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Imaging Tiny Hepatic Tumor Xenografts via Endoglin Targeted Paramagnetic/Optical Nanoprobe Huihui Yan, Xihui Gao, Yunfei Zhang, Wenju Chang, Jianhui Li, Xinwei Li, Qin Du, and Cong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02648 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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ACS Applied Materials & Interfaces

Imaging Tiny Hepatic Tumor Xenografts via Endoglin Targeted Paramagnetic/Optical Nanoprobe Huihui Yan1,a,*, Xihui Gao2,3,4,a, Yunfei Zhang2,a, Wenju Chang5, Jianhui Li6, Xinwei Li2, Qin Du1,*, Cong Li2,*

1

Department of Gastroenterology, The Second Affiliated Hospital, College of Medicine,

Zhejiang University, Hangzhou, Zhejiang Province 310009, China 2

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan

University, Shanghai 201203, China 3

Department of Laboratory Medicine & Central Laboratory, Shanghai Jiaotong University

Affiliated Sixth People's Hospital South Campus, Shanghai 201499, China 4

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix

Composites, Shanghai Jiao Tong University, Shanghai 200240, China 5

Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032,

China 6 a

Ningbo No.2 Hospital, No.41 northwest Street, Ningbo, Zhejiang Province 315010, China These three authors contributed equally to this study.

KEYWORDS: paramagnetic/optical nanoprobe; aptamer; endoglin; tiny hepatocellular carcinoma; image-guided surgery

1

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ABSTRACT Surgery is a mainstay for treating hepatocellular carcinoma (HCC). However, it is a great challenge for surgeons to identify HCC in its early developmental stage. The diagnostic sensitivity for tiny HCC with a diameter less than 1.0 cm is usually as low as 10–33% for computed tomography (CT) and 29–43% for magnetic resonance imaging (MRI). Although MRI is the preferred imaging modality for detecting HCC, with its unparalleled spatial resolution for soft tissue, the commercially available contrast agents, such as Gd3+-DTPA, cannot accurately define HCC due to its short circulation lifetime and lack of tumor targeting specificity. Endoglin (CD105), a type I membrane glycoprotein, is highly expressed both in HCC cells and in the endothelial cells of neovasculature, which are abundant at the tumor periphery. In this work, a novel single-stranded DNA oligonucleotide based aptamer was screened by systematic evolution of ligands in an exponential enrichment (SELEX) assay and showed high binding affinity (KD= 98 pmol/L) to endoglin. Conjugating the aptamers and imaging reporters on a G5 dendrimer created an HCC targeting nanoprobe that allowed the successful visualization of orthotopic HCC xenografts with diameters as small as 1−4 mm. Significantly, the invasive tumor margin was clearly delineated, with a tumor to normal ratio of 2.7 by near-infrared fluorescence imaging and 2.1 by T1-weighted MRI. This multimodal nanoprobe holds promise not only for non-invasively defining tiny HCC by preoperative MRI but also for guiding tumor excision via intra-operative NIR fluorescence imaging, which will probably gain benefit for the patient’s therapeutic response and improve the survival rate. 2


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INTRODUCTION The liver is thought to be a frequent organ of tumorigenesis and metastases because it serves as a “friendly soil” for tumor cells, with its well-vascularized tissue, low blood flow shear rates and plenty of capillaries with nutrients.1 Hepatic cancer is the second leading cause of cancer-related death currently,2 and more than 90% of hepatic cancers are hepatocellular carcinoma (HCC).2 The World Health Organization (WHO) reports that 788 000 deaths were caused by HCC worldwide in 2015.2 Over the next 10–20 years, the incidence is estimated to increase and will peak in approximately 2030.2 Given the progression rate of HCCs (89.9– 100%)3-4 and the improved prognosis of HCC patients if they receive intervention in an early stage5-7, having an accurate diagnosis of HCC is crucial when a single lesion’s diameter is less than 1 cm to improve the therapeutic response. Surgical resection is a mainstay of HCC treatment.2 Patients who receive interventions in an early period achieve a 5-year survival rate as high as 70% after timely resection or transplant, but the median survival for advanced HCC patients who are only suitable for palliative treatments is less than one year. 8Thus, there is an urgent demand in the clinic for precisely delineating tiny HCCs before and during surgery. Among the multiple imaging modalities, magnetic resonance imaging (MRI) and computed tomography (CT) have been widely used for HCC visualization in the clinic due to their high spatial resolution.2 Whereas both CT and MRI have 100% accuracy in defining HCCs larger than 2.0 cm with the assistance of extracellular contrast agents, these values decrease to 44–47% for 3



