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Chelator-Free Conjugation of 99mTc and Gd3+ to PEGylated Nanographene Oxide for Dual-modality SPECT/MR Imaging of Lymph Nodes Tianye Cao, Xiaobao Zhou, Yingying Zheng, Yuyun Sun, Jian Zhang, Wei Chen, Jianping Zhang, Zhiguo Zhou, Shi-Ping Yang, Yingjian Zhang, Hong Yang, and Ming-Wei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14836 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 26, 2017

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ACS Applied Materials & Interfaces

Laboratory of Nuclear Physics and Ion-beam Application (MOE), Fudan Univeristy; Shanghai Engineering Research Center for Molecular Imaging Probes; The Key Laboratory of Resource Chemistry of Ministry of Education, Shanghai Key Laboratory of Rare Earth Functional Materials, and Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Shanghai Normal University, Zhang, Jianping; Department of Nuclear Medicine, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University; Center for Biomedical Imaging, Fudan University; Key Laboratory of Nuclear Physics and Ion-beam Application (MOE), Fudan Univeristy; Shanghai Engineering Research Center for Molecular Imaging Probes Zhou, Zhiguo; Shanghai Normal University, Yang, Shi-Ping; Shanghai Normal University, Department of Chemistry Zhang, Yingjian; Fudan University Shanghai Cancer Center, Yang, Hong; Shanghai Normal University, Department of Chemistry Wang, Ming-Wei; Fudan University Shanghai Cancer Center, PET Center, Department of Nuclear Medicine

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Chelator-Free Conjugation of 99mTc and Gd3+ to PEGylated Nanographene Oxide for Dual-modality SPECT/MR Imaging of Lymph Nodes

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Tianye Cao†ǁ, Xiaobao Zhou†‡ǁ, Yingying Zheng†, Yuyun Sun†, Jian Zhang†‡, Wei

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Chen†‡, Jianping Zhang†, Zhiguo Zhou‡, Shiping Yang‡, Yingjian Zhang†, Hong

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Yang‡* and Mingwei Wang†*

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†

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Department of Oncology, Shanghai Medical College, Fudan University; Center for

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Department of Nuclear Medicine, Fudan University Shanghai Cancer Center;

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Biomedical Imaging, Fudan University; Key Laboratory of Nuclear Physics and

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Ion-beam Application (MOE), Fudan University; Shanghai Engineering Research

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Center for Molecular Imaging Probes, No. 270 Dong’An Road, Shanghai 200032,

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China.

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‡

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Laboratory of Rare Earth Functional Materials, and Shanghai Municipal Education

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Committee Key Laboratory of Molecular Imaging Probes and Sensors, Shanghai

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Normal University, No. 100, Guilin Road, Shanghai 200234, China.

The Key Laboratory of Resource Chemistry of Ministry of Education, Shanghai Key

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KEYWORDS: nanographene oxide, SPECT, MR, molecular imaging, lymph node

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imaging

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ABSTRACT: PEGylated ultra-small nanographene oxide (usNGO-PEG) has

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exhibited great potential in nanotheranostics due to its newly-discovered

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physicochemical properties derived from the rich functional groups and bonds. Herein,

7

we developed a general, simple and kit-like preparation approach for

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Gd-anchored

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[99mTcI(CO)3(OH2)3]+ (abbreviated to

NGO-PEG

using

a

chelator-free

strategy.

In

99m

TcI- and

this

strategy,

99m

TcI) and GdCl3 were mixed with

99m

Tc- and Gd-usNGO-PEG via the synergistic coordination of

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usNGO-PEG to yield

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N and O atoms from NGO and PEG with

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exogenous chelators. Under optimized conditions, the nanoprobes

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Gd-usNGO-PEG were reliably prepared with high yields and good stability. Serial

14

comparative experiments of the labeling yield, the measurements of –NH2 density and

15

ζ-potentials, various characterizations including energy-dispersive X-ray analysis

16

spectroscopy, X-ray photoelectron spectroscopy and Fourier-transform infrared

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spectroscopy demonstrated that both usNGO and PEG synergistically provide the

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electron-donating atoms O and N to coordinate with

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nanocomplexes. Furthermore, both 99mTc- and Gd-usNGO-PEG exhibited excellent

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in vivo imaging of lymph nodes using single photon emission computed

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tomography/computed tomography (SPECT/CT) and magnetic resonance (MR) 2

99m

TcI- and Gd3+ without additional 99m

Tc- and

99m

TcI and Gd to form stable

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imaging after local injection. Therefore, these results showed the successful

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establishment of

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and the potential of multi-modality SPECT/CT and MR imaging of lymph nodes.

99m

Tc- and Gd-anchored usNGO-PEG using a chelator-free strategy

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■ INTRODUCTION

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Nanoscale graphene oxide (NGO) and its derivatives, which have many fascinating

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physicochemical properties have been a focus of increasing attention in the research

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fields of molecular imaging,1-2 drug/gene delivery carriers,3-4 photodynamic therapy

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(PDT)5-6 and photothermal therapy7-9. Notably, NGO-based nanotheranostics have

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shown promising application potential for the imaging, diagnosis and therapy of

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cancer in preclinical settings as NGO itself has been proved to be a versatile

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multi-functional nanocarrier for multi-modality imaging and synergistic combination

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therapy.10-11 Molecular imaging allows the real-time observation and study of

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biological processes from the molecular and cellular levels to organ and whole-body

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levels in vivo, and thus plays a critical role in both basic research and clinical practice.

