Targeted Phototherapy by Niobium Carbide for Mammalian Tumor Models Similar to Human Being


 BackgroundAlthough nanomaterial-mediated phototherapy has been extensively studied, the major antitumor success is limited to treating subcutaneous tumor on nude, lacking of clinically-relevant big animal study. Therefore, it is urgent to make further investigation on the typical big model, which is more closely related to the human body. ResultsIn this work, niobium carbide (NbC) is selected as photoactive substance in virtue of its outstanding near infrared (NIR) absorption properties and resultantly NIR-triggered hyperthemia and reactive oxygen species generation for the synergetic photothermal and photodynamic effect. Moreover, macrophage is used as bio-carrier for the targeted delivery of NbC and the phagocytosis of macrophages is proved to be able to retain the photothermal/photodynamic effect of NbC. Resultantly, macrophage loaded NbC could realize complete removal of solid tumor on both of nude mice and big animal of rabbits. Meanwhile, two-dimensional ultrasound, shave wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) have been applied for monitoring the physiological evolutions of in vivo tumor post treatment, which clearly disclose the photoablation process of tumor and provide a new way for the surveillance of tumor on the big animal study. Conclusion﻿Hence, large animal model study in this work presents higher clinical significance than the previous studies.


Introduction
Cancer is widely regarded as a major threat to the public health and it lacks a global solution [1]. Cancer diseases, such as liver cancer and breast cancer, remain to be the leading causes of death or disability worldwide [2,3]. The poor therapeutic e cacies of traditional antitumor therapies (like surgery or chemotherapy and radiotherapy) in clinical practice are related to the signi cant side effects and inevitable secondary actions [4,5]. Up to now, scientists are working on developing a more e cient way to improve the accuracy and the controllability of oncotherapy. To achieve better treatment outcomes for the patients, it requires to develop novel therapeutic techniques to surmount above issues [6]. Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), emerged in recently years as a promising alternative to the traditional antitumor therapies by virtue of high spatio-temporal selectivity, low complications, minimal damage, and deeper penetration with acid of near-infrared (NIR) light [7][8][9][10].
Phototherapy works through the light-triggered photoactive materials for the generation of local hyperthermia (PTT) or reactive oxygen species (ROS) [11,12] (PDT) to induce cancer cell death. According to this modality, phototherapy could be helpful to achieve high spatiotemporal accuracy. So far, although lots of researches have illustrated the advantage and superiority of PTT or PDT, these two methods are still in the developing stage as they can just realize antitumor attempt on the mice modal [13]. Obviously, the mice with a subcutaneous tumor modal is quite different from the clinical tumor in tumor size, depth, and physiological characteristics and so forth. The original intention of NIR-mediated phototherapy is to treat deep-site tumor relying on the excellent penetration depth of NIR, whereas the limited volume of mice and shallow subcutaneous tumor evidently could not examine the advantages of phototherapy. The subcutaneous tumor on mice with a thin layer of skin only is almost no barrier for the NIR arrival. Hence, the current mice modal fails in evaluation of phototherapy e ciency. Prior to the clinical trials, the big animal modal study is very necessity but seldom was done.
Rabbits are frequently-used big animal modal and the subcutaneous fat layer is also very suitable for simulating deep-site tumor. What is more, VX2 carcinoma modal on rabbits also shows great similarities to the human tumor. VX2 carcinoma could be implanted in many tissues of rabbits and shows great similarities to the human orthotopic tumor in many aspects [14] such as vascularization, histological and biological characteristics. Furthermore, thick skin of rabbits and subcutaneous lipid layers are supposed to simulate the treatment on deep tumors of human to an extreme degree. Better than mice model, rabbits are more suitable to establish the orthotopically xenografted tumor and the tumor could also be larger. To sum up, orthotopic tumors on rabbits are more similar to the clinical human tumor [15]. However, the fast development of tumor and high-rate of tumor recurrence on rabbits hinders the phototherapy from big animal modal. That is why nearly no reports on big animal studies in phototherapy. To achieve a success on big animal modal, an enhanced antitumor outcome is highly needed. The synergetic PTT and PDT treatment could give a dual-punch on tumor and make the overall stage of phototherapy to be effective. It is reported that the PDT action is strong in early stage and becomes weaker as oxygen depletion, while the PTT is weaker in early stage and increases with temperature elevation. Consequently, synergetic action of PTT/PDT treatment is promising for the effective tumor removal, especially for the singlematter mediated one. Compared with PTT/PDT system with multicomponents, it could avoid mutual interference and absorption mismatch between photothermal agent and photosensitizer. As a consequence, photoactive material that behaves as both of photothermal agent and photosensitizer should be explored and served for the big animal attempt.
In this study, we present a novel photoactive material of NbC, which is an NIR-harvesting material that has both PTT and PDT effects. Meanwhile, cell cargo of macrophage has been used for loading NbC nanoparticles. Macrophage could pursue tumor by recognizing cytokines of cancer cells. Resultantly, NbC loaded into macrophages (NbC@M) via endocytosis of macrophages could realize targeted delivery to the tumor site. In this work, successful photoablation of solid tumor was realized on the mice with a subcutaneous tumor and rabbits with a breast cancer of VX2 carcinoma with NbC@M (Scheme 1). What is noteworthy, the treatment on rabbits provides the possibility of further clinical application on human.
To provide more convincing evaluation on the treatment outcome of PTT/PDT, this work used shave wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) for orthotopic rabbit tumor detection, monitoring, and diagnosis, which has seldom been investigated but is of great signi cance[16, 17].

