Magnetic Semiconductor Gd-Doping CuS Nanoparticles as

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Magnetic Semiconductor Gd-Doping CuS Nanoparticles as Activatable Nanoprobes for Bimodal Imaging and Targeted Photothermal Therapy of Gastric Tumors Hua Shi, Yidan Sun, Runqi Yan, Shunli Liu, Li Zhu, Song Liu, Yuzhang Feng, Peng Wang, Jian He, Zhengyang Zhou, and Deju Ye Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04179 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Magnetic Semiconductor Gd-Doping CuS Nanoparticles as Activatable Nanoprobes for Bimodal Imaging and Targeted Photothermal Therapy of Gastric Tumors Hua Shi,£,† Yidan Sun,£,‡ Runqi Yan, £,‡ Shunli Liu,† Li Zhu,† Song Liu,† Yuzhang Feng,§ Peng Wang,§ Jian He,*,† Zhengyang Zhou,*,† and Deju Ye*,‡ †

Department of Radiology, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing

University Medical School, Nanjing, 210008, China ‡

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

Chemical Engineering, Nanjing University, Nanjing, 210093, China § National Laboratory of Solid State Microstructures, College of Engineering and Applied Sciences

and Collaborative Innovation Center of Advanced Microstructures, Nanjing University, 210093, China. Corresponding authors: Prof. Jian He, E-mail: [email protected]; Phone number: +862583597518 Prof. Zhengyang Zhou, E-mail: [email protected]; Phone number: +862583597518 Prof. Deju Ye, E-mail: [email protected]; Phone number: +862589681905

ACS Paragon Plus Environment

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ABSTRACT: Targeted delivery of enzyme-activatable probes into cancer cells to facilitate accurate imaging and on-demand photothermal therapy (PTT) of cancers with high spatiotemporal precision promises to advance cancer diagnosis and therapy. Here, we report a tumor-targeted and matrix metalloprotease-2 (MMP-2)-activatable nanoprobe (T-MAN) formed by covalent modification of Gd-doping CuS micellar nanoparticles with cRGD and an MMP-2-cleavable fluorescent substrate. T-MAN displays a high r1 relaxivity (~60.0 mM-1s-1 per Gd3+ at 1 T) and a large near-infrared (NIR) fluorescence turn-on ratio (~185-fold) in response to MMP-2, allowing high-spatial-resolution magnetic resonance imaging (MRI) and low-background fluorescence imaging of gastric tumors as well as lymph node (LN) metastasis in living mice. Moreover, TMAN has a high photothermal conversion efficiency (PCE, ~70.1%) under 808 nm laser irradiation, endowing it with the ability to efficiently generate heat to kill tumor cells. We demonstrate that T-MAN can accumulate preferentially in gastric tumors (~23.4% ID%/g at 12 h) after intravenous injection into mice, creating opportunities for fluorescence/MR bimodal imaging-guided PTT of subcutaneous and metastatic gastric tumors. For the first time, accurate detection and laser irradiation-initiated photothermal ablation of orthotopic gastric tumors in intraoperative mice was also achieved. This study highlights the versatility of using a combination of dual biomarker recognition (i.e., αvβ3 and MMP-2) and dual modality imaging (i.e., MRI and NIR fluorescence) to design tumor-targeting and activatable nanoprobes with improved selectivity for cancer theranostics in vivo.

KEYWORDS: CuS nanoparticles, activatable probe, molecular imaging, photothermal therapy, gastric tumor


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Activatable molecular imaging probes that produce enhanced signals upon interaction with a specific molecular target have emerged as essential tools for the early diagnosis of diseases as well as the rapid monitoring of therapeutic response.1-3 A number of molecular imaging probes with controllable activation by different stimuli in tumor tissues, including acidic microenviroment, hypoxia, redox potential and enzymes, have been actively reported.4-7 Considering that the expression and activity of some enzymes are significantly upregulated in many cancer cells, enzyme-activatable molecular imaging probes capable of switching “on” fluorescence from an “off” state through continuous enzymatic catalysis have shown promising results for cancer detection.8-10 To date, many prominent fluorescent probes that can be selectively activated by cancer-associated enzymes, such as matrix metalloproteases (MMPs),11-13 γ-glutamyl transpeptidase14,15 and cathepsins,16-18 have been developed, allowing for noninvasive and realtime detection of cancers in living subjects. Nevertheless, most of these enzyme-activatable fluorescent probes are subject to limited resolution and tissue penetration depth due to the absorption and scattering of light by in vivo tissues.19,20 In addition, they are generally designed to target a single cancer-associated enzyme and thus suffer from low specificity for in vivo cancer imaging due to cross-reactivity with some normal (noncancerous) cells. Well-designed activatable probes capable of combining enzymatic activation with receptor-mediated uptake and of integrating sensitive fluorescence signals with high spatial resolution and unlimited tissue penetration depth offered by MRI are therefore desirable. This type of bimodal probes will allow not only the accurate detection of deep-seated cancers through MRI but also fluorescence-guided therapy to ablate cancers. Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer death globally.21,22 It has been reported that greater than 90% of GC patients diagnosed at an early


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stage can be cured by endoscopic or surgical resection, while the survival rate of GC patients diagnosed at an advanced stage is less than 20% due to the high risk of invasion and lymph node (LN) metastasis. Accurate monitoring of GC and LN metastasis is important to guide treatment and determine prognosis. To date, several methods, such as gastroscopic exams, computed tomography (CT),23 and MRI,24 have been developed for the diagnosis of GC in clinics, but their use for the non-invasive detection of small GC at early stage lesions and distant LN metastasis remains very challenging. In the clinic, surgery to resect both primary GC and metastatic LNs is still the only curative approach to GC therapy, but it has a high rate of failure, as surgical management performed by visual inspection and palpation is often limited by inappropriate intraoperative tumor delineation. Moreover, extended dissection of LNs by surgery can often cause postoperative

complications

and

increase

the

morbidity

and

mortality

of

the

lymphadenectomy.25,26 Therefore, it is demanded to develop a new approach allowing not only pretreatment diagnosis to guide GC therapy but also efficient ablation both primary and metastatic GC with minimal damage to normal tissues. In the past decades, photothermal therapy (PTT) has gained tremendous attention as an effective therapeutic approach for cancer therapy.27-30 It involves the use of light-absorbing materials (i.e., photothermal agents) that generally exert low dark toxicity within cancer cells but generate heat upon NIR light irradiation, causing local hyperthermia and thereby inducing irreversible cancer cell death.31,32 Compared with traditional cancer therapies such as surgery, chemotherapy or radiotherapy, PTT has the advantages of noninvasiveness and high spatiotemporal precision, enabling the realization of localized therapy and reduced harm to normal tissues. Accordingly, many prominent photothermal materials, including small organic dyes,33-35 gold nanomaterials,3639

graphene,40 carbon nanotubes,41 transition metal oxide/sulfide nanoparticles,42-44 and