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MRI and 40–44% for CT when the tumor diameters decrease to 1–2 cm.2 These values further decrease to 29–43% for MRI and 10–33% for CT when the diameters of HCCs are less than 1.0 cm.2 Therefore, visualization of tiny HCCs with diameters below 1.0 cm, especially for those with diameters 1−4 mm, is crucially important to improve the prognosis of HCC patients.9-10 The non-specificity and transient circulation lifetime of diethylenetriamine pentaacetic acid (Gd3+-DTPA), a commercially available MR contrast agent,2 limit its ability to detect HCCs. Although gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) and gadobenate dimeglumine (Gd-BOPTA) demonstrated higher hepatic tumor uptake than Gd3+-DTPA did, particularly for small HCCs2, they showed low specificity for HCC diagnosis11-12 Therefore, there is a necessity to develop novel MR contrast agents to image small-volume HCCs with high sensitivity and speciﬁcity. Angiogenesis plays an important role during the development, invasion, migration and metastasis of malignant tumors.13-14 Imaging angiogenesis is a promising approach for visualizing cancers in their early developmental stages because tumors can hardly grow to 5 mm without nourishment from new blood vessels.14 Angiogenesis biomarkers, including endoglin, αvβ3 integrin and VEGFR-2, are widely used for tumor diagnosis. Among these marks, endoglin shows higher expression levels on the vascular endothelium in numerous types of solid tumors than αvβ3 integrin and VEGFR-2.14-15 Previous work reported that endoglin could promote the invasion of HCC cells by increasing VEGF expression;16 and the expression of αvβ3 integrin usually occurs at a later stage than VEGF/VEGFR axis during the process of angiogenesis.17 Therefore, endoglin targeted nanoprobe holds the promise to delineate HCC invasive margin 4


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with high target to background ratio.18 Recently, imaging probes functionalized with endoglin antibody (TRC105, 148 kDa) or the Fab fragment of endoglin antibody (TRC105-F(ab')2, 97 kDa) were reported to visualize murine breast tumors with high specificity by positron emission tomography (PET).19-20 However, the high cost, transient circulation lifetime induced by non-specific reticulo-endothelial system (RES) uptake and potential immunogenicity compromise the clinical applications of these antibody-based imaging probes. An aptamer is a kind of single-stranded DNA or RNA oligonucleotide that has receptor binding affinity comparable to that of an antibody but has lower immunogenicity and better stability. Moreover, aptamers can be prepared with much lower cost compared to that of monoclonal antibodies. We hypothesized that an aptamer with a high targeting affinity and specificity to endoglin would be valuable to develop as an imaging probe to visualize tiny HCCs with improved sensitivity and specificity. In this work, a novel aptamer with high binding affinity to endoglin was screened via a systematic evolution of ligands using the exponential enrichment (SELEX) assay. Conjugating this aptamer with the paramagnetic agent Gd3+-DTPA and near-infrared fluorophore IR783 on the G5 dendrimer created the aiming nanoprobe that successfully delineated orthotopic HCC xenografts with diameters as low as 4 mm. To the best of our knowledge, this is the first multimodal imaging probe for detecting tiny HCCs that uses an aptamer as a receptor targeting domain. This nanoprobe holds promise to guide tiny HCC surgery by preoperatively locating the neoplastic tissue by MRI and intra-operatively assisting 5



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tiny HCC excision via fluorescence imaging.

