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Smart Bacterial Magnetic Nanoparticles for Tumor-Targeting Magnetic Resonance Imaging of HER2-Positive Breast Cancers Yunlei Zhang, Qianqian Ni, Chaoli Xu, Bing Wan, Yuanyuan Geng, Gang Zheng, Jun Tao, Zhenlu Yang, Ying Zhao, Jun Wen, Junjie Zhang, Shouju Wang, Yuxia Tang, Yanju Li, Qirui Zhang, Li Liu, Zhaogang Teng, and Guangming Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15838 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Smart Bacterial Magnetic Nanoparticles for Tumor-Targeting

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Magnetic Resonance Imaging of HER2-Positive Breast Cancers

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Yunlei Zhang,*,† Qianqian Ni,† Chaoli Xu,┬ Bing Wan,|| Yuanyuan Geng,

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Zheng,† Zhenlu Yang,† Jun Tao, § Ying Zhao,† Jun Wen,† Junjie Zhang,§ Shouju Wang,†

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Yuxia Tang,† Yanjun Li,† Qirui Zhang,† Li Liu,† Zhaogang Teng,*,† Guangming Lu*,†,‡

Gang

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Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing

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†

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University, Nanjing, 210002 Jiangsu, P. R. China State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

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‡

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and Chemical Engineering, Nanjing University, Nanjing 210093, P.R. China

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

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Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, China

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§

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Advanced Materials (IAM), Jiangsu National Synergetic Innovation Centre for Advanced

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Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan

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Road, Nanjing 210023, P.R. China

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┬

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Medicine, Nanjing, 210002 Jiangsu, P.R. China

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|| Department

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of Nanjing Medical University, Nanjing 210002, 2, 2, P.R.China

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*Corresponding Author. Fax: +86 25 8480 4659. Tel: +86 25 8086 0185. E-mail:

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[email protected]; [email protected]; [email protected]

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China

Key Laboratory for Organic Electronics and Information Displays & Institute of

Department of Ultrasound Diagnostics, Jinling Hospital, Nanjing University School of

of Respiratory and Critical Care Medicine, the Affiliated Jiangning Hospital

1


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ABSTRACT: Supersensitive magnetic resonance (MR) imaging requires contrast with

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extremely high r2 values. However, synthesized magnetic nanoparticles generally have a

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relatively low r2 relaxivity. Magnetosomes with high saturation magnetization and good

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biocompatibility have shown potential value as MR imaging contrast agents.

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Magnetosomes that target human epidermal growth factor receptor-2 (HER2) were

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prepared using genetic technology and low-frequency sonication. Anti-HER2 affibody of

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the ability to target HER2 was displayed on membrane surface of the magnetosomes

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through the anchor protein MamC, allowing the bacterial nanoparticles to target tumors

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overexpressing HER2. The prepared nanoparticles exhibited a very high relaxivity of

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599.74 mM-1 s-1, better dispersion and their ability to target HER2 was demonstrated both

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in vitro and in vivo. Also, the HER2-targeting magnetosomes significantly enhanced MR

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imaging of orthotopic breast cancer models with or without HER2 expression using a 7.0

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T scanner. In particular, tumors overexpressing HER2 demonstrated better MR imaging

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than HER2-negative tumors after i.v. administration of HER2-targeting magnetosomes,

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and the MR signals of the augmented contrast could be detected from 3 h to 24 h. The

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magnetosomes did not cause any notably pathogenic effect in the animals. Therefore, we

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expect that non-invasive imaging of tumors using HER2-targeting magnetosomes has

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potential for clinical applications in the near future.

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KEYWORDS: Magnetic resonance imaging (MRI), magnetosomes, HER2, breast

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cancer, cancer imaging

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1. INTRODUCTION

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Magnetic resonance imaging (MRI) with non-invasive characteristics and high spatial

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resolution is one of the most powerful imaging tools available in diagnostic imaging.

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Development of contrast agents, such as magnetic nanoparticles, has greatly expanded

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the strength of MRI in characterizing physiological and molecular changes of cancers,

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providing precise information to determine tumor prognosis and treatment.1-3 For

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supersensitive MRI, the contrast agents should have extremely high r2 relaxivity.4 In

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contrast, synthetic superparamagnetic iron oxide nanoparticles (SPIOs) generally have

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relatively low r2 values. In fact, r2 relaxivity of SPIOs can be increased by controlling

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the magnetic core,5-6 increasing particle size

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However, regulation of the magnetic core and iron oxidation state in SPIOs does not

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greatly augment their r2 relaxivity, and size increases generally caused a

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superparamagnetic-ferromagnetic transition,9-10 so the resulting magnetic nanoparticles

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were no longer suitable as MRI contrast agents. In addition, to increase biocompatibility

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and stability of SPIOs in vivo, the surfaces have to be modified with polyethylene glycol

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(PEG),11 dextran,12, chitosan13-14 and other polymers,15-16 which are indispensable parts of

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the synthetic magnetic nanoparticles.

7-8

and regulating the iron oxidation state.7

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Magnetosomes of Magnetospirillum gryphiswaldense MSR-1 (M. gryphiswaldense

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MSR-1) consisting of ferromagnetic iron oxide crystals were characterized by

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high-saturation magnetization, uniform size (35 - 42 nm), good dispersion and 3


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biocompatibility,17-18 demonstrating potential value as an MRI contrast agent. The

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magnetosomes are smaller than a magnetic Weiss domain,19 indicating that they could

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have extremely high r2 values; the high regularity of magnetic monocrystals in the

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magnetosomes also signified potentially high r2 relaxivity.20 Actually, the magnetosomes

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extracted from Magnetospirillum magneticum AMB-1 and Magnetovibrio blakemorei

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strain MV-1 displayed extremely high r2 values with a 17.2 T Biospec preclinical

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scanner, which was four times higher than the commercial MRI contrast agent Feridex.21

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Due to this excellent characteristic, the ferromagnetic iron oxide nanocubes that

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mimicked magnetosomes were developed, and they achieved highly sensitive magnetic

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resonance (MR) imaging of single cells and transplanted pancreatic islets.22 However, the

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r2 relaxivity of the synthetic nanoparticles mimicking magnetosomes was only slightly

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higher than that of Feridex and still much lower than that of magnetosomes.22 So far, the

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r2 relaxivity of synthesized SPIOs still fall far behind of natural magnetosomes.