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12-13

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single photon emission computed tomography (SPECT), has emerged as the most

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powerful molecular imaging technique for the diagnosis and therapy evaluation of

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various serious diseases, especially cancers, in clinical settings, due to the sensitive

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and quantitative detection of targets.14-15 Recently, radionuclide-based PET and

Radioactive nuclear imaging, including positron emission tomography (PET) and


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SPECT imaging have been included in the areas of nanomedicine, nanoscience and

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nanotechnology to investigate and optimize the in vivo biological behaviors of

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nanomaterials and nanocarriers, and to develop molecular imaging probes derived

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from nanocomplexes, such as NGO, using appropriate radiolabeling strategies for

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nanoparticles.16-18

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Currently, the radiolabeling strategies for nanoparticles can be classified into

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chelator-based methods and chelator-free methods.19-20 The former methods are the

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most widely-used radiolabeling strategies for nuclide-labeled nanoparticles, which

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involve the introduction of exogenous chelators into the surface of nanoparticles to

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coordinate with certain radioisotopes to form stable nanocomplexes.21-22 These

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methods have been used for decades to produce radiolabeled nanoparticles for

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molecular imaging and theranostics for cancer due to the available chemistry and

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wide selectivity of chelators. For example, radiolabeled NGO analogs have been

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explored for tumor-targeted molecular imaging using the nuclides 64Cu, 66Ga and 111In.

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23-25

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of nanoparticles has become an increasing issue. In contrast, the latter strategies are

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emerging radiolabeling approaches, which employ the specific physicochemical

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interactions between radionuclides and nanoparticles without the addition of chelators.

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The current progress in chelator-free radiolabeling focuses mainly on inorganic metal

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nanoparticles, such as 64Cu-CuS, 18F-NaYF4:Yb,Tm and 153Sm-NaLuF4:Yb,Gd,Tm,

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which have been comprehensively reviewed by the Chen and Cai groups.19-20 In

However, the effect of chelators on the size, surface charge and pharmacokinetics



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summary, chelator-free radiolabeling has the unique advantages of yielding

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radiolabeled nanocomplexes as it is an easy, fast and efficient kit-like method.

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Chelator-free radiolabeling of non-metal nanoparticles has also emerged, which

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largely facilitates the mutual application of new radionuclides and nanomaterials for

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cancer imaging and theranostics. In 2015, Grimm and colleagues reported

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chelator-free radiolabeling of 6 medical nuclides with one general substrate of

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amorphous silica nanoparticles.26 They demonstrated high radiochemical yield, good

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in vivo stability and feasible lymph node imaging of radiolabeled Si nanoparticles. In

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2017, Cai’s group developed the new theranostic nuclide *As-labeled thiolated

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mesoporous silica nanoparticles and reported effective radiolabeling, good stability

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and whole-body PET imaging (*As: 72As for PET, 76As for therapy).27 During the

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same period, they pioneered the chelator-free radiolabeling of nanographene with

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64

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with abundant  bonds.28 This is a significant finding as it breaks the stereotype of

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chelation. However, it is unclear whether other nuclides can be labeled onto graphene

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using a chelator-free strategy.

Cu2+ via the interactions between Cu2+ and  electrons of reduced graphene oxide

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For the current chelator-free labeling strategy, there are some potential limitations.

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Firstly, it mainly focuses on introducing radionuclides into the core of nanoparticles,

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and thus pays little attention to the surface of nanoparticles for radiolabeling,

20

especially after surface functionalization (e.g. PEGylation). Secondly, it restricts the


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use of radionuclides, and has not included non-radioactive metal isotopes. Thirdly,

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one type of nanoparticle platform is restricted to specific radionuclides, and does not

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involve radionuclide or non-radioactive isotopes for multi-modality molecular

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imaging.

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As PEGylated NGO (NGO-PEG) holds more abundant electron-donating atoms

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from functional groups, such as -NH2 and -COOH, than NGO, we herein prepared

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99m

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SPECT/CT and MR dual-modality imaging of lymph nodes. 99mTc is the most widely

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used radionuclide in nuclear medicine for SPECT/CT imaging of bone metastasis and

Tc- and Gd-NGO-PEG using a chelator-free strategy and investigated the

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organ (e.g. kidney, heart, thyroid) function due to its favorable low-energy -ray

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emission (140 keV), suitable half-life (t1/2 = 6.02 h), diverse 99mTc-labeled

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radiopharmaceuticals (e.g. 99mTc-DTPA, 99mTc-MDP, etc.) and wide availability from

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commercial 99Mo/99mTc generators. Gadolinium (Gd), in the form of Gd3+ and related

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complexes (e.g. Gd-DTPA and derivatives), is now the dominant contributor to MR

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contrast agents for the diagnosis of various diseases in the current clinic setting.

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Furthermore, from the view of coordination chemistry, 99mTc and Gd share similar

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chelating features via the coordination bonds with O and N atoms.29-30 Therefore,

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based on our previous study where ultra-small sized (< 50 nm), PEGylated (six-arm

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branched, with terminal -NH2) NGO (usNGO-PEG) was reported31, we applied

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abundant functional groups including -NH2, -COOH, and -OH, providing

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coordinating N and O atoms, to fabricate 99mTc- and Gd-labeled usNGO-PEG using a 6 ACS Paragon Plus Environment


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chelator-free strategy without additional exogenous chelators. Considering the

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possible 99mTc-colloid from the direct reduction of SnCl2, we started from

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[99mTcI(CO)3(OH2)3]+ (abbreviated to 99mTcI) to achieve chelator-free radiolabeling of

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usNGO-PEG with 99mTcI, as 99mTcI is easily prepared from 99mTcO4- in the presence of

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NaBH4 and CO. To obtain Gd-usNGO-PEG, aqueous solutions of usNGO-PEG and

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GdCl3 were mixed in a sonicator bath at room temperature for 12 h. Overall, 99mTc-

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and Gd-usNGO-PEG were prepared with a high yield in a short time, and both

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exhibited high stability in vitro and in vivo. Importantly, high contrast SPECT/CT and

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MR images of lymph nodes were produced using small animal imaging scanners after

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subcutaneous injection of 99mTc- and Gd-usNGO-PEG into the footpads of mice,

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respectively. We then developed a simple and stable conjugation of 99mTc- and

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Gd-usNGO-PEG using the chelator-free approach via the synergistic coordination of

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N and O atoms (from NGO and PEG) with 99mTc and Gd, and demonstrated their

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multi-modality SPECT/CT and MR imaging capabilities of lymph nodes. We believe

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that the findings in this study could facilitate the research and development (R&D) of

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multi-modality molecular imaging probes and multifunctional nanotheranostics from

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PEGylated GO and similar-structured nanoparticles, such as other carbon-based

18

nanomaterials.