MATERIALS AND METHODS Materials. All reagents were of analytical grade and purchased from Aladdin Reagent (Shanghai, China), unless otherwise specified. Rabbit anti-mouse/human endoglin antibody, rabbit anti-human CD31 antibody and mouse anti-human CD31 antibody were purchased from Abcam (Cambridge, MA, USA). Fetal bovine serum, penicillin and streptomycin, high glucose DMEM, Goat anti-rabbit HRP-labeled secondary antibody, Alexa Fluor 633-labeled goat anti-rabbit secondary antibody, Alexa Flour 488-labeled goat anti-rabbit secondary antibody and Alexa Flour 647 goat anti-mouse secondary antibody were purchased from Invitrogen (Calsbad, CA, USA). Tissue cell rapid lysate was purchased from Solarbio (Beijing, China). A bicinchoninic acid (BCA) protein quantitative kit, Nde I/BamH endonucleases and rhodamine succinimide ester were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidenedifluoride membranes were purchased from Millipore (MA, USA). 5% skimmed milk powder and purified human endoglin antibody, functional grade, were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). The Supersignal WestPico Chemiluminescent Substrate Kit was purchased from Pierce Biotechnology (Rockford, IL, USA). Other reagents for the western blot assays were purchased from JRDUN Biotechnology (Shanghai, China). Rabbit anti-mouse/human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Cell Signaling Technology (Boston, MA, USA). The 6


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pET 15b vector was purchased from Novagen (Madison, WI, USA). E.coli, DH5α strain, was purchased from New England Biolabs (Beverly, MA, USA). HRP labeled avidin and biotin were purchased from Vector Labs (Burlingame, CA, USA). CNBr, Sepharose 4B bead, 3, 3′, 5, 5′-Tetramethylbenzidine Liquid Substrate (TMB), Tween 20, polyamidoamine (PAMAM) G5 dendrimer (77.35 mg/mL in methyl alcohol, containing 128 primary amino groups, MW: 28,826 Da), GdCl3, trypsin and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Activated polyethylene glycol (PEG) derivatives, Malemide (Mal)-PEG2k- N-hydroxysuccinimidyl (NHS) ester and PEG2K-NHS ester were purchased from JenKem Technology Co. Ltd. (Beijing, China). Diethylenetriaminepentaacetic acid (DTPA)-NHS ester was purchased from Macrocyclics (Plano, TX, USA). Isoflurane (AERRANE) was purchased from Baxter Healthcare Corporation (Deerfield, IL, USA). Cell Counting Kit-8 was purchased from YEASEN Biotechonology Co., Ltd. (Shanghai, China). Patients. The research program was in line with the ethics guidelines of the Helsinki Declaration (1975) and was approved by the Ethics Committee of the Second Affiliated Hospital of School of Medicine, Zhejiang University. All patients provided informed consent prior to the study. In total, there were 6 male (mean age: 63 years, age range: 41-77 years) patients. The HCC lesions ranged from 1 to 10 cm in diameter. Animals. The animal experiments complied with the rules established by the Institutional Ethical Committee of Animal Experimentation and were executed in strict accordance with the government and international animal experiment guidelines. All nude mice were supplied by 7



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Shanghai Slac Lab Animal Ltd. (Shanghai, China). They were housed in the animal facility at the School of Pharmacy, Fudan University, under pathogen-free barrier conditions. The room temperature and humidity were controlled (23 °C and 50%, respectively). The photoperiod was a 12-h light and 12-h dark cycle. Histopathological Staining. The tumor tissues excised from three HCC patients were fixed in 10% neutral buffered formalin for 24 h, processed into paraffin and sectioned into slices with a thickness of 5.0 µm. Tumor sections were first stained with primary rabbit anti-human CD31 antibody

or

primary

rabbit

anti-human

endoglin

antibody.

