Targeted and Multifunctional Technology for Identification between

Feb 19, 2019 - ... light and releases heat that effectively and accurately kills tumor cells, thus improving patient's survival and postoperative qual...
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Biological and Medical Applications of Materials and Interfaces

A targeted and multifunctional technology for identification between hepatocellular carcinoma and liver cirrhosis Han Deng, Wenting Shang, Guanhua Lu, Pengyu Guo, Ting Ai, Chihua Fang, and Jie Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20600 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

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A targeted and multifunctional technology for identification between hepatocellular carcinoma and liver cirrhosis Han Denga, b, d, #, Wenting Shangb #, Guanhua Lua,b, Pengyu Guob, Ting Aia, b, d ,Chihua Fanga, d*, Jie Tianb, c, **

a. Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China. b. CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China. c. Beihang Uiversity, Beijing, 100080, PR China d. Provincial Clinical and Engineering Center of Digital Medicine, Guangzhou, 510280, China # These authors contributed equally to this work #Ms. Han [email protected]; #Dr. Wenting Shang, [email protected]. * Corresponding author. Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, No. 253, Gongye Avenue, Guangzhou 510280, PR China. ** Corresponding author. Beijing Key Laboratory of Molecular Imaging. Zhongguancun East Road #95, Haidian Dist. Beijing 100190, PR China. Email addresses: [email protected] (H. Deng); [email protected] (W. Shang); [email protected] (C. Fang); [email protected] (J. Tian)

Abstract Continuously updated diagnostic methods and advanced imaging methods have led to an increase in the early detection rate of small liver cancer , however, even with current diagnosis methods, it is still challenging to accurately judge a nodule with a diameter less than 2 cm whether it is hepatocellular carcinoma or liver cirrhosis. To solve this issue, a new technology is needed to distinguish above two kinds of liver nodules. There is an emerging imaging method that improves tissue resolution and sensitivity to detect micro-nodules with diameters less than 2 cm. To detect micro-nodules, photoacoustic imaging (PAI) was used to provide noninvasive images at depths of several centimeters with a resolution of approximately 100 m.

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To improve specificity, we developed a probe that specifically targets hepatocellular carcinoma by recognizing the biomarker GPC3 on the hepatocellular carcinoma cell membrane. The probe not only has a strong photoacoustic signal, but also have magnetic resonance signal. Furthermore, the material own photothermal effect which absorbs longer wavelength light and releases heat, effectively and accurately kills tumor cells, thus improving patient survival and postoperative quality of life. Herein, we present a new technology that uses photoacoustic imaging to image and target micro-hepatocellular carcinoma biological processes derived from liver cirrhosis with high spatial resolution.

Keywords: small hepatocellular carcinoma, liver cirrhosis, differential diagnosis, photoacoustic imaging, GPC3, photothermal therapy

Introduction Liver cancer is the fourth most common cause of cancer deaths worldwide1-4. Approximately 90% of liver cancers are Hepatocellular carcinoma (HCC), and 80% 90% of HCC are coupled with liver cirrhosis (LC)

5-7.

Although HCC cases are well

identified in the final stages, early diagnosis of HCC in high-risk populations remains a challenge due to early recessive clinical symptoms and difficulties posed by the differential diagnosis of concomitant LC 8. Therefore, early diagnosis and treatment of liver cancer with sizes less than 2 cm is of paramount importance9, 10. However, the diagnosis is extremely difficult due to indistinct boundaries between LC and HCC lesions that can only be defined by pathological examination11. The ideal time to effectively treat HCC is when the tumor nodule is less than 2 cm in size because smaller tumors are more amenable to potential curative treatments, such as resection, ablation, and liver transplantation, instead of just doing a palliative treatment10. Early detection and identification of small HCC are the key to early treatment and consequently good prognosis for liver cancer12, 13. Therefore, novel technology must be designed to distinguish small HCC from LC to devise an early and precise disease intervention.

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In the clinic, HCC and LC are often distinguished by serological detection and preoperative imaging methods, such as ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI). However, these methods have limited abilities in differentiating between HCC and LC. The most important serum test for liver cancer is serum alpha-fetoprotein (AFP) detection. AFP is a traditional serum and histochemical exosome biomarker14 and is the first step in early clinical surveillance of HCC. However, serological detection fails to accurately detect HCC tumors that are less than 2 cm, due to the fact that LC can transform into HCC nodules without any increase in AFP levels. In previously reported studies, 40% of HCC patients were reported to have normal levels (< 20 ng/ml) of AFP4, 5, resulting in inaccurate diagnosis of the disease. Furthermore, determining the precise location of HCC lesions is not possible even if small HCC is detected early by a sudden increase in AFP. Other imaging methods must be used to help determine tumor size and location. Ultrasound, as the first imaging choice for screening and surveillance for HCC, is unable to clearly distinguish between normal liver tissue, hepatic vasculature, and liver nodules due to its low-resolution. It has also been reported that the accuracy of ultrasonography may greatly depend on the experience of the doctor and performance of the ultrasonography apparatus15. It is difficult to accurately detect HCC nodules less than 2 cm in diameter with the CT method without contrast-enhanced agents. Even when using contrast-enhanced agents, the CT method could only detect 68% of HCC tumors with a diameter of 1-2 cm16, 17, which indicates that the detection of small tumors remains challenging using CT. Although MRI has a high spatial resolution, identifying micro-nodules with diameters less than 0.5 cm is still challenging18. Therefore, a novel technique is urgently required to circumvent the above challenges. Photoacoustic imaging (PAI) is an emerging whole-body imaging modality that has been developed for scouting small cancer tumors19. This process eliminates the disadvantages of ultrasound and optical techniques, while taking advantage of the low ultrasound scattering of tissue instead of optical scattering, and the high-sensitivity of optical imaging instead of low-resolution acoustic imaging20-23. During this process, different optical wavelengths can be excited by specific