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In addition to the r2 relaxivity of magnetic nanoparticles, an accumulated

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contrast-enhancing agent in a tumor site is another key factor in obtaining supersensitive

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MR imaging.23-24 Differing from the naked or small molecular modified surfaces of

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SPIOs, the natural lipid bilayers on the magnetosomes provide good biocompatibility and

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ample functional groups for conjugation of tumor targeting or therapeutic agents.20, 25 For

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example, depending on abundant membrane proteins of lipid bilayers, tumor-targeting

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peptides were successfully displayed on surface of the magnetic particles via chemical

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reaction or genetically modification. The peptide-modified magnetosomes exhibited a

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remarkable enhancement of MR imaging of brain and breast cancers, demonstrating

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clinical value of the bacterium-derived nanoparticles. However, the membrane proteins 4



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on magnetosomes are generally recognized as foreign antigens by immune system, which

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could stimulate inflammatory response after intravenous (i.v.) administration and produce

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adverse effects; the chemical reaction could result in the introduction of toxic substances

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into magnetosomes; genetically manipulation in Magnetospirillum is complex, especially

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for obtaining active foreign proteins. Importantly, the valuable tumor-targeting peptides

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remain scarce, falling behind the requirement of molecular imaging for more specific

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imaging agents. In contrast to tumor-targeting peptides, affibody with similar binding

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sites as antibody could be obtained through in vitro technique for high affinity (up to 22 26

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pM) and specificity to any protein target.

In particular, affibody as small protein

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molecular (6.5 KDa) was characterized with low immunogenicity, high biocompatibility,

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excellent biodegradability and robust physical properties (extreme pH and elevated

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temperature).27 They can be largely produced at a low cost or form new functional

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protein through using biological methods in the type strain of Escherichia coli (E.coli).28

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Because of their hydrophilicity and high-affinity to antigens, several affibodies are under

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investigation for cancer imaging and therapy in clinical trials.26, 29-30

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Depending on the excellent characteristics of affibody, we introduce a new strategy

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to prepare tumor-targeting magnetosomes with low immunogenicity and high affinity to

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tumor cells for MR imaging of human epidermal growth factor receptor-2

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(HER2)-positive breast cancers via genetic technology and low-frequency sonication.

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The anti-HER2 affibody fused with MamC protein were first expressed and purified from

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the type strain of E.coli BL21 through genetic technology. Simultaneously, membrane

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proteins in phospholipid bilayers of the magnetosomes (M. gryphiswaldense MSR-1)

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were totally removed using low concentration of SDS solution. Depending on the anchor 5


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protein of MamC, anti-HER2 affibody were successfully displayed on the magnetosomes

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via low-frequency sonication. The anti-HER2 affibody-modified magnetosomes

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exhibited highly sensitive MR-enhanced contrast for breast cancer detection, especially

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HER2 overexpressed breast cancers. Theoretically, many kinds of affibodies and other

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targeting proteins can be decorated on the magnetosomes to construct different T2 MR

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contrasts through this technology and advance the development of molecular imaging.

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2. MATERIALS AND METHODS

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2.1. Materials: Anti-HER2 antibody, horseradish peroxidase (HRP)-labelled anti-6 ×

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histags antibody and recombinant HER2 protein with histags were obtained from Abcam

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(Cambridge, UK). Bovine serum albumin (BSA), isopropyl β-D-thiogalactoside (IPTG),

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mammalian cell total protein lysis buffer, 4% paraformaldehyde and tween 20 were

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purchased from Sangon Biotech (Shanghai, China). Bugbuster® master mix and

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tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich (St. Louis,

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MO, USA). Counting kit-8 was from Dojido (Kumamoto, Japan). Dulbecco’s modified

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Eagle’s medium (DMEM), 0.5% trypsin-EDTA, L-glutamine and penicillin-streptomycin

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solution were obtained from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). Fetal

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bovine serum was bought from Gibco/ThermoFisher scientific (Waltham, MA, USA).

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Neodymium-iron-boron (Nd-Fe-B) magnets were provided by NingboJinke Co., Ltd.

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(Ningbo, China). HisTrapTM FF crude and polyvinylidene fluoride (PVDF) membrane

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were obtained from GE Healthcare Life Science (Piscataway, USA). Immobilon western

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chemiluminescent HRP substrate and skimmed milk were purchased from BIO-RAD

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company (Hercules, CA, USA). Maleimide derivative cyanine dyes (Cy5.5) was provided

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by Seebio Biotechnology Co., Ltd. (Shanghai, China). Neutral balsam was obtained from 6



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Haoran Co., Ltd. (Shanghai, China). Perls stain A and B was bought from Solarbio Co.,

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Ltd. (Beijing, China).

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2.2. Magnetosome extraction: M. gryphiswaldense MSR-1 was passaged for three

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times in sodium lactate medium at 30 °C before moving to the amplification culture

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medium. Ferric citrate was added to the fermentation medium at a final concentration of

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30 μM for magnetosome synthesis. The bacteria were collected by centrifugation at 6000

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rpm for 20 min and washed thrice using Millipore water. Then the bacterial pellets were

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broken through sonication in phosphate buffer saline (PBS) (pH 7.4) to release

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magnetosomes. Finally, the magnetic particles were gathered using Nd-Fe-B magnets and

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freeze-dried for storage.

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2.3. Purification of MamC-Anti-HER2 affibody: The nucleotides of MamC

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(GenBank: CDK99608.1) and anti-HER2 affibody were optimized using JAVA codon

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adaptation tool for soluble expression in E.coli BL21 cells. The recombinant nucleotides

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of the two proteins (MamC and anti-HER2 affibody) containing linker peptides, six

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histidines and two cysteines were synthesized and colonized into the pET22b plasmid

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with Kanamycin resistant gene (see the gene sequence of the recombinant protein in the

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Supporting Information). The protein expressed by the recombinant gene was named as

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MamC-HAF. In addition, 30 amino acids at the C-terminal of anti-HER2 affibody were

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deleted from MamC-HAF to prepare the truncated MamC-HAF protein (abbreviated as

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MamC-THAF), which was used as a control group in the study. E.coli BL21 with the

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plasmids containing the genes of MamC-HER2 or MamC-THAF were cultured in

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lysogeny broth (LB) media, and the protein expression was induced by adding IPTG at a

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final concentration of 0.5 mM. The bacteria containing MamC-HAF or MamC-THAF 7


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were gathered by centrifugation at 10000 g for 10 min. Next, they were mixed with

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Bugbuster® master mix and shaken at room temperature for 20 min for lysing cells and

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releasing cytoplasmic proteins. After centrifugation at 16000 g for 20 min, the

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MamC-HAF and MamC-THAF proteins contained in the supernatants were purified

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using HisTrapTM FF crude following the instruction.