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semiconducting polymer nanoparticles,45-47 have been developed and utilized for cancer PTT. Among them, semiconductor copper sulfide (CuS) nanoparticles are particularly attractive due to their strong and broad absorbance in the NIR region, high photothermal conversion efficiency (PCE), high photostability and low toxicity, permitting consecutive and repetitive irradiation for effective tumor ablation.48-50 Moreover, recent studies on cancer theranostics have prompted the integration of imaging tags into well-designed CuS nanoparticles, facilitating to optimize therapeutic approach and avoid overtreatment through imaging-guided PTT of cancers.51-53 We have previously reported tumor-targeting CuS nanoparticles that contain cRGD ligands and Cy5.5 fluorophores, which can offer NIR fluorescence and computed tomography (CT) contrast for bimodality imaging-guided PTT of LN metastasis of GC cells.49 However, this type of CuS nanoparticles exhibit “always-on” fluorescence and CT signals, which will produce high background incapable of detecting GC in real time. In addition, they can only target a single cancer-associated receptor (i.e., αvβ3) which may also suffer from low specificity incapable of guiding precise PTT of GC in vivo. To improve the accuracy for real-time detection and treatment of both primary and metastatic GC, in this work, we report a novel magnetic semiconductor CuS nanoparticle doped with Gd3+, and further engineer it into a tumor-targeting and MMP-2activatable fluorescent nanoprobe (denoted T-MAN) for efficient fluorescence/MR bimodal imaging and on-demand therapy of GC through NIR light-mediated PTT. Our demonstration of the successful detection and PTT of orthotropic GC and LN metastasis in living mice suggests the great potential of T-MAN for advanced cancer theranostics in biomedical research. Scheme 1 Scheme 1a illustrates the design of T-MAN, consisting of novel Gd-doping CuS nanodisks (Gd/CuS), a polyethylene glycol (PEG2000)-decorated amphiphilic phospholipid polymer (DSPE-


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PEG2000) layer, a tumor-targeting ligand (cRGD), and an NIR fluorophore (Cy5.5) together with a QSY21-labeled MMP-2-cleavable peptide substrate ((QSY21)-GGPLGVRGK(Cy5.5)-SH). The Gd/CuS nanodisks are used as photothermal agents because of their small size, strong NIR absorption and high PCE. Moreover, the integration of paramagnetic Gd3+can offer T1-shortening effect that allows bright MR contrast for high-resolution imaging of deep-seated tumors (e.g., orthotopic GC) in vivo. The use of DSPE-PEG2000 to encapsulate Gd/CuS nanodisks into micelles can help improve water solubility and biocompatibility, prolong blood circulation and reduce immunogenic and antigenic responses.54,55 More importantly, the micellar encapsulation can also facilitate close package of the flat Gd/CuS nanodisks inside the core, which may initiate geometrical confinement effect; this can restrict molecular rotation of the Gd/CuS nanodisks, thus increasing the rotational correlation time (τR) and augmenting r1 relaxivity.56-58 Cy5.5 is chosen as the fluorophore because of its strong fluorescence in the NIR region, and QSY21 was used as the quencher because of its high efficiency to quench the NIR fluorescence of Cy5.5.59 The application of peptide GPLGVRG as the MMP-2-specific substrate is because it has an excellent ability to be cleaved by MMP-2, which subseqently triggers the release of QSY21 from T-MAN, resulting in the recovery of Cy5.5 fluorescence. cRGD has been demonstrated as an efficient tumor-targeting ligand capable of selective binding to integrin αvβ3 overexpressed on the membrane of many tumor cells, facilitating active delivery into tumor cells.60 In addition, integrin αvβ3 has also been reported to bind with MMP-2 at its hemopexin domain and regulate the activation of MMP-2;61 therefore, the binding of cRGD in T-MAN to αvβ3 on tumor cells can help orient the peptide substrate for efficient cleavage by MMP-2.62,63 Scheme 1b illustrates the mechanism by which T-MAN detects and induces GC cell death upon irradiation with an 808 nm laser. T-MAN initially exhibits a high MRI signal, but its NIR fluorescence is turned “off” due to the quenching effect of QSY21. After


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intravenous administration, T-MAN can be efficiently delivered into GC tissues through αvβ3 integrin-mediated active delivery. Once inside the GC tissues, the MMP-2 overexpressed in the extracellualr matrix and αvβ3-bound MMP-2 overexpressed on the GC cell surface can specifically recognize and cleave the peptide substrate, affording T-MAN-cle with recovery of Cy5.5 fluorescence. T-MAN-cle can then enter into GC cells via αvβ3 integrin-mediated endocytosis and accumulate preferentially in the lysosomes. The bright MR contrast offered by T-MAN can provide high resolution and deep tissue penetration capable of noninvasively monitoring the accumulation of T-MAN in GC tissues; the activatable NIR fluorescence can offer a highly sensitive signal for real-time imaging of MMP-2 activity associated with GC cells. Guided by fluorescence/MR bimodal imaging, NIR laser (808 nm) irradiation at GC locations can produce hyperthermia to ablate GC cells both in vitro and in vivo. As a result, the combination of dual biomarker recognition (i.e., αvβ3 and MMP-2) and dual modality imaging (i.e., MRI and NIR fluorescence) allows T-MAN to accurately delineate primary GC tissues and LN metastasis, which can further guide the efficient PTT of GC in vivo. Figure 1 We first synthesized the Gd/CuS nanodisks by a high-temperature chemical reaction of CuCl2 and GdCl3 with sulfur using oleylamine (OA) as the solvent.64 After optimizing the conditions, the molar ratio of Gd and Cu in the as-prepared OA-coated Gd/CuS nanodisks was found to be approximately 0.15 : 1 according to inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The results of X-ray powder diffraction (XRD) analysis in Figure S1 showed that the Gd/CuS nanodisks shared a similar XRD pattern to that of Gd-free CuS nanodisks, matching well to the pattern of the hexagonal CuS covellite phase (JCPDS-06-0464). An X-ray photoelectron spectroscopy (XPS) spectrum of the Gd/CuS nanodisks showed the respective peaks assigned to