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molecules. PAI can clearly differentiate between blood vessels and nodules by optical identification23. Therefore, we can take full advantage of PAI to detect small HCC with diameters less than 2 cm. Contrast agents are usually employed to enhance image signal24. Several materials have been explored as PAI agents, such as FeS nanoplates and Co9Se8 nanoplates. Recently, several types of PAI nanoparticles, such as Fe3O4 and CuS nanoplates have been presented as multifunctional contrast agents for photothermal therapy (PTT). However, these materials are relatively susceptible to oxidization in solutions and might have potential long-term toxicity25, 26. Some reports have shown that the toxicity of the material will decrease after the appropriate PEG modification27, 28.

FeSe2-PEG is considered a good PAI and PTT contrast agent29, 30. Additionally, the

material can yield positive MRI signal for auxiliary diagnosis. Furthermore, multiple bio-markers have been discovered to improve specificity. In particular, Glypican-3 (GPC3) is an oncofetal proteoglycan anchored in the cell membrane that is overexpressed in more than 50% of HCC patients14, 31, but not in the healthy adult liver. Therefore, GPC3 is being developed as a therapeutic target32-35. Importantly, the levels of GPC3 is markedly increased in small HCC tumors compared to LC36, 37, indicating that the transition from premalignant lesions to small HCC is usually associated

with

increased

GPC338,39.

A

12-mer

peptide

(sequence:

DHLASLWWGTEL) was identified by screening a phage display peptide library that demonstrated ideal GPC3 binding affinity40. In this study, as represented in the schematic diagram, FeSe2-PEG-Peptide was designed by the oleic acid method (Figure 1). We injected the synthesized probe into the mouse model tail vein (Scheme 1A). The probe is transported to the liver tissue through the blood circulation. We used PAI to detect HCC and LC and found that the probe was most concentrated in the tumor at twelve hours post-injection. HCC models emitted strong focal signal in the hepatic nodules while LC models exhibited low or no signal in the hepatic nodules. The PAI procedure was followed by MRI as an auxiliary diagnosis. Finally, after diagnosis by the above imaging methods, we performed photothermal therapy (PTT) on the liver cancer model and tested the

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therapeutic effect after treatment41.

Scheme1. (A)Schematic illustration of the diagnosis between Liver cirrhosis (LC) and hepatocellular carcinoma (HCC) by photoacoustic imaging (PAI) after injecting targeted probe (FeSe2-PEG-peptide) (B)The photothermal therapy (PTT) of HCC by 785 nm laser, and then verified by bioluminescence images.

Methods 2.1.1 Synthesis and Characterization of FeSe2 nanoparticles The probe was composed of FeSe2, PEG, and peptide. FeSe2 nanoparticles were synthesized by the following process. To ensure the whole reaction was performed in an oxygen-free environment, oleylamine (OM, 15 ml, Acros) solution combined with 1-octadecene (ODE, 10 ml, Acros) was heated to 120°C, which was protected by inert nitrogen. Then, the solution underwent vigorous magnetic stirring for 30 min in a three-necked flask. Meanwhile, selenium powder (2 mmol, Acros) was dissolved in OM (4 ml) under nitrogen followed by the addition of FeCl2·4H2O (1 mmol, Acros) powder into the flask for 30 min, then the solution turned pale yellow. Selenium solution was injected into the flask and incubated for 10 min, then the mixture turned black. Subsequently, the temperature was raised to 150°C and maintained for 30 min.

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All of the above reactions are required to be carried out strictly under nitrogen protection. After the final product was cooled to 25°C, excess absolute ethyl alcohol was added to wipe off superfluous surfactants and solvents followed by centrifugation and repeated ethyl alcohol washes to obtain FeSe2 nanoparticles. The synthesis method of FeSe2 is based on a previous publication31.

Figure 1. Schematic illustration of the synthetic route of targeted probe (FeSe2-PEG-Peptide).

2.1.2 Synthesis of FeSe2-PEG-peptide Following the synthesis of FeSe2, the surface of FeSe2 was modified by polyethylene glycol (PEG). Initially, Poly (acrylic acid) (PAA, ~1800 MW, Sigma-Aldrich) solution was mixed with an ethanol solution of FeSe2 by ultrasonication for 30 min, and stirred for 6 h. This was followed by centrifugation at 14800 rpm for 5 min and dialysis for 2 h to eliminate excess PAA and ethanol, and to obtain water-soluble PAA modified FeSe2 nanoparticles. Next, COOH-PEG-NH2 (MW = 3350, Chemgen Pharma, China) was added to the FeSe2-PAA solution and mixed by ultrasonication for 30 min. After adjusting

to

pH

7.4,

5

mg

of

N-(3-dimethylaminopropyl-N’-ethylcarvodiimide)-hydrochloride (EDC) was added to connect the carboxyl group on the FeSe2-PAA to the amino group on the PEG. The mixture was stirred overnight with a magnetic stirrer to obtain FeSe2-PEG. Finally, the targeted probe was synthesized to provide the carboxyl group to connect the amino

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group on the peptide at a steady rate. The amino acid sequence of the peptide connecting the probe to GPC3 expressed on the HCC was DHLASLWWGTEL. FeSe2-PEG and the peptide mixture were stirred overnight at 4°C and centrifuged the next day to obtain the precipitated probe.