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2.4. Modification of magnetosomes with MamC-HAF and MamC-THAF:

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Magnetosomes were first resuspended in 1% SDS and boiled at 100 °C for 5 min to

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remove proteins from their phospholipid bilayers. The gathered magnetosomes were

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collected using Nd-Fe-B magnets and washed thrice using PBS. 100 μg of the

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magnetosomes was individually incubated with 200 μL of MamC-HAF (1 mg/mL),

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MamC-THAF (1 mg/mL) or BSA (1 mg/mL) on ice for 1 h. Next, the solutions were

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sonicated (20 W, 0.5 s/0.5 s) for 3 min in an ultrasonic cell disruptor (Xinyi JY92-IIN,

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Ningbo, China) and incubated on ice for another 30 min. The magnetosomes with the

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MamC-HAF or MamC-THAF were gathered using magnets and immersed in 100 μL of 5

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mM TCEP (100 mM Stock) for 10 min to open the disulfides of cysteines at the

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C-terminal of the proteins. The magnetosomes decorated with the foreign proteins were

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collected by magnets and washed using PBS three times. Further, the modified

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magnetosomes were mixed with 200 μL of 0.1 mg/mL Cy5.5 and shaken at room

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temperature for 3 h. The conjugated near-infrared fluorescence on the magnetosomes was

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identified using the IVIS Spectrum System (Xenogen Corporation–Caliper, Alameda,

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CA, USA). The magnetosome with MamC-HAF protein was abbreviated as BMW-HAF;

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MamC-THAF decorated magnetosome was named as BMW-THAF. The BMW and its

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derivatives were dissolved in water and mounted on 3 mm carbon-coated copper mesh 8



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for observation using transmission electron microscopy (TEM). The TEM images were

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captured using an HT7700 microscope (Hitachi, Tokyo, Japan) at an accelerating voltage

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of 100 kV. Simultaneously, zeta potentials of the water-dissolved magnetosomes were

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recorded through a Brookhaven analyzer (Brookhaven Instruments Co., Holtsville, NY,

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USA)

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2.5. Identification of MamC-HAF and MamC-THAF on magnetosomes: The

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presence of MamC-HAF or MamC-THAF on the magnetosomes was identified using

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sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western

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blot. In brief, 1 μg of BSA, 3 μg of MamC-HAF, 3 μg of MamC-THAF, 20 μg of

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magnetosomes with BSA, 20 μg of magnetosomes with MamC-HAF and 20 μg of

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magnetosomes bearing MamC-THAF were mixed with SDS-PAGE loading buffer and

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boiled for 10 min at 100 °C. After centrifugation, the supernatants of the samples were

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loaded to 12% gels and conducted at 120 volts for 1 - 2 h in a Mini-Protean Tetra

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Electrophoresis System (BIO-RAD, USA). After washing using PBS thrice, the gels were

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stained by coomassie brilliant blue G250 for directly protein imaging or transferred to

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PVDF membrane in Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (BIO-RAD,

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USA) for Western blot. After washing using TPBS (PBS containing 0.05% (v/v) tween

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20), the PVDF membrane were first blocked using 5% skimmed milk and incubated with

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HRP-labelled anti-6 × histags antibody at a dilution of 1 : 2000 for 12 h. Next, after

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washing thrice using TPBS, the proteins on the membrane were displayed through

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immobilon western chemiluminescent HRP substrate and imaged using an imaging

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system 5200 Multi (Tanon, Shanghai, China).

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2.6. Identification of HER2 in breast cancer cells and evaluation of HAF binding 9


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ability to HER2 protein: SK-BR-3, MCF-7 and MDA-MB-468 breast cancer cells were

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bought from the American Type Culture Collection (USA). The cells were cultured in

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L-glutamine and 100 U/mL penicillin/streptomycin-containing DMEM medium

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supplemented with 10% fetal bovine serum at 37 °C in a cell incubator. The cells at

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exponential phase were collected by centrifugation at 300 g for 5 min, and 25000 cells

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per well were seeded in a six-well-plate. After overnight incubation, the media were

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removed and washed thrice with PBS. Subsequently, 100 μL of the mammalian cell total

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protein lysis buffer were added into each well and incubated on ice for 15 min. After

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centrifugation, the supernatant was collected and analyzed using Western blot, in which

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anti-HER2 antibody was used at a dilution of 1 : 400. The bands on the membrane were

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captured under an imaging system 5200 Multi. Alternatively, the HAF protein was used

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as antibody to confirm the binding ability of the protein to HER2 protein. Because of

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histags in the C-terminal end of the HAF, HRP-labelled anti-6 × histags antibody was

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used as the second antibody to detect HAF following the above protocol of Western blot.

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The recombinant HER2 protein with histags was used as positive control in the

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experiment. In addition, the tumor tissues of SK-BR-3, MCF-7 and MDA-MB-468 were

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excised from the tumor-bearing mice and fixed in 4% paraformaldehyde overnight at

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room temperature. Further, the fixed tissues were processed following the standard

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protocol of immunochemistry, in which the optimized concentration of anti-HER2

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antibody was at 1 : 150 of dilution. The tissue sections were captured under the Olympus

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microscope (Olympus IX71, Tokyo, Japan).

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2.7. Evaluation of BMW-HAF internalization by breast cancer cells with

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HER2-expression difference using atomic absorption spectrometry (AAS): SK-BR-3, 10



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MCF-7 and MDA-MB-468 cells (25000 cells per well) were cultured in a six-well-plate.

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After the cells growing to 90% confluence, 30 μg/mL of BMW-HAF or BMW-THAF

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were added into the wells and incubated for 1 h, 3 h, 6 h and 12 h, respectively. At a

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definite time, the cells were harvested through centrifugation after digestion using 0.5%

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trypsin-EDTA. The gathered cells were washed using PBS for three times and numbered

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through a hemocytometer. Next, some of the cells were used for total protein extraction

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and protein quantification. Simultaneously, equal number of the cells were immersed in 2

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mL of hydrochloric acid, and the solutions were heated to 250 °C on a high-temperature

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heater (DragonLab, Shanghai, China) until the liquid was completely evaporated. Finally,

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4 mL of millipore water was added to dissolve the irons for the further analysis in an

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atomic absorption spectrometry (180-80, Hitachi Inc., Japan). For the in vivo

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experiments, the tumor-bearing mice (n = 3) were i.v. administrated of 125 μg/300 μL of

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BMW-HAF and BMW-THAF, respectively. After 24 h treatment, the mice were

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sacrificed, and the organs (tumor, liver, spleen, lung, heart and kidney) were weighted

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and homogenated. 5 mL of hydrochloric acid was added to the homogenate and

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processed like the former experiments. In the parallel experiments, the organs were fixed

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in 4% formaldehyde for overnight, and the irons in the serial sections of the organs were

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stained by Prussian blue staining.

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2.8. Targeted cell uptake evaluation of BMW-HAF using flow cytometry and

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confocal microscopy: SK-BR-3, MCF-7 and MDA-MB-468 cells at 90% confluence

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were collected, respectively, and seeded in a six-well-plate. After overnight, the media

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were replaced with the fresh media containing 30 μg/mL of BMW-HAF or BMW-THAF,

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and further incubated for 1 h, 3 h and 6 h. And then, the cells were collected by 11


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centrifugation after treatment using 0.5% trypsin-EDTA and washed thrice by PBS. In

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the end, the cells were resuspended in 500 μL PBS, and the Cy5.5 fluorescence of the

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cells was immediately analyzed using a flow cytometry (CytoFLEX, Beckman Coulter

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Inc., USA). Simultaneously, the cells were incubated with 30 μg/mL of BMW-HAF or

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BMW-THAF for 2 h. After treatment, the cells were washed thrice with PBS and fixed in

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4% paraformaldehyde for 15 min at room temperature. The cells were washed again by

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PBS for three times. Finally, the coverslip was immersed by one drop of mounting

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medium with DAPI to counter stain nuclei. The cellular uptake and distribution of

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BMW-HAF or BMW-THAF with Cy5.5 fluorescence signals were imaged through an

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inverted confocal laser scanning microscope (LSM 710, Carl Zeiss, Germany).

