Renal Clearable Peptide Functionalized Ba2GdF7 Nanoparticles for

Subscriber access provided by Kaohsiung Medical University

Functional Inorganic Materials and Devices

Renal Clearable Peptide Functionalized Ba2GdF7 Nanoparticles for Positive Tumor-targeting Dual-mode Bioimaging Yang Feng, Hongda Chen, Baiqi Shao, Shuang Zhao, Zhenxin Wang, and Hongpeng You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07129 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Renal

Clearable

Peptide

Functionalized

Ba2GdF7

Nanoparticles for Positive Tumor-targeting Dual-mode Bioimaging Yang Feng,

a, c

Hongda Chen,

b, c

Baiqi Shao,

a

Shuang Zhao,

a, c

Zhenxin Wang,

b,

* and

Hongpeng You a, * a State Key Laboratory of Rare Earth Resource Utilization , Changchun Institute of Applied Chemistry, University of Chinese Academy of Sciences, Changchun 130022, P. R. China b State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, University of Chinese Academy of Sciences, Changchun 130022, P. R. China c University of Science and Technology of China, Hefei 230026, P. R. China

KEYWORDS: Ba2GdF7, tumor-targeting, renal clearance, MR imaging, CT imaging ABSTRACT: Considering the dilemma between the effective tumor targeting and the avoidance of potential toxicity, it is desired to design nanoprobes with positive tumor-targeting and good renal clearance ability. In present work, we developed the epidermal growth factor receptor (EGFR)-targeted peptides functionalized Ba2GdF7 nanoparticles (termed as pEGFR-targeted Ba2GdF7 NPs) for positive tumor-targeting magnetic resonance imaging and X-ray computed tomography (MRI/CT) dual-mode bioimaging. The positive tumor-targeting ability of pEGFR-targeted Ba2GdF7 NPs is achieved by conjugation of EGFR-targeted peptides on 6.5 nm Ba2GdF7 nanoparticle surface through formation of Gd-phosphonate coordinate bonds. The pEGFR-targeted Ba2GdF7 NPs display desirable cytocompatibility in the test concentration range, and high binding affinity with lung cancer cells. In vivo MR and CT imaging results demonstrate that the pEGFR-targeted Ba2GdF7 NPs are able to be accumulated and detained within an engrafted A549 lung carcinoma, which enhances both MR and CT contrast in the tumor tissue. Systematic in vivo experimental results further demonstrate that the pEGFR-targeted Ba2GdF7 NPs have favorable in vivo renal clearance kinetics as well as reasonable in vivo biocompatibility.

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. INTRODUCTION Bioimaging techniques have hitherto received significant attention owing to the remarkable accuracy of disease diagnosis, specifically for cancer imaging.1-4 As the powerful noninvasive diagnostic tools, MR and CT imaging have achieved great advancement.5-7 However, it is difficult to obtain precise information from single-mode diagnostic imaging because each imaging mode has its inherent advantages and drawbacks.8 It is proven to be that the accuracy and sensitivity of disease diagnosis can be improved by integrating strengths of different imaging modes through dual or multi-modality contrast agents.9-11 It is well known that Gd3+ is a promising T1 contrast agent owing to its largest number of unpaired electrons with parallel spin.12 In addition, barium (Ba) is most preferred for preparing CT contrast agent owing to large K-edge values and high X-ray mass absorption coefficients. The large K-edge value of Ba is 37.4 keV and the absorption coefficients of Ba are 8.51 and 3.96 cm2 g-1 at 60 and 80 keV, respectively.13 Therefore, the NPs based on Ba and Gd elements are the better materials for dual-modality imaging (MRI and CT imaging). Although enormous efforts have been made in the field of development of Ba and Gd elements-based multimodal imaging nanoprobes, such as BaGdF5,14 and Ba2GdF715 during the past decades, the applications of NP-based contrast agents are still severely limited due to their long-term retention and accumulation in the healthy tissue, leading to potential toxicity.16,17 Recently, researchers have demonstrated that the NPs with ultra-small size can be rapidly excreted by the renal route.18 For example, 3 nm cysteine-CdSe/ZnS quantum dots can be excreted about 75 % injection dose by the urine within 4 h,19 while 3 nm glutathione-coated AuNPs were cleared out nearly 70 % injection dose by the urine at 60 min post-injection.20 Unfortunately, most of these ultra-small NPs have difficulties in precisely transportation and accumulation into tumor lesion site, causing weakened enhanced permeability and retention (EPR) effect.21-23 Therefore, it is of significance to overcome this dilemma by designing NPs with appropriate size for efficient renal clearance and enhancing contrast effect.24-27 Besides the performance of materials themselves, targeting ability is another effective factor for increasing contrast enhancement.28-30 EGFR, as a class of membrane receptor tyrosine kinases, plays pivotal roles in many cellular processes including proliferation and apoptosis of cell, activation of angiogenesis, and development of metastatic capacity.31-33 In particular, EGFR is normally overexpressed in 70 % of non-small cell lung cancers patients,34 suggesting that targeting