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Gd 4d (141.5 eV), S 2p1/2 (161.3 eV) and 2p3/2 (162.5 eV), and Cu 2p1/2 (951.2 eV) and 2p3/2 (931.3 eV), revealing the presence of Gd, Cu and S on the surface, with Gd : Cu ratio similar to that obtained from ICP-MS analysis (Figure S2). Transmission electron microscopy (TEM) analysis displayed a uniform structure of the Gd/CuS nanodisks with an average size of ~13 nm in diameter and ~3 nm in thickness (Figure 1a). The high-resolution TEM (HRTEM) images (Figure S3a) revealed the fringes of hexagonal Gd/CuS (102) and (006) planes with lattice spacings of ~0.30 and ~0.27 nm, respectively, which were close to those of previously reported CuS nanodisks.64 The components of the Gd/CuS nanodisks were further analyzed through elemental mapping (Figure S3b). Moreover, it was found that the UV-Vis-NIR absorption spectrum of the Gd/CuS nanodisks was nearly identical to that of CuS in cyclohexane, exhibiting broad absorption ranging from 700 to 1000 nm (Figure S4). These results demonstrated that the Gd/CuS nanodisks were a single crystal and possessed strong localized surface plasmon resonance (LSPR). With the novel Gd/CuS nanodisks, we next employed amphiphilic DSPE-PEG2000 polymers to encapsulate them into micelles to improve their aqueous solubility and biocompatibility for biological applications (Scheme 1a). We found that ~95.2% Gd/CuS nanodisks could be readily encapsulated by a mixture of DSPE-PEG2000-OMe and DSPE-PEG2000-NH2 (9/1 by mass) to yield monodisperse micellar nanoparticles (Gd/CuS@DSPE-PEG-NH2) (Figure S5 and Note S1). Zeta potential measurements showed that the Gd/CuS@DSPE-PEG-NH2 possessed a positive value of 31.8 mV, which is attributable to the presence of free NH2 groups on the nanoparticle surface (Figure S6). The subsequent coupling of free NH2 with NHS-PEG4-MAL afforded MALfunctionalized Gd/CuS@DSPE-PEG-MAL micelles, which were then covalently conjugated with cRGD-SH and synthesized (QSY21)-GGPLGVRGK(Cy5.5)-SH (Scheme S1) to produce the cRGD-functionalized and MMP-2-activatable multifunctional nanoparticle (T-MAN). The


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numbers of cRGD and (QSY21)-GGPLGVRGK(Cy5.5) molecules per T-MAN were estimated to be 140 ± 46 and 67 ± 22, respectively (Figure S7 and Note S2). TEM revealed the shape of TMAN, in which the Gd/CuS nanodisks were packed in close proximity (Figure 1b). Dynamic light scattering (DLS) analysis demonstrated that T-MAN had good monodispersity in aqueous solution with an average hydrodynamic size of ~61 nm (Figure 1c), which was slightly larger than that of Gd/CuS@DSPE-PEG-MAL (~56 nm). The increased hydrodynamic size of T-MAN could be due to the introduction of (QSY21)-GGPLGVRGK(Cy5.5) and cRGD on the surface (Figure S6). As both (QSY21)-GGPLGVRGK(Cy5.5) and cRGD possessed negative charges (e.g., -SO3H, COOH), a negative zeta potential (-15.20 mV) was observed for T-MAN, which was in contrast to the positive zeta potential of Gd/CuS@DSPE-PEG-MAL (10.33 mV). The UV-Vis-NIR absorption spectra showed that T-MAN exhibited a strong NIR absorption band from 700 to 1000 nm (Figure 1d), which was ascribed to the strong LSPR from the encapsulated Gd/CuS nanodisks (Figure S4). Moreover, due to the presence of (QSY21)-GGPLGVRGK(Cy5.5), T-MAN also showed two characteristic absorption peaks at 625 nm and 685 nm (Figure 1d). To evaluate the ability of T-MAN as a fluorescence/MR dual-modality imaging-guided PTT agent, we first investigated the fluorescence response of T-MAN toward MMP-2. T-MAN initially displayed very weak NIR fluorescence (Figure 1e), indicating that the fluorescence of Cy5.5 in TMAN was significantly quenched by QSY21 via an efficient Förster resonance energy transfer (FRET) process. Upon incubation with MMP-2, the fluorescence at 690 nm increased gradually and reached a plateau after 2 h. The maximum fluorescence turn-on ratio was found to be ~185fold, similar to that of free (QSY21)-GGPLGVRGK(Cy5.5)-SH after incubation with MMP-2 (Figure S8). High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis revealed the cleaved product