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2.9. Prussian blue staining: Cells were subcultured and seeded on the coverslip in a

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six-well-plate. After overnight culture, the cells treated by 30 μg/mL of BMW-HAF or

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BMW-THAF for different times were washed thrice using PBS and fixed in 4%

266

paraformaldehyde for 15 min. The cells were then washed twice using water and

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Millipore water for 4 min, respectively. The iron staining in the fixed cells and organ

268

sections were performed following the protocol of Prussian blue staining. In detail, the

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equal volume of perls stain A and B were mixed and added to the fixed cells and tissue

270

sections, and they were incubated for 20 min at room temperature. Afterwards, the

271

samples were washed using Millipore water for 5 min and immersed in nuclear fast red

272

solution for 10 min. Further, the cells and sections were washed using water for 5 s and

273

processed following the general protocol of dehydration and transparency. The coverslip

274

with the cells or tissue sections were mounted using neutral balsam. The cellular uptake

275

and tissue distribution of BMW-HAF were captured under an Olympus microscope 12



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(Olympus IX71, Tokyo, Japan).

277

2.10. In vitro and in vivo toxicity assay: Normal breast cell line (MCF-10A) were

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cultured in DMEM/F12 medium with 5% horse serum, 20 ng/mL of epidermal growth

279

factor, 0.5 μg/mL of hydrocortisone, 100 ng/mL of cholera toxin and 10 μg/mL of insulin

280

at 37 °C in a cell incubator; the culture condition of human embryonic kidney cells

281

(293T) was the same as the breast cancer cells of SK-BR-3, MCF-7 and MDA-MB-468.

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The cells were collected at 90% confluence, and 8000 cells per well were seeded in a

283

96-well-plate. After overnight, the media were replaced with the fresh media containing

284

BMW-HAF at the concentrations of 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/mL and further

285

incubated for 48 h. Finally, 100 μL of fresh media with 10 µL of cell counting kit-8 were

286

added to each well of the 96-well plate and incubated for 1 - 4 h under cell culture

287

condition. The absorbance of each well of the plate was recorded at 450 nm through a

288

spectrophotometer (Infinite M200 Pro, Tecan Magellan, Switzerland). Next, the

289

hemolysis assay was conducted following the general protocol. In brief, 1 mL of human

290

whole blood was diluted in 2 mL of 0.9% NaCl and centrifuged at 400 g for 5 min. The

291

collected red blood cells (RBCs) were resuspended in 10 mL of 0.9% NaCl, 0.2 mL of

292

which was added to 0.8 mL of BMW-HAF in 0.9% NaCl at 0.05, 0.1, 0.2, 0.4, 0.8 and

293

1.6 mg/mL. Next, the suspensions were gently vortexed and incubated at 37 °C for 2 h in

294

a cell incubator. A 100 μL of the supernatant from each sample were transferred into a

295

96-well-plate after centrifugation at 400 g for 5 min. The absorbance of the hemoglobin

296

released from red blood cells was determined by a microplate reader at 490 nm (Infinite

297

M200 Pro, Tecan Magellan, Switzerland). In addition, female BALB/c mice (n = 10),

298

aged 6 to 8 weeks, were i.v. injected with 300 μL PBS of 300 μg BMW-HAF or PBS 13


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299

every week. Body weight was documented every 3 days for one month. Simultaneously,

300

1 day or 3 days after BMW-HAF or PBS administration, the serum biochemical level of

301

alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase

302

(ALP), creatinine (CR) and blood urea nitrogen (BUN) in the mice (n = 3) were tested.

303

Finally, the mice (n = 3) were sacrificed by cervical dislocation, and the livers, spleens,

304

lungs, hearts and kidneys were fixed in 4% paraformaldehyde overnight at room

305

temperature. The pathological changes of the tissues were analyzed through

306

hematoxylin-eosin (H&E) staining. All the animal experiments were conducted following

307

the National Institutes of Health Guide for the Care and Use of Laboratory Animals under

308

the supervision of the Animal Ethics Committee of Jinling Hospital.

309

2.11. Magnetic resonance imaging: BMW-HAF and BMW-THAF was serially

310

diluted and suspended in an agar matrix; the iron concentrations range from 0 to 0.20 ×

311

10-3 mM. Transverse r2 relaxivity of the magnetosome materials was measured using T2

312

mapping sequence in a 7.0 T Bruker Biospec 7T/20 USR (BRUKER BIOSPEC,

313

Germany). BALB/c nude mice bearing MDA-MB-468 and SK-BR-3 breast tumors were

314

i.v. administrated with 300 μL PBS of 125 μg BMW-HAF, BMW-THAF or PBS. At

315

defined time points, MRI was performed and analyzed in the 7.0 T MRI scanner by using

316

a T2 RARE sequence (TR = 1500 ms, TE = 25 ms; echoes = 8.33; flip angle = 90 deg;

317

FOV = 35 mm; slice thickness = 1 mm).

318

2.12. Statistical analysis: Statistical significance for all the experimental results was

319

determined by Student’s t test. If the P values below 0.05, the differences between the

320

groups were considered significant.

321

3. RESULTS AND DISCUSSION 14



322

This study aimed to develop a new strategy based on the high relaxivity of magnetosomes

323

and antibody-mimicking affibody for preparing tumor-targeting T2 MRI contrast agents

324

through using genetic technology and low-frequency sonication. In detail, MamC is the

325

most abundant membrane protein in magnetosomal lipid bilayers that was proposed to

326

anchor foreign proteins,31 and anti-HER2 affibody, which specifically binds to the HER2

327

protein, has achieved great success in tumor imaging and therapy.26, 32 Here, the MamC

328

protein was used to anchor the anti-HER2 affibody on the membrane bilayers of

329

magnetosomes to prepare an HER2-targeting magnetite nanoparticle. The nucleotides of

330

MamC and anti-HER2 proteins were first optimized using software for soluble

331

expression in E.coli BL21 cells. The peptide of glycine-glycine-glycine-glycine-cysteines

332

was generally used as linker for constructing recombinant protein, favoring the flexibility

333

of conjugated proteins.33-34 Here, three-repeat units of the linker peptide were used to

334

increase the binding ability of anti-HER2 affibody in MamC-HAF protein to HER2 on

335

breast cancers (Figure 1a). Six-histidines and two cysteines at the C-terminal of the

336

fusion protein were used for protein detection and labeling with Cy5.5 fluorescence,