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■ RESULTS AND DISCUSSION 7 ACS Paragon Plus Environment

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Preparation and Characterization of 99mTc-usNGO-PEG. To obtain

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99m

Tc-radiolabeled usNGO-PEG, the low-valence labeling core [99mTcI(H2O)3(CO)3]+

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(abbreviated to 99mTcI) was first prepared. Briefly, small amounts of NaBH4 (5 mg) as

4

a reducing agent was freshly flushed with CO for 5 min in a vial. Then, [99mTcO4]−

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(30 mCi) in saline solution (1 mL, 0.9% NaCl/H2O) was added to the sealed vial and

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heated at 80 °C for 30 min. According to the radioactive thin layer chromatography

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(Radio-TLC) measurement, the yield of 99mTcI was >85% (Figure S1a).32

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Subsequently, 99mTcI (100 µL) and usNGO-PEG (1 mg/mL, 0.4 mL) were mixed to

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yield 99mTc-usNGO-PEG and the radiolabeling reaction was further optimized under

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different conditions. For this purpose, we included various incubation times (1, 5, 15,

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30 and 60 min, ), temperatures (25, 40 and 80 ºC) and pH values (2, 7.3 and 12)

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(Figure 1 a, b, c), respectively. The radiochemical yield in each radiolabeling test was

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assessed both by Radio-TLC and centrifugal nanoparticle purification. As shown in

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Figure 1a, the labeling efficiency increased to 81.4 ± 6% at 5 min and then rapidly

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declined to 10.9 ± 4% at 60 min. The reason for this may have been due to the

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residual NaBH4 in the solution, which could have further reduced usNGO-PEG to

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gradually remove carbonyl and hydroxyl groups along with reaction time at 80 °C.33

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This phenomenon was further confirmed by the low labeling rate (only 7.8 ± 1%) of

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99m

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vital contribution of the carbonyl or hydroxyl groups on the usNGO-PEG to the

21

labeling reaction. The radiochemical yield improved with increased temperature from

Tc- with NaBH4-reduced usNGO-PEG (Figure 1d) within 5 min, which proved the



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gure 1b), sugggesting thaat the influeence of the llabeling 25 tto 80 °C forr 5 min (Fig

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tem mperature is to overcom me the activaation energy y required fo or stable 99mmTcI binding g.26

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For the pH-deppendent influ uence, the llabeling yieeld at pH 2 or o 12 markeedly decreassed

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to aapproximateely 50% (80 °C, 5 min)) (Figure 1c)). This indiccated that nneutral pH

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benefits the higgh labeling rate r of 99mT TcI with usN NGO-PEG. In I comparisson, the

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radiiolabeling reeactions of 99mTcI withh usNGO an nd PEG, the componentts of

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NGO-PEG, were w carried d out, respe ctively. How wever, the radiochemic r cal yields of usN

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m bothh 99mTc-usN NGO and 99m Tc-PEG w were only ap pproximately y 40% (Figuure 1d). These

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resuults indicateed that both usNGO andd PEG syneergistically contributed c d to the efficcient

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coorrdination off 99mTcI with h usNGO-P PEG. Thereffore, the opttimized connditions for

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prepparing 99mTcc-usNGO-P PEG were neeural pH media m at 80 °C for 5 minn using 99mTc T I

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as thhe labeling core.

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Figure 1. The influence factors of the radiolabeling reactions of [99mTc(OH2)3(CO)3]+

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with usNGO-PEG. The influence of time (a), temperatures (b) and pH (c) on the

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radiolabeling rate of [99mTc(OH2)3(CO)3]+ with usNGO-PEG. The radiolabeling rate

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of [99mTc(OH2)3(CO)3]+ with PEG, usNGO, usNGO-PEG and NaBH4-reduced

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usNGO-PEG (d). The radiolabeling rate was measured by Radio-TLC (developing

6

solution CH3OH:HCl = 95: 5).

7

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Furthermore, the physicochemical properties of 99mTc-usNGO-PEG were examined

9

for subsequent in vivo tests. The ζ-potential values before and after the introduction of

10

99m

TcI into usNGO-PEG, shown in Table S1, were found to be very similar.

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Compared with usNGO-PEG 31, the size and morphology of 99Tc-usNGO-PEG did

12

not significantly alter and no aggregates appeared after the labeling process, as shown

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by the TEM image in Figure 2a, which was obtained 3 days later when the

14

radioactivity had fully decayed. Importantly, the high stability (>95%) of

15

99m

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plasma (50%) at 37 °C within 24 h (Figure 2e). These results enabled the following in

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vivo lymph node imaging using 99mTc-NGO-PEG SPECT/CT.

Tc-NGO-PEG was confirmed by incubation experiments in normal saline or serum

18



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Figu ure 2. The characteriza c ations and sstability testts of 99Tc-ussNGO-PEG G and

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Gd--usNGO-PE EG.

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DXA (c) andd both size distribution n histogramss (d). The withh the corresponding ED

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stabbility test off 99mTc-usNG GO-PEG (ee) and Gd-u usNGO-PEG G (f) studiedd in serum

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soluution (50%, pH 7.3, red d line) and nnormal salin ne (black lin ne).

The TEM T imagess of 99Tc-usN NGO-PEG (a), Gd-usN NGO-PEG (b) (

8

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P Preparation n and Charracterizatioon of Gd-ussNGO-PEG G. In order tto prepare

16

Gd--usNGO-PE EG, the saturated amounnt of GdCl3 to react wiith a specifiic amount of

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usN NGO-PEG was w determin ned, which was the dom minant factor in this reeaction proccess.