Page 2 of 20

Page 3 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EGFR is an effective strategy to increase the binding affinities of tumor cells for nanoprobes. Thus, the positive tumor-targeting contrast agents can be achieved by engineering the NPs with EGFR-targeting ligands including peptides and antibodies35-38. Among them, the peptides with short amino acid sequences have gained extensive interest for many applications. They have been divided into different species according to the functions including cell-penetrating, tumor-targeting, antibiosis, antioxidation, anticancer, and the others.39 Tumor targeting peptides are not only have high affinities with the receptors overexpressed on the tumor cell surface,40 but also prevent nonspecific uptake by the reticuloendothelial system, resulting in improvement of tumor tissue penetration of NPs.41 For example, the cancer cell internalization capability of Au NPs can be effectively enhanced through conjugating cyclic arginine-glycine-aspartic acid (c(RGDyC)) peptides on their surfaces, and it further improves the Au NP-based radiotherapy efficacy on tumor.42 In our previous work, peptides functionalized NaGdF4 nanodots have been successfully synthetized for tracking orthotopic colorectal tumor.43 Moreover, the peptides can be easily synthesized by solid-phase peptide synthesis strategy and the physicochemical properties of peptides can be reiteratively adjusted for satisfying requirements of different bioapplications through predesigning the amino acid sequences of peptides. For example, the phosphopeptides (named as pPeptides) are the peptides modified with the phosphorylation of amino acid possessing the ability of phase transfer through the Gd-phosphate coordination reaction. Therefore, the characteristics of peptides facilitate significant reduction of the time- and labor-cost of the synthesis of multifunctional nanoprobes. In this work, we developed a renal-clearable contrast agent (termed as pEGFR-targeted Ba2GdF7 NPs) for positive tumor-targeting MR/CT dual-mode imaging through conjugating EGFR-targeted peptides on 6.5 nm Ba2GdF7 NPs surface. Taking advantage of Gd-phosphate coordination reaction, the hydrophobic oleate ligands of Ba2GdF7 NPs are easily replaced by the hydrophilic phosphorylated EGFR-targeted peptides through peptide-mediated phase transfer. In vivo experiments on mouse-bearing A549 tumor demonstrate that renal clearable pEGFR-targeted Ba2GdF7 NPs exhibit good biocompatibility, effective renal clearance capability, positive tumor targeting ability, and strong MR and CT contrast enhancement. MATERIALS AND METHODS 2.1. Materials. Gd2O3 (99.99 %) was obtained from Shanghai Yuelong Non-Ferrous Metals Ltd.



Page 4 of 20

(Shanghai, China). GdCl3 solution was prepared by dissolving Gd2O3 in dilute HCl under stirring and

heating.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT)

and

paraformaldehyde (4 %) were obtained from Beijing Ding guo Biotechnology Ltd. (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), and trypsin-EDTA solution were provided by Gibco Inc. (New York, USA). EGFR-targeted pPeptides (sequence: G(p-S)GYHWYGYTPQNVI, molecular weight (MW): 1.8 kDa) were purchased from China Peptides Co. Ltd. (Shanghai, China). The A549 cell line was supplied by the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The nude mice (6 weeks old, 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd.. Other chemical reagents were analytical grade and provided by Beijing Chemical Reagent Co. (Beijing, China). 2.2. Characterization. The phase structure of the samples was recorded on a X-ray powder diffraction (XRD) with a D8 Focus diffractometer (λ= 0.15406 nm, 5° min−1, from 10° to 85°, Bruker Co., Germany). The transmission electron microscopy (TEM) images were characterized by a JEOL-2010 transmission electron microscope (JEOL Co. Japan) at 200 kV. The infrared spectra were conducted with a Bruker Vertex 70 Fourier transform infrared (FTIR) spectrometer (Bruker Co., Germany). Energy-dispersive X-ray spectra (EDS) were obtained by an energy dispersive spectroscopy (Oxford instrument Ltd., UK). Zetasizer Nano ZS (Malvern Instruments Ltd., UK) was employed to inspect zeta potential (ζ) and dynamic light scattering (DLS) of the as-prepared products. The X-ray photoelectron spectrometer (XPS) spectra were carried out on a VG ESCALAB MKII spectrometer (VG Scientific Ltd., UK). The analysis of element were characterized by an ELAN 9000/DRC ICP-MS system (Perkin Elmer, USA). Thermogravimetric analyses (TGA) were evaluated by a TGA-2 analyzer (PerkinElmer Co., USA, 10 oC min−1, from 20 o

C to 800 oC). Nitrogen adsorption/desorption curves were carried out on Brunauer–Emmett–Teller

(BET) measurements using an ASAP 2020 surface area analyzer. (Micromeritics, USA) T1-weighted MR images were recorded on a Philips Achieva 3.0 T MRI scanner (Magnetom Avanto, Philips, Netherlands). CT images were obtained by a 64-detector row CT unit (General Electric, Milwaukee, WI). 2.3. Synthesis of Ba2GdF7 NPs. First, aqueous solution (2 mL) containing NaOH (9 mmol) was added into12 mL of ethanol solution and 8 mL of oleic acid (90 % (v/v)) mixture. After stirring for 30 min, GdCl3 solution (1 mL, 1 mmol mL-1) and BaCl2 solution (2 mL, 1 mmol mL-1) were added


Page 5 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


into the above mixture. 30 min later, aqueous solution (8 mL) containing NaF (6 mmol) was introduced at room temperature. After stirring for 30 min, the as-obtained mixing solution was sealed in a 50 mL Teflon-lined autoclave and heated at 180 oC for 24 h. After the Teflon-lined autoclave was cooled to room temperature, the products were washed with cyclohexane and ethanol twice. 2.4. Synthesis of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs. The pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs were prepared via ligand exchange. First, the pPeptides (10 mg) were added into the 50 mL round flask. Then, glacial acetic acid solution (2 mL, 10 %) was added to dissolve the pPeptides. After that, aqueous solution (8 mL) was added into the above mixture. Then, cyclohexane (10 mL) containing oleate-capped Ba2GdF7 NPs (Gd3+：10 mg) was mixed with above solution under stirring at 25 oC for 24 h. For tryptone-Ba2GdF7 NPs, aqueous solution (10 mL) of the tryptone (10 mg) and cyclohexane (10 mL) of the oleate-capped Ba2GdF7 NPs (Gd3+：10 mg) were mixed under stirring at 25 oC for 24 h. Subsequently, the pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs were collected by liquid separation method and centrifugated at 10000 rpm for 15 min, respectively. The pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs were stored in water at 4 oC for further experiments. 2.5. Cell Cytotoxicity and Cellular Internalization of pEGFR-targeted Ba2GdF7 NPs. A549 cells were cultured in DMEM medium containing 10 % FBS and 1 % penicillin/streptomycin at 37 o