Second,

the

glucose

oxidase-diaminobenzidine (DAB) method was used to achieve CD31 or endoglin staining with a brown color. Third, sections were counterstained with hematoxylin staining solution. After completely rinsing, sections were dehydrated with 70%, 80% and 100% ethanol successively, transferred into xylene, and mounted. The tumor tissues excised from another three HCC patients were embedded in optimal cutting temperature compound, frozen and sectioned with a thickness of 10 µm. After incubationg with rabbit anti-human endoglin (1:100 dilution) and mouse anti-human CD31 (1:20 dilution) primary antibodies, respectively, at 4 °C for overnight, They were incubated with Alexa Fluor 488 -labeled goat anti-rabbit secondary antibody (1:400 dilution) and Alexa Fluor 647 -labeled goat anti-mouse secondary antibody (1:200 dilution) at room temperature for 1 h followed the nucleus staining by 4’,6-diamidino-2-phenylindole (DAPI, 0.5 mg/mL). The immunofluorescence images were collected on a Zeiss LSM 710 META confocal laser scanning microscope (Carl Zeiss, Oberkochen, Baden-wurttemberg, Germany). 8


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Cell Cultures. Human umbilical vein endothelial cells (HUVECs), human hepatocellular carcinoma SMMC-7721 cells, mice normal hepatic NCTC 1469 cells，human breast cancer MCF-7 cells and human normal liver LO2 cells were purchased from the Chinese Academy of Sciences Cell Bank. All the cells utilized in this research were cultured in Dulbecco’s modified Eagle’s medium with 1% streptomycin, 1% penicillin and 10% fetal bovine serum (FBS) (37 °C, 5% CO2). Western Blotting. Typically, the cultured cells at 80% confluence or obtained tissue blocks （tumor tissues and normal tissues） were added to a lysis buffer containing proteases and phosphatase inhibitors. The proteins were extracted, and the protein concentrations were quantified with the bicinchoninic acid (BCA) method. Then, 30 µg of total protein from each were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) within 1.5 h. Then, gels were transferred to polyvinylidene difluoride membranes (25 V, 30 minutes) via the semi-dry electro-transfer method. 5% skimmed milk powder was used to block the membranes that were incubated with endoglin (1:1000) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000) primary antibodies for 4 h at room temperature. The membranes were incubated with HRP-labeled secondary antibody (1:3000) at 37 °C for 1 h. Visualization was performed using a Supersignal WestPico Chemiluminescent Substrate Kit (Pierce Biotechnology, Rockford, IL, USA). Densitometry studies were calculated by ImageJ software (NIH, Bethesda, MD, USA). Screening Endoglin Targeted Aptamer. The nucleotide sequence of the extracellular terminal 9



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of human endoglin protein comprises the 26th-136th amino acids on the N end. SELEX was used to screen for an aptamer with high affinity to the extracellular terminal of human endoglin protein from a random single nucleotide sequences library. The detailed steps were as follows: (1) Recombinant human endoglin extracellular terminal protein was coupled with Sepharose 4B beads, which were activated by CNBr, in accordance with the requirements of the specification. On the next day, the endoglin-Sepharose 4B beads suspension was sealed for 2 h with Tris-HCl buffer at room temperature. After washing with NaHCO3 buffer, Tris-HCl buffer and acetate buffer, the suspension was precipitated and diluted with 20% equal volume of glycerol for storage. (2) A DNA random sequence library (self-constructed, 1012) was mixed with the endoglin-Sepharose 4B beads and reacted at room temperature for 1 h. The beads were centrifuged and washed with a low-salt buffer, high-salt buffer and LiCl buffer. Finally, the beads precipitation was added to a phenol: chloroform: isoamyl alcohol mixture to extract conjugated DNA fragments. (3) The DNA fragments were extracted and amplified by PCR, with the upstream primer: 5’-AGTCCATATGTAGACTATGTCAGTTACG-3’ and downstream primer: 5’-GACTTGGATCCGCTTAGTCAGATTCGGATCT-3’. (4) The amplified PCR products were collected, quantified and diluted. Then, the above products were mixed with endoglin-Sepharose 4B beads and reacted at room temperature for 1 h. Next, the beads were centrifuged and washed with low-salt buffer, high-salt buffer and LiCl buffer. Finally, the beads precipitation was added to a phenol: chloroform: isoamyl alcohol mixture to extract the conjugated DNA fragments. Then, PCR amplification was performed. The procedure was repeated 8 times. (5) The final extracted DNA fragments were digested with Nde I and BamH 10