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Diffferent amouunts of GdC Cl3 (5, 25, 500, 100 and 150 1 µg) werre added to uusNGO-PE EG

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soluution (1.0 mg, m 1 mL tottal volume) and the reaaction mixtu ure was stirrred in a

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soniicator bath for f 10 h. When W the reaaction was complete, ceentrifugationn and size

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excllusion mem mbrane filtraation were pperformed to o remove free Gd3+ andd possible 11 ACS Paragon Plus Environment

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precipitate. Centrifugal separation was repeated 3–5 times with intervals of washing

2

with water to purify the obtained product Gd-usNGO-PEG. According to the

3

determination by inductively coupled plasma optical emission spectrometry

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(ICP-OES), the saturated amount of element Gd within usNGO-PEG was

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approximately 4 wt% (2.5×10−4 mmol per 1 mg usNGO-PEG), as shown in Figure S2.

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According to these conditions, Gd-usNGO-PEG was reliably prepared using a

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saturated amount of Gd.

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Gd-usNGO-PEG was then fully characterized using various analytical

9

measurements. According to ICP-OES measurements, Gd-usNGO-PEG in

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physiological solution demonstrated high stability (Figure 2f), and no aggregation in

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normal saline or serum solution was observed within 48 h. Based on TEM images,

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Gd-usNGO-PEG remained in an unfolded shape with a lateral width of 63 ± 9 nm,

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larger than that of 99mTc-usNGO-PEG (51 ± 8 nm) (Figure 2 a, b, d). This may have

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been due to the amount of non-radioactive Gd in the former compared with that in the

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latter. This could be explained by the changes in the ζ-potential values (Table S1).

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Starting from usNGO-PEG with a negative charge, the ζ-potential of the former

17

became positive due to the introduction of Gd3+, whereas that of the latter remained

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negative after the coordination of 99mTcI (Table S1). As shown in Figure 2c,

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energy-dispersive X-ray analysis spectroscopy of Gd-usNGO-PEG showed the

20

presence of C, O and Gd, which indicated that Gd was present on Gd-usNGO-PEG.

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The successful formation of Gd-usNGO-PEG was further verified by the 12 ACS Paragon Plus Environment


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Fourier-transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS)

2

spectra. The FT-IR spectrum (Figure 3a) of usNGO-PEG showed multiple

3

characteristic absorbance peaks at 3425 cm−1 (νN–H), 2877 cm−1 (νC-H), 1105 cm−1

4

(νC-O-C), 1720 cm–1 and 1651 cm–1 (different types of carboxylic groups νc=o stretch

5

signified the presence of polyamides formed by condensation between the amino

6

group and carboxylic group). For Gd-usNGO-PEG, the FT-IR spectra of vibration

7

peak νC=O shifted to 1633 cm−1 and νC-N shifted from 1457 cm−1 to 1428 cm−1,

8

respectively, indicating that the C=O and C-N stretches were influenced by chelation

9

with Gd3+. 34 In the XPS spectrum, Gd-usNGO-PEG showed that the binding energy

10

value of the gadolinium peak (Gd 4d) was 142.8 eV, which confirmed successful

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Gd3+ chelation (Figure 3b). Furthermore, the binding energy values of both N 1s and

12

O 1s in Gd-usNGO-PEG shifted to 400.0 eV and 532.9 eV, compared with 401.2 eV

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and 533.3 eV in usNGO-PEG, respectively (Figure 3c and d).35 The XPS spectra of C

14

1s in usNGO-PEG included peak 1 (C=C/C–C, 285.6 eV), peak 2 (C–O/C–O–C,

15

~286.8 eV), and peak 3 consisting of C=O (carbonyl, ~288.3 eV) and O–C=O

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(carboxyl, ~290.2 eV). In comparison, peak 3 in the XPS spectra of C 1s in

17

Gd-usNGO-PEG significantly shifted from 289.7 eV to 288.7 eV.33, 36 From these

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changes in the XPS spectra, we speculated that the coordination interaction occurred

19

between Gd3+ and the electron donors N, O and C=O. In addition, the amine density

20

of Gd-usNGO-PEG (detected by the OPA method37) decreased to 2.2×10−4 mmol per

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1 mg usNGO-PEG. These results indicated that Gd3+ coordination may require 13 ACS Paragon Plus Environment

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H2) of usNG GO-PEG (T Table S2). T Thus, the ressults equivalent amoounts of the amine (–NH

5

o Gd3+ withh functional groups (−C C=O/ O−C= =O and −NH H2) suppport the cooordination of

6

of uusNGO-PEG G.

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Figu ure 3. The spectroscop pic characteerizations off Gd-usNGO O-PEG, com mpared with h

10

thatt of usNGO-PEG. FTIR R (a) and XP PS spectra with w Gd 4d region (b), N 1s region n (c),

11

O 1s region (d)) and C 1s peak p (e, f) oof usNGO-P PEG and Gd d-usNGO-PE EG. The C 1s

12

peakk curve wass fitted by considering c the followin ng contributions: peak 1 (C=C/C-C C, 14 ACS Paragon Plus Environment


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1

285.6 eV), peak 2 (C-O/C-O-C, ~286.8 eV), and peak 3 consists of C=O (carbonyl,

2

~288.3 eV) and O-C=O (carboxyl, ~290.2 eV).

