI Bimodal Probes for in vivo Imaging by Enzyme-Mediated Fluorogenic Reaction and SelfAssembly Runqi Yan†,‖, Yuxuan Hu†,‖, Fei Liu†, Shixuan Wei†, Daqing Fang‡, Adam J. Shuhendler§, Hong Liu‡, Hong-Yuan Chen†, Deju Ye†,Ф* †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China ‡ State

key Laboratory of Drug Research and Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China §

Department of Chemistry & Biomolecular Sciences, University of Ottawa, Ottawa, ON, K1N 6N5, Canada

Ф Research

Center for Environmental Nanotechnology (ReCent), University, Nanjing, 210023, China

KEYWORDS: In situ Self-assembly, multimodality imaging, activatable probe, fluorogenic reaction, in vivo ABSTRACT: Stimuli-responsive in situ self-assembly of small molecules to form nanostructures in living subjects has produced promising tools for molecular imaging and tissue engineering. However, controlling the self-assembly process to simultaneously activate multimodality imaging signals in a small-molecule probe is challenging. In this paper, we rationally integrate a fluorogenic reaction into enzyme-responsive in situ self-assembly to design small-molecule-based activatable near-infrared (NIR) fluorescence and magnetic resonance (MR) bimodal probes for molecular imaging. Using alkaline phosphatase (ALP) as a model target, we demonstrate that probe (P-CyFF-Gd) can be activated by endogenous ALP overexpressed on cell membranes, producing membrane-localized assembled nanoparticles (NPs) that can be directly visualized by cryo-SEM. Simultaneous enhancements in NIR fluorescence (> 70-fold at 710 nm) and r1 relaxivity (~2.3-fold) enable real-time, high-sensitivity, high-spatial-resolution imaging and localization of the ALP activity in live tumor cells and mice. P-CyFF-Gd can also delineate orthotopic liver tumor foci, facilitating efficient real-time, image-guided surgical resection of tumor tissues in intraoperative mice. This strategy combines activatable NIR fluorescence via a fluorogenic reaction and activatable MRI via in situ self-assembly to promote ALP activity imaging, which could be applicable to design other activatable bimodal probes for in vivo imaging of enzyme activity and locations in real time.

■ INTRODUCTION Multimodality molecular imaging probes are essential to advancing biomedical and clinical research;1-3 however, bimodality imaging probes with a synergetic combination of magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence represent an unmet need in early diagnosis malignant tumors. MRI can produce anatomic images with unlimited tissue-penetration depth and high spatial resolution to promote preoperative detection of deep-seated tumors;4-7 NIR fluorescence can generate images with high sensitivity, useful for detecting low concentrations of tumor-associated biomarkers and for image-guided tumor surgery.8-13 Several NIR fluorescence/MRI bimodal probes have been reported that offer high sensitivity and high spatial resolution for molecular imaging and surgical guidance.14-15 However, most probes display ‘always-on’ signals regardless of interactions with tumor cells, which can cause low tumor-

to-background ratios (TBR). Although several activatable NIR fluorescence/MR bimodal probes with ‘off–on’ signals upon interaction with biological targets have enabled realtime imaging, they are often built as NPs, which suffer from poor reproducibility and low tumor penetrability due to their large size.16,17 Experts have suggested developing small-molecule-based activatable probes; the well-defined chemical structure and small size of these molecules enable them to extravasate and diffuse deeply into tumor tissues to reach molecular targets and become activated.1820 Yet the design of small-molecule probes capable of simultaneously switching on NIR fluorescence and MRI signals upon interaction with tumor cells remains challenging. In addition, probes’ small size can lead to fast washout from the target site and decrease tumor accumulation and TBR, thus compromising long-term molecular imaging. Therefore, a new strategy to design activatable fluorescence/MR bimodality imaging

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Figure 1. Schematic illustration shows an ALP-activatable NIR fluorescence (FL)/MR bimodal probe for in vivo imaging. (a) Chemical structure of P-CyFF-Gd, and proposed ALP-mediated fluorogenic reaction and in situ self-assembly of P-CyFF-Gd into NPs that show increased NIR FL and r1 relaxivity. (b) Proposed mechanism of P-CyFF-Gd for NIR FL/MR bimodality imaging of ALP-positive tumor cells in vivo. Following systemic administration into mice, P-CyFF-Gd as a small molecule may easily across blood vessel and diffuse into tumor tissues. In tumor cells that express high levels of ALP, P-CyFF-Gd is dephosphorylated by membrane-bound ALP and converted into fluorescent CyFF-Gd, which subsequently self-assemble into fluorescent and magnetic NPs. These assembled NPs are mainly retained on cell membrane capable of reporting the ALP activity and locations; some of them can further enter into tumor cells through endocytosis. The accumulation on cell membrane or in lysosomes can prolong retention in tumor tissues, thereby leading to high NIR FL and MRI contrast. (c) The chemical structure of designed non-assembled control probe, P-Cy-Gd.

probes capable of high tumor accumulation is urgently needed.