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(QSY21)-GGPLG (HPLC retention time, TR = 17.4 min) in the filtrate (Figure S9a,b). This result demonstrated that the increase in Cy5.5 fluorescence was due to the MMP-2-mediated cleavage of the peptide substrate in T-MAN, which slightly reduced the hydrodynamic size while obviously increased the zeta potential from -15.2 to -8.2 mV (Figure S9c,d). The kinetic parameters of the reaction of T-MAN with MMP-2 were subsequently measured using the Michaelis-Menten equation, and the apparent Michaelis constant, Km, and the catalytic constant, kcat, were determined to be ~1.67 μM and 0.12 s-1, respectively (Figure S10). The kcat/Km value was calculated to be approximately 7.2 × 104 M-1 s-1, which was close to that of other previously reported peptide-based fluorescent probes for MMP-2, indicating good enzymatic-cleavage efficiency of T-MAN toward MMP-2 (Table S1). Subsequent evaluation of the fluorescence of T-MAN following incubation with varying concentrations of MMP-2 demonstrated a high sensitivity for MMP-2 detection in solution (detection limit: ~0.64 pM) (Figure 1f and S11). Moreover, T-MAN was also found to be a specific fluorescent probe for MMP-2 (Figure S12). The longitudinal relaxivity (r1) of T-MAN was measured to be 60.0  1.7 mM−1 s−1 per Gd3+ ion at 1 T, which was ~15-fold higher than that of the clinically used contrast agent Dotarem (4.0  0.2 mM−1 s−1) under the same conditions (Figure 1g). The large r1 relaxivity was probably due to the geometrical confinement of the closely packed Gd/CuS nanodisks within the micellar core, which could ultimately restrict rotation and increase the tumbling time (τR).56-58 T1-weighted MRI at 1 T clearly showed that the images became brighter as the concentration of T-MAN increased. The MR signals of T-MAN were significantly stronger than those of Dotarem at the same Gd3+ concentration (Figure 1h). These results suggested that T-MAN could act as a highly efficient T1 contrast agent for MRI.


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We then investigated its photothermal properties following irradiation with an 808 nm laser. As shown in Figure S13, the temperature of the T-MAN solution was dependent on the probe concentration or laser power density. The temperature of the T-MAN solution (50 μg/mL) increased rapidly from ~25.2 to ~63.9 ºC after 808 nm laser (0.8 W/cm2) irradiation for 5 min, and the thermal image of the solution was much brighter than before irradiation. In contrast, no obvious temperature increase was observed upon irradiation of DI water. T-MAN possessed good photothermal stability, with the temperature increasing similarly after irradiation for five cycles (Figure 1i). Notably, the PCE of T-MAN was measured to be ~70.1% (Figure S14 and Note S3), which was much higher than those of most other previously reported CuS nanoparticles (Table S2). This high value of PCE was presumably due to the increased size of T-MAN51,65 and enhanced electric fields caused by the closely packaged Gd/CuS nanodisks within T-MAN.66,67 In addition, the geometrical confinement of Gd/CuS nanodisks within the core of T-MAN could restrict rotation of the nanodisks, which might help reduce the energy dissipation through motion and also contribute to a high PCE.68,69 We also found that both the photothermal conversion efficiency and r1 relaxivity of T-MAN were negligibly changed following incubation with MMP-2 (Figure S15). Moreover, T-MAN was stable in physiologically relevant environments (Figure S16). These results suggest that T-MAN holds great potential as a theranostic probe for biological applications. Figure 2 Encouraged by the above results, we next applied T-MAN for imaging and PTT of gastric tumor cells in vitro. We first evaluated the expression levels of αvβ3 and MMP-2 in human gastric MKN45 tumor cells and normal gastric GES-1 cells. Western blotting analysis showed that both αvβ3 and MMP-2 were highly expressed in MKN45 cells but not in GES-1 cells (Figure S17). The ability of T-MAN to detect MKN45 cells was then demonstrated through both fluorescence and


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MR imaging. Fluorescence images showed that the NIR fluorescence in MKN45 cells increased gradually upon incubation with T-MAN (1.5 μM), with the maximum fluorescence reached after 4 h (Figure S18). Colocalization studies showed that the enhanced NIR fluorescence was distributed mainly in the lysosomes, with the Pearson's correlation coefficient of 0.91, indicating that T-MAN could be efficiently activated and taken up by MKN45 cells (Figure S19). When MKN45 cells were pretreated with an MMP-2 inhibitor (SB-3CT, 20 µM) or free cRGD (10 μM), the strong lysosomal fluorescence was efficiently suppressed (Figure 2a). In addition, significantly weaker intracellular fluorescence was also observed when either MKN45 cells were incubated with cRGD free T-MAN-ctrl or GES-1 cells were incubated with T-MAN. These results suggested that MMP-2 and αvβ3 integrin overexpressed on MKN45 cells played key roles in the activation and uptake of T-MAN, thus resulting in strong intracellular NIR fluorescence capable of differentiating MKN45 cells from MMP-2- and αvβ3-deficient CES-1 cells. Along with the enhanced intracellular fluorescence, remarkably enhanced T1-weighted MR contrast was observed in MKN45 cell pellets after incubation with T-MAN (Figure 2b). The percentage signal enhancement (% SE) relative to blank MKN45 cell pellets was found to be ~200%, which was significantly higher than that in MKN45 cells pretreated with free cRGD (77%) or incubated with T-MAN-ctrl (43%) (Figure 2c). The strong MR contrast was slightly reduced when the MMP-2 activity in MKN45 cells was inhibited by SB-3CT, while significantly lower MR contrast was observed in T-MAN-incubated GES-1 cell pellets (% SE = ~30%). Subsequent ICP-MS analysis demonstrated a significantly increased intracellular Gd3+ concentration (~0.6 fmol/cell) in MKN45 cells incubated with T-MAN, indicating the high efficiency with which TMAN enters MKN45 cells via αvβ3-mediated cellular uptake (Figure 2d).


