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Biological and Medical Applications of Materials and Interfaces
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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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%
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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
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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
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sections, and they were incubated for 20 min at room temperature. Afterwards, the
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samples were washed using Millipore water for 5 min and immersed in nuclear fast red
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solution for 10 min. Further, the cells and sections were washed using water for 5 s and
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processed following the general protocol of dehydration and transparency. The coverslip
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with the cells or tissue sections were mounted using neutral balsam. The cellular uptake
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and tissue distribution of BMW-HAF were captured under an Olympus microscope 12
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(Olympus IX71, Tokyo, Japan).
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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
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factor, 0.5 μg/mL of hydrocortisone, 100 ng/mL of cholera toxin and 10 μg/mL of insulin
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at 37 °C in a cell incubator; the culture condition of human embryonic kidney cells
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(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
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96-well-plate. After overnight, the media were replaced with the fresh media containing
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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),
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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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every week. Body weight was documented every 3 days for one month. Simultaneously,
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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.
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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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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
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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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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
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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
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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
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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
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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
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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
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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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