permeability

and

Recently, stimuli-triggered in situ self-assembly of small molecules to form nanostructures has become a promising strategy to build molecular imaging probes.21-25 Instead of directly using preformed nanomaterials, this strategy begins with small molecules (or peptides) with welldefined chemical structures, which may easily cross blood vasculature and penetrate tumors; when reacting with tumor-related stimuli (e.g., enzyme), molecules are converted into nanostructures at the tumor site to prolong retention and amplify imaging signals. This approach leverages the strong tumor permeability of small molecules and the prolonged tumor retention of nanomaterials, showing promise for imaging of biomolecules in tumors. Rao and colleagues proposed a target-enabled in situ ligand self-assembly strategy based on a firefly luciferininspired biocompatible reaction,26 which could build NIR

fluorescence,27 positron emission tomography (PET),28 or MRI29-31 probes to noninvasively image caspase-3/7 activity in mice. Xu and colleagues developed an enzymeinstructed self-assembly strategy to enable molecular selfassembly of fluorescent Phe-Phe dipeptide-based hydrogelators into nanofibers in culture cells.32-34 Recently, this strategy was utilized by Wang’s,35 Liang’s,36 and our group,37 respectively, to build activatable photoacoustic imaging probes for in-vivo studies. However, limited reports have investigated the potential of this strategy in building activatable multimodality imaging probes. To our knowledge, no research has developed small-moleculebased probes which can be activated to turn on NIR fluorescence and MRI signals after self-assembly into NPs. Herein, we design and synthesize small-molecule-based activatable NIR fluorescence/MRI bimodal probes (fluorogenic MRI probes) for in vivo


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Figures 2. Characterization of P-CyFF-Gd in vitro. (a) HPLC and (b) DLS analysis of P-CyFF-Gd (200 μM) incubating with the ALP (2 U/mL) at 37 °C for indicated time. (c) TEM and AFM images of NPs formed by incubating P-CyFF-Gd (200 μM) with ALP (2 U/mL, 37 °C) for 30 min. Scale bars: 200 nm. (d) UV-Vis absorption and (e) fluorescence spectra of P-CyFF-Gd (5 μM) incubating with the ALP (0.1 U/mL, 37 °C) for 0 to 40 min. λex = 680 nm. Inset: photographs (d) and fluorescence images (e) of P-CyFF-Gd (5 μM) pre (-) and post (+) incubation with the ALP (0.1 U/mL). (f) T1-weighted MR images and T1 values (0.5 T) of P-CyFF-Gd (200 μM) in Tris buffer upon incubation with ALP (2 U/mL) for 0 to 40 min. (g) Fluorescence spectra of P-CyFF-Gd (5 μM) following incubation with varying concentration (0, 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 50, 75 and 100 U/L, 37 °C) of ALP for 30 min. (h) Fluorescence spectra and (i) T1-weighted MR images of P-CyFF-Gd incubated with trypsin, BSA, lysozyme, ACP, ALP or ALP together with its inhibitor Na3VO4 in Tris buffer (pH 8.0).

imaging through an enzyme-triggered fluorogenic reaction and in situ self-assembly strategy. This strategy leverages the catalytic activity of an enzyme that facilitates amplification of NIR fluorescence via an ongoing fluorogenic reaction along with in situ self-assembly, enabling augmentation of MR properties and enhanced accumulation in target tissue. Using alkaline phosphatase (ALP) as a model enzyme, we show that the designed activatable bimodal probe allows for noninvasive, highsensitivity, spatial-resolution imaging of subcutaneous ALP-positive tumors along with NIR fluorescence-guided real-time surgical resection of orthotopic liver tumors in mice. This novel demonstration of small-molecule-enabled amplification of NIR fluorescence and MRI signals in living

animals highlights the potential of this strategy for tumor imaging. ■ RESULTS Design and synthesis of ALP-activatable bimodal probe. Figure 1a depicts the design of an ALP-activatable bimodal imaging probe, P-CyFF-Gd, consisting of (1) a prequenched NIR fluorophore (merocyanine, Cy-Cl) capped with an ALP recognition phosphate group (-PO3H); (2) a paramagnetic DOTA-Gd chelate for MRI; and (3) a hydrophobic dipeptide Phe-Phe (FF) linker to promote self-assembly. With the presence of hydrophilic -PO3H and DOTA-Gd groups, P-CyFF-Gd is initially a water-soluble small-molecule probe that exhibits off NIR fluorescence and low r1 relaxivity. After systemic administration,



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Table 1. Effective r1 relaxivities of Gd-based MR probes.a MR Probe

r1 (mM-1 s-1 )

MR Probe

r1 (mM-1 s-1 )

P-CyFF-Gd

8.9 ± 0.3

P-Cy-Gd

5.8 ± 0.3

P-CyFF-Gd + ALP b

20.1 ± 0.5

P-Cy-Gd + ALP b

5.9 ± 0.3

Preformed NPs c

20.7 ± 0.3

Dotarem

5.4 ± 0.3

P-CyFF-Gd + ALP + Na3VO4

9.5 ± 0.3

T1 values (0.5 T) were measured in Tris buffer (pH 8.0), using an inversion recovery spin-echo sequence, and the effective r1 relaxivities were obtained by plotting 1/T1 versus the concentration of Gd3+ (see Figure S6). Values denote mean ± SD. b 2 U/mL ALP was used for the reaction. c Preformed NPs were obtained by dispersing chemically synthesized CyFF-Gd in Tris buffer (pH 8.0).