Characterization of the NbC nanoparticles
In this work, the NbC nanoparticles have been employed as photoactive material for the phototherapy. Firstly, the morphological features have been checked with transmission electron microscopy (TEM). As shown in Figure. 1a, TEM image clearly shows NbC nanoparticle of 10-30 nm, and meanwhile the aggregation of particles was concerned. The average hydrodynamic size of NbC was con rmed to be 255 nm according to the dynamic light scattering (DLS) results ( Figure. 1b), which is bigger than TEM observation and further veri es the aggregation between particles. The crystal phase and crystallinity of powder sample were characterized by X-ray diffraction (XRD). In the Figure. 1c, all the diffractive peaks are consistent with JCPDS No. 65-7964 of NbC species, and no impurities are found. The XPS survey on core-level of Nb 3d orbit reveals the presence of two spin-orbit coupling doublets, corresponding to Nb 4+ and Nb 2+ ions after tting. Above results veri ed the pure phase of NbC nanoparticles.
Then, the optical absorbance of NbC was examined and showed in the Figure. 1e-f. Powder absorbance of NbC detected by spectrophotometer discloses strong and wide photoabsorption in the range of 300-2000 nm, and thus it covers the biological window of 700-1200 nm for the phototherapy ( Figure. 1e). Meanwhile, the absorbance of NbC aqueous solution presents a similar wide optical absorption band, and the absorbance is positively correlated with the concentration of NbC ( Figure. 1f). Next, the photothermal conversion effect was carried out. As shown in Figure. 1g, NbC aqueous solution of various concentration exhibits a sharp temperature increment under 808 nm NIR irradiation with a safe power density of 1 W/cm 2 , being in contrast to the deionized (DI) water control that gives a slight temperature elevation only. In brief, the higher concentration it is, the higher temperature rise. For calculating the photothermal conversion e ciency, the curve of temperature change for 20 min was detailed in the Figure. 1h, which includes 808 nm (1W/cm 2 ) irradiation for 10 min and natural cooling for another 10 min. According to previously reported method [18,19], the photothermal conversion e ciency (η) value of NbC was evaluated to be 47.5% ( Figure. 1i), being much higher than that of most commonly used PTT agent. These evidences indicate that NbC has excellent photothermal properties and could be used for the PTT treatment of tumor.

Characterization of NbC uploaded by macrophages
In this work, we employed the macrophage as a bio-cargo for the targeted delivery of NbC to the tumor site. Firstly, we examined the payload capability of macrophage towards NbC via endocytosis. The NbC nanoparticles were incubated with macrophages to form an NbC@M complex under different incubation time and NbC concentration, and then the uptake of NbC by macrophages as incubation time and concentration of NbC was studied by inductively coupled plasma mass spectrometry (ICP-MS) measurement. Figure. 2a and b show the micrographs of macrophages before and after loading of NbC, which clearly indicate the endocytosis of NbC by macrophages. The quantitative study has con rmed that optimal incubation conditions were incubation time = 12 h and NbC concentration = 0.1 mg/mL ( Figure. 2c-d), wherein macrophages play the best role in endocytosis. When the concentration of the NbC is too high, the intracellular content of NbC will be signi cantly reduced, probably because it exceeds the maximum amount of macrophage endocytosis and leads to the cell death. In short, macrophage is an ideal drug carrier as expected. Whether macrophage loading affects the photothermal behavior is another issue we were concerned. As shown in the Fig. 2e and f, there is no signi cant difference in the temperature rising trend between equivalent NbC and macrophage-engulfed NbC (NbC@M), indicating that the endocytosis of macrophages did not affect the photothermal capacity of NbC.