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endonucleases, cloned into the pET15b vector and transformed into E.coli, DH5α strain. Finally, the plasmid was extracted and sequenced. The 5' end of the final sequence was functionalized with a primary amine for conjugating with PEG. Binding Affinity Measurement of Endoglin Targeted Aptamer. An ELISA assay was performed to measure the receptor-binding affinity of the screened aptamers. The recombinant endoglin extracellular terminal protein was added to the enzyme plate at the corresponding coating concentrations (at least two concentrations). The plate was sealed with 10% FBS. Then, the aptamer labeled with biotin (at 3’ terminal, aptamer-biotin) was prepared with a ratio dilution and added to the plate. 1: 1000 HRP labeled avidin, 3, 3′，5, 5′-Tetramethylbenzidine Liquid Substrate (TMB) and termination liquid sequentially were added to react. Colorimetry was performed at 450 nm with an enzyme-labeled instrument (Tecan-200 Infinite Pro reader, Nuremberg, Bavaria, Germany). For the affinity calculation, a scatter plot, with the optical density (OD) value as the ordinate and aptamer-biotin dilution as the abscissa, was created, with the inflection point set to OD 100% to find the OD 50%. The negative logarithm of the aptamer-biotin dilution was calculated using the OD450 value as the ordinate and the negative logarithm as the abscissa to obtain a regression curve, regression equation and correlation coefficient r. The value of OD 50% was substituted into the regression equation, the negative logarithm of the corresponding dilution of the aptamer-biotin solution was obtained, and finally, the corresponding dilution of aptamer-biotin was obtained. The already known aptamer-biotin concentration was substituted, and the corresponding aptamer-biotin dilution was used in the 11



affinity calculation formula ( K =

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Ag n −1 , n = 2,4,6,8... , ) to obtain the K value. ( n = Ag ' n × [Ab'] − [Ab]

where Ag and Ag' are different coating concentrations, and Ab and Ab' are the corresponding concentrations of aptamer-biotin at 50% OD450 for the coating concentrations of Ag and Ag', respectively). Synthesis and Characterization of Nanoprobes. The control nanoprobe without the modified aptamer (denoted as Den-PEG) was prepared as previously described.21-23 The targeted dendrimer particle marked with the modified aptamer (denoted as Den-Apt1) was also prepared as before. Briefly, the fifth-generation dendrimer (PAMAM G5 dendrimer, diameter: 7 nm) was selected as the platform for the particle. In Den-Apt1, the aptamer was labeled on the surface of the dendrimer by a flexible PEG linker. Both Den-Apt1 and Den-PEG were labeled with rhodamine and IR783 fluorophores as well as paramagnetic Gd3+-DTPA chelators. The nanoprobes were characterized as previously described.21-23 Briefly, the molar ratio between the aptamer, PEG, DTPA and dendrimer was quantified by detecting the proton integrals of aptamer (1.2, 3.2 ppm), PEG (3.7 ppm, O-CH2), DTPA (3.3-2.4 ppm) and dendrimer (3.3-2.2 ppm) in an 1

H NMR spectrum. The molar weights of the nanoprobes were determined by integrating the

characteristic protons of dendrimer, PEG, DTPA and aptamer in the 1H NMR spectra. The hydrodynamic diameter, the polydispersity index (PDI), and the Zeta potential of the nanoprobes were measured by a Malvern Zetasizer instrument (Malvern Instruments Inc., Westborough, MA, USA) at room temperature. To measure the surface charges, the instrument was calibrated with a standard solution, for which the Zeta potential was -50 mV. The Gd3+ concentrations of 12