C and 5 % CO2. The A549 cells (8×103 cells per well) were seeded into 96-well plate for 12 h. Then,

the cells were treated with various concentrations of pEGFR-targeted Ba2GdF7 NPs for 24 h. The cells of control group were treated with tryptone-Ba2GdF7 NPs, and the concentrations were the same as pEGFR-targeted Ba2GdF7 NPs. Afterward, MTT (10 µL, 5 mg mL-1) solution was added into per well and incubated for 4 h. The absorbance of per well was performed on a microplate reader at 490 nm, calculating the relative cell viabilities. To evaluate the cellular internalization efficiency of the pEGFR-targeted Ba2GdF7 NPs, A549 cells were incubated with pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs for 24 h, respectively. Subsequently, the NPs-stained cells were washed with PBS (three times). Then, the cells were detached from the culture plates by trypsin-EDTA solution, centrifuged (1000 rpm, 5 min) and measured by ICP-MS. 2.6. In vitro T1-Weighted MR and CT Imaging. The A549 cells (1×105 cells per well) were co-cultured with different amounts (0, 0.25, 0.5, 1, 2 mg mL-1) of pEGFR-targeted Ba2GdF7 NPs



and tryptone-Ba2GdF7 NPs at 37 oC for 24 h, respectively. Then, the cells stained with pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs were washed, centrifuged, and collected into the Eppendorf tube (1.5 mL), respectively. The cells were immobilized by agarose solution (1 %) for T1-Weighted MR and CT imaging. 2.7. In vivo T1-Weighted MR and CT Imaging. All animal experiments were performed under the guideline which was approved by the Local Ethics Committee for Institutional Animal Care and Use of Jilin University. To engraft the tumors in mice, 100 µL of PBS solution containing A549 cells (1×106 cells) were injected into the flank region of the right of the each nude mouse. The nude mice bearing tumors were anesthetized by isoflurane, then, 200 µL (20 mg Gd kg−1 for MRI; 40 mg Gd kg−1 for CT) of the pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs in 0.9 wt % NaCl solution were injected into the nude mice via the tail vein, respectively. The MR and CT images were obtained at different time points. 2.8. Pharmacokinetic Behaviors of the pEGFR-targeted Ba2GdF7 NPs. To invest pharmacokinetic behaviors of the pEGFR-targeted Ba2GdF7 NPs, pEGFR-targeted Ba2GdF7 NPs (20 mg Gd kg−1) in 200 µL of 0.9 wt % NaCl solution were injected into mice through the tail vein. The urine and blood were gathered at fixed time points and dissolved in aquaregia solution. Then, the content of Gd3+ ion was measured by ICP-MS. 2.9. In Vivo Cytotoxicity Evaluation. The healthy mice were randomly assigned to two groups. The mice of treated group were intravenously injected pEGFR-targeted Ba2GdF7 NPs (20 mg Gd kg−1) and the mice of control group were untreated. These mice were hosted in the case for 30 days post-injection, respectively. The mice were anesthetized using chloral hydrate (10 %), and the major organs including lung, liver, spleen, heart, and kidney were harvested from treated and control groups for hematoxylin and eosin (H&E) staining. 2.10. Hemolysis Assay. The Ethylenediaminetetraacetic acid stabilized mouse whole blood (1 mL) was obtained and diluted with PBS (pH=7.4, 2 mL). The blood solution was centrifuged (8000 rpm for 10 min) and washed with PBS five times. Subsequently, the purified red blood cells sank to the bottom of the Eppendorf tubes were resuspended in PBS (10 mL). 200 µL of the resultant suspension was incubated with 800 µL of pEGFR-targeted Ba2GdF7 NPs solution with various concentrations (Gd3+: 10, 25, 50, 100 and 200 ppm) and kept 3 h under room temperature. 200 µL of resultant suspension incubated 800