Phototherapeutic Effect of NbC@M in vitro
The focus of this work was not only using NbC as photothermal agent for the PTT, but also expecting NbC as photosensitizer to generate reactive oxygen species (ROS) for the PDT as well [20]. Two methods were used to detect the production of ROS. The rst one was realized with a DPBF probe, which could be decomposed by ROS, resulting in a decrement of its typical absorption peak at 420 nm. Thus, the determination of DPBF absorption loss by spectrophotometry could be used for the evaluation of ROS level. As shown in Figure. 3a, NbC@M and NbC could mediate faster and higher DPBF loss as compared with control group and thus indicated the production of aplenty ROS under 808 nm NIR irradiation.
Moreover, the degradation curve of DPBF for the NbC@M and NbC is nearly the same, suggesting that macrophage loading also has no affection on the photosensitive properties of NbC. Then, another probe of 2',7'-dichlorodihydro uorescein diacetate (DCFH-DA) was employed for the detection of intracellular ROS level. ROS can oxidize the non-uorescent DCFH-DA to produce green uorescent 2',7'dichloro orescein (DCF), which can determine the level of ROS in cells by visual uorescence. Compared with other control groups, bright green uorescence appeared in the NIR-excited NbC@M group, which is similar to that in positive control of H 2 O 2 treated group, indicating the generation of ROS within cells.
( Figure. 3b-f) Therefore, the phagocytosis of macrophages made no affection on PTT and PDT effects of NbC@M under irradiation.
The biocompatibility of NbC@M was examined with CCK-8 assay and the results proved the low cytotoxicity of NbC@M to the HepG2 cells without irradiation. The cell viability was over 85% after 12 h and 80% after 24 h incubation even at a concentration of as high as 2 mg/mL ( Figure. 4a & b). Then, the in vitro phototherapy evaluation was performed under NIR irradiation. In consideration of dual-role of NbC for simultaneous generation of ROS and hyperthermia, it may give both of PDT and PTT effect to the cancer cells. For investigating the single contribution from PDT or PTT, the ice bath or ROS quencher of NaN 3 was introduced to eliminate heat or ROS respectively. CCK-8 method was used also to analyze the cell viability of HepG2 after different treatments. The results in Figure. 4c show that the single role of PDT realized 22% of cancer cell death, while the PTT gave 64% cancer cell death. In contrast, combination of PDT/PTT (without introduction of NaN 3 and ice bath) show the best cancer cell killing effect of 74 % cell death. At the same time, the killing effect of PTT is much better than that of PDT. Thus, above results veri ed the synergetic action of PDT and PTT to the ablation of cancer. Besides, uorescence staining method with Calcien AM (green uorescence for labeling live cells) and propidium Iodide (PI, red uorescence for labeling dead cells) was applied to visually evaluate NbC@M-triggered phototherapy effect. As shown in the uorescence microscopic images ( Figure. 4d-f, Fig. S1a-c), the control group, NbC@M treated cells or NbC treated cells, and 808 nm NIR irradiated cells all present green uorescence only, indicating that neither NIR irradiation nor NbC@M kills HepG2 cells. In sharp contrast, macroscopic cancer cell death was con rmed in NbC@M or NbC meditated phototherapy groups as shown by distinct red uorescence ( Figure. 4g-I and Figure. S1d-f). Moreover, the range of dead cells is positively correlated to the duration of irradiation. In this work, NbC plays a major role in killing tumor cells regardless of whether loaded in macrophage. To sum up, these results suggest that NbC@M is an effective NIRtriggered synergetic PTT/PDT agent for the cancer treatment.