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nanoprobes were determined by a Hitachi P-4010 Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) system (Tokyo, Japan) with nebulizer gas flow at 0.9 L/min and RF power at 1100 W. The longitudinal relaxivity r1p values of Den-Apt1 and Den-PEG were calculated via plotting the 1/T1 versus the concentration of Ga3+ in two nanoprobes. The Den-Apt1 and Den-PEG were characterized through transmission electron microscopy (Tecnai G2 Spirit BioTwin, FEI, Hillsboro, OR, USA). Den-PEG and Den-Apt1 were evaluated by migration by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to verify their purity. Then, 30 µL of the treatment buffer [100 mM Tris, pH 6.8, 2% (w/v) SDS, 12% (v/v) glycerol, 0.01% (w/v) bromphenol blue] with 50 µg nanoprobes was loaded on a 7% polyacrylamide gel. After 2 h of electrophoresis at 100 V, fluorescent images of the SDS-PAGE gels were obtained using an In Vivo Multispectral Imaging System (Kodak, Rochester, NY, USA, FOV = 12.8 cm, f/stop = 4, bin = high resolution, exposure time = 2 s). The λex/λem for acquiring rhodamine and IR783 fluorescence images were 543 nm/ 560 nm and 745 nm/ 780 nm, respectively. We evaluated the serum stability of the nanoparticles by monitoring the dynamic light scattering (DLS) diameters and the fluorescence intensities (excited at 507 nm and 755nm, detected at 583 nm and 785nm, Shimazu RF-5301PC, Tokyo, Japan) of Den-Apt1 and Den-PEG before (0 h) and after (1 h, 2h , 4 h, 8h, 16h 24 h) addition of FBS. Cell Viability. The biocompatibility of Den-Apt1 and Den-PEG were evaluated by means of CCK-8 assay with human normal hepatic LO2 cells. After incubation with two nanoprobes (0.5, 1, 2, 5, 10, 20, 50 µM) and PBS (control group) for 24 h，the cell viability was calculated through 13



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detecting the optical density by enzyme-labeled instrument (Tecan-200 Infinite Pro reader, Nuremberg, Bavaria, Germany). Confocal Fluorescence Microscopy Studies. SMMC-7721 cells were isolated by trypsin digestion, seeded onto 35-mm glass dishes, cultured to a confluence of approximately 50%, and incubated for 2 h or 24 h with 2.0 mL of a solution containing 4.0 µM of Den-Apt1 or Den-PEG at 37 °C. Immediately thereafter, the SMMC-7721 cells were washed with PBS and imaged by a Zeiss LSM 710 META confocal laser scanning microscope (Carl Zeiss, Oberkochen, Baden-wurttemberg, Germany). For blocking assays, cells were pre-incubated with 30 µg/ml purified human endoglin antibody at 4 °C for 30 min, washed with PBS and incubated with Den-Apt1 solution (4 µM) at 37 °C for 2 h or 24 h. Flow Cytometry Studies. SMMC-7721 cells in the logarithmic growth phase were digested with trypsin, counted under a microscope, and seeded on a six-well plate at a density of 300,000 cells per well. Then, 2 mL of medium was added to each well, cultured. The binding assay was performed when each well reached 80% confluence. SMMC-7721 cells were incubated with 2.0 µM of Den-Apt1 or Den-PEG for 0 min, 15 min, 30 min, 1 h, 4 h or 24 h. For the 0-min groups, DMEM complete medium was added instead of the probes. After incubation, the SMMC-7721 cells were washed, centrifuged with 1,776 g of centrifugation at 4 °C, and fixed with 4% paraformaldehyde. The cell nanoparticle uptake efficiency was quantified immediately using a BD Accuri C6 flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Development of Orthotopic Hepatic Tumor Xenograft. Briefly, a solution of approximately 14