Page 16 of 36

3

4

MR Properties and Cytotoxicity in vitro. Prior to examining the imaging

5

capability and in vivo applications of 99mTc- and Gd-usNGO-PEG, phantom studies

6

and in vitro tests were performed. MR images of Gd-usNGO-PEG at various

7

concentrations of Gd were acquired to demonstrate the enhanced T1 MR contrast

8

using our 7.0T MR scanner. A decrease in T1 relaxation time (from 2.5 to 0.45 ms)

9

was observed with increasing Gd concentration (from 0.001 to 0.125 mM determined

10

using ICP-OES), which was consistent with the corresponding T1-weighted MR

11

images (Figure 4a and inset). Thereafter, the r1 relaxivity was calculated by the linear

12

fit of Gd3+ concentration vs 1/T1. The calculated r1 value was 18.0 mM−1 S−1, which

13

was much higher than that of the commercially available MRI agent Magnevist (r1 =

14

3.6 mM−1 S−1, Figure 4b).34 This enhancement effect was most likely due to the strong

15

interaction between a significant portion of Gd3+ ions and the entrapped usNGO,

16

which limited water accessibility of the Gd3+ ions 38. To determine the in vitro

17

cytotoxicity of Gd-usNGO-PEG and 99Tc-usNGO-PEG, the cell viability of both were

18

found to >90% (Figure 5), even at concentrations up to 200 µg/mL at 48 h

19

post-incubation, with no obvious toxicity. Our results demonstrated that the strong

20

coordination of Gd3+ with usNGO-PEG effectively blocked the leakage of Gd3+ into


Page 17 of 36

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4

wo nanoprobbes 99mTc- and a the surroundinggs to inducee serious cyytotoxicity399, and the tw

5

EG demonsttrated good biocompatiibility, whicch is beneficcial for in vivo Gd--usNGO-PE

6

bioiimaging.

5

6 12

Figu ure 4. The magnetic m reelaxation ratte and MR imaging i of Gd-usNGO O-PEG,

13

com mpared withh that of Mag gnevist. Thee Relaxation rate (1/T1 1) versus vaarious

14

concentrations of (a) Gd-u usNGO-PEG G (0.001, 0.005, 0.024, 0.052, 0.1 25 mM of Gd G 3+)

15

3 and (b) Magnevvist (0.1, 0.5, 1, 2.5, 5 mM of Gd3+ ), conducted at the 7.00 T MRI

16

scannner for preeclinical smaall animal sstudies. Inseet: T1-weigh hted MR im mages of

17

Gd--usNGO-PE EG and Mag gnevist withh different concentrations.

13



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1

Figure 5. The in vitro cytotoxicity of 99Tc-usNGO-PEG and Gd-usNGO-PEG. The

2

relative viability of 4T1 cells incubated with 99Tc-usNGO-PEG (a) and

3

Gd-usNGO-PEG (b) at different concentrations for 24 and 48 h, respectively. Error

4

bars were based on quintuplicate samples.

Page 18 of 36

5 6

SPECT/CT and MR Imaging of Lymph Nodes. As NGO-PEG has served as a

7

robust platform for cancer theranostics, 99mTc- and Gd-usNGO-PEG nanoprobes

8

generated by the chelator-free kit-like protocol may promote many biomedical

9

applications of NGO-based nanoparticles. As a proof-of-concept study, we applied

10

99m

11

lymphatic system is an important first line of defense against infection, and a common

12

route for cancer metastasis.40-41 Following a subcutaneous injection of

13

99m

14

normal BALB/c mice, serial SPECT scans were performed at various times. As

15

shown in Figure 6a, 99mTc-usNGO-PEG SPECT/CT imaging mapped the popliteal

16

node (1) in the leg, then the sacral node (2) and caudal node (3), and finally the

17

mesenteric nodes (4)(5).42-43 The uptake of 99mTc-usNGO-PEG in the five lymph

18

nodes gradually increased from 1 h to 24 h (Figure  6b). Notably, the popliteal node

19

showed the highest signals at each time point and the accumulation of

20

99m

21

post-injection (Figure 6b) with the uptake values of 0.35, 0.42, 0.98 and 1.78 %ID/g,

Tc- and Gd-usNGO-PEG in lymph node imaging using SPECT/CT and MR, as the

Tc-usNGO-PEG solution (200 μCi, 50 μL, 1 mg/mL) into the left footpad of

Tc-usNGO-PEG in the popliteal node was clearly seen at 1, 3, 6 and 24 h


Page 19 of 36

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13

p al structure of resppectively. Inn order to acccurately loocate the possition and physiologica

14

the popliteal noode, coronall SPECT/CT T and T1-w weighted MR R imaging w were furtherr

15

perfformed togeether (Figurre 7). All strructures werre clearly viisible with m minimal

16

backkground siggnals as 99mTc-usNGOT -PEG had not diffused or drained iinto the

17

m at this tim me point. Onn MR imagiing, the accu umulation oof vasccular system

18

Gd--usNGO-PE EG (50 μL, 1 mg/mL, 1 mM of Gd d) in the pop pliteal node (white arro ow

19

Figure 7b) also appeared 1 h after iinjection off the nanoprobe into thee footpad off in F

20

n was ob bserved for uup to 24 h with w 99mTc-u usNGO-PEG G SPECT/C CT micce, and the node

21

imaaging (Figurre 7a). As an n internal coontrol, the contralatera c l lymph nodde showed no n

22

conttrast enhanccement. In summary, s oour results demonstrate d d that 99mTcc- and

23

Gd--usNGO-PE EG exhibit great g potentiial as multi--modality SPECT/MRI S I probes forr

24

lym mph-node maapping.

14

15 17

Figu ure 6. 99mTcc- usNGO-P PEG SPECT T/CT imagiing of lymph h nodes witth quantitatiive

18

anallysis. SPEC CT/CT MIP images (a) and the corrresponding quantitativve analysis (b) ( 18 ACS Paragon Plus Environment


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Page 20 of 36

4

five differennt lymph nodes (1: poplliteal node; 2: sacral no ode; 3: cauddal node; 4 and a of fi

5

5: m mesenteric nodes) n weree mapped affter subcutan neous administration oof

6

99m

T Tc-usNGO-P PEG (200 μCi, μ 50 μL ,,1 mg/mL) in i left footp pad at 1, 3, 6 and 24 h.