337

respectively (Figure 1a). The two recombinant genes were cloned into the pET22b

338

plasmids and expressed in E. coli B21 cells (Figure 1b). The purity of the extracted

339

proteins from the strains was greater than 95%, compared to the BSA, and the protein

340

bands on the SDS-PAGE were totally consistent with the calculated molecular weights of

341

the proteins (Figure 1c). The Western blot, using the anti6 × histags antibody, further

342

demonstrated the successful expression of the fusion proteins, and they could then be

343

used to decorate the magnetosomes to construct HER2-targeting nanoparticles (Figure

344

1d). To evaluate the binding ability of HAF to HER2 protein on membrane surface of 15


Page 16 of 44

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345

cancer cells, three breast cancer cell lines, SK-BR-3 with HER2 overexpression, MCF-7

346

with HER2 expression and MDA-MB-468 without HER2 expression, were used during

347

the in vitro experiments. Figure 1e clearly demonstrated that the expression of HER2 in

348

SK-BR-3 was significantly higher than MCF-7, and there was no HER2 expression in

349

MDA-MB-468 cells. The binding ability of HAF to HER2 proteins of the breast cancer

350

cells was also evaluated by Western blot. Differ from the Figure 1e, HAF was instead of

351

commercial HER2 antibody to detect HER2 expression levels, and it showed the similar

352

binding ability to HER2 protein as the commercial antibody (Figure 1f). The use of

353

recombinant HER2 protein (named HER2 protein in Figure 1f) directly displayed the

354

binding ability of the HAF to the targeted proteins.

16



355 356

Figure 1. Plasmid construction and heterologous expression of MamC-HAF and MamC-THAF

357

proteins. (a) Schematic diagram of recombinant genes of MamC-HAF and MamC-THAF. (b) Plasmid

358

mapping of pET22b-MamC-HAF and pET22b-MamC-THAF. (c) Detection of purified MamC-HAF

359

and MamC-THAF using SDS-PAGE. (d) Confirmation of MamC-HAF and MamC-THAF through

360

Western blot. (e) Evaluation of HER2 expression in different breast cancer cells. (f) Binding ability of

361

HAF protein to HER2 of the breast cancer cells. The recombinant HER2 protein with six histidines

362

(HER2 protein) was used as positive control.

363 17


Page 18 of 44

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364

Magnetosomes extracted from the strain of M. gryphiswaldense MSR-1 demonstrated

365

an elongated hexagonal-prismatic morphology, uniform particles size (35 - 42 nm) and

366

good dispersion (Figure 2a). To lower the immunogenicity, proteins in the phospholipid

367

bilayers of the magnetosomes were removed using 1% SDS, providing spaces for foreign

368

proteins in phospholipid bilayers of the magnetosomes. Compared to the original

369

nanoparticles, adding the foreign proteins (MamC-HAF) did not change the morphology

370

or phospholipid bilayer (red rectangle) of the magnetosomes (Figure 2b). However, the

371

removal of membrane proteins significantly reduced the zeta potential of the

372

magnetosomes (Figure 2c). And, the ligation of negatively-charged cy5.5 on the proteins

373

further lowered electric charges of the MamC-HAF or MamC-THAF modified

374

magnetosomes (Figure 2c). Considering the negatively charged membranes of

375

mammalian cells,35 this could significantly reduce the non-specific binding of bacterial

376

nanoparticles to normal cells,36 which is necessary for imaging agents in clinical

377

applications. In addition, the presence of MamC-HAF or MamC-THAF on the

378

magnetosomes was further confirmed by SDS-PAGE. Compared to the control group of

379

magnetosomes with BSA (abbreviated as BMW-BSA) and affibody (data not shown),

380

released MamC-HAF/MamC-THAF from the constructed magnetosomes could be clearly

381

identified on the SDS-PAGE gels (Figure 2d). The results indicated that MamC is a key

382

factor for anchoring foreign proteins in the membrane bilayers of magnetosomes. In

383

addition, the lack of additional protein bands on the SDS-PAGE gel demonstrated that the

384

original proteins on the membrane of the magnetic particles could be completely removed

385

using 1% SDS. The proteins on the magnetosomes were further quantified by comparing

386

the band intensity of the purified MamC-HAF or MamC-THAF. The calculated results 18



387

demonstrated that there were approximately 1.16 μg fresh proteins per 20 μg of

388

BMW-HAF or BMW-THAF. Simultaneously, the near-infrared fluorescence (NIFR) dye

389

Cy5.5 was successfully conjugated on the magnetosomes using the thiol groups of

390

cysteines at C-terminals of the foreign proteins (Figure S1, Supporting Information). This

391

enabled the detection of internalized magnetosomes by cancer cells. In particular, the

392

diameter of the magnetosomes enables fitting into the narrow regions of magnetic single

393

domains of magnetite, with potentially high r2 relaxivity.19 The experimental results

394

certified that nanoparticles constructed with BMW-HAF and BMW-THAF had high

395

relaxivity r2 and reached 599.74 mM-1 s-1 and 579.6 mM-1 s-1, respectively, which was

396

three times higher than the commercial MR reagent, feridex (r2 =130-170 mM-1 s-1)

397

(Figure 2e).21-22

398

to the high regularity of monocrystals of their iron oxide core, the mineralization process

399

of which was precisely regulated by multiple genes in the magnetotactic bacterium.37 The

400

high r2 value of the magnetosome constructs indicates their potential application as a

401

biogenic contrast agent for molecular MR imaging.

In fact, the high r2 values of the magnetosomes should be also ascribed

19


Page 20 of 44

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402 403

Figure 2. Magnetosomes characterization. (a) Magnetosomes of Magnetospirillum gryphiswaldense

404

MSR-1. (b) Magnetosomes modified with the proteins of MamC-HAF (the red rectangle indicates the

405

phospholipid bilayers on the magnetosomes). (c) Zeta potentials of the magnetosomes and its

406

derivatives. (d) Identification of MamC-HAF and MamC-THAF on the membrane of magnetosomes

407

using SDS-PAGE. (e) Estimation of transverse relaxivity r2 by a linear fit of 1/T2 versus

408

concentration of magnetosomes with the proteins of MamC-HAF (BMW-HAF) or MamC-THAF

409

(BMW-THAF). BMW@ indicates the membrane-protein removed BMW.

410 411

To evaluate the biocompatibility of BMW-HAF, normal cells, blood cells and

412

immunocompetent BALB/c mice were used to investigate possible toxicity of the

413

nanoparticles. The in vitro results demonstrated that high concentrations of BMW-HAF

414

slightly inhibited the growth of MCF-10A and 293T cells, and this effect gradually

415

increased with greater concentrations (Figure 3a). However, cell viability was still above

416

80%, even they incubated with the 1.6 mg/mL BMW-HAF for 48 h. The inhibition of cell 20



417

growth by the high concentrations of nanoparticles should be ascribed to the

418

corresponding high concentration of iron on the membranes of the cells, which prevent

419

cells from being nourished and blocks signals among cells, which finally delays cell

420

growth. Importantly, low concentrations of BMW-HAF did not exhibit substantial

421

toxicity against the cells. Additionally, a hemolysis assay was carried out to determine

422

whether BMW-HAF affected the integrity of red blood cell membranes. The results

423

demonstrated that the hemoglobin concentration rises slightly but does not exceed 3.67%

424

after treatment with BMW-HAF, even at the maximum concentration of 1.6 mg/mL,

425

further demonstrating the good biocompatibility of the nanoparticles (Figure 3b).