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5×106 of SMMC-7721-GFP (Shanghai Aolu Biotechnology Co. Ltd., Shanghai, China) cells concentrated in 100 µL of cell culture medium was prepared. The solution was subcutaneously injected into the right leg of a nude mouse (18−20 g, male). The mice were fed in a specific pathogen free (SPF) level animal room for 2 weeks. Then, the subcutaneous neoplasms were excised, cut into pieces (size: 1 mm×1 mm×1 mm), and inoculated into the livers (usually the upper lobes, which were close to the abdominal wall) of another group of nude mice via microsurgical operation. Another 2−3 weeks later, the mice bearing orthotopic hepatic cancer xenografts in a size range of 3−4 mm were used for further research. In Vivo MRI Studies. A Biospec 70/20 MRI scanner (Bruker Inc., Billerica, MA, USA) was used to capture in vivo MR images at 7.0 T. The mice bearing orthotopic hepatic tumor xenografts were divided into four groups (n=4) and were injected with Den-Apt1, Den-PEG, Gd-DTPA, or purified human endoglin antibody as an endoglin blocking agent, followed by Den-Apt1. Den-Apt1, Den-PEG, or Gd-DTPA, dissolved in PBS solution to a total volume of 0.25 mL, was intravenously injected at a dosage of 0.05 mmol/kg of [Gd3+], and purified human endoglin antibody was injected at a dosage of 100 µg per mouse, 2 h before injecting Den-Apt1. As the mouse was placed in the MR coil, it inhaled isoflurane (0.5−2%) in 100% oxygen, and the gas flow was continuously regulated by monitoring mouse respiration. The mouse temperature in the magnet was maintained by a heating pad regulated by a thermostat. Continuous monitoring of respiration was accomplished by a Bruke PhysioGard system. A gating device was applied to reduce the respiration interference to the liver MR image. The mouse tail vein was cannulated to 15



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improve the convenience of the intravenous administration of various solutions. Dynamic T1-weighed images of mice liver bearing orthotopic hepatic tumor xenografts were taken before and after injection at 15, 30, 60 and 120 min. After these processes, the mice were returned to their cages and further imaged after 24 h. Transverse section images of 1-mm-thick liver sections were obtained using an MSME sequence (TR = 600 ms, TE = 11 ms, and number of average = 4). The signal intensity of the liver tumor region (T1 (tumor)) in the MR images was quantified using eFilm Workstation 1.5.3 software. To calculate the T/N ratio = T1 (tumor) / T1 (normal), the signal intensity of normal liver tissue (T1 (normal)) in the same slice was measured. For each mouse, three T1-MR images within the maximum tumor range were selected to quantify the signal intensity of tumor tissue and normal liver tissue at each time point. Then, the average value of the T/N ratio was calculated. Ex Vivo Optical Imaging Studies. Optical imaging studies were carried out on Kodak's in vivo optical imaging system (Rochester, NY, USA). The mice bearing orthotopic hepatic tumor xenografts were divided into two groups (n=6) and injected with Den-Apt1 or Den-PEG. The mice were anesthetized at 2 h or 24 h post injection (n=3). After heart perfusion with saline, the organs, including heart, liver, spleen, lung, kidney, brain and muscle, of the mice were excised and arranged on the glass plates in succession. Then, near-infrared fluorescence imaging was performed (λex/λem=745/780 nm). Additionally, the livers of the mice were taken out and arranged on the glass plates in succession. White light imaging, GFP imaging (λex/λem=395/509 nm) and the near-infrared fluorescence imaging (λex/λem=745/780 nm) were performed. NIR 16


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fluorescence images and GFP images were captured with a 0.5-s exposure time (FOV = 6.4 or 12.8 cm; f/stop, 4; bin, high resolution). The fluorescence intensities were calculated by ImageJ software. Immunofluorescent Staining. Immunofluorescent staining showed the localization of Den-Apt1 in normal and tumor tissues as well as the colocalization of endoglin and Den-Apt1. Primary rabbit anti-mouse endoglin antibody (1:50) and Alexa Fluor 633-conjugated secondary antibodies (1:100) were utilized for fluorescent imaging. 1% Triton X-100 was applied to increase the permeability, and DAPI was applied for nuclear counterstaining. Next, immunofluorescent imaging was performed with a Zeiss LSM 710 META confocal laser scanning microscope (Carl Zeiss, Oberkochen, Baden-wurttemberg, Germany). The percentage of merged area (yellow) in each slice relative to the total Den-Apt1 deposition area (red) was analyzed by the ImageJ software. Ten randomly selected amplification fields (400×) were evaluated for each slice. Statistical Analysis. Statistical Product and Service Solutions (SPSS) 16.0 software (Chicago, IL, USA) was used for statistical analysis. Quantitative data were expressed as the mean ± standard deviation (SD). Student’s t test was applied to evaluate the significance of the data. If the P value was less than 0.05, it was considered to have a significant difference. The data denoted by * for P

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