Phototherapeutic Effect of NbC@M in nude mice tumor
On the basis of excellent phototherapeutic properties of NbC@M in vitro, it gives us con dence to explore it for the in vivo studies, which were performed with HepG2-tumor-bearing nude mice as a typical model animal rstly. The twenty mice were randomly divided into four groups on average: 1. control group (PBS was only injected intravenously), 2. mice treated with 808 nm NIR irradiation, 3. mice treated with NbC@M injection intravenously, 4. mice treated with NbC@M + 808 nm NIR irradiation. An infrared thermal camera was used to record temperature changes in the tumors location. As shown in the Figure.  During this period, there was no signi cant difference in body weight among groups of mice (Fig. 5c). It can be observed intuitively that the volume of tumors in the group 1-3 was getting bigger and bigger, while the tumors in the group 4 were gradually shrank until almost complete disappearance ( Figure. 5d-e, Figure. S2), leaving scars only on the skin. The scabs without blood supply would appear in the group 4 after treatment, while tumors in the other three groups were not destroyed and retained blood vessels, whose the high blood perfusion could be seen by CEUS (white dotted line outline, Figure. 6a). In contrast, scabs show no blood perfusion area (yellow dotted line outline, Figure. 6a). In addition to the blood perfusion, the SWE value of the tumor site in all groups was changing continuously with the passage of time. The SWE values of tumors in the group 1, group 2 and group 3 showed a continuous upward trend in terms of both mean and maximum values. On the contrary, the SWE values of scabs in the group 4 increased signi cantly at the 3rd day, but decreased at the 7th and 14th day as shown in the Figure. 6b-c and Figure. S3. These results demonstrated that the tumors in the three control groups were continuously progressing and neither 808 nm laser irradiation nor NbC@M only can ablate the tumors, while an effective antitumor treatment is NbC@M mediated PTT/PDT. The results of CEUS and SWE showed that compared to the control group, the tumor area in the NbC@M mediated PTT/PDT group had been cured, scabbed and gradually improved as no blood perfusion and low SWE value. To evaluate the in vivo toxicity of NbC@M, the main organs (heart, liver, spleen, lung and kidney) and blood of mice in group 1-4 were extracted at the 14th day. The hematoxylin-eosin (H&E) staining results showed no obvious pathological changes, meanwhile, the hemanalysis indicated no blood abnormalities in the group 1-4 ( Figure. S4 and Figure. S5), thus illustrating that NbC@M is bio-safe for the phototherapy.

Phototherapeutic Effect of NbC@M in rabbits tumor
In this study, rabbit VX2 carcinoma, a squamous epithelioid carcinoma with rapid growth, was used to build breast tumors on rabbits mainly from consideration of similar tumor microenvironment between rabbits and humans. Similar to the nude mice experiment, rabbits were also divided into four groups. Because of the thicker skin of rabbits, the volume of tumors could not be measured with vernier calipers and B-mode ultrasonography was applied for this. Firstly, the temperature changes of NIR 808 group and NbC@M + NIR 808 group, as shown in Figure. 7a, were similar to that of nude mice experiment. In sharp contrast to the control group, the temperature of tumor area can rise rapidly up to 65 ℃ in the NbC@M + NIR 808 group ( Figure. 7b). It is well known that the insu cient internal blood supply may lead to the appearance of necrotic areas in the tumors, which can break through the skin and drain pus ( Figure. 7c). Hence, the size of the tumors seemed not grow, but in fact they were progressing. As time went by, the SWE values of the tumors in the control, NIR 808 and NbC@M groups increased only slightly, while the SWE value of the tumors in the NbC@M + NIR 808 group increased signi cantly due to the presence of scab ( Figure. S6). Moreover, the expanding necrotic areas in the control, NIR 808 and NbC@M groups were characterized by abundant blood supply within the tumors and no blood supply in the necrotic areas observed by CEUS. The tumor areas in the NbC@M + NIR 808 group were not enhanced as a whole, which was different from the other three groups. The mutual con rmation of SWE and CEUS results indicated that the tumors were completely cured by the PDT/PTT effects of NbC@M. Furthermore, no signi cant pathological change was observed in all groups of rabbits ( Figure. S7), showing negligible biological toxicity. In this part, we have successfully realized phototherapy on big animal of rabbits.

Conclusions
In conclusion, this work presented a phototherapy study on both of mice modal and large animal model of rabbits in a more clinically meaningful way. NbC photoactive material exhibited excellent NIRharvesting performance in terms of the hyperthermia and ROS generation. Signi cant photothermal and photodynamic capabilities could be retained even after loaded within macrophages, which served as a carrier for the targeted enrichment in tumor. The SWE and CEUS results showed that the effect of NbC@M-mediated PTT/PDT method successfully excised subcutaneous tumor on mice and orthotopic tumor on rabbits. Meanwhile, NbC@M demonstrated negligible hematotoxicity and systemic toxicity. Therefore, this study showed that NbC@M-mediated PTT/PDT has taken a further step on clinical treatment from expanding experiments on nude mice to rabbit.