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To investigate the PTT efficacy of T-MAN, MKN45 cells were incubated with varying concentrations of T-MAN, followed by irradiation with an 808 nm laser (0.8 W/cm2) for 5 min. Figure 2e showed that T-MAN had negligible dark cytotoxicity against both MKN45 and GES-1 cells, while a dose-dependent reduction in cell viability was observed in MKN45 cells upon laser irradiation. The relative cell viability was reduced to ~15.6% in MKN45 cells after PTT with 100 μg/mL T-MAN, which was significantly lower than the T-MAN-treated CES-1 cells (~75.3%) or T-MAN-ctrl-treated MKN45 cells (~58.1%) (Figure S20). The PTT-induced cell death was further validated via fluorescence staining of live and dead cells with calcein AM and propidium iodide (PI) (Figure S21). These results imply that T-MAN features promising PTT properties enabling it to effectively kill MKN45 tumor cells upon 808 nm laser irradiation. Figure 3 We next investigated the capacity of T-MAN for fluorescence/MR dual-modality imaging–guided PTT of gastric tumors in vivo. The blood half-life (t1/2) of T-MAN after intravenous (i.v.) injection into healthy mice was found to be ~4.5 h, which was appropriate for blood circulation and elimination (Figure S22). Subsequently, T-MAN (75 μL, 25 μM Cy5.5) was i.v. administered into subcutaneous (s.c.) MKN45 tumor-bearing mice, and whole-body fluorescence images were acquired longitudinally. MKN45 tumors became fluorescent gradually, and the maximum fluorescence was observed at 12 h (Figure 3a and S23). This strong tumor fluorescence could be efficiently reduced by intratumorally (i.t.) injecting the mice with SB-3CT (~3.5-fold lower) or free cRGD (~1.5-fold lower) (Figure 3c). In addition, a significant ~2-fold decrease in tumor fluorescence was observed in mice that received i.v. injection of T-MAN-ctrl relative to those treated with T-MAN. The significant difference in tumor fluorescence was also validated by ex vivo imaging of tumor tissue slices resected 12 h after each treatment (Figure S24). Consistent


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with the fluorescence imaging results, T1-weighted MR contrast in MKN45 tumors was obviously enhanced in mice at 12 h following i.v. injection of T-MAN (0.05 mmol Gd/kg) (Figure 3b). The % SE in tumors was found to be ~130%, which was significantly ~2.0-fold higher than that in tumors blocked with cRGD and ~2.7-fold higher than that in tumors treated with T-MAN-ctrl (Figure 3d). The MR contrast in T-MAN-treated tumors was slightly reduced when treated with SB-3CT, suggesting that the inhibition of MMP-2 activity in tumors did not affect the accumulation of T-MAN in tumor tissues. As such, the reduction in tumor fluorescence by SB3CT was mainly due to the inhibition of MMP-2 activity, which prevented the activation of TMAN. Moreover, a biodistribution study using ICP-MS analysis revealed that T-MAN accumulated mainly in the reticuloendothelial system (e.g., liver and spleen), at 4 h after i.v. injection into mice (Figure 3e). However, at 12 h, the tumor held the highest uptake (~ 23.4% ID%/g) among the main organs, which matched well with the significantly enhanced fluorescence and MR contrast in tumors. The high uptake of T-MAN in MKN45 tumor was presumably attributed to the enhanced permeability and retention (EPR) in combination with the cRGDmediated active delivery. These results demonstrated that T-MAN could be efficiently delivered into MKN45 tumors to enhance MR contrast and could be selectively activated by MMP-2 to turn on NIR fluorescence. Notably, the achievement of a large NIR fluorescence enhancement (~185fold) and preferential accumulation in tumors could allow T-MAN to accurately detect tiny s.c. MKN45 tumors with a size of only ~3.5 mm3 (2.4 × 1.7 mm) in mice (Figure S25). Moreover, we found that both the fluorescence intensity and tumor-to-background ratio (TBR) increased with tumor size, suggesting potential applications of T-MAN in monitoring tumor growth. Guided by efficient fluorescence and MR imaging, PTT of s.c. MKN45 tumors with T-MAN was next evaluated in mice that received an i.v. injection of PBS buffer, T-MAN or T-MAN-ctrl. After


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12 h, the tumors were irradiated with or without the 808 nm laser (0.8 W/cm2) for 10 min. The real-time thermal images recorded by an infrared (IR) thermal camera showed that the temperature in the tumors treated with T-MAN rapidly increased to 55.2 ºC after irradiation for 5 min (Figure 3f,g), which was high enough to kill tumor cells.70,71 In contrast, the temperatures in the T-MANctrl-treated and PBS-treated groups were only ~44.4 ºC and ~38.2 ºC, respectively. The tumor growth was remarkably suppressed in mice receiving T-MAN plus laser irradiation, while the tumors in the control groups treated with PBS only (group i), PBS with laser irradiation (group ii), T-MAN only (group iii), T-MAN-ctrl only (group iv) and T-MAN-ctrl with laser irradiation (group v) still exhibited rapid growth (Figure 3h). The difference in tumor volumes on day 16 after each treatment was also confirmed from the photographs of dissected tumors, showing that three of five tumors were completely eradicated after treatment with T-MAN and laser irradiation (Figure S26a). Subsequent histological examination of tumor tissue slices by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed significant tumor cell death in the T-MAN and laser irradiation-treated groups compared to the other five control groups (Figure S26b). These results suggested that T-MAN possessed strong PTT efficiency against MKN45 tumors in vivo. The body weight of the mice in all six groups was minimally changed after treatment (Figure S26c). Moreover, H&E staining of the major organ tissue slices (e.g., heart, liver, spleen, lung and kidney) resected from the mice on the 16th day after the respective treatment showed no apparent pathological changes (Figure S27). These results suggested that T-MAN had low in vivo systemic toxicity but displayed high antitumor efficacy under 808 nm laser irradiation. Figure 4