5

6 13

Figu ure 7. The coronal c SPE ECT/CT andd MR imaging of popliiteal node. T The SPECT T/CT

14

imaaging (a) witth the schem me of coronnal plane mo ouse and T1 1-weighted MR imagin ng (b)

15

nd after the injection off of ppopliteal nodde (white arrrow) in thee same mouse before an

16

99m

17

o the left foootpad at diffferent time points (1, 33, 6 and 24 h) mg//mL, 1 mM of Gd ) into

18

usinng small aniimal SPECT T/CT and 7..0 T MRI sccanner (TR = 206.4 mss, TE = 1.4 ms), m

19

resppectively.

T Tc-usNGO-P PEG (50 μL L, 200 μCi, 1 mg/mL) and a Gd-usN NGO-PEG ((50 μL, 1

14 16 17

T This proof-oof-concept study establiished a chellator-free, kit-like, k simpple preparattion T and Gd-labeled usN NGO-PEG, and demon nstrated theiir promising g metthod for 99mTc-


Page 21 of 36

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1

applications for dual-modality SPECT/MR imaging of lymph nodes with the

2

advantages of high sensitivity due to SPECT and exquisite soft-tissue contrast due to

3

MRI. As a result of the rich functional groups on the surface of usNGO-PEG, further

4

conjugation of specific targeting ligands, such as proteins, antibodies, or peptides, can

5

be readily achieved, which would promote the R&D of NGO-PEG-based nanoagents

6

and carbon-based nanocarriers for future cancer-targeted SPECT/MR imaging,

7

simultaneous radiotherapy, and imaging-guided surgical resection of lymph nodes.

8

9

■ CONCLUSIONS

10

In conclusion, we developed a simple, stable and kit-like method for the conjugation

11

of 99mTc- and Gd-usNGO-PEG using a chelator-free approach via the synergistic

12

coordination of N and O atoms (from NGO and PEG) with 99mTcI and Gd without

13

additional exogenous chelators. Under optimized conditions, the two nanoprobes

14

99m

15

stability by direct mixing of usNGO-PEG with [99mTcI(CO)3(OH2)3]+ (99mTcI) and

16

GdCl3, followed by centrifugal membrane separation. Serial experimental

17

measurements and characterizations showed that both usNGO and PEG

18

synergistically provided the electron-donating atoms O and N to coordinate with

19

99m

20

then demonstrated their multi-modality imaging capabilities of lymph nodes using

Tc- and Gd-usNGO-PEG were reliably prepared with high yields and good

TcI and Gd to form the stable nanocomplexes 99mTc- and Gd-usNGO-PEG. We



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Page 22 of 36

1

SPECT/CT and MR as SPECT has the advantage of high sensitivity and MRI imaging

2

has the advantage of exquisite soft-tissue contrast. 99mTc-usNGO-PEG SPECT/CT

3

imaging, along with lymph vessel drainage from the injection site in mice, showed

4

that the footpad demonstrated five lymph nodes with high signal intensity and long

5

imaging time up to 24 h. For Gd-usNGO-PEG with high relaxivity r1, the MR images

6

only visualized the popliteal node with clear contrast. Moreover, this general

7

99m

8

ligand-modified NGO-PEG due to the rich functional groups on the surface of

9

NGO-PEG. This could facilitate the R&D of NGO-PEG-based nanoagents and

Tc/Gd-conjugation approach could be readily applied in biotargeted

10

carbon-based nanocarriers for future cancer-targeted SPECT/MR imaging,

11

simultaneous radiotherapy, and imaging-guided surgical resection of lymph nodes.

12

13

■ EXPERIMENTAL SECTION

14

Reagents and Chemicals All reagents were of analytical grade and used

15

without further purification. Cyanine5.5 NHS ester (Cy5.5® NHS ester) was

16

purchased from Lumiprobe Corporation. Graphite powder (~50 µm, 99.99+%),

17

sulfuric acid (H2SO4, 98%), hydrogen peroxide solution (H2O2, 30 wt% in H2O),

18

sodium carbonate (Na2CO3),

19

N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride (EDC-HCl,

20

99%), sodium persulfate (Na2S2O8, >98%), potassium permanganate (KMnO4, 21 ACS Paragon Plus Environment

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1

99%), GdCl3(99.99%), and SnCl2.2H2O were obtained commercially from

2

Aldrich. RPMI 1640 and DMEM from GIBCO/Invitrogen, and cell counting

3

kit-8 (CCK-8) were from Beyotime Institute of Biotechnology. 99mTcO4 was

4

bought from Shanghai GMS Pharmaceutical Co., Ltd. Six-armed PEG with six

5

hydroxyl end groups (6-armed PEG-OH, Mn = 5300 g/mol, determined with

6

Matrix-Assisted Laser Desorption/ionization Time-of-Flight

7

(MALDI-TOF-MS), Mw/Mn = 1.04, determined with gel permeation

8

chromatography) was synthesized via anionic polymerization of ethylene oxide

9

using D-mannitol as the initiator. Six-armed PEG–OH was converted to

10

6-armed PEG with six amino end groups (6-armed PEG-NH2) based on the

11

previous protocol.44

12

Characterization Instruments. Radiolabeling rate and radiochemical purity

13

were determined via the radioactive thin layer chromatography (Radio-TLC,

14

Ray test, Germany) scanner with CH3OH:HCl = 95:5 as the developing solution.

15

Transmission electron microscope (TEM) and Energy-dispersive X-ray analysis

16

(EDXA) were determined at 200 kV with a JEM-2010 (JEOL). The samples

17

were dispersed in H2O and dripped onto a copper grid for the TEM tests.