Page 6 of 20

Page 7 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


µL of H2O and PBS was served as positive control and negative control, respectively. Then, the samples were centrifuged (12000 rpm, 5 min) and the absorbance values of supernatants were determined. The hemolysis percentage of purified red blood cells was calculated based on this formula: Hemolysis percentage = [(sample absorbance–negative control absorbance) / (positive control absorbance–negative control absorbance)]×100. 3. RESULTS AND DISCUSSION 3.1. Characterization of pEGFR-targeted Ba2GdF7 NPs. The Ba2GdF7 NPs were synthesized via a solvothermal method, and oleate ligands were capped on the surface of Ba2GdF7 NPs. The oleate-Ba2GdF7 NPs exhibit good monodispersity and the average size of oleate-Ba2GdF7 NPs is 6.5±2 nm with the lattice spacing of 0.264 nm (Figure 1a). The XRD pattern of as-synthesized products is presented in Figure S1, which is well indexed as the Ba0.625Er0.375F2.375 NPs (JCPDS No.78-1449) without any other additional peak. The EDX spectrum of as-synthetized NPs shows that the stoichiometric ratio of Ba: Gd is 2:1, indicating the stoichiometric composition of the Ba2GdF7 NPs (as shown in Figure S2). It is known that the oleate ligands on the NPs can be exchanged by pPeptides via the formation of coordination bond of Gd3+ with phosphate.44 To achieve its positive tumor-targeting capability, EGFR-targeted pPeptides have been used to replace the original hydrophobic oleate ligands on the Ba2GdF7 NPs surface since the tumor cells normally have high expression of EGFR.45 In this case, the tryptone (casein-derived phosphopeptide mixture) modified Ba2GdF7 NPs (tryptone-Ba2GdF7 NPs) have been used as control NPs because tryptone has poor affinity with EGFR as well as the average MW of tryptone is similar to that of used pPeptides. In addition, the tryptone modified Gd nanodots have been demonstrated to have efficient EPR-based passive tumor targeting capability.43 After ligand exchange, P element (1.99 keV) can be detected in the EDS spectrum of pEGFR-targeted Ba2GdF7 NPs and the P peak (143.2 eV, P 2p) and N peak (401.2 eV, N 1s) belong to the pPeptides in the XPS spectrum of NPs (Figure S3), indicating that the surface of Ba2GdF7 NPs have been conjugated with pPeptides. As shown in Figure 1b, after pPeptides conjugation, there is negligible change in morphology and size of the NPs. The absorbance bands at 544 and 1128 cm−1 in FTIR spectrum of pEGFR-targeted Ba2GdF7 NPs are attributed to the symmetric stretching vibration and antisymmetric bending mode of PO43−, revealing that the oleate ligands have been substituted by pPeptides (as shown Figure S4). The TGA curves of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs suggest that the total weight



loss of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs are 7 and 50%, respectively (Figure S5). Furthermore, the specific surface area of Ba2GdF7 NPs is 0.5789 m2 g-1, and it can be calculated that the density of pPeptides on NPs is 7.1×1013 mol nm2. The zeta potential of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs in the culture medium (DMEM supplemented with 10 % FBS) are -7.2 mV and -4.3 mV, respectively. The hydrodynamic (HD) size of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs are 148.9 and 27.8 nm, respectively. The results are consistent with the structure of pEGFR-targeted Ba2GdF7 NPs wherein the pPeptides have relative complex secondary structure and high isoelectric points (PI). Longitudinal relaxivity (r1) is tested for evaluating the MRI enhancement effect of the pEGFR-targeted Ba2GdF7 NPs by a 3T clinical MRI scanner. The r1 value of pEGFR-targeted Ba2GdF7 NPs (5.72 mM−1 S−1) is higher than those of the tryptone-Ba2GdF7 NPs (2.46 mM−1 S−1) in Figure 2a and clinical Gd-based contrast agent (Gd-DTPA, 3.8 mM−1 S−1).46 Because the tryptone mainly consist of three kinds of peptides, and the polar amino acids occupy 31.2 %, 41.7 %, and 60 %, respectively, but the proportion of polar amino acids of EGFR-targeted pPeptides is up to 60 %. Therefore, the high r1 value of pEGFR-targeted Ba2GdF7 NPs is attributed to the existence of polar amino acids, which contribute to the strong hydrogen-bonding, accelerating water molecules exchange rate around Gd3+ ions.47,48 The signal intensity of MR is remarkably enhanced through increasing pEGFR-targeted Ba2GdF7 NPs concentration (Figure 2c). Under the same experimental conditions, the MR signal intensity of pEGFR-targeted Ba2GdF7 NPs is higher than that of tryptone-Ba2GdF7 NPs. Figure 2d shows the CT images of the pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs with different concentrations. The CT signal value is increased linearly with the concentrations of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs, respectively (Figure 2b), and the HU value (200) of 2 mg L−1 pEGFR-targeted Ba2GdF7 NPs is equivalent to that of 8 mg L−1 iodine in Omnipaque (a clinical CT contrast agent). These results indicate that pEGFR-targeted Ba2GdF7 NPs could be severed as efficient dual-mode imaging contrast agent for MR and CT imaging. 3.2 The interactions of pEGFR-targeted Ba2GdF7 NPs with cells. For in vivo bioapplications, the cytotoxicity of pEGFR-targeted Ba2GdF7 NPs was first assessed. The A549 cells were incubated with pEGFR-targeted Ba2GdF7 NPs in a broad concentration range. In the presence of as high as 200 µg mL-1 pEGFR-targeted Ba2GdF7 NPs, the A549 cells still have 85 % viability which is similar to the viability of tryptone-Ba2GdF7 NPs treated cells (Figure S6), demonstrating the pEGFR-targeted


