Bacterial Magnetosomes Release Iron Ions and Induce Regulation of Iron Homeostasis in Endothelial Cells.

Magnetosomes (MAGs) extracted from magnetotactic bacteria are well-defined membrane-enveloped single-domain magnetic nanoparticles. Due to their superior magnetic and structural properties, MAGs constitute potential materials that can be manipulated via genetic and chemical engineering for use in biomedical and biotechnological applications. However, the long-term effects exerted by MAGs on cells are of concern in the context of in vivo applications. Meanwhile, it remains relatively unclear which mechanisms are employed by cells to process and degrade MAGs. Hence, a better understanding of MAGs' degradation and fundamental signal modulations occurring throughout this process is essential. In the current study, we investigated the potential actions of MAGs on endothelial cells over a 10-day period. MAGs were retained in cells and found to gradually gather in the lysosome-like vesicles. Meanwhile, iron-ion release was observed. Proteomics further revealed a potential cellular mechanism underlying MAGs degradation, in which a group of proteins associated with vesicle biogenesis, and lysosomal enzymes, which participate in protein hydrolysis and lipid degradation, were rapidly upregulated. Moreover, the released iron triggered the regulation of the iron metabolic profiles. However, given that the levels of cell oxidative damage were relatively stable, the released iron ions were handled by iron metabolic profiles and incorporated into normal metabolic routes. These results provide insights into the cell response to MAGs degradation that may improve their in vivo applications.

In vitro magnetosome remineralization for silver-magnetite hybrid magnetosome biosynthesis and used for healing of the infected wound.

BACKGROUND: Magnetosomes (BMPs) are organelles of magnetotactic bacteria (MTB) that are responsible for mineralizing iron to form magnetite. In addition, BMP is an ideal biomaterial that is widely used in bio- and nano-technological applications, such as drug delivery, tumor detection and therapy, and immunodetection. The use of BMPs to create multifunctional nanocomposites would further expand the range of their applications. RESULTS: In this study, we firstly demonstrate that the extracted BMP can remineralize in vitro when it is exposed to AgNO3 solution, the silver ions (Ag+) were transported into the BMP biomembrane (MM) and mineralized into a silver crystal on one crystal plane of Fe3O4. Resulting in the rapid synthesis of an Ag-Fe3O4 hybrid BMP (BMP-Ag). The synergy between the biomembrane, Fe3O4 crystal, and unmineralized iron enabled the remineralization of BMPs at an Ag+ concentration ≥ 1.0 mg mL-1. The BMP-Ag displayed good biocompatibility and antibacterial activity. At a concentration of 2.0 mg/mL, the BMP-Ag and biomembrane removed Ag-Fe3O4 NPs inhibited the growth of gram-negative and gram-positive bacteria. Thus using BMP-Ag as a wound dressing can effectively enhance the contraction of infected wounds. CONCLUSIONS: This study represents the first successful attempt to remineralize organelles ex vivo, realizing the biosynthesis of hybrid BMP and providing an important advancement in the synthesis technology of multifunctional biological nanocomposites.

Self-Confirming Magnetosomes for Tumor-Targeted T1 /T2 Dual-Mode MRI and MRI-Guided Photothermal Therapy.

Nanomaterials as T1 /T2 dual-mode magnetic resonance imaging (MRI) contrast agents have great potential in improving the accuracy of tumor diagnosis. Applications of such materials, however, are limited by the complicated chemical synthesis process and potential biosafety issues. In this study, the biosynthesis of manganese (Mn)-doped magnetosomes (MagMn) that not only can be used in T1 /T2 dual-mode MR imaging with self-confirmation for tumor detection, but also improve the photothermal conversion efficiency for MRI-guided photothermal therapy (PTT) is reported. The MagMn nanoparticles (NPs) are naturally produced through the biomineralization of magnetotactic bacteria by doping Mn into the ferromagnetic iron oxide crystals. In vitro and in vivo studies demonstrated that targeting peptides functionalized MagMn enhanced both T1 and T2 MRI signals in tumor tissue and significantly inhibited tumor growth by the further MRI-guided PTT. It is envisioned that the biosynthesized multifunctional MagMn nanoplatform may serve as a potential theranostic agent for cancer diagnosis and treatment.

Strategy for Avoiding Protein Corona Inhibition of Targeted Drug Delivery by Linking Recombinant Affibody Scaffold to Magnetosomes.