2.2 Characterization of FeSe2 and FeSe2-PEG-peptide The hydrodynamic diameters and zeta potentials of FeSe2 and FeSe2-PEG-peptide were measured by Zetasizer Nano (ZS) (Malvern Instruments, UK). The images and diameters were measured by transmission electron microscopy (TEM) (JEOL, JAPAN). A UV-3600Plus spectrophotometer (SHIMADZU, JAPAN) was used to analyze the absorbance spectrum and measure the material stability of the samples. Serum stability was tested by dissolving the sample (0.8 mg/ml) in serum to simulate the in vivo environment and placing the solution in 4°C for 1 h, 3 h, 6 h, 12 h, 24 h, 3 days, and 7 days, followed by a UV test. To characterize the absorbance spectrum, the UV-vis-NIR absorption spectrum intensities of FeSe2-PEG at different concentrations were collected when the wavelength was 785 nm until the value plateaued. The UV spectrum provided guidance for the photoacoustic signal. Before FeSe2-PEG was injected into mice for imaging, we determined if the material was positive for photoacoustic or MRI signals in vitro by performing a phantom experiment. Different concentrations of FeSe2-PEG were scanned by a 9.4T MRI scanner to obtain T2-weighted images and the relaxation rates were calculated at the same concentration gradient as mentioned above to simultaneously measure signal intensity. Using agar as a carrier, the photoacoustic signals of FeSe2-PEG at different concentrations were measured by multispectral optoacoustic tomography (MSOT) (iTheraMedical, Germany).

2.3.1 Probe in human HCC cells Toxicity toward Hep-G2, HUH7, and SK-Hep1 cancer cells was tested by the MTT cell survival assay. Hep-G2 cancer cells were chosen to test cytotoxicity by confocal microscopy and to test the liver-targeting ability of the probe by transmission

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electron microscopy (TEMP). Cells were cultured in DMEM (Gibco) media supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin at 37°C, 5% CO2. All reagents in the cell culture experiments were purchased from Gibco. To test the effect of the probe on cell viability, 1 × 104 cells per well were cultured in 96-well plates for 24 h, followed by incubation with different concentrations of FeSe2-PEG for 24 h. Finally, the MTT assay was used to determine cell growth and survival rates. To assess probe toxicity in cells, cell morphology was assessed by confocal microscopy. Effect of the probe on cytoskeleton and cell nucleus was assessed by first incubating Hep-G2 cells with different concentrations of FeSe2-PEG for 24 hours, followed by staining the cytoskeleton with rhodamine phalloidin and counterstaining the nucleus with DAPI. Cell images were captured by confocal microscopy. Tumor-bearing mice were anaesthetized and fixed with glutaraldehyde by perfusion-fixation. Liver tumor tissues from fixed mice were dissected, fixed, embedded, strained, and observed by TEMP for ultrastructural changes of morphology.

2.3.2 Probe in vitro PTT The Hep-G2 cancer cells were seeded in 6-well plates at 5 × 105 cells per well for 24 h. Cells in five wells were incubated with FeSe2-PEG (0.1 mg/ml) for an additional 24 h. Four of the five wells were irradiated were irradiated at multiple strengths (0.1 W cm-2, 0.3 W cm-2, 0.5 W cm-2, 0.8 W cm-2) for 5 min, while the control well was not irradiated. The remaining wells were irradiated by 0.8 W cm-2 without probe. The conditions of the six wells were as follows: 1) a. probe with 785 nm laser only (0.1 W cm-2, 5 min); 2) b. probe with 785 nm laser only (0.3 W cm-2, 5 min); 3) c. probe with 785 nm laser only (0.5 W cm-2, 5 min); 4) d. probe with 785 nm laser only (0. 8 W cm-2, 5 min); 5) e. 785 nm laser only (0.8 W cm-2, 5 min); 6) f. probe only. All cells were co-stained with calcein AM and propidium iodide (PI) for 10 min. Cells were imaged by fluorescence microscopy (Leica, Germany).

2.4.1 Animal model

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In this study, we used two animal models, 1) HCC model and 2) LC model. All female mice were acquired from the Beijing Vital River Laboratory Animal Technology Co. Ltd. To generate the HCC model, five-week-old male BALB/c mice were anesthetized with phenobarbital sodium. The nude mice were bolted in the supine position, and the skin was cut at the peritoneum about 0.8 cm below the xiphoid line at the ventral midline, followed by implantation of 1 × 106 Hep-G2 fluc cells into the left lobe of the liver and suturing of peritoneum and skin. The tumor was assessed using a small-animal optical molecular imaging system (IVIS Imaging Spectrum System, America) after 14 days. To generate the LC model, carbon tetrachloride (CCL4) was diluted in olive oil at 40% concentration. CCL4 (2.5 µl/g) was injected intraperitoneally into five-week-old BALB/c white mice, two times a week for 12 weeks. The LC model was confirmed by liver biopsy every fourth week.