Characterization
The morphologies of NbC were observed by TEM (JEOL JEM-2100, Japan). The size distribution analysis was conducted on Zetasizer Nano S90 (Malvern Panalytical, UK). The crystal phase was tested by XRD (Shimadzu XD-D1). The composition and chemical valence of NbC were measured by XPS spectra (PerkinElmer PHI 5600). UV-vis-NIR absorptive spectra of samples were performed on a spectrophotometer (U-4100, Hitachi, Japan). The concentration of Nb element within cells was analyzed by inductively coupled plasma (ICP) atomic emission spectrometer (8300, PerkinElmer, USA). Twodimensional ultrasound (2D US), Color Doppler Flow Imaging (CDFI) and contrast-enhanced ultrasound (CEUS) scans were performed using MyLab twice system (Esaote SpA, Florence, Italy) with LA523 probe.
All the data of shave wave elastography (SWE) were recorded by a real-time US device (Aixplorer; SuperSonic Imagine, Aix-en-Provence, France) with 4-15 MHz liner transducer.

Cell Lines and Cell Culture
The RAW264.7 macrophages and HepG2 cells lines were cultured in DMEM supplemented with 10 % FBS and 1% penicillin/streptomycin in a humidi ed atmosphere of 5% CO 2 at 37 ℃. Hemocytometer (Bürker-Türk, Wertheim, Germany) was used for the total cell counting.

Preparation of the Macrophage-loaded NbC nanoparticles
The RAW264.7 macrophages were incubated with DMEM until the cell coverage reached about 80% 9 0%. Then, the stale DMEM was removed and the fresh DMEM supplemented with various concentrations of NbC (0.125, 0.25, 0.5, 1 and 2 mg/mL) and incubated for varied time (2, 4, 6, 12 and 24 h). After incubation, the supernatant was removed and macrophage-loaded NbC nanoparticles were washed with PBS twice. The macrophages were observed with inverted biological microscope (Olympus CKX41). ICP analysis was used to quantify the uptake amount of Nb content in the macrophage cells. 4.6. Detection of ROS. DPBF solution with NbC or NbC@M was respectively placed in a quartz tube, and then irradiated with 808 nm NIR laser (1 W/cm 2 ) for 10, 20, 40 and 60 min. Then, absorbance at 420 nm was determined by U-4100 spectrophotometer. In order to detect intracellular reactive oxygen species (ROS), HepG2 cells were divided into ve groups, including untreated control (group 1), cells incubated with NbC@M (0.

Cytotoxicity Assay
The standard CCK-8 assay was used to evaluate the viability of cells. HepG2 cells (8×10 3 cells/well) were seeded into 96-well plates and cultured for 12 h. NbC@M solutions of different concentrations were then added into above wells and incubated for another 12 or 24 h, respectively. After washed by PBS solution for three times, 10 µL CCK-8 was added to the samples and incubated for another 1 h. The absorbance at 450 nm was then recorded on a microplate reader (BioTek, Winooski, VT, USA). The CCK-8 method was also applied for analyzing the therapeutic effect of in vitro PDT, PTT, and PDT + PTT experiments.
Similarly, HepG2 cells (8×10 3 cells per well) in each group were cultured in 96-well plates for 12 h. Then, 100 µL NbC@M (500 µg/mL) were added into each well and incubated for another 12 h, respectively. All groups were subjected to 808 nm NIR irradiation except control group. Additional operations are needed to handle PDT and PTT group. The process of PDT should be set in an ice bath, while sodium azide (50 µL, 1×10 − 5 M) was added into the medium of PTT group before NIR irradiation.

In Vitro Phototherapeutic Effect
To evaluate the phototherapeutic effects at the cellular level, HepG2 cells (3×10 5 cell/dish) were cultured with DMEM medium containing NbC or NbC@M (2 mL, 250 µg/mL) in 35 mm culture dishes for 6 h. Redundant NbC or NbC@M was removed by washing with PBS and then 500 µL fresh DMEM medium was added into each dish. Then, above resultant cells were irradiated with 808 nm NIR laser (1 W/cm 2 ) for 2, 5, and 10 min. Several groups were designed as controls, including untreated cells, cells only irradiated by 808 nm NIR laser (1 W/cm 2 ), and incubated with NbC or NbC@M. Finally, dead and live cells were labeled by Calcein-AM (1 µg/mL, 200 µL) and PI (20 µg/mL, 200 µL) for 20 min, and then washed with PBS. The uorescence visualization images were imaged on Olympus BX53 microscope.