18

Fourier transform-infrared (FT-IR) spectra were recorded on AVATAR-360

19

(Nicolet) and FT-IR spectrophotometer with a resolution of 4 cm–1. X-ray

20

photoelectron spectroscopy (XPS) spectra were recorded with RBD 147

21

upgraded Perkin-Elmer PHI5000 system. The Mg Kα (1253.6 eV) anode was 22 ACS Paragon Plus Environment


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Page 24 of 36

1

operated at 14 kV and 20 mA. All binding energies (B.E) were calibrated using

2

the carbon C 1s line at 284.6 eV as a reference. The well-mixed buffer solution

3

was incubated at room temperature for 12 h, and zeta-potential tests were

4

carried out on an ALV-5000 spectrometer-goniometer equipped with an ALV/

5

LSE-5004 light scattering electronic and multiple tau digital correlator and a

6

JDS Uniphase HeNe laser (632.8 nm) with an output power of 22 mW. Relative

7

cell viability was measured at the absorbance of 450 nm using a Tecan GENios

8

Pro microplate reader. Cellular images were obtained under a Leica SP5

9

confocal microscope. Radioactivity was measured using the dose calibrator

10

(CRC®-25R, Capintec, Inc) and radioactive count was measured using the

11

automatic gamma counter (SN695, RiHuan, China).

12

Preparation of usNGO-PEG. NGO@PEG was prepared according to our

13

previous paper.31 Generally, after 500 mg graphite (~50 µm) was added to 18

14

mL H2SO4 (98%) and 6 mL HNO3 (100%), the 3.0 g KMnO4 was gradually

15

added with stirring for 2 h below 0 ºC, then heated to 40 °C for 30 min,

16

following up to 70 °C for 45 min. Next, adding 6 mL H2O maintain the mixture

17

at 105 °C for 10 min and further 40 mL of H2O at 100 °C for 15 min. Finally,

18

140 mL H2O and 10 mL 30% H2O2 were added solution to terminate reaction.

19

After neutralizing with NaOH, 6-armed PEG-NH2 and EDC-HCl was added to

20

NGO solution with sonication for 10 h at room temperature. The final

21

NGO@PEG products was obtained after dialysis (MW cutoff = 14 KDa) 23 ACS Paragon Plus Environment

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1

against double-distilled water for 3 days to remove unbound

2

6-armed-PEG-NH2.

3

Preparation and stability of 99mTc-usNGO-PEG. 99mTc labeling was

4

accomplished in two steps. In the first step, [99mTc(CO)3(H2O)3]+ was prepared

5

according to reference.32

6

with CO for 30 min in the dry vial, 99mTcO4– (1110 MBq, 30 mCi) in saline

7

solution (1 mL, 0.9% NaCl/H2O) was added to the closed vial and the mixture

8

was heated in an oil bath at 80 °C for 30 min. The Radio-TLC analysis of

9

[99mTc(CO)3(H2O)3]+ showed at Rf = 0.1 with acetone as the developing

Typically, after NaBH4 (5 mg) was firstly flushed

10

solution or Rf = 0.8 with CH3OH:HCl = 95: 5 as the developing solution. In the

11

second step, [99mTc(CO)3(H2O)3]+ (100 µL) in a sealed vial was mixed with

12

usNGO-PEG (1 mg/mL, 0.4 mL) heated at 80 °C for 5 min. Then the

13

99m

14

(Millipore). The radiochemical yield of 99mTc-usNGO-PEG was assessed by

15

Radio-TLC at Rf = 0.1 with CH3OH:HCl = 95: 5 as the developing solution.

16

Stability tests of 99mTc-usNGO-PEG were conducted in serum solution (50%, pH 7.4)

17

and normal saline (0.9% NaCl). Mixtures were incubated at 37°C for 0, 1, 3, 6 and

18

24h, and evaluated via Radio-TLC. To prepare NaBH4-reduced usNGO-PEG,

19

NaBH4 solution (5 mg) in water was added to usNGO-PEG. Then, the mixture

20

was heated at 80 oC for 1 h with N2 protect. The yielded NaBH4-reduced

Tc-usNGO-PEG as purified by filtration through 50 KDa MWCO filters



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Page 26 of 36

1

usNGO-PEG solution was purified by filtration through 50 KDa MWCO filters

2

(Millipore).

3

Preparation and stability of Gd-usNGO-PEG To prepare Gd-usNGO-PEG, an

4

aqueous solution of GdCl3 (1 mg in 1 mL) was added to an aqueous dispersion (1

5

mL) of usNGO-PEG (2 mg) and the mixture was sonicated in a bath for 10 h. The

6

mixture was then filtered using a 50 KDa MWCO filters (Millipore) and washed

7

with DI water multiple times until no Gd3+ ions were present in the filtrate (confirmed

8

by inductively coupled plasma optical emission spectrometry (ICP-OES)). After

9

purified, the solution was quantified to 1 mg / mL. The amine density of

10

usNGO-PEG and Gd-usNGO-PEG were detected by OPA method37 with the Primary

11

Amino Nitrogen kit (K-PANOPA). Stability tests on Gd-usNGO-PEG were

12

conducted in serum solution (50%, pH 7.4) and normal saline (0.9% NaCl), incubated

13

at 37°C for 0, 1, 3, 6, 24 and 48 h, and evaluated via ICP-OES.

14

Relaxivity Measurement of Gd-usNGO-PEG. The T1-weighted MR images

15

were obtained using a 7 T MRI scanner (Bruker Biospec 70/20 user). Dilutions

16

of Gd-usNGO-PEG (0.001, 0.005, 0.024, 0.052, 0.125 mM of Gd3+) in water for

17

T1-weighted imaging were placed in a 96 well plate. The following parameters

18

were adopted: a repetition time (TR) of 75, 150, 500, 1000, 2500 ms and ten

19

echo time (TE) of 8.0 ms under the T1 weight spin−echo sequence. The


Page 27 of 36

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1

resulting r1 values were calculated by the curve fitting of 1/T1 versus molar

2

concentration of Gd3+. The slope of the line provides the molar relaxivity r1.