2.4.2 Diagnosis by MRI and PAI To assess hepatic targeting of the probe, we designed two experiments. Firstly, Hep-G2 liver cancer mice were tail vein injected with FeSe2-PEG (200 µl, 2 mg/ml, n = 6) and FeSe2-PEG-peptide (200 µl, 2 mg/ml, n = 6). Secondly, liver cirrhosis mice were injected with FeSe2-PEG-peptide (200 µl, 2 mg/ml, n = 6) in contrast to liver cancer mice (n = 6) for differential diagnosis. The two groups of control mice were placed into MSOT to get photoacoustic data at 0, 1, 3, 6, 12, 24, and 48 h. HCC mice were simultaneously administered with 3D PAI with FeSe2-PEG-peptide at the above fixed time points. The MRI image was obtained before and after 24 h by injecting FeSe2-PEG-peptide into the caudal veins and the animal was viewed on a 1.5T Magnetic Resonance Imager (MRI) equipped with a small animal coil (Aspect Imaging, Israel). We separately collected different MRI images, the comparison involving three models: HCC, LC, and subcutaneous tumor mice. The MRI parameters of T2-weighted images were as follows: repetition time (TR) = 112.0 ms, echo time (TE) = 5.0 ms, slice thickness = 1.0 mm. After the above mice were imaged, liver tumors or cirrhosis nodules were processed for pathological examination.

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2.4.3 In vivo Photothermal therapy We designed four control groups that included one blank control group while the other three groups served as experimental groups. After injection (12 h), PTT was performed on the three experimental groups of mice. Two groups were injected with FeSe2-PEG and FeSe2-PEG-Peptide respectively, while one group was injected with PBS. The same batch of mice were seeded with the same number of Hep-G2 cells with the same conditions on day one. They were fed for two weeks under the same conditions. Nude mice (n = 24) were randomly assigned to four groups: (1) The mice in group A were untreated control; (2) The mice in group B were injected with PBS (200 µl) and irradiated by a 785 nm laser; (3) The mice in group C were injected with FeSe2-PEG (2 mg/ml, 200 µl) and irradiated by a 785 nm laser; (4) The mice in group D were injected with FeSe2-PEG-Peptide (2 mg/ml, 200 µl) and irradiated by a 785 nm laser. The treatment schematic is shown in Figure 7D. After the probe was injected into the mice for 24 h, we first determined orthotopic liver cancer in the mice by IVIS. Mice confirmed to be positive for cancer were subjected to PTT, and the temperature of the tumor before and 5 min after irradiation were recorded. Finally, remaining self-luminescence in mice after PTT was determined by IVIS. After treatment, all nude mice were monitored by the IVIS Imaging Spectrum System and were weighed every three days until the tumor fluorescence signal no longer changed. At the same time, we recorded survival time, which included dates of tumor plantation, treatment, and death for each mouse. Finally, the mouse liver tumors showing no fluorescent signal after therapy were accepted for pathological examination.

3.1 Statistical analysis After experiments, measured data were analyzed by SPSS with independent two-sample t-tests. An experiment was considered significant when P < 0.05.

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Results The scheme for probe synthesis is shown in Figure 1. The probe was constructed using the oleic acid method maintaining strict reaction conditions. The experimental condition requires oxygen-free conditions in order to prevent the generation of iron oxide. Thus, the reaction process was maintained under a nitrogen (N2) atmosphere. The temperature was also strictly controlled to prevent materials from aggregating, when temperatures were increased. To improve biocompatibility, stability, and ability of the carboxyl groups to form connections between FeSe2 nanoparticles and peptide, the surface was modified by polyethylene glycol (PEG). Finally, we added the peptides to the reaction to target the probes.

Figure 2. Characterization of probe. (A) Hydrated particle size of FeSe2 and FeSe2-PEG. (B) UV–vis–NIR

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absorption spectrum of FeSe2 and FeSe2-PEG. (C) Image of FeSe2 was got by Transmission electron microscope (TEM) and High-resolution Transmission electron microscope (HR-TEM). The size of FeSe2 was about 10 nm measured by HR-TEM. (D) Image of FeSe2-PEG was got by TEM and HR-TEM. The size of FeSe2-PEG was about 5 nm measured by HR-TEM. (E) Serum stability of FeSe2-PEG (0.8 mg/ml) measured for 168 h (7 days). Inset: A photo of FeSe2-PEG in various solutions (from left to right: water, DMEM cell medium, PBS, and serum). (F) UV–vis–NIR absorption spectrum intensity of FeSe2-PEG at different concentration when wavelength was 785 nm.

We continued the above experiments with tests to characterize the probe. In particular, we wanted to determine if the probe is effective and safe after injection into the body. To test for effectiveness, we first tested the size of the probe, to determine if it could be excreted via the liver. Hydrated particle size of FeSe2 is approximately 80 nm (Figure 2A) and the diameter is approximately 10 nm (Figure 2C). Transmission electron microscopy confirmed a sheet structure of the nanoparticles and an obvious lattice. We observed that the particle size reduced when PEG was connected to the FeSe2. Hydrated particle size of FeSe2-PEG was approximately 40 nm (Figure 2A) and the diameter was 5 nm (Figure 2D). This was due to the fact that PEG limited the size of the particles. Hydrated particle size of FeSe2-PEG-Peptide was approximately 50 nm modified with peptide, which was compliance with the characterastics of materials modified with biomolecules (Figure S4A)42. For the UV absorption spectrum, FeSe2 and FeSe2-PEG resembled melanoma (Figure 2B) that exhibits good photoacoustic signal43. We then tested the serum stability of the probe to simulate the in vivo environment. We observed the FeSe2-PEG curve to be less volatile and did not fluctuate significantly (Figure 2E), which was a sufficient indication of FeSe2-PEG stability. When measuring the different concentrations of FeSe2 (Figure 2F), we observed a gradual increase in UV intensity with an increase in concentration. The intensity reached its higher limit at a concentration of 5 mg/ml (Figure 2F), marking the maximum absorption intensity. This implied that the photoacoustic signal increases as the concentration increases up to 5 mg/ml.

