Rational Designed Rapid Liver Clearance Rare-earth Core-shell Nanoprobe for Dual-Modal Breast Cancer Imaging in the Second Near-Infrared Window


 BackgroundFluorescence imaging as the beacon for optical navigation has wildly developed in preclinical studies due to its prominent advantages, including noninvasiveness and superior temporal resolution. However, the traditional optical methods based on ultraviolet (UV, 200-400 nm) and visible light (Vis, 400-650 nm) limited by their low penetration, signal-to-noise ratio, and high background auto-fluorescence interference. Therefore, the development of near-infrared-II (NIR-II 1000-1700 nm) nanoprobe attracted significant attentions toward in vivo imaging. Regrettably, most of the NIR-II fluorescence probes, especially for inorganic NPs, were hardly excreted from the reticuloendothelial system (RES), yielding the anonymous long-term circulatory safety issue. ResultsHere, we develop a facile strategy for the fabrication of Nd3+-doped rare-earth core-shell nanoparticles (Nd-RENPs), NaGdF4:5%Nd@NaLuF4, with strong emission in the NIR-II window. What’s more, the Nd-RENPs could be quickly eliminated from the hepatobiliary pathway, reducing the potential risk with the long-term retention in the RES. Further, the Nd-RENPs are successfully utilized for NIR-II in vivo imaging and magnetic resonance imaging (MRI) contrast agents, enabling the precise detection of breast cancer. ConclusionsThe rational designed Nd-RENPs nanoprobes manifest rapid-clearance property revealing the potential application toward the noninvasive preoperative imaging of tumor lesions and real-time intra-operative supervision.

end, the commercially available ICG probe has been widely used for the tracing of lymph nodes in breast and gastric cancers [6,7]. In order to meet the multilevel and diverse demands in clinic, it is essential to exploit the high resolution and precision in vivo imaging technology based on the NIR-II probe [8]. Now various NIR-II uorescence probes, including rare earth-doped nanoparticles (RENPs) [9,10], quantum dots semiconductor [11,12], single-walled carbon nanotubes [13][14][15], and organic molecules [16,17], have been widely developed in recent years. Among these probes, RENPs manifest special advantages, such as large Stokes shift, narrow and multi-peak emission pro les, and excellent photostability, which make them potent for biomedical applications [18][19][20]. For instance, Ren et al. reported an Er-based lanthanide nanoparticle with strong NIR-II uorescence, which enables the imaging-guided surgery of orthotopic glioma [21]. Moreover, Cheng et al. synthesized Nd 3+ -doped rare earth nanoparticles, which were appropriated for NIR-II and T2-weighted MRI dual imaging in an orthotopic hepatocellular carcinoma [22].
These ndings manifest that RENPs have wide prospects for biological imaging.
Nevertheless, extensive studies revealed that RENPs often severely accumulated in the RES organs (liver and spleen) [23] resulting in potential toxicity due to the release of rare earth metal ions, which is a signi cant hindrance to the application of RENPs in vivo imaging [23][24][25] . Therefore, the development of a rapid clearance RENP probe from RES and utilized for accurate diagnosis of diseases is urgent, which requires further investigation.
Here, we report a facile strategy for the synthesis of Nd 3+ -doped RENPs with excellent NIR-II uorescence property and high signal-to-background ratio. After the surface modi cation by polyethylene glycol (PEG) consisting of a methoxy group at one end and a diphosphate group at the other end, the Nd-RENPs present good biocompatibility based on the in vitro and in vivo test, yielding the potential clinical applications. Interestingly, the Nd-RENPs probe could be excreted quickly from the RES system via the hepatobiliary pathway (half-life of 15.8 h in the liver) and rapid clearance from the blood circulation system (Fig. 1a). What's more, the high resolution dual-modal imaging technology, including the NIR-II optical and MRI method, is utilized for sensitive detection of breast tumor with explicit boundary information. Overall, these ndings revealed that the Nd-RENPs with the properties of good uorescence performance and rapid clearance from the liver are potential probes for the real-time imaging of breast cancer in the NIR-II window. Synthesis of NaGdF 4 :5%Nd core nanoparticles First, the ultra-small lanthanide uoride nanocluster (NaLnF 4 (Ln = Gd, Nd, Lu)) precursors were prepared in accordance with the liquid-solid-solution (LSS) strategy [26]. In a typical preparation, sodium hydroxide (1.2 g), deionized water (4 mL), 8 mL ethanol, and 20 mL OA were added in a 50 mL ask successively.