3

Cell viability assay in vitro Mouse breast tumor 4T1 cells were supplied by

4

Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were

5

cultured at 37°C under a humid 5% CO2 atmosphere in DMEM supplemented

6

with 10% FBS and 1% penicillin–streptomycin. 4T1 cells were plated in

7

96-well plates at a density of 5 × 103 cells per well in 100 μL differentiation

8

medium overnight. Gd-usNGO-PEG (diluted in DMED) was subsequently

9

incubated with cells at different concentrations (0, 5, 10, 20, 50, 100 and 200

10

μg/mL). Relative cell viability was measured with the water-soluble tetrazolium

11

salt (WST) assay (cell counting kit-8 (CCK-8) kit, Dojindo, Inc.). After

12

incubated cells at 37 °C for 24 and 48 h, the absorbance was measured at 450

13

nm with a Tecan GENios Pro microplate reader using CCK-8.

14

99m

Tc-usNGO-PEG SPECT/CT imaging of lymph nodes. 99mTc-usNGO-PEG

15

solutions in sterile saline (50 μL, 1 mg/mL, 200–500 µCi injection/mouse) were

16

injected into the left hindpaw pad of the mouse and SPECT/CT imaging was acquired

17

at 1, 3, 6 and 24 h post injection. During the imaging period, mice were anesthetized

18

with 3% isoflurane in a mixture of O2/N2 (25%/75%) at 0.8 L/min in a chamber,

19

followed by 2% isoflurane through a mask placed on the imaging bed. SPECT/CT

20

scans were performed using a NanoSPECT/CT In Vivo Animal Imager (Bioscan,



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Page 28 of 36

1

USA). All imaging data were reconstructed and analyzed using In Vivo Scope

2

(Bioscan, USA). Animal procedures were in agreement with the guidelines of the

3

Institutional Animal Care and Use Committee, Fudan University. Nude mice (female,

4

4 weeks old, 15-20 g) were purchased from Shanghai SLAC laboratory Animal

5

Co., Ltd (Shanghai, China). All animals were housed under standard environmental

6

conditions and acclimated for at least 24 h before experiment.

7

Gd-usNGO-PEG MR imaging of lymph nodes. Before injecting the probe, the

8

mouse was first imaged by the same MR system using a T1_Flash sequence

9

(TR = 206.4 ms, TE = 1.4 ms, 256 × 256 matrix, slice thickness = 1.0 mm FOV

10

3.8*3.07 cm). Then, 50 μL of Gd-usNGO-PEG (1 mg/mL) was injected into the

11

foot pad of the mouse on the left hindpaw and MR images were acquired at 1, 3,

12

6 and 24 h post injection under the same conditions.

13

Statistical Analysis. Quantitative data were presented as means ± SD.

14

One-way analysis of variance was applied to compare differences between

15

groups using SPSS 16.0. P values < 0.05 were considered statistically

16

significant.

17

18

■ ASSOCIATED CONTENT

19

Supporting Information. 27 ACS Paragon Plus Environment

Page 29 of 36

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1

The Supporting Information is available free of charge on the ACS Publications

2

website at DOI:

3

(Figure S1) radiochemical yield of [99mTc(CO)3(OH2)3]+, (Table S1) ζ-potential values,

4

(Figure S2) saturated amount of Gd3+, and (Table S2) amine density (PDF)

5

6

AUTHOR INFORMATION

7

Corresponding Author

8

*E-mail [email protected] (MW Wang) Tel: 86-13761126113；Fax:

9

86-21-64176650

10

*E-mail: [email protected] (H Yang)

11

ORCID

12

Mingwei Wang: 0000-0003-0020-4700

13

Tianye Cao: 0000-0002-0235-0464

14

Author Contributions

15

‖ Tianye Cao and Xiaobao Zhou contributed equally to this work. The manuscript was

16

written based on the contributions of all authors, who have given the final approval to

17

the manuscript.



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1

Notes

2

The authors declare no competing financial interest.

Page 30 of 36

3

4

■ ACKNOWLEDGMENT

5

This study was supported by National Natural Science Foundation of China

6

(11275050, 21771041, 81401514). We gratefully thank from the Education Ministry

7

Key Lab of Resource Chemistry, Shanghai Normal University for the assistance on

8

the characterizations. The authors are gratefully acknowledged to Dr. Yongjun Li

9

from Shanghai Institute of Organic Chemistry, CAS, for the assistance of the

10

preparation and characterizations of NGO.

11

■ REFERENCES

12

(1) Hong, G.; Diao, S.; Antaris, A. L.; Dai, H. Carbon Nanomaterials for Biological

13

Imaging and Nanomedicinal Therapy. Chem. Rev. 2015, 115, 10816-10906.

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(2) Yang, K.; Feng, L.; Shi, X.; Liu, Z. Nano-graphene in Biomedicine: Theranostic

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Applications. Chem. Soc. Rev. 2013, 42, 530-547.

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(3) Wang, J.; Mi, P.; Lin, G.; Wáng, Y. X. J.; Liu, G.; Chen, X. Imaging-guided

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Delivery of RNAi for Anticancer Treatment. Adv. Drug Delivery Rev. 2016, 104,

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44-60.

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(4) Nurunnabi, M.; Parvez, K.; Nafiujjaman, M.; Revuri, V.; Khan, H. A.; Feng, X.;

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Lee, Y. Bioapplication of Graphene Oxide Derivatives: Drug/gene Delivery, Imaging, 29 ACS Paragon Plus Environment

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Polymeric Modification, Toxicology, Therapeutics and Challenges. RSC Adv. 2015, 5,

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42141-42161.

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(5) Yang, Y.; Aw, J.; Xing, B. Nanostructures for NIR Light-controlled Therapies.

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Nanoscale 2017, 9, 3698-3718.

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(6) Rong, P.; Yang, K.; Srivastan, A.; Kiesewetter, D. O.; Yue, X.; Wang, F.; Nie, L.;

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Tabble of Conteents/Abstracct Graphic


Tc and Gd - ACS Publications - American Chemical Society

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