a

P-CyFF-Gd may easily extravasate and diffuse deeply into tumor tissues due to its small molecular size and hydrophilicity (Figure 1b). In ALP-positive tumor tissues, an efficient fluorogenic reaction to uncage P-CyFF-Gd by membrane-bound ALPs is initiated, releasing hydrophobic dephosphorylated product CyFF-Gd that emits NIR fluorescence at 710 nm. CyFF-Gd contains an FF dipeptide

that can offer efficient intermolecular interactions (e.g., hydrophobic interactions, π-π stacking) and promote molecular self-assembly,32,38 generating fluorescent and magnetic NPs. NPs have a substantially larger molecular size than P-CyFF-Gd, potentially restricting molecular rotation and prolonging tumbling time (τR) of Gd-chelates and augmenting r1 relaxivity. On-site assembling NPs are also prone to anchoring on the cell membrane, which can facilitate cellular uptake and localization in lysosomes through endocytosis.39 Prolonged retention in ALP-positive tumors can be achieved, whereas un-activated P-CyFF-Gd molecules are likely washed out from other ALP-deficient normal tissues. Concomitant enhancement of NIR fluorescence and MR contrast can thus be observed, allowing for high-sensitivity and spatial-resolution in vivo imaging of tumors.

The fluorogenic MRI probe P-CyFF-Gd was synthesized according to the procedure illustrated in Scheme S1. The uncaged product CyFF-Gd was also synthesized to validate dephosphorylation and self-assembly (Scheme S2). A control probe (P-Cy-Gd) consisting of a caged Cy-Cl fluorophore and a DOTA-Gd chelate but without the dipeptide FF (Figure 1c), which prevents self-assembly of dephosphorylated product (Cy-Gd) after an ALP-triggered fluorogenic reaction, was synthesized to examine the role of self-assembly in P-CyFF-Gd (Scheme S3). ALP-mediated dephosphorylation and self-assembly of P-CyFF-Gd in vitro. ALP-mediated dephosphorylation and self-assembly of P-CyFF-Gd was investigated upon incubation with ALP (2 U/mL, Tris buffer, pH 8.0). HPLC and MALDI-MS analysis showed that P-CyFF-Gd (200 μM, tR = 12.9 min) was rapidly converted into CyFF-Gd (tR = 15.2 min; halflife less than 10 min), and dephosphorylation was nearly completed after 30 min (Figure 2a, Figure S1). Along with dephosphorylation, dynamic light scattering analysis of the reaction solution revealed a fast and continuous self-assembly process: obvious NPs with hydrodynamic diameter of ~25 nm appeared at 10 min, which increased to and were stabilized at

~66 nm after 30 min (Figure 2b, Figure S2). The polydispersity index (PDI) of the assembled NPs after 30 min was ~0.258 (Table S1). The formation of mono-disperse NPs was verified by atomic force microscope (AFM) and transmission electron microscope (TEM) analysis, and energy-dispersive X-ray spectroscopy confirmed the existent of Gd element (Figure 2c, Figure S3). The size and morphology were similar to those of NPs formed via direct self-assembly of chemically synthesized CyFF-Gd (Figure S4), indicating that the observed NPs were generated through self-assembly of ALP-triggered dephosphorylated products. ALP-activated NIR fluorescence and MRI signals of PCyFF-Gd in vitro. As the presence of capping group -PO3H can prevent internal charge transfer in fluorophore Cy-Cl, PCyFF-Gd (5 μM) initially displayed two absorption peaks at 605 nm and 652 nm along with weak fluorescence in NIR regions (Figure 2d and 2e).40,41 Upon incubation with ALP (100 U/L), the UV-Vis absorption gradually bathochromically shifted to 688 nm, and the NIR fluorescence at 710 nm increased clearly. The UV-Vis absorption and NIR fluorescence each peaked after 30 min, aligning well with the fast dephosphorylation process analyzed by HPLC (Figure 2a). Based on enhanced fluorescence, kinetic parameters of PCyFF-Gd towards ALP were measured using the MichaelisMenten equation. The maximum reaction rate, Vmax, and apparent Michaelis constant, Km, were ~1.05 μM min-1 and ~13.14 μM, respectively (Figure S5). The kcat/Km value was ~3.6 × 104 M-1 s-1, similar to other previously reported probes for ALP detection (Table S2); therefore, P-CyFF-Gd appeared to be an efficient substrate for ALP.