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In addition to the s.c. tumor model, T-MAN was next applied for the fluorescence/MR imaging and PTT of orthotopic gastric tumors in living mice, as orthotopic tumor models are generally preferable because of their ability to replicate the organ-specific microenvironment.72,73 To establish and monitor orthotopic gastric tumor growth, MKN45 tumor cells transfected with luciferase (MKN45/Luc) were used and injected into the gastric serosa layer of nude mice (Figure 4a).74,75 Bioluminescence (BL) imaging revealed that MKN45/Luc tumors successfully grew in the gastric location of mice after two weeks (Figure S28). To evaluate the ability of T-MAN to accumulate in and report the orthotopic gastric tumors, T-MAN was i.v. administered into orthotopic MKN45/Luc tumor-bearing mice, and noninvasive T1-weighted MR images with high spatial resolution and unlimited tissue penetration depth were initially acquired. Figure 4b showed the obviously enhanced MR contrast in the tumor site after 12 h. The % SE in the tumor was found to be ~107% (Figure 4d), indicating that T-MAN could efficiently enter and accumulate in the orthotopic gastric tumors. After that, an abdominal incision was carefully applied to exteriorize the deep-seated stomach to allow accurate delineation of the tumor margin in the stomach through sensitive fluorescence imaging. As shown in Figure 4c, a strong fluorescence signal in the tumor region could be observed in the stomach of mice, agreeing well with the observations made from the BL images (Figure 4c). The tumor fluorescence signal was significantly (~5.2-fold) higher than that of the surrounding stomach tissues (Figure 4e). These results suggested that the NIR fluorescence of T-MAN was efficiently activated and accumulated in the gastric tumor, which could efficiently delineate the tumor margins in the intraoperative stomach. Guided by the imaging results, we employed the 808 nm laser (0.85 W/cm2, 10 min) to precisely irradiate the orthotopic tumors to trigger efficient PTT. The IR images revealed that the temperature of the orthotopic MKN45/Luc tumor rapidly increased from 25.3 to 57.4 ºC after 5


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min (Figure 4f,g). In contrast, the tumor-free stomach showed a much smaller increase in temperature (25.3 to 33.9 ºC) following treatment with T-MAN and laser irradiation (Figure 4f,g). After irradiation, the tumor growth in each mouse was monitored through BL imaging over time. Figure 4h showed that the initial strong BL signal in orthotopic tumors was dramatically reduced after PTT with T-MAN. On day 16, the BL signal in the stomach was close to the background signal, indicating that the orthotopic tumors were efficiently ablated. In sharp contrast, the BL signal in untreated mice (control group) continued to rapidly grow, becoming significantly (~892fold) higher than that of treated mice (Figure 4i). This result was also confirmed by ex vivo BL images, photographs of dissected stomach organs and H&E staining of stomach tissues (Figure 4j,k, S29 and S30). Moreover, taking advantage of the high r1 relaxivity of T-MAN, the PTT efficacy against orthotopic MKN45/Luc tumors was further noninvasively monitored using TMAN-enhanced MR contrast (Figure S31). Taking together, these results demonstrate that T-MAN is promising as a tool to guide the precise PTT of orthotopic gastric tumors in vivo. To the best of our knowledge, T-MAN is the first activatable probe capable of efficient imaging, PTT and noninvasive monitoring of orthotopic GC in vivo. Figure 5 Considering that gastric cancer has a high risk of LN metastasis, accurate diagnosis and removal of LN metastasis of gastric tumors are essential for gastric tumor therapy. To examine the ability of T-MAN for the imaging and PTT of metastatic tumors, an LN metastasis model was established by inoculation of MKN45 tumor cells into the right hind footpad of mice. After three weeks, the metastasis of MKN45 tumor cells to the LN in the inner knee of mice was confirmed by histological examination (Figure S32). Then, T-MAN was applied to detect LN metastasis through both noninvasive MR and fluorescence imaging. To avoid direct activation of T-MAN by primary


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tumors, T-MAN (25 μL, 25 μM) was s.c. injected into the middle site of the right leg in a position between the primary tumor on the footpad and metastatic LN in the inner keen (Figure 5a).76 T1weighted MR images showed bright MR contrast in the metastatic LN after injection of T-MAN, with the maximum % SE of ~163% achieved at 1 h, significantly higher than that of T-MANtreated normal LN in healthy mice (68%, Figure 5b,d). After 4 h, the MR contrast in the metastatic LN remained strong (% SE = ~109%), while the % SE in the normal LN was only ~16.6%, suggesting that T-MAN could efficiently penetrate into and accumulated in the metastatic LN. Furthermore, fluorescence imaging showed that the metastatic LN became fluorescent gradually, with the strongest fluorescence observed at 2 h post injection, which was in contrast to that in the normal LN (Figure 5c,e). At 2 h, the fluorescence intensity in the metastatic LN was found to be significantly (~54-fold) higher than that in the normal LN, which was also observed from ex vivo fluorescence imaging of resected LNs and their tissue slices (Figure S33). Overall, the resulting strong MR contrast and fluorescence could allow T-MAN to easily map LN metastasis in vivo. Guided by imaging, the use of T-MAN and the 808 nm laser (0.5 W/cm2, 10 min) successfully provided a significantly elevated temperature that could trigger effective PTT of metastatic gastric tumors in LNs (Figure S34), further suggesting the high potential of T-MAN for the detection and treatment of gastric tumors and LN metastasis in vivo (Figure 5f-h). In conclusion, we report the development of a tumor-targeted and MMP-2-activatable nanoprobe, T-MAN, through the engineering of a novel type of magnetic semiconductor Cu/CuS nanoparticle, and demonstrated the ability in the fluorescence/MR bimodal imaging and accurate PTT of gastric tumors in vivo. Our studies showed that T-MAN exhibited a large fluorescence turn-on ratio at 690 nm (~185-fold) upon interaction with MMP-2, a high r1 relaxivity (~60.0 mM-1s-1) at 1 T, and preferential tumor accumulation (~23.4% ID%/g) after systemic administration, allowing high-


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sensitivity and high-spatial-resolution imaging of gastric tumors and LN metastasis in living mice. Moreover, T-MAN also has a high photothermal conversion efficiency (~70.1%) under 808 nm laser irradiation, which could efficiently produce hyperthermia to kill gastric tumor cells both in vitro and in vivo. Importantly, accurate detection and laser-initiated ablation of orthotopic gastric tumors through fluorescence/MR bimodal imaging-guided PTT in intraoperative mice following i.v. injection of T-MAN was also realized for the first time. This study reveals the great potential of T-MAN for molecular imaging and PTT of gastric tumors, which may also be adaptable as a precise theranostic for other malignant tumors (e.g., breast cancer and melanoma). In the future, optical fibers plus endoscope techniques instead of abdominal incision may also be uitlized, which will allow to deliver sufficient light dosage for fluorescence imaging and PTT of deap-seated tumors noninvasively.77