Materials and reagents
After stirring for 10 min, 1 mL of gadolinium (III) chloride hexahydrate aqueous solution (0.5 mol/L) and 4 mL of sodium uoride solution (0.5 mol/L) were added dropwise. The resultant solution was stirred at room temperature for 1 h until a yellowish solution formed. The solution was then precipitated with ethanol to collect NaGdF 4 nanoclusters. Following washing with ethanol several times, the NaGdF 4 nanoclusters were dispersed in cyclohexane (2 mL) for further use. The NaNdF 4 and NaLuF 4 nanoclusters were prepared similarly. The well-prepared NaGdF 4 nanoclusters (0.25 mol/L) and NaNdF 4 nanocluster solution (0.0125 mol/L) were later dissolved together in 2 mL cyclohexane. Subsequently, the NaGdF 4 /NaNdF 4 solution was mixed with 6 mL OA and 10 mL ODE in a ask. The mixture was purged with nitrogen (N 2 ) at 70°C for 30 min to remove the cyclohexane thoroughly and then heated to 280°C at a rate of approximately 10°C/min and stirred for 30 min. After cooling to room temperature, the resultant nanoparticles were precipitated by ethanol and collected by centrifugation at 11,000 rpm for 5 min. Finally, precipitation was re-dispersed in cyclohexane for further experiments.
As a typical example, 100 mg of PEG-diphosphate ligand (DP-PEG 2000 ) and 10 mg of OA-coated NaGdF 4 :5%Nd@NaLuF 4 core-shell RENPs were mixed with 5 mL THF. The ligand exchange was then executed under the condition of stirring for 24 h at 40°C. The PEG-coated particles were precipitated by cyclohexane, washed with cyclohexane thrice, and dried in a vacuum at room temperature for 4 h. The dried particles were dispersed in Milli-Q water and puri ed by ultra ltration three times to remove free polymers. The resultant solution was nally dispersed in Milli-Q water for further use.

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The size and morphology of the nanoparticles were captured with an FEI Tecnai G20 transmission electron microscope (TEM, FEI, USA) operating at an acceleration voltage of 200 kV. The hydrodynamic size was measured at 25°C with Zetasizer Nano ZS90 (Malvern, UK) equipped with solid-state He-Ne laser (λ = 633 nm). The concentration of the rare-earth elements was measured by inductively coupled plasmamass spectroscopy (ICP-MS). The down-conversion uorescence spectra were recorded using FLS980 spectra (Edinburgh Instruments, UK) equipped with an 808 nm laser serving as the excitation source. The crystal structures of nanoparticles were characterized with the Shimadzu XRD-6000 X-ray diffractometer (XRD) equipped with Cu Ka1 radiation (λ = 0.15406 nm).
The quantum yields (QYs) of PEGylated Nd-RENPs in water were evaluated via the previously reported method that takes IR-26 dye as a standard (QYs = 0.5%) [27]. All uorescence was collected by FLS980 spectra under excitation of 808 nm laser. The absorption spectra of Nd-RENPs and IR-26 were recorded with Lambda 25 ultraviolet-visible (UV-Vis) spectrometer (Perkin-Elmer, USA). The relative QYs of the RENPs-PEG were calculated in the following formula (1): Фs = Φr (F s / F r ) × (A r / A s ) × (n s 2 / n r 2 ), where Ф represents QYs, and F s and F r respectively denote the integral areas of photoluminescence (PL) of Nd-RENPs and IR-26 under excitation of 808 nm laser [28,29]. A r and A s are the NIR absorbance of the reference and samples at 808 nm, respectively, and n is the refractive index of the solvent (n s = 1.33 for water, n r = 1.44 for dichloroethane).