426

Body weight loss is a key index for evaluating drug toxicity.38 Immunocompetent

427

BALB/c mice who received weekly i.v. administration of BMW-HAF did not exhibit

428

different growth curves, over 30 days, than the group who received PBS (Figure 3c). In

429

the parallel experiments, the blood indices were monitored. However, 1 day after i.v.

430

administration of BMW-HAF, the concentrations of ALT and AST significantly

431

increased (Figure 3d) while ALP obviously decreased. The blood indices of ALT, AST

432

and ALP are closely associated with liver metabolism. The accumulation of

433

magnetosomes in the liver could affect key metabolic processes and result in the

434

alteration of some blood indices. The indices returned to normal levels 3 days after the

435

final i.v. administration, demonstrating that the short-term effect was quickly reversed.

436

Other metabolites, creatinine and blood urea nitrogen, remained at normal levels 1 day

437

and 3 days after i.v. administration of the engineered magnetosomes (data not shown).

438

Notably, no deaths occurred during observation, and mice treated with BMW-HAF

439

behaved normally as judged by comparison with PBS group. Pathogenic changes of key 21


Page 22 of 44

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440

organs in the immunocompetent BALB/c mice were also analyzed using H&E staining

441

after monitoring the grow curves of body weight. As Figure 3e demonstrates, after

442

treatment by BMW-HAF, the organs (livers, spleens, lungs, hearts and kidneys) exhibited

443

no significant differences from the PBS group. The biocompatibility tests further

444

provided supporting evidence for the potential application of BMW-HAF as a contrast

445

agent for MR imaging.

22



446

447

Figure 3. Biocompatibility of magnetosomes with MamC-HAF (BMW-HAF). (a) Inhibitory effects of

448

the BMW-HAF at different concentrations on the growth of MCF-10A and 293T cells. (b) Hemolysis

449

assay. (c) Body weights (n = 10) were recorded every three days after weekly i.v. administration of

450

PBS or BMW-HAF. (d) Serum biochemical levels of alanine aminotransferase (ALT), aspartate 23


Page 24 of 44

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451

aminotransferase (AST), alkaline phosphatase (ALP) on day 1 and 3 after i.v. injection with PBS,

452

BMW-HAF and BMW-THAF, respectively (n = 3). (e) H&E staining of tissues from the BALB/c

453

mice (n = 3) after weekly i.v. administration of PBS, BMW-HAF and BMW-THAF, respectively, for

454

30 days. **, P < 0.01; The data corresponds to the mean ± SD.

455 456

The magnetosomes modified with anti-HER2 affibody were prepared to target HER2

457

expressing cells for MR imaging. The breast cancer cell lines (MDA-MB-468, MCF-7

458

and SK-BR-3) with different HER2 expression were used to evaluate the ability of

459

BMW-HAF nanoparticles to target HER2. Prussian blue staining is the typical method to

460

detect the presence of iron in biological samples.39 It was used in the experiment to find

461

the difference of internalized BMW derivatives by cancer cells. The results of Prussian

462

blue staining demonstrated no significant differences between iron concentration in

463

MDA-MB-468 cells or cells treated with BMW-HAF or BMW-THAF after 6 h

464

incubation (Figure 4a and S2). However, the blue stains being indicative of irons in the

465

group treated with BMW-HAF were much more than that of the BMW-THAF group in

466

the MCF-7 and SK-BR-3 cell lines. This indicates the presence of HER2 proteins on the

467

cell membrane, which can then be specifically targeted by the anti-HER2 affibody on

468

BMW-HAF. In contrast, BMW-THAF with a truncated affibody completely lost the

469

ability to recognize HER2. The ability of BMW-HAF to target HER2 was further

470

confirmed using atomic absorption spectrometry. Figure 4b shows internalized irons per

471

milligram protein in the corresponding cells. HER2-negative cells (MDA-MB-468)

472

treated with BMW-HAF and BMW-THAF did not exhibit significant differences of the

473

internalized irons from 1 h to 12 h. However, in MCF-7 and SK-BR-3 cells, those treated 24



474

with BMW-HAF were significantly higher than those treated with BMW-THAF from 1 h

475

to 12 h after incubation. The difference reached the highest at 6 h in the two cell lines,

476

and this level was maintained, even after 12 h. Specifically, the maximum difference of

477

internalized irons per milligram protein between groups treated with BMW-HAF or

478

BMW-THAF reached 2.02 times in SK-BR-3 cells at 6 h, which was significantly higher

479

than in MCF-7 cells (1.37 times). The AAS results corresponded to those from Prussian

480

blue staining, confirming that BMW-HAF could specifically recognize HER2 and

481

enhance the internalization of magnetosomes by cancer cells expressing HER2.

482 483

Figure 4. BMW-HAF internalization by cancer cells. (a) Prussian blue staining of irons in the cancer

484

cells. BMW-HAF and BMW-THAF were incubated with different cancer cells for 6 h, the irons of

485

which were evaluated using Prussian blue staining. (b) Quantification of irons in the cancer cells

486

through atomic absorption spectrometry. The cancer cells were treated by BMW-HAF and

487

BMW-THAF, respectively, for 1 h, 3 h, 6 h and 12 h. The cells were collected at different time points, 25


Page 26 of 44

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488

and their irons and total proteins were quantified using atomic absorption spectrometry and Bradford

489

method, respectively. The internalized irons by cells were evaluated by dividing protein quantity in

490

the corresponding cells. *, P < 0.05; **, P < 0.01; The data represents the mean ± SD.

491 492

Due to NIFR properties of the bacterial nanoparticles, we could easily quantify the

493

ability of BMW-HAF to target HER2 using flow cytometry. As we found using Prussian

494

blue staining and AAS tests, the mean fluorescence of cells treated with BMW-HAF was

495

the same as HER2-negative MDA-MB-468 cells treated with BMW-THAF (Figure 5a).

496

However, a greater concentration of BMW-HAF/ BMW-THAF was taken up by the

497

cancer cells with extended exposure time. In addition, cell lines expressing HER2

498

(MCF-7 and SK-BR-3) exhibited higher cellular uptake of BMW-HAF than

499

BMW-THAF at 1 h, 3 h and 6 h. However, the fluorescence differences between

500

BMW-HAF and BMW-THAF uptake in SK-BR-3 cells were significantly higher than in

501

MCF-7 cells during the experiment. Fluorescence analysis revealed that the concentration

502

of internalized BMW-HAF in SK-BR-3 cells was 1.87 times higher than that in MCF-7

503

cells, which is consistent with the AAS analysis.