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Figure 3. Photoacoustic images (A) and photoacoustic intensity (C) of FeSe2-PEG solutions at different FeSe2-PEG concentrations. T2-weighted MR images (B) and the relative relaxation rate R2 (D) of FeSe2-PEG solutions at different Fe concentrations.

Before FeSe2-PEG was injected into mice for imaging, we determined if the material was positive for photoacoustic or MRI signals in vitro, testified by a phantom experiment. In terms of the photoacoustic signal and PAI principles, the intensity of the PAI signal depends on the concentration of the probe. While the water control was negative for PAI signal, the PAI signals from FeSe2-PEG gradually increased as concentration increased (Figure 3A). Probe concentration was also linear and correlated (R2 = 0.9938) with the PAI signals (Figure 3C), indicating convincing evidence that FeSe2-PEG was an excellent perfect PAI contrast agent. For T2-weighted MRI signal, as shown in Figure 3B, gradually rising concentration exhibited a marked signal gradient. The T2-weighted imaging signal of water was brighter than all concentrations of the materials, and the signals of FeSe2-PEG

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gradually decreased with the increase in the respective concentrations (Figure 3B). The r2 relaxivity of materials was measured to be 106.72 mM-1 s-1. Linear correlation R2 was 0.999 (Figure 3D), which suggests that probe concentration was linearly correlated to the T2-weighted imaging signals. We concluded that FeSe2-PEG is also a good T2 MRI contrast agent and all of the above-mentioned results indicated that the probe is effective, safe and multi-functional.

Figure4. probe in human hepatic carcinoma cells. (A) Cell viability of Hep-G2 、 HUH7 and SK-Hep1. (B)Targeting ability of probe at the cellular level. The structure of the HepG2 cells was acquired by Transmission electron microscopy under a magnification of × 10000. and the probes were seen around cell membrane of HepG2 (black aggregation). (C) Confocal microscopy images (×100) of Hep-G2 cell.

We next wanted to determine the biosafety and targeting of the FeSe2-PEG probe at the cellular level by two independent methods. Testing for cytotoxicity, we

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observed no significant effect of the different probe concentrations on cells (Figure 4A). The relative cell viability was approximately 100% when the concentration ≤ 0.1 mg/ml and between 90-100% until the concentration reached 0.2 mg/ml (Figure 4A), thus providing sufficient evidence that the probe is non-toxic in vitro. For method of confocal microscopy (Figure 4C), the different probe concentrations showed no effect on cell numbers, cytoskeleton and morphology. The phalloidin bound specifically to F-actin and stained the actin stress fibers in the cell, allowing the intercellular microfilaments to be clearly discernible in Hep-G2 cells incubated with FeSe2-PEG at multiple concentrations. The cell membrane and cell nucleus were not affected by probe. Thus, FeSe2-PEG did not exhibit detectable toxicity towards liver cancer cells. For HCC targeting, we observed an increased aggregate of materials on the cell membrane but not inside the cells, which indicated that the probe is targeted only to the liver cancer cell membrane (Figure 4B).

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Figure 5. Photoacoustic imaging. (A). The cross-section of tumor was acquired from 33.5 mm frames to 37.5 mm frames, at the same time, it could match the diameter that was measured by vernier caliper. (B). Three different group of photoacoustic images were acquired before and after injection of probe and (C) quantitative analysis. (D). Histological examination of cirrhosis nodules

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and tumor. (Red dotted circles represent cirrhosis nodules and tumor area respectively). *P< 0.05, ***P< 0.001.

Next, we wanted to use PAI, MRI, and HCC measurements to evaluate the applicability of the FeSe2-PEG probe in vivo. Furthermore, we wanted to determine whether the probe could distinguish between HCC and LC. We used PAI to measure small HCC since it has the capacity to detect small nodules. MSOT could measure tumors with a diameter of 4 mm, from 33.5-37.5 mm cross sections (Figure 5A). At the same time, we measured the tumor size (3.16 mm) by Vernier calipers. The accuracy of the measurement of the tumor was limited by the caliper layer spacing (5 mm) and may be improved upon in future studies. It is well known that traditional CT and MRI instruments have limited abilities to identify liver nodules with diameters of 1 cm. However, PAI could detect small HCC < 1 cm in diameter. We next wanted to confirm the targeting of the probe in vivo by a comparative study of the targeted and non-targeted probe. Several differences were observed (Figure 5B). Firstly, the photoacoustic signal intensity was different due to selective interaction of the targeted probe with GPC3 expressed on the cell membrane of HCC. This resulted in a higher photoacoustic intensity compared to non-targeted probe in vivo. We observed that the peak value of the targeted probe compared to the peak value of non-targeted probe was 1.9:1.4 (Figure 5C). The photoacoustic intensity was dependent on the depth of penetration and each nude mouse had variable thickness of skin and coating, rendering photoacoustic intensity to be only usable as a reference standard. Secondly, although photoacoustic intensity was not a suitable judgment criterion due to variability, we could still distinguish differences in probe metabolism (Figure 5C). Photoacoustic signal of either probe gradually increased from 0 to 12 h and peaked at the 1 and 12 h timepoints, with the signal at 12 h significantly higher than 1 h. The signal gradually decreased from 12 to 48 h. However, in contrast with the non-targeted probe, the upward trend of the targeted probes was sharper from 1 to 12