Page 8 of 20

Page 9 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ba2GdF7 NPs have low cytotoxicity. The T1-weighted MR and CT signals intensities of the pEGFR-targeted Ba2GdF7 NPs stained A549 cells are increased with increasing the concentrations of Ba2GdF7 NPs in the culture medium (Figure 3a and 3c), showing that the pEGFR-targeted Ba2GdF7 NPs have good MR and CT dual-mode contrast capabilities. It is worthy to see that the MR and CT signal enhancement effects of pEGFR-targeted Ba2GdF7 NPs-stained A549 cells are higher than that of tryptone-Ba2GdF7 NPs stained A549 cells, indicating that pEGFR-targeted Ba2GdF7 NPs have high affinity with A549 cells (Figure 3b and d). 3.3. Dual-mode Imaging Properties and Pharmacokinetic Behaviors of pEGFR-targeted Ba2GdF7 NPs. The xenograft (ectopic) A549 lung tumor mouse models were established to explore contrast capabilities of pEGFR-targeted Ba2GdF7 NPs for in vivo dual-mode imaging. For in vivo MR imaging study (Figure 4), the mouse models bearing A549 tumor were intravenously injected pEGFR-targeted Ba2GdF7 NPs (20 mg Gd kg−1). In Figure 4a, the T1-weighted MR signal is increased with increasing post-injection time. Clearly enhancement (1.9-time) of T1-weighted MR signal of tumor region is observed at 1 h post-injection. In particular, the significant enhancement (3.4-time) of T1-weighted MR signal at tumor site is obtained after injection of 24 h (Figure 4d). The result indicates that pEGFR-targeted Ba2GdF7 NPs can be efficiently accumulated and detained in tumor tissues. The phenomenon could be caused by the interaction of pPeptides with EGFR expressed by tumor cells. The MR signals of major metabolic organs including liver, kidney and bladder were also measured at fixed time points of post-injection (Figure 4c). Generally, the T1-weighted MR signal enhancement effects of the kidney and bladder are higher than that of liver. The maximum T1-weighted MR signal enhancement effects of these organs are obtained at 2 h post-injection, and T1-weighted MR signal intensities of these organs are gradually decreased by prolonging the time from 2 to 24 h post-injection, suggesting that pEGFR-targeted Ba2GdF7 NPs can be excreted by the renal pathway. For in vivo CT imaging study, the mouse models were intravenous injection of pEGFR-targeted Ba2GdF7 NPs (40 mg Gd kg−1). As expected, the CT signal intensity of tumor site is increased with increasing the post-injection time (Figure 5a). In particular, the Hounsfield unit (HU) value of the tumor site at 24 h post-injection is about 3.5-time higher than that of the tumor site at pre-injection, suggesting that the pEGFR-targeted Ba2GdF7 NPs have reasonable CT contrast capability. For comparison, the tryptone-Ba2GdF7 NPs have also been intravenously injected into A549 tumor-bearing nude mice under the same experimental condition.



As shown in Figure 4b and 5b, the maximum T1-weighted MR/CT signal enhancement effects of tumor site are obtained at 2 h post-injection, and the signal intensity of tumor site is decreased by prolonging time from 2 h to 24 h post-injection, indicating that the tryptone-Ba2GdF7 NPs can be accumulated into tumor site through EPR effect. However, retention time of tryptone-Ba2GdF7 NPs in tumor is relative short because of lack of specific interactions between tryptone and cellular expression biomolecules. Comparing with pre-injection, the maximum signal enhancement effects of tryptone-Ba2GdF7 NPs in tumor sites are 2.5-time for T1-weighted MR signal and 3.2-time for CT signal at 24 h, which are lower than those of pEGFR-targeted Ba2GdF7 NPs in tumor sites. After injection of pEGFR-targeted Ba2GdF7 NPs (20 mg Gd kg−1), the blood and urine of mouse were collected to test the element content of Gd at fixed time points. During the corresponding time course, the content of Gd element in blood is gradually decreased while the content of Gd element in urine is increased (Figure 6). About 80 % of injection dose of pEGFR-targeted Ba2GdF7 NPs is founded in the mouse urine, which further demonstrates the good renal clearance of pEGFR-targeted Ba2GdF7 NPs. 3.4. In Vivo Toxicity of pEGFR-targeted Ba2GdF7 NPs. The healthy mice were intravenously injected with single dose (20 mg Gd kg−1) of pEGFR-targeted Ba2GdF7 NPs, and sacrificed at 30 days post-injection. The main organs including heart, liver, spleen, lung, and kidney were collected for histology analysis (Figure 7). Comparing to the control group, there is no noticeable inflammatory lesions or organ damages in the treatment group. Furthermore, the biocompatibility of pEGFR-targeted Ba2GdF7 NPs was also tested by a hemolysis assay in Figure S7. It can be seen that the blood cells have not been destroyed by pEGFR-targeted Ba2GdF7 NPs. In short, the pEGFR-targeted Ba2GdF7 NPs have low toxicity and minimal side effects on normal tissue. 4. CONCLUSIONS In summary, Ba2GdF7 nanoparticles conjugated with the phosphorylated EGFR-targeted peptides have been synthesized for dual-mode MR and CT imaging of tumor. It is found that the as-prepared pEGFR-targeted Ba2GdF7 NPs display high binding capacity to A549 cells and they are preferentially accumulated in the tumor site of mouse-bearing A549 tumor model, resulting in excellent contrast performance in vitro and in vivo MR and CT imaging. Furthermore, the pEGFR-targeted Ba2GdF7 NPs can be efficiently eliminated from mouse body via renal pathway, which decreases the likelihood of toxicity. Moreover, the in vitro cytotoxicity and in vivo toxicity


Page 10 of 20

Page 11 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


measurements of pEGFR-targeted Ba2GdF7 NPs indicate that the pEGFR-targeted Ba2GdF7 NPs have reasonable biocompatibility. Combining the functional diversity of peptides, the Ba2GdF7 nanoparticles could be used as a versatile nanoplatform to design peptide modified nanomedicines for further biomedical applications including tumor diagnosis and therapy through the formation of Gd-phosphonate coordinate bonds. ASSOCIATED CONTENT Supporting Information Characterization of XRD pattern, EDS spectra, XPS survey spectra, FTIR spectra, TGA curves, Viability of A549 cells, Hemolysis assay. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. (Z. Wang) *E-mail: [email protected]. (H. You) ORCID Zhenxin Wang: 0000-0002-1908-9848 Hongpeng You: 0000-0003-2683-6896 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This study is financially supported by Key Program of the Frontier Science of the Chinese Academy of Sciences (Grant No. YZDY-SSW-JSC018), the National Natural Science Foundation of China (Grant No. 51472236), the National Basic Research Program of China (973 Program, Grant No. 2014CB643803), and the Fund for Creative Research Groups (Grant No. 21521092). REFERENCES (1)