In Vivo Phototherapy on mice
All experimental female Balb/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. HepG2 cells were digested and dispersed in DMEM to form a uniform suspension, which were then injected into the subcutaneous tissue of the upper left hind leg of nude mice to establish tumor-bearing mice model (tumor size is about 150 mm 3 ). Then, tumor-bearing mice were randomly divided into four groups (n = 5 for each group): group 1 (100 µL PBS was injected intravenously as control), group 2 (808 nm NIR irradiation for 10 min, 1 W/cm 2 ), group 3 (NbC@M was injected intravenously) and group 4 (intravenous injection of NbC@M plus 808 nm NIR irradiation for 10 min). NbC@M (100 µL) was injected intravenously into nude mice at a concentration of 1 mg/mL in the group 3 and 4, respectively. In the group 2 and 4, 808 nm NIR irradiation (1 W/cm 2 ) was required to irradiate the tumor area for 10 min. Notably, the mice in the group 4 should be irradiated at 12 hours post injection of NbC@M. The change of temperature was monitored and recorded every 30 s by an FL-IR System E6 infrared camera. The data of tumor size and body weight of nude mice were recorded in detail for 14 days. The tumor volume was calculated as V = length × width 2 / 2. Relative body weight was designed as dividing instant weight by initial weight (W/W 0 ), and similarly relative tumor volume was de ned as V/V 0 . 4.11. In Vivo Phototherapy on rabbits VX2 tumor-bearing rabbits were divided into four groups randomly, including injection of PBS (1 mL) as control, receiving only NIR irradiation, receiving only injection of NbC@M (1 mL, 1 mg/mL), and receiving NIR irradiation at 8 h post-injection of NbC@M (1mL, 1 mg/mL). For the real-time thermal imaging, tumor regions were irradiated by an 808 nm laser with a power density of 2 W/cm 2 for 20 min, and then monitored with an infrared imaging device (FL-IR System E6). All injections of PBS or NbC@M are performed through the posterior auricular vein.

Ultrasound on mice and rabbits
Ultrasound tests as a general term, including two-dimensional ultrasound (2D US), Color Doppler Flow Imaging (CDFI), SWE and CEUS, were completed by two radiologists with more than ve years of experience. After con rming the tumor area and applying enough ultrasonic coupling gel during SWE process, the probe was kept in a stable position with no pressure for about 3 seconds and was vertical to better reduce compression artifacts. A suitable region of interest (ROI) and scale ruler were identical for the elastographic quantitative analysis (SWE max and SWE mean ). CEUS examinations were performed with intravenous vein injection of SonoVue (Bracco SpA, Milan, Italy) followed by a saline ush. 100 µL SonoVue was injected into the caudal vein for mice, while 1 mL SonoVue was injected into the posterior auricular vein for rabbits. Video clips of the examinations, including the process of enhancement and washout, were immediately recorded over a time period of 1 min after injection of SonoVue.

Histological and Blood Biochemistry Analysis
To perform histological analysis, all the mice and rabbits were sacri ced at 14th day after treatment. Hematoxylin and eosin (H&E) staining of major organs including heart, liver, spleen, lung, kidney was done for histopathologic analysis. Slices were observed by a digital microscope (magni cation: ×100; DM3000; Leica, Germany). The blood (20 µL) of mice were collected and tested at 14th day after treatment by automatic blood analyzer (HF-3800).

Declarations Author Contributions
Zhao Liu worked at concept design, experiment arrangement and manuscript drafting. Chongshen Guo and Yuhang Tian carried out the synthesis and characterization of theranostic agent. Shan Jiang, Haitao Shang and Haoyan Tan participated in the cell mice and rabbit experiment. Kexin Chen was responsible for diagnosis and analysis of pathological section. Zhao Liu and Lei Zhang were in charge of summarizing and analyzing experimental data. Hui Jing and Wen Cheng joined the study's design and performed review and editing. All authors read and approved the nal manuscript.

Con ict of Interest
The authors declare no con ict of interest.