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h, while the downward trend was more gradual from 12 to 48 h. Finally, we evaluated the ability of the probe to distinguish between HCC and LC. As shown in Figure 5B, we observed several differences between HCC and LC. HCC presented as focal nodules before the injection of the targeted probe, while the rest of the liver was normal. In contrast, LC presented as extensive nodules and the overall presentation was not homogeneous. However, the above observation was not sufficient for identification, and PAI imaging at different time points and quantitative analysis of PAI signals were required. Moreover, after injection, the targeted probe metabolism in the two models was different. In HCC mice, the PAI signal rapidly increased from 0 to 12 h, and peaked at 1 and 12 h with the signal at 12 h significantly higher than that at 1 h. The signal gradually decreased from 12 to 48 h. In LC mice, the PAI signal gradually increased from 0 to 12 h, and subsequently decreased gradually from 12 to 48 h, but the peak signal was inconspicuous. Thirdly, the signal intensity was strikingly different between the two models (Figure 5B). Due to cell-specific targeting of the target probe, nanoparticles, which are capable of recognizing HCC, could not recognize the liver LC cells. Probe accumulated at 12 h in the nodule region for HCC models, while less probe in the nodule region at 12 h was detected in the LC nodule regions for LC models, which was caused by pathogeny structure. Intricate blood vessels in LC lesions lead to the growth of pseudolobuli. We concluded that the probe could sufficiently distinguish between HCC and LC lesions and adequately measure tumor size. Pathological examination results further supported that the diagnosis made by PAI was correct. To conclude, the diagnosis of HCC, exclusion of LC, and PAI with FeSe2-PEG-Peptide was successful.

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Figure 6. MRI imaging. Comparison of tumor regions before and after 24h injection of targeted probes in three different model mice. Liver cirrhosis( LC) model: cross-section plane contrast image (a, b) and coronal plane contrast image (c, d) of liver; Subcutaneous transplanted tumor model: cross-section plane contrast image (e, f) and coronal plane contrast image (g, h) of subcutaneous transplanted tumor; Hepatocellular carcinoma (HCC) model: cross-section plane contrast image (i, j) and coronal plane contrast image (k, l) of liver.

While the probe was positive for PAI, the probe was also positive for MRI signal, which was used for an auxiliary diagnosis. Based on our above phantom experiment results (Figure 3D), FeSe2-PEG had a strong signal based on T2-weight. We wanted to determine the applicability of this property in vivo to distinguish between HCC and LC lesions (Figure 6). The HCC model presented as a focal point in the liver area before injection. This focal point would become darker after injection. Meanwhile, the LC model presented as diffuse tuberosity, which showed considerable heterogeneity with nodules, and was lighter in color after injection compared to normal liver due to alcoholic fatty depositions as verified by pathological examination. Secondly, for mice with subcutaneous tumors, the tumors exhibited strong signals 12 h after injection and turned darker. At the same time, we observed dispersed dark areas in the middle of the tumor that represented necrotic cells within the tumor. We concluded that MRI could be used as an auxiliary diagnostic method.

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Ablation treatment is the general choice for treatment in the clinic at very early stage liver cancer (single nodule with diameter < 2 cm with Child-Pugh A). In this study, we administered PTT based on the photothermal effect of materials. Below we explain the photothermal effect from the perspective of material properties, cells, and in vivo observations. Firstly, we tested the photothermal characteristic of FeSe2-PEG. Increasing temperatures were found to match material concentration and irradiation time (Figure S2A). The sample temperature increased with irradiation, with the water temperature rising from 28.5°C to 29.1°C after irradiation for 5 min. Meanwhile, the FeSe2-PEG (0.8 mg/ml) temperature increased from 28.5°C to 74.5°C, which was high enough to kill cells. The photothermal stability of FeSe2-PEG (0.15 mg/ml) was validated by repeated heat absorption and dissipation (Figure S2B). The photothermal stability was still maintained after five rounds of the above process. We concluded that FeSe2-PEG has a good photothermal effect The FeSe2-PEG photothermal conversion efficiency of 785nm continuous laser can be calculated to be 34.76% (Supplementary Note 1). Next, we tested the effect of PTT at the cellular level (Figure S2C, S2D). Photos e and f were used as blank controls to exclude single influencing factors. We observed a significant reduction in survivability in response to laser irradiation. However, laser irradiation (0. 8 W cm-2, 5 min) or probe incubation alone did not affect the cell survival. The above results indicated that FeSe2-PEG could be an efficient photothermal agent for focal ablation of HCC cells under near infrared (NIR) laser irradiation and thus suitable as a PTT. Especially, when irradiated by laser (0.8 W cm-2) for 5 min, we observed all the cells died. In summary, the material has a good PTT effect.

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Figure 7. Post-treatment effect. (A) The mice survival after treatments in different groups. (B) The mice body weights after treatments in different groups. (C) Bioluminescence images was used to continuously monitor the signal intensity of tumor every 5d in different groups. (D)Photothermal therapy process flow chart. (E) HCC treated by PTT after injecting FeSe2-PEG-Peptide and FeSe2-PEG, then pathological examination was performed. Necrosis was found in FeSe2-PEG-peptide and FeSe2-PEG treated tissues, but relapses tumor cells were found in FeSe2-PEG treated tissues. ns:P> 0.05; *P< 0.05; **P< 0.01; ***P< 0.001.