Hou, M.; Lu, X.; Zhang, Z.; Xia, Q.; Yan, C.; Yu, Z.; Xu, Y.; Liu, R. Conjugated Polymer Containing Organic

Radical for Optical/MR Dual-Modality Bioimaging. ACS Appl. Mater. Interfaces 2017, 9, 44316-44323. (2)

Ge, X.; Song, Z.; Sun, L.; Yang, Y.; Shi, L.; Si, R.; Ren, W.; Qiu, X.; Wang, H. Lanthanide (Gd3+ and Yb3+)

Functionalized Gold Nanoparticles for inVivo Imaging and Therapy. Biomaterials 2016, 108, 35-43. (3)

Zeng, L.; Luo, L.; Pan, Y.; Luo, S.; Lu, G.; Wu, A. In Vivo Targeted Magnetic Resonance Imaging and

Visualized Photodynamic Therapy in Deep-tissue Cancers Using Folic Acid-functionalized Super Paramagnetic



Upconversion Nanocomposites. Nanoscale 2015, 7, 8946-8954. (4)

Barreto José, A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Nanomaterials: Applications

in Cancer Imaging and Therapy. Adv. Mater. 2011, 23, H18-H40. (5)

Louie, A. Multimodality Imaging Probes: Design and Challenges. Chem. Rev. 2010, 110, 3146-3195.

(6)

Ma, M.; Chen, H.; Chen, Y.; Wang, X.; Chen, F.; Cui, X.; Shi, J. Au Capped Magnetic Core/mesoporous Silica

Shell Nanoparticles for Combined Photothermo-/chemo-therapy and Multimodal Imaging. Biomaterials 2012, 33, 989-998. (7)

Yang, S.; Li, Z.; Wang, Y.; Fan, X.; Miao, Z.; Hu, Y.; Li, Z.; Sun, Y.; Besenbacher, F.; Yu, M. Multifunctional

Bi@PPy-PEG Core–Shell Nanohybrids for Dual-Modal Imaging and Photothermal Therapy. ACS Appl. Mater. Interfaces 2018, 10, 1605-1615. (8)

Su, F.; Agarwal, S.; Pan, T.; Qiao, Y.; Zhang, L.; Shi, Z.; Kong, X.; Day, K.; Chen, M.; Meldrum, D.;

Kodibagkar, V. D.; Tian, Y. Multifunctional PHPMA-Derived Polymer for Ratiometric pH Sensing, Fluorescence Imaging, and Magnetic Resonance Imaging. ACS Appl. Mater. Interfaces 2018, 10, 1556-1565. (9)

Wei, R.; Xi, W.; Wang, H.; Liu, J.; Mayr, T.; Shi, L.; Sun, L. In Situ Crystal Growth of Gold Nanocrystals on

Upconversion Nanoparticles for Synergistic Chemo-photothermal Therapy. Nanoscale 2017, 9, 12885-12896. (10) Sun, L.; Wei, R.; Feng, J.; Zhang, H. Tailored Lanthanide-doped Upconversion Nanoparticles and Their Promising Bioapplication Prospects. Coord. Chem.Rev. 2018, 364, 10-32. (11) Ge, X.; Song, Z.; Sun, L.; Yang, Y.; Shi, L.; Si, R.; Ren, W.; Qiu, X.; Wang, H. Lanthanide (Gd3+ and Yb3+) Functionalized Gold Nanoparticles for In Vivo Imaging and Therapy. Biomaterials 2016, 108, 35-43. (12) Li, H.; Wei, R.; Yan, G.; Sun, J.; Li, C.; Wang, H.; Shi, L.; Capobianco, J. A.; Sun, L. Smart Self-Assembled Nanosystem Based on Water-Soluble Pillararene and Rare-Earth-Doped Upconversion Nanoparticles for pH-Responsive Drug Delivery. ACS Applied Materials & Interfaces 2018, 10, 4910-4920. (13) Zeng, S.; Tsang, M.; Chan, C.; Wong, K.; Hao, J. PEG Modified BaGdF5:Yb/Er Nanoprobes for Multi-modal Upconversion Fluorescent, In Vivo X-ray Computed Tomography and Biomagnetic Imaging. Biomaterials 2012, 33, 9232-9238. (14) Yi, Z.; Li, X.; Lu, W.; Liu, H.; Zeng, S.; Hao, J. Hybrid Lanthanide Nanoparticles as A New Class of Binary Contrast Agents for In Vivo T1/T2 Dual-weighted MRI and Synergistic Tumor Diagnosis. J. Mater. Chem. B 2016, 4, 2715-2722. (15) Feng, Y.; Chen, H.; Ma, L.; Shao, B.; Zhao, S.; Wang, Z.; You, H.: Surfactant-Free Aqueous Synthesis of Novel Ba2GdF7:Yb3+, Er3+@PEG Upconversion Nanoparticles for in Vivo Trimodality Imaging. ACS Appl. Mater.