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FIGURES

Scheme 1. General design of T-MAN for fluorescence/MR bimodal imaging and targeted PTT of tumors. (a) Illustration of the structure and synthesis of T-MAN through amphiphilic DSPE-PEG-assisted micellar encapsulation of OA-coated magnetosemiconductor Gd/CuS nanodisks. (b) Schematic illustration of the mechanism of T-MAN for fluorescence/MR imaingguided PTT of gastric tumors in vivo. Following intravenous administration, T-MAN can selectively enter and accumulate in the gastric tumors via αvβ3 integrin-mediated active delivery. Once inside the gastric tumor tissues, it can be efficiently activated by αvβ3-bound MMP-2 and MMP-2 overexpressed in the extracellular matrix, producing remarkably enhanced NIR fluroescence and strong T1-weighted MR contrast to accuratly delineate the gastric tumors. Guided by fluorescence/MR bimodal imaging, precise irradiation of the gastric tumor tissues with an 808 nm laser can produce hyperthemia, ultimately inducing irreversible tumor cell death .


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Figure 1. Characterization of Gd/CuS nanodisks and T-MAN. (a) TEM image of Gd/CuS nanodisks. (b) TEM image of T-MAN. Inset: enlarged TEM image of single T-MAN. Scale bar: 50 nm. (c) DLS analysis of T-MAN. (d) UV-Vis spectra of T-MAN (67 g/mL Cu2+) (red), Gd/CuS@DSPE-PEG-MAL (67 g/mL Cu2+) (black) and (QSY21)-GGPLGVRGK(Cy5.5) (1.14 M) (blue). (e) Fluorescence (FL) spectra of T-MAN (0.5 μM MMP-2 substrate) following incubation with MMP-2 (10 nM) at 37 ºC for 0, 5, 10, 15, 20, 30, 40, 60, 80, 100, 110, and 120 min. λex = 670 nm. (f) FL spectra of T-MAN (0.5 μM MMP-2 substrate) following incubation with 0, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 3.0, 5.0, 8.0, 10, 12 and 15 nM of MMP-2 at 37 ºC for 2 h. (g) Plots of 1/T1 of T-MAN and Dotarem versus Gd3+ concentration at 1 T. Values


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are mean ± standard deviation (SD) (n = 3). (h) T1-weighted (upper) and false-color-mapped (lower) MR images (1 T) of T-MAN and Dotarem with Gd3+ concentration ranging from 0.03 to 0.33 mM. TE/TR = 5.0/276.1 ms. (i) Photothermal stability of T-MAN (50 μg/mL) in DI water upon irradiation for five cycles (808 nm, 0.8 W/cm2).

Figure 2. Cellular uptake and in vitro cytotoxicity studies. (a) Fluorescence images of MKN45 cells incubated with (I) T-MAN (1.5 μM MMP-2 substrate), (II) T-MAN and free cRGD (10 μM), (III) T-MAN-ctrl (1.5 μM), (IV) T-MAN and MMP-2 inhibitor SB-3CT (20 μM), or (V) GES-1 cells incubated with T-MAN for 4 h. The images were acquired with excitation at 650 ± 22.5 nm


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and emission from 670 - 750 nm. Scale bar: 20 μm. (b) T1-weighted MR images (1 T, TE/TR = 5.0/276.1 ms) of MKN45 cell pellets after incubation with (I) T-MAN (500 μM Gd3+), (II) T-MAN and free cRGD (10 μM), (III) T-MAN-ctrl (500 μM Gd), (IV) T-MAN and SB-3CT (20 μM), or (V) GES-1 cells incubated with T-MAN for 4 h. (c) The average % MR signal enhancement (% SE) of cell pelletes as shown in (b). (d) ICP-MS analysis of Gd uptake in cells with indicated treatments in (b). (e) Viabilities of MKN45 cells and GES-1 cells incubated with different concentrations of T-MAN (0, 1, 10, 25, 50, 100, 150, 200 μg/mL Cu2+) in the absence or presence of laser irradiation (808 nm, 0.8 W/cm2, 5 min). Values were mean ± SD (n = 3). **p< 0.01, ***p< 0.001.


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Figure 3. Imaging and PTT of s.c. gastric tumors in vivo. (a) FL images (λex/em = 660/710 nm) and (c) average FL intensities (12 h) of s.c. MKN45 tumors in living mice receiving: (I) i.v. injection of T-MAN (25 μM MMP-2 substrate, 75 μL), (II) i.t. injection of free cRGD (2 mM, 100 μL) followed by i.v. injection of T-MAN 1 h later, (III) i.v. injection of T-MAN-ctrl (25 μM based on MMP-2 substrate, 75 μL), (IV) i.t. injection of MMP-2 inhibitor SB-3CT (1 mM, 100 μL) followed by i.v. injection of T-MAN 1 h later. (b) T1-Weighted MR images (1 T) and (d) average % SE (12 h) of s.c. MKN45 tumors in living mice receiving: (I) i.v. injection of T-MAN (0.05 mmol/Kg Gd3+), (II) i.t. injection of cRGD (2 mM, 100 μL) followed by i.v. injection of T-MAN 1 h later, (III) i.v. injection of T-MAN-ctrl (0.05 mmol/Kg Gd3+), (IV) i.t. injection of SB-3CT (1 mM, 100 μL) followed by i.v. injection of T-MAN 1 h later. Images in (a) and (b) were acquired pre and 12 h post injection of T-MAN. Red arrows indicate the location of tumors. White arrows indicate the internal standard of Dotarem (1 mM) in each image. Values were mean ± SD (n = 3). **p< 0.01, ***p< 0.001. (e) Biodistribution of T-MAN in MKN45 tumors and main organs of mice at 4 h, 12 h and 24 h after i.v. injection. Values were mean ± SD (n = 3). (f) IR thermal images and (g) average temperature changes of s.c. MKN45 tumors in living mice at 12 h post i.v.injection of T-MAN (5 mg/Kg Cu2+), T-MAN-ctrl (5 mg/Kg Cu2+) or PBS, followed by irradiation with the 808 nm laser (0.8 W/cm2) for 0-5 min. (h) Changes in tumor volumes in living mice following treatment with either (i) PBS, (ii) PBS + laser, (iii) T-MAN, (iv) T-MAN-ctrl, (v) T-MAN-ctrl + laser, or (vi) T-MAN + laser. Values were mean ± SD (n = 5). ***p< 0.001.