After diagnosis by imaging, HCC should be treated by PTT as observed in our

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experiments on the photothermal effect of FeSe2-PEG-Peptide. One group was injected with FeSe2-PEG-Peptide. During treatment, the surface temperature was measured by an infrared thermal imaging and increased from 31.7°C to 56.5°C, which was high enough to ablate the tumors. In the group injected with FeSe2-PEG, the surface temperature of the tumor also increased from 31.6°C to 51°C (Figure 7D). However, this dramatic increase was not observed in the group injected with PBS (31.6°C to 39°C). To prove PTT availability for small HCC after treatment, we monitored survival time, weight, fluorescence signal value, and pathology. For survival time, we observed that tumors of groups C and D survived longer than groups A and B (Figure 7A). Group D tumors survived a little longer than those in Group C. This indicated that the PTT with targeted probes is the most effective treatment. Liver cancer usually results in significant loss of body weight in mice over time. In our weight-monitoring studies, we observed a significant improvement in body weight of the animals compared to the control after treatment (Figure 7B). The weight of mice in group B was reduced daily. However, the weight of groups C and D increased after therapy with group D exhibiting a faster increase in weight than group C. Fluorescence signal intensity measurement showed gradual increase in the tumor fluorescence signal in group B mice (Figure 7C). The tumor fluorescence signal of groups C and D gradually decreased until 20 days, which suggested that photothermal effect of FeSe2-PEG and FeSe2-PEG-Peptide was enough to cause apoptosis. However, the tumor fluorescence signal of group C decreased first and then later increased. Group C was injected with a non-targeting probe. Therefore, the probe did not bind to HCC cells and thus did not respond to PTT, resulting in unirradiated HCC cells and subsequent relapse at later stages. Finally, the liver tissue was harvested from treated mouse evaluated for PTT effects by pathological examination. Necrosis was found in groups C and group D, but relapse tumor cells were found in group C (Figure 7E). In summary, the data presented in this study show that PTT with targeted probes such as the FeSe2-PEG-Peptide, is an effective therapeutic option in not only identifying and distinguishing early HCC tumor lesions from LC nodules, but also in

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selective ablation of the tumor.

Discussion Currently, for patients with primary liver cancer, we usually use gadoxetic acid disodium injections (Gd-EOB-DTPA) to enhance MRI for the detection of small HCC less than 2 cm in size44, and then inject ICG into the patient to guide the surgical removal of liver cancer tissue clinically45. To reduce the potential long-term toxicity caused

by

multiple

injections,

we

developed

a

multifunctional

agent,

FeSe2-PEG-Peptide, which can be used for both treatment and imaging. We designed FeSe2-PEG-peptide as a multifunctional probe. It has both PAI signals and MRI signals for imaging, and also has a PTT effect for therapy. Moreover, the probe specially targeted HCC. This results in a large number of probes on the surface of HCC cells, which leads to higher PAI signals in the tumor nodules of HCC mice than in LC nodules of LC mice. Liver nodules of LC mice have weak PAI signal because of temporary arrest in intricate blood vessels of LC lesions. Therefore, we can easily distinguish between HCC and LC. Our imaging approach to detect tumors has many benefits compared to other imaging methods.

It is still a challenge for traditional imaging methods to detect

small nodules less than 0.5 cm18. In our experience, we can detect the nodules with 3.16 mm without contrast agent. MSOT as a PAI instrument that we have used is photoacoustic computed tomography (PACT), which can reach an imaging depth up to 7 cm, with lateral resolution of 720 m20. Further improvement in depth and speed of interest can make the above tumor size and boundary more accurate21. At the same time, we can design a targeted probe like FeSe2-PEG-Peptide to create high contrast imaging of tumor in liver, which is a blood-rich environment21. Furthermore, PAI can also image hemodynamics. Different optical wavelengths can be excited by different molecules. According to other reports, PAI can image myoglobin, hemoglobin, melanin, etc46. PAI can differentiate blood vessels and nodules due to its clear optical identification22. Similar to the above PAI of different groups of mice, green represents the spectrum of the probe, red represents the spectrum of hemoglobin, and blue

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represents the spectrum of deoxyhemoglobin. We found a large amount of oxidized hemoglobin signal around the tumor, which is consistent with previous results that the tumor is rich in small blood vessels47, 48. In the clinic, tumor recurrence is a major problem for patients with HCC after surgery or other treatment49.Though the mechanism is still unclear, an important factor for recurrence is that there are still some remaining tumor cells after treatment50. As in this study (Figure 7C), the group injected with non-targeted probes (FeSe2-PEG), two of the six HCC models recurred later. The reason for this may be that there are still some residual tumor cells after non-targeted PTT. Meanwhile, the targeted group (FeSe2-PEG-Peptide) did not recur later. This may be because the material is targeted to the surface of liver cancer cells, which causes precise PTT treatment at the cell level and avoids excessive killing of normal liver cells. Therefore, targeted PTT provides a new strategy to cure early stage, small-sized liver cancers51.

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Conclusions In this work, we synthesized a targeted probe FeSe2-PEG-Peptide conjugated with GPC3 for contrast-enhanced PAI and MRI for HCC identification and PTT. In the early stage, we repeatedly demonstrated the targeting, non-toxicity, and strong photoacoustic signal of the probe in terms of chemistry and cells. Then, we applied the probe with PAI, which could detect small nodules in vivo. We found that it could successfully distinguish small-sized HCC and LC, though they were both liver nodules by appearance. The probe acted differently in small-sized HCC and LC in terms of aggregation time and enrichment concentration of the probe. The probe was much more enriched in small-HCC than LC. Moreover, the targeted probes showed a trend of "fast-forward and fast-out" in HCC. However, the targeted probes show a trend of "slow-forward and slow-out" in LC mice, which may be attributed to the disordered blood vessels around the nodules. After accurate differential diagnosis, we performed PTT targeting small-sized HCC. Based on the results, the targeted probe is more effective than the non-targeted probe. After 45 days of observation, all mice with targeted PTT were cured, while two out of the mice injected with the non-targeted probe relapsed. Here, we show that the probe designed in this study effectively demarcates early HCC lesions with exceedingly small tumor sizes by PAI. Furthermore, the probe is able to distinguish between HCC and LC nodules and can be used to introduce photothermal therapy by laser irradiation specifically by increasing the temperature of the tumor cells, making this an exciting novel therapeutic strategy.