Along with activation of NIR fluorescence, r1 relaxivity was substantially enhanced in P-CyFF-Gd after incubation with ALP in Tris buffer (Figure S6, Table 1). The r1 relaxivity of P-CyFF-Gd at 0.5 T was 8.9 ± 0.3 mM-1 s-1. Upon ALP activation, it increased to 20.1 ± 0.5 mM-1s-1, ~3.8-fold higher compared to that of Dotarem (5.4 ± 0.3 mM-1s-1). The large r1 relaxivity of P-CyFF-Gd after activation was similar to that of preformed NPs generated from direct dispersion of CyFF-Gd in Tris buffer (20.7 ± 0.3 mM-1s-1), which concurred with our previously reported self-assembling Gd-containing NPs.42 The higher r1 relaxivity of P-CyFF-Gd in response to ALP could be ascribed to the formation of Gd-NPs, which could progressively shorten the T1


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Figure 3. Imaging of ALP activity in tumor cells. (a) NIR fluorescence imaging of HeLa cells incubated with P-CyFF-Gd (100 μM) for 10 to 60 min. (b) Representative cryo-SEM image of self-assembled NPs anchoring on the membrane of a HeLa cell following incubation with P-CyFF-Gd (100 μM) for 1 h. (c) Enlarged cryo-SEM image of the NPs in (b). (d) Enlarged cryo-SEM image of the NPs in (c). The red boxed indicate the enlarged areas. (e) TEM image of the self-assembled NPs collected from membranes of live HeLa cells following incubation with P-CyFF-Gd (100 μM) for 1 h. (f) Fluorescence (up) and T1-weighted MR (down) (TE/TR = 5/100 ms, 1 T) images of indicated cell pellets. λex/em = 670/(750 ± 50) nm. (g) Comparison of the average FL intensity (red) and T1 value (0.5 T, black) of cell pellets in (f). (n=3). (h) ICP-MS analysis of the Gd contents that are resided on cell membrane or intracellularly after indicated treatment. (n=3). I: HeLa cells incubated with P-CyFF-Gd (100 μM, 1 h); II: HeLa cells pretreated with Na3VO4 (10 mM, 20 min), followed by incubating with P-CyFF-Gd (100 μM, 1 h); III: HEK293T cells incubated with P-CyFFGd (100 μM, 1 h); IV: HeLa cells incubated with P-Cy-Gd (100 μM, 1 h). Values denote mean ± SD.

of water protons to produce brighter T1-weighted MR images (Figure 2f). Although the incubation of control probe P-Cy-Gd with ALP could produce dephosphorylated product Cy-Gd and significantly enhance NIR fluorescence at 710 nm, no NPswere detected in the reaction solution (Figure S7). Subsequent measurements of the T1 value and r1 relaxivity showed little change in P-Cy-Gd solution before and after incubation with ALP (Figure S7d, Table 1). These findings suggest that the dipeptide linker FF was essential to drive molecular self-assembly, producing Gd-containing NPs with significantly higher r1 relaxivity compared with nonaggregation molecules (e.g., P-CyFF-Gd, P-Cy-Gd, Cy-Gd). Given the distinct r1 relaxivity between NPs and monomeric molecules, the critical micelle concentration (CMC) of CyFF-Gd was approximately 16 μM in Tris buffer (pH 8.0) (Figure S8). To examine whether CyFF-Gd selfassembly could induce NIR fluorescence quenching, we incubated 25 μM of P-CyFF-Gd (greater than the CMC of CyFF-Gd) with ALP (100 U/L) for 30 min. CyFF-Gd (25 μM) absorption shifted red compared with Cy-Gd; a new

shoulder peak at ~775 nm appeared, signaling the formation of nanoaggregates in CyFF-Gd solution (Figure S9). A ~70-fold enhancement in NIR fluorescence was achieved for P-CyFF-Gd after dephosphorylation. This enhancement was only slightly weaker than that of Cy-Gd (25 μM), suggesting non-obvious quenching of NIR fluorescence within NPs (Figure S10). The ALP-induced fluorogenic reaction and self-assembly of P-CyFF-Gd could thus turn on NIR fluorescence and MRI signals. Next, we examined the sensitivity of P-CyFF-Gd to detect ALP in solution; fluorescence increased with ALP concentration (Figure 2g). A plot of fluorescence intensity (710 nm) compared with ALP concentration revealed a linear correlation within 0.5–5 U/L. The detection limit was ~17 mU/L (single-to-noise = 3; Figure S11), similar to the range of other reported probes and indicating good sensitivity for ALP (Table S2). A subsequent test of P-CyFFGd selectivity towards ALP over other representative enzymes (e.g., Trypsin, BSA, Lysozyme, acid phosphatase (ACP)) showed that only ALP could obviously augment NIR fluorescence and produce brighter T1-weighted MR