Cytotoxicty of Nd-RENPs
The cytotoxicity of core-shell Nd-RENPs was assessed by MTT assays. Brie y, 4T1 cells were seeded into 96-well plates at a concentration of 6 × 10 3 cell/well and cultured under the condition of 37°C and 5% CO 2 for 24 h. The cells were then incubated with media containing different concentrations of Nd-RENPs (0, 5, 10, 25, 50, 100, and 200 µg/mL), which were quanti ed with the concentration of gadolinium (Gd).
The cells were added with MTT (5 mg/mL, 10 µL/well) after incubation for 24 h and incubated at 37°C for 4 h. Thereafter, 100 µL of DMSO was added to each well. Following 15 min shaking, the 96-well plate was detected by EnSpire® Multimode Plate Reader (PerkinElmer, USA) at 490 nm. Cell viability was calculated in accordance with the formation: (A sample -A blank / A control -A blank ) × 100% [30].

Hemolysis test
First, the collected red blood cells (RBCs) were puri ed by centrifugation and washed with phosphatebuffered saline (PBS). Then, different concentrations of Nd-RENPs were added into freshly isolated RBCs. After incubation of 3 h under the condition of 37°C, RBCs were centrifuged at 10,000 rpm for 5 min.
Hemolysis capability was appraised by measuring the absorbance of supernatants at 540 nm. PBS treatment was acted as the negative control, and deionized water treatment was adopted as the positive control. The hemolysis ratio was calculated by the following formula: hemolysis % = (sample absorbance-negative control absorbance )/(positive control absorbance -negative control absorbance) × 100% [31].

Animal tumor model construction
All animal experiments were executed in accordance with guidelines approved by the ethics committee of Soochow University (Soochow, China). Speci c pathogen-free grade BALB/c female mice (4-5 weeks old) were selected and fed adaptively for one week before tumor-bearing. Tumor-bearing was conducted by subcutaneous inoculation of 5 × 10 6 4T1 cells into mice at the right ank region on the back of the mice.

NIR-II uorescence imaging
The nude mice were anesthetized with iso urane, and then DP-PEG 2000 -modi ed Nd-RENPs were administered through intravenous injection (15 mg of Gd 3+ per kilogram body weight). In vivo NIR-II uorescence imaging was performed by the NIR-II Imaging System (Serious II 900-1700) under the excitation of 808 nm laser (45 mW/cm 2 ). NIR-II photoluminescence images were obtained by using the long-pass lter of 1250 and 1000 nm. The tumor-to-background ratio (TBR) of two signal channels was calculated using the following equation

Biodistribution of Nd-RENPs
The biodistribution of Nd-RENPs was analyzed by uorescent quantitation and SPECT/CT imaging. The BALB/c mice (n = 3) were imaged under the NIR-II imaging system at different time points after intravenous administration for uorescent quantitation. Subsequently, the mice were sacri ced at 1, 24, 48, and 72 h post-injection. The major organs and tissues, including heart, liver, spleen, lung, kidneys, small intestine, large intestine, feces, and stomach, were taken and imaged ex vivo under the NIR-II imaging system. Finally, uorescence was quantitated to analyze the distribution of Nd-RENPs.
Radioactive Technetium-99m ( 99m Tc) was labeled on Nd-RENPs for SPECT/CT imaging through the chelating effect between the phosphate group and 99m Tc according to the reported method [32,33]. The