Acknowledgements The authors thank Huijuan You, Ziyu Han, Yaqin Wang, and Kun Su for assistance to finish the experiments. This work was supported by Major Instrument Project of National Natural Science Fund under grant No. 81627805; National Key R&D Program of China Grant under No. 2017YFA0205200; The National Natural Science

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Foundation of China grant number [81601576, 81501540, 81227901, 61231004, 61671449, 81401471, 81527805]; NSFC-GD Union Foundation (U1401254) ; The Science and Technology Plan Project of Guangzhou (No. 201604020144); Digital Theranostic Equipment Research Special Program of The “13th five-year” National Key Research Plan (No. 2016YFC0106500); The United Fund of National Natural Science Foundation of China and Government of Guangdong Province (Grant No. U1401254).

Supporting information Supporting information for this article including supplementary data of nanoparticle characterization (Hydrated particle size, UV-Vis spectrum, size stability), pathological examination and bioluminescence images of two different animal models, photothermal effect of probe in vitro, MRI imaging of comparison of tumor regions before and after 12h injection of targeted probes in three different model mice, non-targeting ability of FeSe2-PEG at the cellular level, the photothermal conversion efficiency of FeSe2-PEG.

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41. Liang, X.; Shang, W.; Chi, C.; Zeng, C.; Wang, K.; Fang, C.; Chen, Q.; Liu, H.; Fan, Y.; Tian, J. Dye-Conjugated Single-Walled Carbon Nanotubes Induce Photothermal Therapy Under the Guidance of Near-Infrared Imaging. CANCER LETT 2016, 383 (2), 243-249. 42. Wu, F.; Su, H.; Cai, Y.; Wong, W.; Jiang, W.; Zhu, X. Porphyrin-Implanted Carbon Nanodots for Photoacoustic Imaging and in Vivo Breast Cancer Ablation. ACS Applied Bio Materials 2018, 1 (1), 110-117. 43. Galanzha, EI.; Shashkov, EV.; Spring, PM.; Suen, JY.; Zharov, VP. In vivo, Noninvasive, Label-Free Detection and Eradication of Circulating Metastatic Melanoma Cells Using Two-Color Photoacoustic Flow Cytometry with a Diode Laser. CANCER RES 2009, 69 (20), 7926-7934. 44. Hammerstingl, R.; Huppertz, A.; Breuer, J.; Balzer, T.; Blakeborough, A.; Carter, R.; Fusté, LC.; Heinz-Peer, G.; Judmaier, W.; Laniado, M.; Manfredi, RM.; Mathieu, DG.; Müller, D.; Mortelè, K.; Reimer, P.; Reiser, MF.; Robinson, PJ.; Shamsi, K.; Strotzer, M.; Taupitz, M.; Tombach, B.; Valeri, G.; van Beers, BE.; Vogl, TJ. Diagnostic Efficacy of Gadoxetic Acid (Primovist)-enhanced MRI and Spiral CT for a Therapeutic Strategy: Comparison with Intraoperative and Histopathologic Findings in Focal Liver Lesions. EUR RADIOL 2008, 18 (3), 457-467. 45. Yamamoto, J. Treatment Strategy for Small Hepatocellular Carcinoma: Comparison of Long-Term Results After Percutaneous Ethanol Injection Therapy and Surgical Resection. HEPATOLOGY 2001, 34 (4), 707-713. 46. Tang, J.; Xi, L.; Zhou, J.; Huang, H.; Zhang, T.; Carney, PR.; Jiang, H. Noninvasive High-Speed Photoacoustic Tomography of Cerebral Hemodynamics in Awake-Moving Rats. J CEREBR BLOOD F MET 2015, 35 (8), 1224-1232. 47. N, F, HP, G, J, L. The Biology of VEGF and its Receptors. NAT MED 2003, 9 (6), 669-676. 48. Carmeliet, P.; Jain, RK. Molecular Mechanisms and Clinical Applications of Angiogenesis. NATURE 2011, 473 (7347), 298-307. 49. Wu, C.; Chen, Y.; Ho, HJ.; Hsu, Y.; Kuo, KN.; Wu, M.; Lin, J. Association Between Nucleoside Analogues and Risk of Hepatitis B Virus-Related Hepatocellular Carcinoma Recurrence Following Liver Resection. JAMA-J AM MED ASSOC 2012, 308 (18), 1906-1913. 50. Poon, R.; Fan, ST.; Wong, J. Risk Factors, Prevention, and Management of Postoperative Recurrence After Resection of Hepatocellular Carcinoma. ANN SURG 2000, 232 (1), 10-24. 51. Peng, H.; Tang, J.; Zheng, R.; Guo, G.; Dong, A.; Wang, Y.; Yang, W. Nuclear-Targeted Multifunctional Magnetic Nanoparticles for Photothermal Therapy. ADV HEALTHC MATER 2017, 6 (7), 1601289.

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