images in Tris buffer (pH 8.0), which were greatly suppressed when ALP activity was inhibited by Na3VO4 (100 µM) (Figures 2h and 2i). Hence, P-CyFF-Gd appeared specific for ALP in enzyme reaction buffer (Tris, pH 8.0). The pH effect on P-CyFF-Gd activation by ALP was also examined. The optimum pH to turn on P-CyFF-Gd fluorescence was within 7.0-8.0 (Figure S12). Stability test revealed that P-CyFF-Gd was stable in culture medium (DMEM), but it could be activated slowly in the DMEM medium containing 10% FBS or mouse whole blood due to the existent of ALP (Figures S13). In addition, the assembled NPs exerted high stability in Tris buffer under different pH or Tris buffer containing 10% serum (Figures S14, Figures S15). Fluorescence and MR imaging of ALP in living cancer cells. Before applying P-CyFF-Gd to detect ALP activity in living cells, we evaluated its cytotoxicity against normal human embryonic kidney cells (HEK293T), human cervical cancer cells (HeLa) and liver cancer cells (HepG2) using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay. Figure S16 indicates that P-CyFF-Gd had little effect on cell viability against tested cell lines, suggesting P-CyFF-Gd as a biocompatible probe for cell studies. We then applied P-CyFF-Gd to detect ALP activity in living HeLa cells, which were reported to overexpress ALP on the cell surface.43 P-CyFF-Gd is a fluorogenic probe and can image ALP activity in real time. When HeLa cells were incubated with P-CyFF-Gd (100 μM), NIR fluorescence gradually lit up and was distributed mainly on the cell membrane, where ALP molecules tended to locate. After 45 min, NIR fluorescence appeared in the interior of HeLa cells, which became brighter upon prolonging the incubation time to 60 min (Figure 3a). Distribution of NIR fluorescence on the cell membrane and intracellularly was verified using confocal z-scan imaging of HeLa cells (Figure S17). Colocalization studies with a lysosomal tracker revealed punctate intracellular fluorescence mainly distributed in the lysosomes (Figure S18). By contrast, negligible fluorescence was found in Na3VO4-treated HeLa cells and ALP-deficient HEK293T cells following incubation with P-CyFF-Gd (Figure S19). When phosphate (10 mM) was added into the culture medium to inhibit membrane-bound ALP activity,44 little fluorescence on cell membranes or lysosomes could be observed (Figure S20). When incubation of HeLa cells with P-Cy-Gd, gradually enhanced NIR fluorescence was distributed only in the culture medium (Figure S21). Moreover, neither the cell membrane nor lysosomes displayed obvious NIR fluorescence when we incubated HeLa cells with preformed NPs or ALP-deficient HEK293T cells with PCyFF-Gd and ALP (Figure S22, S23). We used HPLC to confirm the production of dephosphorylated CyFF-Gd in HeLa cells (Figure S24). Following incubation of P-CyFF-Gd (100 μM) with HeLa cells for 1 h, P-CyFF-Gd and CyFF-Gd were detected at a ratio of ~1.5: 1 in the culture medium. After removing the medium, only CyFF-Gd was clearly observed in lysed cell pellets. Conversely, upon incubation with Na3VO4-treated

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HeLa cells or HEK293T cells, little CyFF-Gd appeared in cell pellets. As with P-CyFF-Gd, HeLa cells incubated with P-Cy- Gd (100 μM, 1 h) produced a substantial amount of dephosphorylated Cy-Gd; however, nearly all Cy-Gd were distributed in the culture medium with little in cell pellets. We then employed cryo-SEM to image in-situ-formed NPs directly on the HeLa cell membrane. Figures 3b–d display many uniform NPs on the surface of a HeLa cell upon incubation with P-CyFF-Gd (100 μM, 1 h), and the average diameter was ~60 nm; no similar NPs were found on blank HeLa cells (Figure S25). These membrane-bound NPs were collected from the membranes of HeLa cells and analyzed by TEM (Figure 3e, S26), confirming the formation of spherical NPs with sizes similar to those of NPs formed via incubation of P-CyFF-Gd with ALP in Tris buffer (Figures 2b and 2c). In addition, TEM analysis of the culture medium revealed that some in-situ-formed NPs were also distributed extracellularly (Figure S27), correlating to that of CyFF-Gd detected by HPLC (Figure S24). Encouraged by the above results, we investigated NIR fluorescence and MR bimodality imaging of ALP activity in HeLa cell pellets. Figure 3f indicates that HeLa cells incubated with P-CyFF-Gd (100 μM, 1 h) displayed significantly brighter NIR fluorescence than the other controls, which correlated well with confocal fluorescence imaging of individual cells on dishes (Figure S19). Remarkably enhanced MR contrast was produced in PCyFF-Gd-treated HeLa cell pellets but was significantly lower in Na3VO4-treated HeLa cells, ALP-deficient HEK293T cells, and HeLa cells incubated with P-Cy-Gd. This difference may be attributable to enhanced r1 relalxivity and NP accumulation in HeLa cells, thus shortening water protons’ T1 relaxation time in these cells (Figure 3g, Figure S28). Subsequent ICP-MS analysis verified that HeLa cells incubated with P-CyFF-Gd contained large Gd3+ contents (∼4.09 fmol/cell on membrane and ∼0.59 fmol/cell intracellularly), whereas the other controls had negligible amounts (∼0 to ~0.1 fmol/cell on membrane and ~0.02 to ~0.05 fmol/cell intracellularly) (Figure 3h). These results imply that PCyFF-Gd could be efficiently dephosphorylated by membrane-bound ALP, producing CyFF-Gd that underwent efficient molecular self-assembly. These insitu-formed NPs could reduce diffusion and enhance accumulation near the ALP site, producing strong NIR fluorescence and MR contrast to report on ALP activity. Membrane-adherent NPs could also facilitate clathrindependent endocytosis (Figure S29), prolonging retention in HeLa cells for long-term imaging. Bimodality Imaging of ALP in living mice. To elucidate the ability of P-CyFF-Gd for in-vivo imaging, ALP-triggered activation of NIR fluorescence and MR contrast were investigated in living mice through subcutaneous (s.c.) injection of P-CyFF-Gd with or without ALP. Longitudinal fluorescence imaging showed rapid fluorescence turn-on at the injection site containing PCyFF-Gd and ALP (Figure S30a). Fluorescence intensity