Page 12 of 20

Page 13 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


& Interfaces 2017, 9, 15096-15102. (16) Wei, Q.; Chen, Y.; Ma, X.; Ji, J.; Qiao, Y.; Zhou, B.; Ma, F.; Ling, D.; Zhang, H.; Tian, M.; Tian, J.; Zhou, M. High-Efficient Clearable Nanoparticles for Multi-Modal Imaging and Image-Guided Cancer Therapy. Adv. Funct. Mater. 2018, 28, 1704634. (17) Yu, M.; Zheng, J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS Nano 2015, 9, 6655-6674. (18) Bourquin, J.; Milosevic, A.; Hauser, D.; Lehner, R.; Blank, F.; Petri‐Fink, A.; Rothen‐Rutishauser, B. Biodistribution, Clearance, and Long-term Fate of Clinically Relevant Nanomaterials. Adv. Mater. 2018, 0, 1704307. (19) Soo Choi, H.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.; Frangioni, J. V. Renal Clearance of Quantum Dots. Nat. Biotechnol. 2007, 25, 1165. (20) Peng, C.; Gao, X.; Xu, J.; Du, B.; Ning, X.; Tang, S.; Bachoo, R. M.; Yu, M.; Ge, W.; Zheng, J. Targeting Orthotopic Gliomas With Renal-clearable Luminescent Gold Nanoparticles. Nano Res. 2017, 10, 1366-1376. (21) Kim, H. J.; Yi, Y.; Kim, A.; Miyata, K. Small Delivery Vehicles of siRNA for Enhanced Cancer Targeting. Biomacromolecules 2018. (22) Ehlerding Emily, B.; Chen, F.; Cai, W. Biodegradable and Renal Clearable Inorganic Nanoparticles. Adv. Sci. 2015, 3, 1500223. (23) Yang, Y.; Liu, Y.; Cheng, C.; Shi, H.; Yang, H.; Yuan, H.; Ni, C. Rational Design of GO-Modified Fe3O4/SiO2 Nanoparticles with Combined Rhenium-188 and Gambogic Acid for Magnetic Target Therapy. ACS Appl. Mater. Interfaces 2017, 9, 28195-28208. (24) Ambarish, P.; Sasmit, S.; Kelly, C.; Poulomi, S.; Anne-Laure, P.; Sudipta, B.; Shiladitya, S. Anti-platelet Agents Augment Cisplatin Nanoparticle Cytotoxicity by Enhancing Tumor Vasculature Permeability and Drug Delivery. Nanotechnology 2014, 25, 445101. (25) Liu, R.; Xiao, W.; Hu, C.; Xie, R.; Gao, H. Theranostic Size-reducible and No Donor Conjugated Gold Nanocluster Fabricated Hyaluronic Acid Nanoparticle with Optimal Size for Combinational Treatment of Breast Cancer and Lung Metastasis. J. Controlled Release 2018, 278, 127-139. (26)

Hu, C.; Cun, X.; Ruan, S.; Liu, R.; Xiao, W.; Yang, X.; Yang, Y.; Yang, C.; Gao, H. Enzyme-triggered Size

Shrink and Laser-enhanced NO Release Nanoparticles for Deep Tumor Penetration and Combination Therapy. Biomaterials 2018, 168, 64-75. (27)

Ruan, S.; Hu, C.; Tang, X.; Cun, X.; Xiao, W.; Shi, K.; He, Q.; Gao, H. Increased Gold Nanoparticle

Retention in Brain Tumors by in Situ Enzyme-Induced Aggregation. ACS Nano 2016, 10, 10086-10098.



(28) Verwilst, P.; Park, S.; Yoon, B.; Kim, J. S. Recent Advances in Gd-chelate Based Bimodal Optical/MRI Contrast Agents. Chem. Soc. Rev. 2015, 44, 1791-1806. (29) Seidi, K.; Neubauer, H. A.; Moriggl, R.; Jahanban-Esfahlan, R.; Javaheri, T. Tumor Target Amplification: Implications for Nano Drug Delivery Systems. J. Controlled Release 2018, 275, 142-161. (30) Li, J.; Du, Y.; Jiang, Z.; Tian, Y.; Qiu, N.; Wang, Y.; lqbal, M. Z.; Hu, M.; Zou, R.; Luo, L.; Du, S.; Tian, J.; Wu, A. Y1 Receptor Ligand-based Nanomicelle As A Novel Nanoprobe for Glioma-targeted Imaging and Therapy. Nanoscale 2018, 10, 5845-5851. (31) Cui, Z.; Chen, S.; Wang, Y.; Gao, C.; Chen, Y.; Tan, C.; Jiang, Y. Design, Synthesis and Evaluation of Azaacridine Derivatives as Dual-Target EGFR and Src Kinase Inhibitors for Antitumor Treatment. Eur. J. Med.Chem. 2017, 136, 372-381. (32) Merrick, D. T.; Kittelson, J.; Winterhalder, R.; Kotantoulas, G.; Ingeberg, S.; Keith, R. L.; Kennedy, T. C.; Miller, Y. E.; Franklin, W. A.; Hirsch, F. R. Analysis of c-ErbB1/Epidermal Growth Factor Receptor and c-ErbB2/HER-2 Expression in Bronchial Dysplasia: Evaluation of Potential Targets for Chemoprevention of Lung Cancer. Clin. Cancer Res. 2006, 12, 2281. (33) Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal Growth Factor Receptor Mutations in Lung Cancer. Nat. Rev. Cancer 2007, 7, 169. (34) Sadhukha, T.; Wiedmann, T. S.; Panyam, J. Inhalable Magnetic Nanoparticles for Targeted Hyperthermia in Lung Cancer Therapy. Biomaterials 2013, 34, 5163-5171. (35) Chen, G.; Roy, I.; Yang, C.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826-2885. (36) Yun, M.; Kim, D. Y.; Lee, Pyo A.; Ryu Y.; Kim TY.; Zheng JH.; Yoo SW.; Hyun H.; Oh G.; Jeong J.; Moon M.; Min JH.; Kwon SY.; Kim JY.; Chung E.; Hong Y.; Lee W.; Kim HS.; Min JJ. A High-Affinity Repebody for Molecular Imaging of EGFR-Expressing Malignant Tumors. Theranostics 2017, 7, 2620-2633. (37) Morshed, R. A.; Muroski, M. E.; Dai, Q.; Wegscheid, M. L.; Auffinger, B.; Yu, D.; Han, Y.; Zhang, L.; Wu, M.; Cheng, Y.; Lesniak, M. S. Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer. Mol. Pharmaceut. 2016, 13, 1843-1854. (38) Zhang, Y.; Hong, H.; Orbay, H.; Valdovinos, H. F.; Nayak, T. R.; Theuer, C. P.; Barnhart, T. E.; Cai, W. PET Imaging of CD105/endoglin Expression with a 61/64Cu-labeled Fab Antibody Fragment. Eur. J. Nucl. Med. Mol. I. 2013, 40, 759-767. (39) Hamley, I. W. Small Bioactive Peptides for Biomaterials Design and Therapeutics. Chem. Rev. 2017, 117,