Histological analysis
Major organs (heart, liver, spleen, lung, and kidney) of mice were harvested on day 7 after intravenous injection to assess the histological toxicity caused by Nd-RENPs. The histological toxicity was evaluated by optical microscope following placement in 10% neutral buffered formalin, routine processing into para n, sectioning into thin slices, and performing hematoxylin-eosin staining (H&E) which is based on general phase transfer and separation mechanism [34,35] . NaGdF 4 , NaNdF 4 , and NaLuF 4 nanoclusters were respectively employed as core, dopant, and shell to achieve Nd-RENPs through reaction. TEM images showed that the average diameter of the core RENPs (NaGdF 4 :5%Nd) and coreshell RENPs (NaGdF 4 :5%Nd@NaLuF 4 ) were approximately 14.7 ± 1.9 and 25.7 ± 2.3 nm, respectively ( Fig. 1b-d). Some Nd-RENPs were spherical, while the others were short-rod shaped. The XRD peaks of Nd-RENPs ( Fig. S1a and b) were displayed consistent with the standard card of NaGdF 4 (JCPDS: 27-0699) and NaLuF 4 (JCPDS: 27-0726), indicating the good crystallinity of core-shell Nd-RENPs without signi cant changes with the adding of Nd 3+ dopants. Thereafter, the uorescence property of NPs was analyzed by FLS980 spectra. As indicated from the uorescence spectra in Fig. 1e, the core RENPs (NaGdF 4 :5%Nd) and core-shell RENPs (NaGdF 4 :5%Nd@NaLuF 4 ) possessed two emission peaks (1060 and 1340 nm) in the NIR-II region under the excitation of 808 nm laser. The uorescence intensity of coreshell Nd-RENPs at the same molar concentration increased 3.4 times at 1060 nm comparing with that of the core NPs. This phenomenon is attributed to the NaLuF 4 shell coating, which can effectively decrease the non-radiative transition process of Nd 3+ ions [36]. The result was also veri ed by uorescence imaging. Figure 1f shows that unlike H 2 O without uorescence, core RENPs and core-shell RENPs demonstrated strong uorescence under the excitation of 808 nm laser. The core-shell RENPs exhibited stronger photoluminescence intensity compared with core RENPs, which should be facilitated for the in vivo bioimaging. However, the oleate ligands on the surfaces of the RENPs contributed to the reduced solubility of particles in water, which was not conducive to their biomedical applications. The functional PEG polymer was adopted to replace the oleate ligand to improve the water solubility and biocompatibility of Nd-RENPs. The PEGylated Nd-RENPs (Fig. S2) displayed the similar morphology to that of unmodi ed RENPs. The uorescence spectra result revealed that the uorescence intensity at 1060 nm was decreased by nearly 4.2 times after surface modi cation (Fig. 1g), namely, the ligands exchange step could damage the passivation effect and expose more surface defect resulting in the quenching of the PL of the synthesized NPs. The relative photoluminescence QYs were quanti ed to further verify the photoluminescence intensity of Nd-RENPs. The result (Fig. S3) showed that the relative QY of Nd-RENPs was 0.89%, which is higher than the similar nanoprobe reported in previous literature [18,34,37].

Synthesis and Characterization
The dynamic light scattering (DLS) and uorescence spectra of Nd-RENPs at different time points were analyzed to evaluate the physicochemical stability and photostability of the PEGylated Nd-RENPs, which were important for their applications. The results of DLS displayed that PEGylated core-shell Nd-RENPs possessed a single scattering peak located at 32.7 nm in water, PBS (pH = 7.4), and the peak remained the same within three days (Fig. S4a, b). Additionally, the NIR-II uorescence intensity did not decrease in three days (Fig. S4c, d), suggesting the high photostability of the NIR-II Nd-RENPs in water and PBS. Moreover, as demonstrated in Fig. S5, the zeta potential of Nd-RENPs remained nearly unchanged in 72 h.
Those results demonstrated the good physicochemical stability of the Nd-RENPs. Herein, the excellent uorescence property and stability indicate the potential application in bioimaging.

Toxicity Assays
Excellent biocompatibility is essential for the application of imaging probes. The potential cytotoxicity of PEGylated core-shell Nd-RENPs was investigated in this study by MTT and hemolysis assays. Figure 2a and b showed that the cell viabilities (4T1 and MCF-7 cells) exceeded 80% after treatment with the Nd-RENPs for 24 h, even when the concentration of Gd 3+ is as high as 200 µg/mL. Additionally, the hemolysis percentage of the RBCs was remarkably lower than 5% (limiting value) [38] after Nd-RENPs incubation, demonstrating the remarkable hemocompatibility of Nd-RENPs (Fig. 2c, d).