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Figure 4. Bimodality imaging of endogenous ALP in living mice of HeLa tumor. (a) Longitudinal FL imaging and (c) normalized FL intensity of mice receiving i.v. injection of P-CyFF-Gd (I), P-Cy-Gd (II) or P-CyFF-Gd (50 μM, 200 μL) together with i.t. injection of 10 mM Na3VO4 (50 μL) at 0, 1, 2, 4 and 8 h (III). (b) T1-weighted MR images and (d) % SE of HeLa tumor-bearing mice receiving i.p. injection of P-CyFF-Gd (I), P-Cy-Gd (II) or P-CyFF-Gd (0.015 mmol kg-1 Gd3+) together with i.t. injection of Na3VO4 (10 mM, 50 μL). Images were acquired before (Pre), 2, 4, 6 and 10 h after injection, using TE/TR = 15/446 ms at 1 T. (e) HPLC trace of P-CyFFGd (black), CyFF-Gd (blue), or tumor lysate resected from mice of HeLa tumor at 2 h following injection of P-CyFF-Gd (50 μM, i.v.) (red). (f) Biodistribution (% ID/g) of P-CyFF-Gd (red) or P-Cy-Gd (blue) in HeLa tumors and main organs (H: heart, Li: liver including gallbladder, Lu: lung, Sp: spleen, Ki: kidneys, St: stomach, In: intestines, T: tumor) at 4 h after i.p. injection into mice (0.015 mmol kg-1 Gd3+). The amount of Gd3+ in tumors and main organs were determined by ICP-MS. White arrows indicate the Dotarem solution (1 mM) as the internal standard. Red arrows point the tumor locations in mice. Data denote mean ± SD (n = 3, *P < 0.05, ***P < 0.001).

peaked at 1 h, more than 2.4-fold higher than that with PCyFF-Gd alone (Figure S30b). As with fluorescence, a timedependent enhancement in T1-weighted MR contrast at the injection site containing P-CyFF-Gd (400 μM) and ALP (4 U/mL) was clearly observed; the MRI intensity peaked at 1 h. By contrast, the MRI intensity declined immediately for P-CyFF-Gd alone, presumably due to fast clearance of unactivated small molecules of P-CyFF-Gd (Figure S30c and d). Significant enhancements in NIR fluorescence and MR contrast at the ALP-containing site compared to the ALP-free site demonstrated that P-CyFF-Gd could be efficiently activated by ALP in vivo. We then applied P-CyFF-Gd for the detection of endogenous ALP in subcutaneous HeLa-tumor-bearing mice via NIR fluorescence and MR bimodality imaging. Figure 4a reveals that after intravenous injection of PCyFF-Gd (200 μL, 50 μM), imaging mice displayed gradually increasing tumor fluorescence, which peaked at 2 h and could be reduced when Na3VO4 (10 mM) was injected into tumors to inhibit ALP activity (Figure 4c).

Although mice injected with P-Cy-Gd exhibited strong tumor fluorescence at 1 h, fluorescence declined rapidly to the level of Na3VO4-treated tumors after 2 h―~2.8-fold lower than in P-CyFF-Gd-treated mice. When receiving i.p. injection of P-CyFF-Gd (0.015 mmol kg-1 Gd), gradually enhanced T1-weighted MR contrast in HeLa tumors was also observed (Figure 4b). The maximum signal enhancement (%SE) in tumors was ~58% at 4 h, ∼2.8-fold and ~4.1-fold higher than in those treated with P-Cy-Gd (~21%) and P-CyFF-Gd together with Na3VO4 (~14%), respectively (Figure 4d). As MRI can produce highresolution multi-slice images capable of mapping wholetumor tissue (Figure S31), the enhanced MR contrast was clearly distributed within the tumor tissues, verified by fluorescence imaging of tumor tissue slices (Figure S32). HPLC analysis of tumors resected from mice at 2 h postinjection of P-CyFF-Gd indicated that most P-CyFF-Gd was converted into CyFF-Gd in tumors, confirming efficient dephosphorylation in living