Page 14 of 20

Page 15 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14015-14041. (40) Bartczak, D.; Muskens, O. L.; Nitti, S.; Millar, T. M.; Kanaras, A. G. Nanoparticles for Inhibition of In Vitro Tumour Angiogenesis: Synergistic Actions of Ligand Function and Laser Irradiation. Biomater. Sci. 2015, 3, 733-741. (41) Bray, B. L. Large-scale Manufacture of Peptide Therapeutics by Chemical Synthesis. Nat Rev. Drug Discov. 2003, 2, 587. (42) Liang, G.; Jin, X.; Zhang, S.; Xing, D. RGD Peptide-modified Fluorescent Gold Nanoclusters as Highly Efficient Tumor-targeted Radiotherapy Sensitizers. Biomaterials 2017, 144, 95-104. (43) Chen, H.; Li, X.; Liu, F.; Zhang, H.; Wang, Z. Renal Clearable Peptide Functionalized NaGdF4 Nanodots for High-Efficiency Tracking Orthotopic Colorectal Tumor in Mouse. Mol. Pharmaceut. 2017, 14, 3134-3141. (44) Liu, F.; He, X.; Zhang, J.; Zhang, H.; Wang, Z. Employing Tryptone as a General Phase Transfer Agent to Produce Renal Clearable Nanodots for Bioimaging. Small 2015, 11, 3676-3685. (45) Mao, J.; Ran, D.; Xie, C.; Shen, Q.; Wang, S.; Lu, W. EGFR/EGFRvIII Dual-Targeting Peptide-Mediated Drug Delivery for Enhanced Glioma Therapy. ACS Appl. Mater. Interfaces 2017, 9, 24462-24475. (46) Xing, H.; Zhang, S.; Bu, W.; Zheng, X.; Wang, L.; Xiao, Q.; Ni, D.; Zhang, J.; Zhou, L.; Peng, W.; Zhao, K.; Hua, Y.; Shi, J. Ultrasmall NaGdF4 Nanodots for Efficient MR Angiography and Atherosclerotic Plaque Imaging. Adv. Mater. 2014, 26, 3867-3872. (47) Ponsiglione, A. M.; Russo, M.; Netti, P. A.; Torino, E. Impact of biopolymer matrices on relaxometric properties of contrast agents. Interface Focus 2016, 6. (48) Ni, D.; Bu, W.; Ehlerding, E. B.; Cai, W.; Shi, J. Engineering of Inorganic Nanoparticles as Magnetic Resonance Imaging Contrast Agents. Chem.Soc. Rev. 2017, 46, 7438-7468.

Figure 1. TEM images of (a) oleate-capped Ba2GdF7 NPs, and (b) pEGFR-targeted Ba2GdF7 NPs.



Figure 2. (a) R1 relaxivities of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs as a function of the molar concentration of Gd3+ (Gd3+: 0, 0.2, 0.5, 0.6, and 0.7 mg mL−1). (b) HU value of aqueous solution of pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs as functions of the molar concentration of Gd3+ (Gd3+: 0, 0.25, 0.5, 1, and 2 mg mL−1), respectively. (c) MR images of (c1) pEGFR-targeted Ba2GdF7 NPs solutions and (c2) tryptone-Ba2GdF7 NPs solutions with different concentrations (Gd3+: 0, 0.25, 0.5, 1, and 2 mg mL−1). (d) CT images of (d1) pEGFR-targeted Ba2GdF7 NPs solutions and (d2) tryptone-Ba2GdF7 NPs solutions with different concentrations (Gd3+: 0, 0.25, 0.5, 1, and 2 mg mL−1).

Figure 3. (a) MRI of (a1) pEGFR-targeted Ba2GdF7 NPs and (a2) tryptone-Ba2GdF7 NPs stained A549 cells in the


Page 16 of 20

Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


presence increasing concentration of Gd3+. (b) Relative MR signals of pEGFR-targeted Ba2GdF7 NPs stained A549 cells and tryptone-Ba2GdF7 NPs stained A549 cells. (c) CT images of (c1) pEGFR-targeted Ba2GdF7 NPs and (c2) tryptone-Ba2GdF7 NPs stained A549 cells in the presence increasing concentration of Gd3+. (d) Relative CT signals of pEGFR-targeted Ba2GdF7 NPs stained A549 cells and tryptone-Ba2GdF7 NPs stained A549 cells.

Figure 4. (a)Time-dependent T1-MR images of the nude mouse bearing A549 tumor injected with pEGFR-targeted Ba2GdF7 NPs. (b) tryptone-Ba2GdF7 NPs. (tumors marked with green circles and white circles). (c) Corresponding representative MR signal values of major organs. (d) Time-dependent MR signal values of the tumors (pEGFR-targeted Ba2GdF7 NPs and tryptone-Ba2GdF7 NPs). The error bars indicate the s.d. (*P>0.05, **P

Renal Clearable Peptide Functionalized Ba2GdF7 Nanoparticles for

Recommend Documents