NIR-II imaging of subcutaneous tumor
Optical imaging is a promising method for diagnosis because of the absence of radiation and high temporal resolution. Traditional uorescence imaging in the visible light region is used in vitro due to its poor tissue penetration and signal to noise ratio (SNR). The Nd-RENPs that emit in the NIR-II region of 1060 and 1340 nm, which are characterized by deep tissue penetration and weak tissue auto uorescence, might be suitable for the diagnosis of in vivo imaging. Therefore, a simple subcutaneous model was constructed to evaluate the imaging performance of Nd-RENPs in the NIR-II region. Following the injection of Nd-RENPs (15 mg of Gd 3+ per kilogram body weight) into the tumorbearing mice via tail vein, NIR-II uorescence images were acquired at different time points (15 min, 2, 4, 8, and 24 h). The multiplexed intravital NIR-II uorescence imaging was conducted with the aid of different types of lters (long-pass lter of 1250 and 1000 nm) due to the uorescence emission of Nd-RENPs at 1060 and 1340 nm. Figure 3a and b indicated the presence of uorescence signal after 15 min post-injection. The NIR-II uorescence signal increased in the rst 4 h post-injection, and the signal reached the maximum at 4 h (Fig. 3c, d). Subsequently, the signal decreased signi cantly. Moreover, 1340 nm uorescence has a lower background (TBR = 8.2) and higher resolution than that of 1060 nm (TBR = 2.5). Owing to the minimal auto uorescence, low scattering and absorbance of NIR-II emission at 1340 nm afford high SNR and deep tissue penetration depth comparing with that of uorescence at 1060 nm, which facilitates the NIR-II optical imaging of tumor. Real-time visualization of tumors also opens the possibility of Nd-RENPs for accurate detection during surgery. Thus, Nd-RENPs are promising nanoprobes for the potential applications in surgical navigation of NIR-II imaging. Notably, the uorescence signal in the liver rapidly decreased, and the LBR dropped from a peak of 21.7 at 4 h to 5.1 after administration for 24 h, suggesting the rapid clearance of Nd-RENPs from the liver. It is distinct from most of the NIR-II inorganic uorophores that lingered in the liver [9,39].

In Vivo Pharmacokinetics of Nd-RENPs
Further experiments were performed to investigate the metabolism of the Nd-RENPs. Figure 4a demonstrated the gradual increase of the uorescence signals in the liver after administration and peaking at 4 h. Subsequently, the signal in the liver began to decrease, and it was invisible after injection for 72 h, indicating the clearance of almost all particles from the liver. The in vivo half-life of Nd-RENPs in the liver was calculated as 15.8 h (Fig. 4b), which is signi cant short than the similar nanoprobes reported previously [9,25,39] and close to that reported by Yang and co-workers [40]. In addition, the pharmacokinetic results presented in Fig. 4c revealed that the blood half-life of nanoparticles is around 10.5 min, which contributed to rapid clearance in vivo. The high-magni cation NIR-II imaging of hind-limb vessels was recorded after intravenous administration of core-shell Nd-RENPs due to the outstanding optics properties (Fig. S6a). The Full Wave at Half Maximum (FWHM) was 804 µm (Fig. S6b), which was measured by plotting the red line of vessel pro les in Fig. S6a. The NIR-II signal intensity measured by the same vessel in the hind limb (Fig. S6c) gradually declined during the 60 min post-injection, which illustrated the rapid blood clearance of Nd-RENPs.
The biodistribution of Nd-RENPs was conducted by uorescence quantitation in the NIR-II region. It revealed that Nd-RENPs were mainly distributed in the liver and spleen after administration (Fig. 5a, b, respectively). The uorescence signals in the liver and spleen signi cantly decreased with the prolongation of administration. The signal in the liver at 24 h post-injection was around ve folds lower than that at 1 h and dropped over 90% at 72 h (Fig. 5a, b). Meanwhile, the uorescence intensity in feces and large intestine contents was analyzed. Figure 5 shows that the signal was observed to be elevated at 24 h, which indicated that the Nd-RENPs might excrete through the pathway from bile to feces.
Additionally, SPECT/CT was employed to further obtain comprehensive and real-time biodistribution information of Nd-RENPs [21,32] . The results shown in Fig. 6a implicated that the Nd-RENPs were quickly cleared from the circulatory system and then taken up by the RES system organs (liver and spleen). Subsequently, intense signal in intestine was gradually captured, suggesting the excretion of Nd-RENPs through enterohepatic circulation from the liver to intestines. Moreover, the mice were sacri ced after injection for 24 h. The biodistribution of Nd-RENPs in vital organs was analyzed by a gamma counter. As indicated in Fig. 6b, the ID% g − 1 value of the feces was signi cantly higher than that in the other organs (except liver and spleen), which also suggest the excretion pathway of Nd-RENPs through enterohepatic circulation. We speculated that the rapid excretion of Nd-RENPs in vivo might be related to the surface modi cation of PEG and the suitable short-rod shape of Nd-RENPs, which have been proven to facilitate nanomaterial escape from the capture of RES system and promote the excretion of nanoparticles through the hepatobiliary route [32,[35][36] .