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Figure 5. Bimodality imaging and fluorescence-guided surgery of orthotopic liver tumors. (a) Whole-body fluorescence (down) and bioluminescence (up) imaging of normal mice and orthotopic HepG2/Luc liver tumor xenograft mice. The fluorescence images were acquired in living mice following i.v. injection of P-CyFF-Gd (50 μM, 200 μL) at 4 h (λex/em = 660/710 nm). (b) T1weighted MR imaging of orthotopic HepG2/Luc liver tumor xenograft mice. Images were acquired before (Pre-contrast) and 4 h post i.p. injection of P-CyFF-Gd (0.015 mmol kg-1) at 1 T. Red dotted circles indicate the locations of tumor in liver. White arrow indicates the gallbladder. (c) Imaging-guided surgical resection of orthotopic HepG2/Luc liver tumor in an intraoperative mouse 30 min after directly spraying P-CyFF-Gd (10 μM) on liver. White arrow indicates the resected tumor tissue, and red arrow indicates the tumor foci in liver tissue detected by NIR fluorescence imaging.

mice (Figure 4e). These results demonstrate that P-CyFFGd, as a small molecule, has good permeability to readily diffuse into tumor tissues and become activated. The extensive NIR fluorescence distributed in the interior of multicellular spheroids (MCSs) of HeLa cells confirms this finding (Figure S33). The biodistribution of P-CyFF-Gd and P-Cy-Gd was examined via ex vivo fluorescence imaging of main organs and tumors resected from mice. Figure S34 shows that PCyFF-Gd-treated tumors exhibited the brightest fluorescence among all resected tissues at 2 h post i.v. injection; the fluorescence intensity was approximately two-fold higher than that of livers including gallbladder. Moreover, P-CyFF-Gd-treated tumors displayed ~3.1-fold higher fluorescence intensity than P-Cy-Gd-treated tumors. ICP-MS analysis confirmed that the ID% g-1 in PCyFF-Gd-treated tumors (~23.5%) was much higher than that of other organs and ~2.3-fold higher than that of P-CyGd-treated tumors (~11.1%) (Figure 4f). These results corroborate the significantly brighter fluorescence and MR

contrast observed noninvasively in living mice (Figure 4a and 4b), suggesting that ALP-mediated fluorogenic reaction and in situ self-assembly were efficient to activate P-CyFF-Gd and enhance accumulation in ALP-related tumors. Efficient fluorescence and MR bimodality imaging of ALP-related tumors in vivo were thus achieved. Figure 4f also illustrates that the ID% g-1 in livers (including gallbladder) was much higher in P-CyFF-Gd-treated relative to P-Cy-Gd-treated mice but opposite in kidneys. This discrepant biodistribution could be due to different elimination pathways between dephosphorylated products as evidenced by coronal T1-weighted MR images (Figure S35). Imaging-guided surgery of orthotopic liver tumors. Considering that the ALP-mediated fluorogenic reaction and in-situ self-assembly could facilitate localization of activated NPs in tumor tissues, P-CyFF-Gd was applied to visualize and guide resection of orthotopic HepG2 liver tumors. ALP expression in HepG2 cells was investigated by incubating cells with P-CyFF-Gd, revealing similarly strong


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NIR fluorescence as in HeLa cells (Figure S36). Then, we injected luciferase-transfected HepG2 cells (HepG2/Luc) into the left lobe of livers to establish orthotopic liver tumors in mice. After 2 weeks, strong bioluminescence (BL) indicated successful growth of HepG2/Luc tumors in the livers. Following i.v. administration of P-CyFF-Gd, bright NIR fluorescence was found in the left liver of mice implanted with a HepG2/Luc tumor, agreeing well with the BL imaging region; mice with a normal liver showed much weaker fluorescence and BL (Figure 5a). P-CyFF-Gd also produced higher T1-weighted MR contrast in the tumor region relative to surrounding normal liver tissues (Figure 5b). These results indicate that P-CyFF-Gd could report the location of orthotopic liver tumors in living mice through noninvasive imaging with NIR fluorescence and MRI. Given these results, we performed fluorescence-guided dissection of orthotopic HepG2/Luc tumors in intraoperative mice by directly spraying P-CyFF-Gd on the liver. After 30 min, strong fluorescence enabling accurate delineation of tumor margins in the liver was produced and could guide efficient surgical resection of tumor tissues (Figure 5c). BL imaging of intraoperative mice confirmed successful dissection of tumors from the liver, highlighting P-CyFF-Gd as a promising molecular imaging tool to guide orthotopic liver tumor surgery. ■DISCUSSION Fluorogenic probes are crucial analytical tools in biology, as in situ formation of fluorescent products via fluorogenic reactions can offer ‘off-on’ fluorescence for sensitive and specific detection of biomolecules.45,46 However, due to tissue-based scattering and absorption of photons, fluorogenic probes are prone to low spatial resolution and shallow penetration in in-vivo imaging. In this work, we combined a fluorogenic reaction with enzyme-mediated in situ self-assembly, providing a novel small-molecule-based fluorogenic MRI probe that elicited large enhancements in NIR fluorescence and in r1 relaxivity upon reacting with an enzyme of interest (i.e., ALP). The synergetic combination of activatable MRI and NIR fluorescence signals facilitated real-time, high-sensitivity, high-spatial-resolution in vivo imaging of enzyme activity and locations. This strategy is particularly promising for in-vivo imaging. First, the probe consists of a small-molecule organic compound with a well-defined chemical structure that is biocompatible and likely to react with the enzyme, ensuring fast enzymatic reaction kinetics (kcat/Km = ~3.6 × 104 M-1 s-1 towards ALP; Figure S5). Second, this small probe is prone to diffuse into interstitial spaces and penetrate tissues (e.g., tumors) deeply, ensuring greater activation to enable detection of enzyme activity in MCSs (Figure S33) and tumor xenograft mice (Figure 4 and 5). Third, in situ self-assembly helps NPs, which are normally diffusionresistant due to their increased size, to localize activated probes near enzyme locations (e.g., the cell membrane where ALP is located) and provide localized imaging signals that can spatiotemporally visualize enzymes in living cells (Figure 3a) and mice (Figure 5).44,47