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Figure 4. Imaging and PTT of orthotopic gastric tumors in vivo. (a) Schematic illustration for the FL/MR biomodal imaging, PTT and BL monitoring of orthotopic MKN45/Luc gastric tumors in living mice. (b) T1-weighted MR images and (d) average MR signals of orthotopic MKN45/Luc gastric tumors in living mice before (Pre-contrast) and 12 h post i.v. injection of T-MAN (0.05 mmol/kg Gd3+). White arrows indicate the locations of tumors, and yellow dash circles indicate the locations of stomachs. Values were mean ± SD (n = 3). *p< 0.05. (c) FL (left) and BL (right) images of orthotopic MKN45/Luc gastric tumors in intraoperative mice at 12 h post i.v. injection of T-MAN (25 μM MMP-2 substrate, 75 μL). BL images were acquired immediately upon i.p. injection of firefly luciferin. Red arrows indicate the tumors, and red dash circles indicate adjacent stomach tissues. (e) Average FL and BL intensities in tumor sites and adjacent stomach tissues as mentioned in (c). Values were mean ± SD (n = 3). ***p< 0.001. (f) IR thermal images and (g) average temperature changes of orthotopic gastric tumors and normal stomachs in living mice at 12 h post i.v.injection of T-MAN (5 mg/Kg Cu2+), followed by irradiation with the 808 nm laser


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(0.8 W/cm2). (h) BL images and (i) average BL intensities of orthotopic MKN45/Luc gastric tumors in living mice that were untreated (Control) or treated with T-MAN plus laser irradiation. (j) Ex vivo BL images and (k) BL intensities of orthotopic MKN45/Luc gastric tumors resected from mice that were untreated (Control) or treated with T-MAN plus laser irradiation on day 16. Values were mean ± SD (n = 5). **p< 0.01.

Figure 5. Imaging and PTT of LN metastasis of gastric tumors in vivo. (a) Schematic illustration for the fluorescence/MR biomodal imaging and PTT of LN metastasis in living mice. (b) T1-weighted MR images and (d) mean % SE of metastatic and normal LNs in living mice before (0 h), and 0.5, 1, 2, 4 h post s.c. injection of T-MAN (0.025 mmol/kg Gd3+) into the right leg. Red and yellow arrows indicate the metastatic and normal LNs, respectively. Dotarem (1 mM) phantoms as internal standards are indicated by white arrows. Values were mean ± SD (n = 3). **p< 0.01. (c) Fluorescence images and (e) average fluorescence intensities of metastatic and normal LNs in living mice before (0 h), and 0.5, 1, 2, 4 h post s.c. injection of T-MAN (25 μM


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MMP-2 substrate, 25 μL) into the right leg. Red and yellow arrows indicate the metastatic and normal LN, respectively. Values were mean ± SD (n = 3). ***p< 0.001. (f) Photographs and (g) average weights of metastatic LNs resected from mice 16 days after treatment with (I) PBS only, (II) PBS + laser, (III) T-MAN (2.5 mg/Kg Cu2+) or (IV) T-MAN + laser (2.5 mg/Kg Cu2+). (g) Values were mean ± SD (n = 5). ***p< 0.001. (h) H&E (up) and TUNEL staining (bottom) of metastatic LN tissue slices resected from mice 1 day after indicated treatments. Scale bars: 40 μm.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website Detailed synthesis and characterization, experimental procedures, supplementary figures, tables and notes AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Phone Number: +862583597518 * E-mail: [email protected]; Phone Number: +862583597518 * E-mail: [email protected]; Phone Number: +862589681905 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. £ H. Shi, Y. Sun and R. Yan contributed equally.


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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 (81671751, 81871410, 21632008, 21775071), the Natural Science Foundation of Jiangsu Province (BK20150567, BK20171118), Social Development Foundation of Jiangsu Province (BE2015605), Outstanding Youth supported by Medical Science and technology development Foundation Nanjing (JQX16022), Jiangsu Province Key Medical Young Talents, “13th Five-Year” Health Promotion Project Of Jiangsu Province (QNRC2016041). Hua Shi and Yidan Sun were supported by the Scientific Research Foundation of Graduate School of Nanjing University (2017CL05). REFERENCES (1) Razgulin, A.; Ma, N.; Rao, J. Chem. Soc. Rev. 2011, 40, 4186-4216. (2) Yan, R.; Ye, D. Sci. Bull. 2016, 61, 1672-1679. (3) Kim, T.; Zhang, Q.; Li, J.; Zhang, L.; Jokerst, J. V. ACS Nano 2018, 12, 5615-5625. (4) Li, F. Y., Lu, J. X., Kong, X. Q., Hyeon, T., Ling, D. S. Adv. Mater. 2017, 29, 1605897. (5) Hu, X., Li, F. Y., Wang, S. Y., Xia, F., Ling, D. S. Adv. Healthc. Mater. 2018, 7, 1800359. (6) Li, F.Y., Du, Y., Liu, J. A., Sun, H., Wang, J., Li, R. Q., Kim, D., Hyeon, T., Ling, D. S. Adv. Mater. 2018, 30, 1802808. (7) Lu, J. X., Sun, J. H., Li, F. Y.; Wang, J.; Liu, J. N.; Kim, D.; Fan, C. H.; Hyeon, T.; Ling, D. S. J. Am. Chem. Soc. 2018, 140, 10071-10074.. (8) Kamiya, M.; Kobayashi, H.; Hama, Y.; Koyama, Y.; Bernardo, M.; Nagano, T.; Choyke, P. L.; Urano, Y. J. Am. Chem. Soc. 2007, 129, 3918-3929.


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