Magnetic Resonance Imaging of Subcutaneous Tumor
Although NIR-II imaging possesses superior temporal resolution, its low tissue resolution remains a problem. MRI, which is widely used in clinical diagnosis, has high tissue resolution. The integration of the two imaging technologies can effectively improve the diagnosis e ciency of the diseases. The design of NIR-II and MRI dual-functional probe is critical to the realization of the aforementioned integration. Rareearth ions, such as Gd 3+ , Dy 3+ , and Ho 3+ , are potent agents used to relax the water protons for MRI because they have either a large number of unpaired electrons in the 4f orbitals and/or a large magnetic moment [41,42]. In addition to the outstanding uorescence performance in the NIR-II region, the coreshell Nd-RENPs exhibit interesting magnetism due to the existence of Gd 3+ . The longitudinal proton relaxation times (T1) relaxivity coe cient of the Gd-based Nd-RENPs was measured via a small MRI scanner under a 3T magnetic eld to investigate the MRI capability. Figure 7b shows that 1/T1 depended on the concentration of Gd 3+ , and the r 1 value of Nd-RENPs was 1.09 mM − 1 s − 1 . T1-weighted MRI was performed on breast tumor-bearing mice to evaluate the dual-modal imaging capacity of Nd-RENPs in vivo. The results displayed that the MRI signals of subcutaneous tumors gradually increased and progressively moved toward the center of the lesion (Fig. 7a). The signal enhanced by 1.46-fold at 240 min compared with the previous one (Fig. 7c). Hence, as a potential MRI contrast agent, Nd-RENPs can provide complementary information for NIR-II imaging and improve the diagnosis effect of breast cancer.

Biocompatibility of Nd-RENPs
Finally, H&E staining of major organs (heart, liver, kidney, spleen, and lung) was employed to further evaluate the biosafety of the Nd-RENPs. No clear pathological injury was observed from mice in the experimental groups after Nd-RENP administration for seven days compared with mice from the control group (Fig. 8). In addition, blood biochemical indices including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cre) were analyzed, and none of these blood biomarkers were signi cantly altered compared with the control groups (Fig. S7). These results revealed that the Nd-RENPs, as dual-imaging contrast agents, possess excellent biocompatibility, which further illustrated their potential application in clinical diagnosis and imaging-guided surgery.

Conclusions
A kind of excretable NIR-II rare-earth nanoparticles, NaGdF 4 :5%Nd@NaLuF 4 core-shell Nd-RENPs, has been successfully synthesized in this study with high biocompatibility. The nanoparticles enabled opticalguided tumor detection without invasion and high spatial resolution sensing in vivo for intraoperative identi cation and navigation with the help of NIR-II imaging modality. Additionally, a comprehensive examination by several methods veri ed that most of the Nd-RENPs could be eliminated in vivo within 72 h via hepatic clearance route, which might be relative to the surface modi cation of Nd-RENPs and their short-rod shape which have been shown to be easily cleared from RES. Meanwhile, as multifunctional nanoprobes, Nd-RENPs could provide comprehensive MRI information pre-operatively. Moreover, in vitro and in vivo assays demonstrated the excellent biocompatibility of Nd-RENPs. Consequently, this study reveals that the core-shell Nd-RENPs with the properties of excellent uorescence and rapid metabolism have considerable value for clinical application in the accurate diagnosis of breast cancer.

Figure 2
In vitro toxicity of Nd-RENPs. a, b) Cytotoxicity caused by Nd-RENPs in 4T1 (a) and MCF-7 (b) cell lines was analyzed by MTT after incubation for 24 h. c, d) Hemolysis activity of Nd-RENPs was appraised by color observation (c) and spectrophotometry (d).    In vivo MRI of tumor-bearing mice. a) T1-weighted MRI of breast tumor after injection with Nd-RENPs for 0, 5, 15, 30, 60, 90, 120, and 240 min. b) T1 relaxivity plot of the aqueous suspension of Nd-RENPs. c) Mean intensity of MRI signals of tumors.