In-situ self-assembled NPs anchored on the membrane of HeLa cells were observed with cryo-SEM (Figures 3b-d) and TEM (Figure 3e). The nanoparticle size aligned with that of preformed NPs (Figure S4). These in-situ selfassembled NPs could also greatly improve cell entry via clathrin-dependent endocytosis (Figure S29), triggering efficient uptake of Gd-chelates (~0.59 fmol/cell intracellularly; Figure 3h) unlike the non-assembled probe (i.e., P-Cy-Gd) or preformed NPs (Figure 3h, Figure S22). This enhanced cellular uptake via in situ self-assembly could contribute to longer residence in target tissues, permitting efficient cell labeling and long-term molecular imaging. The P-CyFF-Gd reported herein is useful for detecting ALP, an important biomarker in serum, which is often assayed as an indicator of liver dysfunction in clinics.48-50 Because ALP is upregulated in many other tissues (e.g., bone, intestine, kidneys) under pathological conditions (e.g., inflammation, malignant tumors), mere detection of ALP activity in serum does not necessarily indicate liver disorders.51-54 Therefore, noninvasive detection of ALP activity in localized tissue is needed in addition to serum. P-CyFF-Gd possesses high sensitivity, high spatial resolution, and deep tissue imaging offered by NIR fluorescence and MRI, rendering it suitable for real-time imaging of ALP activity in tumors following systemic administration (Figure 4). For the first time, P-CyFF-Gd was found capable of precisely positioning orthotopic liver tumor foci in living mice. Localized imaging contrast effectively delineated tumor margins in livers and guided efficient surgical tumor resection (Figure 5). It is anticipated that the synergetic combination of MRI and NIR fluorescence imaging within P-CyFF-Gd may be applicable to improve cancer diagnostics and surgery in clinics. MRI’s high spatial resolution, deep tissue penetration and low sensitivity can be well-compensated by NIR fluorescence’s high sensitivity and easy operation.55,56 These benefits permit P-CyFF-Gd to produce: (1) exquisite structural images that can report the location of ALP-positive tumor tissues in a whole body through noninvasive MRI previous surgery; (2) sensitive NIR fluorescence images that can delineate the tumor margins and guide tumor surgery in intraoperative body. Thus, P-CyFF-Gd may serve as a useful contrast agent that allows preoperative diagnosis and real-time intraoperative guidance of ALP-related tumor surgery in clinics. These results suggest that integrating a fluorogenic reaction and in-situ self-assembly is advantageous for in vivo imaging and localization of ALP activity in real time, a benefit that can be extended to other enzyme-activatable NIR fluorescence and MRI bimodal probes (e.g., βgalactosidase,50,57 γ-glutamyl transpeptidase58,59). ■ CONCLUSION We report a novel strategy of integrating an enzymemediated fluorogenic reaction with in-situ self-assembly, applied to design a small-molecule-based activatable NIR fluorescence/MRI bimodal probe for real-time in vivo imaging of ALP activity. The ALP-triggered fluorogenic



reaction and self-assembly of P-CyFF-Gd into monodisperse NPs were validated through a series of in vitro and in vivo experiments, and self-assembled NPs on the cell membrane were directly examined using cryo-SEM. Given simultaneous enhancement in NIR fluorescence and r1 relaxivity, P-CyFF-Gd appears suitable for noninvasively measuring and localizing ALP activity in live tumor cells and living mice. Moreover, P-CyFF-Gd was successfully applied to map orthotopic liver tumor margins in intraoperative mice, allowing for real-time image-guided resection of liver tumors. This work demonstrates the ability of P-CyFF-Gd for in vivo imaging of ALP in tumors, which could be amenable to detect the activity of ALP in other diseases (e.g., liver disorders, inflammation). In the future, this strategy could be adopted to design activatable probes with synergetic combinations of NIR fluorescence and other imaging modalities (e.g., photoacoustic imaging, PET, CT) and may be useful in controlling drug delivery for cancer theranostics.

■ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details and data; Figures S1−S36 (PDF)

■AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions ‖These authors contributed equally.

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENT Financial supports from the National Key R&D Program of China (2017YFA0701301), National Natural Science Foundation of China (21775071 and 21632008), the Fundamental Research Funds for the Central Universities (020514380185) and CAS Key Laboratory of Receptor Research (SIMM1904YKF-03).

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MRI Bimodal Probes for in vivo Imaging

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