504

Confocal microscopy provided an intuitive way to observe the internalization of the

505

BMW-HAF by the cancer cells. Cells in all groups exhibited Cy5.5 (green color)

506

fluorescence after incubation with BMW-HAF or BMW-THAF for 2 h (Figure 5b). As

507

observed in the above experiments, the SK-BR-3 cells treated by BMW-HAF exhibited

508

greater fluorescence intensity than cells in the BMW-THAF group; HER2-negative

509

MDA-MB-468 cells demonstrated the same fluorescence intensity in the BMW-HAF and

510

BMW-THAF groups. These results demonstrated that BMW-HAF has high r2 relaxivity 26



511

and specificity for HER2 proteins, with potential as an enhanced contrast for in vivo MR

512

imaging of cancers overexpressing HER2.

513

514

Figure 5. Analysis of cellular taken up of the BMW-HAF through flow cytometry and confocal

515

microscopy. (a) Quantification of the internalized BMW-HAF by flow cytometry based on Cy5.5

516

fluorescence. Cancer cells were incubated with BMW-HAF or BMW-THAF for 1 h, 3 h and 6 h. At

517

specific time intervals, the cells were collected and analyzed through flow cytometry. The mean

518

fluorescence intensity of the cells was obtained by computing against the PBS group. The solid line

519

represents BMW-HAF while the dotted line stands for BMW-THAF. (b) Confocal imaging of

27


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520

BMW-HAF in the cancer cells. The cells were treated by BMW-HAF or BMW-THAF for 2 h and

521

imaged under confocal microscopy. Mean fluorescence intensity is abbreviated as MFI; The results

522

correspond to the mean ± SD; *, P < 0.05; **, P < 0.01.

523 524

To verify the ability of BMW-HAF nanoparticles to target HER2 in vivo, mice bearing

525

MDA-MB-468/MCF-7/SK-BR-3 tumors were used. The HER2 expression levels in the

526

tumor sections were first evaluated using immunochemistry. The SK-BR-3 tumor slides

527

clearly demonstrated positive HER2 expression (brown yellow color), while the MCF-7

528

and MDA-MB-468 tumors displayed very weak staining, which was almost the same as

529

in vitro HER2 detection (Figure 6a). This indicated that established tumor models could

530

be used to evaluate the ability of BMW-HAF to target HER2 because of the significant

531

differences of HER2 expression among the tumor models. Actually, the accumulation of

532

irons in SK-BR-3 tumors treated by BMW-HAF was two times higher than that of the

533

BMW-THAF group at 24 h after i.v. administration (Figure 6b). In contrast, there was no

534

significant difference between the two groups in the MDA-MB-468 and MCF-7 tumors.

535

This signified that BMW-HAF with the anti-HER2 affibody could selectively target

536

tumors that highly expressed HER2 (SK-BR-3). In addition, the iron concentration in

537

tumors in the BWM-HAF and BMW-THAF treated groups was twice that in the PBS

538

group in the MDA-MB-468 and MCF-7 tumors. These results indicate that both

539

BMW-HAF and BMW-THAF could enter tumors, even those tumor cells that do not

540

overexpress HER2. The non-selective accumulation of iron nanoparticles in tumors is

541

probably due to the enhanced permeability and retention (EPR) effects of solid tumors.40

542

Furthermore, BMW-HAF had a different in vivo distribution than BMW-THAF. 28



543

According to the iron content in the organs, more BMW-THAF accumulated in the livers

544

and spleens than BMW-HAF in the tumor model mice. However, the concentration of

545

BMW-HAF nanoparticles in the lungs was significantly higher than that of BMW-THAF

546

in the tumor models. The truncated affibodies in BMW-THAF are lacking 30 amino

547

acids, compared to the anti-HER2 affibody on BMW-HAF, which may make them

548

exhibit a different in vivo distribution. Therefore, the in vivo distribution should be

549

carefully considered when BMW is modified to obtain additional functionality. To

550

intuitively observe the in vivo distribution of iron particles, Prussian blue staining was

551

employed. As shown in the Figures 6c and S3, the iron nanoparticles (blue points

552

indicated by red arrows) were clearly observed in tumor sections after 24 h of treatment

553

by BMW-HAF or BMW-THAF. Also, the constructed magnetosomes were partly

554

retained in the livers, spleens, lungs, hearts and kidneys (Figure S4 and S5). Contrarily,

555

no blue stain was observed in the organ sections of the group treated with PBS. Prussian

556

blue staining also demonstrated that there were more irons in SK-BR-3 tumors treated

557

with BMW-HAF than BMW-THAF, which was similar to the in vivo AAS results.

558

Moreover, iron was localized in the viable region of tumors and also in the necrotic

559

tissue, especially in BMW-HAF group (Figure S3). The uniform distribution of

560

BMW-HAF in the tumors provides a solid foundation for MR imaging of the breast

561

cancers to define boundaries.

29


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562

563

Figure 6. HER2 expression in the tumors and in vivo distribution of BMW-HAF. (a) HER2

564

expression in different tumor models was evaluated using immunochemistry. Every group contains

565

three mice. (b) In vivo distribution of BMW-HAF. The irons in the tumors, livers, spleens, hearts,

566

lungs and kidneys were quantified using atomic absorption spectrometry 24 h after i.v. injection with

567

BMW-HAF or BMW-THAF. Three mice in each group were analyzed. (c) Distribution of

568

BMW-HAF in tumors (three mice in every group). 24 h after i.v. administration of BMW-HAF or

569

BMW-THAF, the irons in the tumor sections were stained through using Prussian blue staining. The

570

data stands for the mean ± SD; *, P < 0.05; **, P < 0.01.

571 572

The high r2 relaxivity and HER2-targeting ability of BMW-HAF demonstrated its

573

potential as a contrast agent for MR imaging of breast cancers overexpressing HER2. 30



574

Therefore, BALB/c mice with a single SK-BR-3 tumor or double tumors containing

575

MDA-MB-468 and SK-BR-3 were used to evaluate the effects of BMW-HAF on the

576

sensitivity of MR imaging (Figure 7a). The results of AAS and Prussian blue staining

577

revealed that the engineered magnetosomes could be retained in the tumors for 24 h

578

(Figure 6b and c). Therefore, the mice were imaged 3 h and 24 h after i.v. administration

579

of PBS or the modified magnetosomes (BMW-HAF or BMW-THAF). Upon comparison

580

with the PBS group, BMW-HAF and BMW-THAF obviously increased the sensitivity of

581

T2 MR imaging in both the MDA-MB-468 and SK-BR-3 tumors (Figure 7b). Both

582

BMW-HAF and BMW-THAF darkened the tumor regions compared with the pre-tumors

583

after 24 h treatment. However, SK-BR-3 tumors exhibited a deeper color than

584

MDA-MB-468 tumors after treatment by BMW-HAF; mice treated with BMW-THAF

585

did not exhibit the same phenomenon. This was further confirmed by measuring

586

contrast-noise ratio （ CNR ） of tumor regions. For example, CNR values of

587

BMW-THAF treated SK-BR-3 and MDA-MB-468 tumors only reached 0.8 and 0.77 of

588

the pre-tumors, respectively. In the BMW-HAF treated group, the CNR values in

589

SK-BR-3 tumors was only 0.64 of pre-tumors, which is lower than MDA-MB-468

590

tumors (0.74) after 24 h treatment. This should be due to that anti-HER2 affibody

591

decorated on magnetosomes (BMW-HAF) enhanced the internalization of the bacterial

592

nanoparticles by SK-BR-3 tumors with high HER2 expression, and the subsequent

593

accumulation in the tumors greatly increased the sensitivity of T2 MR imaging.23-24 To

594

determine how long after i.v. administration the engineered magnetosomes could enter

595

tumors, T2 MR images was captured 3 h after i.v. administration of BMW-HAF or

596

BMW-THAF. Figure 7c demonstrates that BMW-HAF displayed enhanced T2 MR 31


Page 32 of 44

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597

signals in the SK-BR-3 tumors at the defined time point. The CNR values of the

598

BMW-HAF treated SK-BR-3 tumors were only 0.76 of the pre-tumors, which exhibited

599

significant differences. However, the tumors treated with BMW-THAF did not show the

600

phenomenon. This further confirmed the selective targeting ability of BMW-HAF against

601

the tumors overexpressing HER2. Taking into account the high r2 relaxivity and ability of

602

BMW-HAF to target tumors overexpressing HER2, it has the potential to be used as a

603

clinical contrast agent for T2 MR imaging in cancer diagnosis and defining the tumor

604

boundaries during surgical treatment.

32



33

605


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606

Figure 7. BMW-HAF as contrast agent for enhancing MR imaging. (a) Schematic illustration of

607

BMW-HAF as contrast agent for in vivo MR imaging of tumors overexpressing HER2. (b) MR

608

imaging of tumors using BMW-HAF and BMW-THAF as contrast agents and contrast-noise ratio

609

(CNR) of tumor regions. Mice (n = 3) bearing SK-BR-3 tumors or double tumors (SK-BR-3 and

610

MDA-MB-468) were imaged before i.v. administration of PBS, BMW-HAF or BMW-TAF, and the

611

T2 MR signals were captured again after 24 h. (c) BMW-HAF as contrast agent enhances MR

612

imaging of HER2-overexpressed breast tumors in a short time. The mice (n = 3) were imaged

613

pre-treatment, and the MR signals were recorded at 3 h after i.v. injection of BMW-HAF or

614

BMW-THAF. The values on the CNR histogram stand for the ratio of the CNR values of BMW-HAF

615

or BMW-THAF treated tumors to the pre-tumors. The CNR data represents the mean ± SD; The

616

tumor location was marked with red circle; *, P < 0.05; **, P < 0.01.

617 618

4. CONCLUSION

619

Herein, we fabricated an HER2-targeting contrast agent to enhance MR imaging using

620

bacterium-derived magnetosomes and antibody-mimicking anti-HER2 affibody. The total

621

proteins in membrane bilayers of the magnetosomes were carefully removed by SDS

622

detergent to make spaces for the foreign proteins and lower magnetosomal

623

immunogenicity. The MamC protein, as the primary anchor protein of lipid bilayers on

624

magnetosomes, was co-expressed with the anti-HER2 affibody for constructing

625

recombinant protein of MamC-HAF. Due to the anchor activity of the MamC, many

626

MamC-HAF proteins were inserted into the lipid bilayers of the magnetosomes to form

627

HER2-targeting magnetic nanoparticles (BMW-HAF) through low-frequency sonication. 34



628

Natural magnetosomes modified with the HER2-targeting affibody exhibited high r2

629

relaxivity, better dispersion and good biocompatibility. Furthermore, the bacterial

630

nanoparticles decorated with the anti-HER2 affibody could selectively target breast

631

cancer cells overexpressing HER2 both in vitro and in vivo, and they significantly

632

enhanced T2 MR imaging of orthotopic breast cancers, especially cancers highly

633

expressing HER2. This study developed a new method for constructing a tumor-targeting

634

T2 MR contrast based on magnetosomes, which will greatly advance the development of

635

naturally magnetic nanoparticles and noninvasive cancer imaging.

636

ASSOCIATED CONTENT

637

Supporting Information

638

Gene sequence, fluorescence intensity, Prussian blue staining of in vitro and in vivo

639

AUTHOR INFORMATION

640

Corresponding Authors

641

*Email: [email protected]. Fax: +86 25 8480 4659. Tel: +86 25 8086 0185

642

*E-mail: [email protected].

643

*E-mail: [email protected]

644

Present Addresses

645

†Department

646

Jiangsu, P.R. China

647

Author Contributions

of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing, 210002

35


Page 36 of 44

Page 37 of 44 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


648

The manuscript was prepared through contributions of all authors. All authors have given

649

approval to the final version of the manuscript.

650

Notes

651

The authors declare no competing financial interest.

652

ACKNOWLEDGEMENTS

653

This investigation was supported by the National Key Basic Research Program of the

654

PRC (2014CB744504 and 2014CB744501), the National Natural Science Foundation of

655

China (81530054, 81501538, 81401469), the China Postdoctoral Science Foundation

656

(2016M593035, 2018T111163) and the Jiangsu Planned Projects for Postdoctoral

657

Research Funds (1501122c).

658

We appreciate the advice and technical supporting from Prof. Ying Li and Wei Jiang

659

(State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China

660

Agricultural University) for the study.

661

Abbreviations

662

Alanine aminotransferase (ALT); alkaline phosphatase (ALP); aspartate aminotransferase

663

(AST); atomic absorption spectrometry (AAS); blood urea nitrogen (BUN); bovine serum

664

albumin (BSA); creatinine (CR); Escherichia coli (E.coli); hematoxylin-eosin (H&E);

665

horseradish Peroxidase (HRP); human embryonic kidney cells (293T); human epidermal

666

growth factor receptor-2 (HER2); intravenous (i.v.); isopropyl β-D-thiogalactoside

667

(IPTG); lysogeny broth (LB); maleimide derivative cyanine dyes (Cy5.5); magnetic

668

resonance imaging (MRI); near-infrared fluorescence (NIFR); neodymium-iron-boron

669

(Nd-Fe-B); phosphate buffer saline (PBS); polyethylene glycol (PEG); polyvinylidene 36



Page 38 of 44

670

fluoride (PVDF); red blood cells (RBCs)

671

sodium

672

superparamagnetic iron oxide nanoparticles (SPIOs); supersensitive magnetic resonance

673

(MR); tris(2-carboxyethyl)phosphine (TCEP).

674

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