PURPOSE: Nanoparticles (NPs) decorated with functional ligands are promising candidates for cancer diagnosis and treatment. However, numerous studies have shown that chemically coupled targeting moieties on NPs lose their targeting capability in the biological milieu because they are shielded or covered by a "protein corona". Herein, we construct a functional magnetosome that recognizes and targets cancer cells even in the presence of protein corona. METHODS: Magnetosomes (BMPs) were extracted from magnetotactic bacteria, M. gryphiswaldense (MSR-1), and decorated with trastuzumab (TZ) via affibody (RA) and glutaraldehyde (GA). The engineered BMPs are referred to as BMP-RA-TZ and BMP-GA-TZ. Their capacities to combine HER2 were detected by ELISA, the quantity of plasma corona proteins was analyzed using LC-MS. The efficiencies of targeting SK-BR-3 were demonstrated by confocal laser scanning microscopy and flow cytometry. RESULTS: Both engineered BMPs contain up to ~0.2 mg TZ per mg of BMP, while the quantity of HER2 binding to BMP-RA-TZ is three times higher than that binding to BMP-GA-TZ. After incubation with normal human plasma or IgG-supplemented plasma, GA-TZ-containing BMPs have larger hydrated radii and more surface proteins in comparison with RA-TZ-containing BMPs. The TZ-containing BMPs all can be targeted to and internalized in the HER2-overexpressing breast cancer cell line SK-BR-3; however, their targeting efficiencies vary considerably: 50-75% for RA-TZ-containing BMPs and 9-19% for GA-TZ-containing BMPs. BMPs were incubated with plasma (100%) and cancer cells to simulate human in vivo environment. In this milieu, BMP-RA-TZ uptake efficiency of SK-BR-3 reaches nearly 80% (slightly lower than for direct interaction with BMP-RA-TZ), whereas the BMP-GA-TZ uptake efficiency is <17%. CONCLUSION: Application of the RA scaffold promotes and orients the arrangement of targeting ligands and reduces the shielding effect of corona proteins. This strategy improves the targeting capability and drug delivery of NP in a simulated in vivo milieu.

Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1.

BACKGROUND: Magnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil. RESULTS: Three recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78-101%. CONCLUSIONS: The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

Drug-internalized bacterial swimmers for magnetically manipulable tumor-targeted drug delivery.

Tumor-targeted drug carriers are becoming attractive for precise drug delivery in anti-tumor therapy. However, a lot of the reported drug delivery systems are complicatedly designed and their destiny in vivo is beyond our control, which limited their clinical applications. Hence, it is urgently needed to develop spatio-manipulable self-propelled nanosystems for drug delivery in a facile way. Here, we have successfully constructed drug-internalized bacterial swimmers, whose movement can be manually controlled by an external magnetic field (MF). We demonstrate that the swimmers maintain the mobility to align and swim along MF lines. Further studies reveal that the doxorubicin (DOX-) internalized bacterial swimmers are able to navigate toward tumor sites under the guidance of MF, rendering enhanced anti-tumor efficacy compared with that of dead ones and free DOX. Therefore, the MF-guided bacterial swimmers hold great promise for spatio-manipulable drug delivery in precision medicine.

Novel Protein Mg2046 Regulates Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1 by Modulating a Proper Redox Status.

Magnetotactic bacteria (MTB) are a large, polyphyletic group of aquatic microorganisms capable of absorbing large amounts of iron and synthesizing intercellular nano-scaled nanoparticles termed magnetosomes. In our previous transcriptomic studies, we discovered that a novel gene (MGMSRv2_2046, termed as mg2046) in Magnetospirillum gryphiswaldense strain MSR-1 was significantly up-regulated during the period of magnetosome synthesis. In the present study, we constructed a MSR-1 mutant strain with deletion of mg2046 (termed Δmg2046) in order to evaluate the role of this gene in cell physiological status and magnetosome formation process. In comparison with wild-type MSR-1, Δmg2046 showed similar cell growth, but much lower cell magnetic response, smaller number and size of magnetosomes, and reduced iron absorption ability. mg2046 deletion evidently disrupted iron uptake, and redox equilibrium, and strongly inhibited transcription of dissimilatory denitrification pathway genes. Our experimental findings, taken together with results of gene homology analysis, indicate that Mg2046 acts as a positive regulator in MSR-1 under microaerobic conditions, responding to hypoxia signals and participating in regulation of oxygen metabolism, in part as a co-regulator of dissimilatory denitrification pathway with oxygen sensor MgFnr (MGMSRv2_2946, termed as Mg2946). Mg2046 is clearly involved in coupled regulation of cellular oxygen, iron and nitrogen metabolism under micro-aerobic or anaerobic conditions. Our findings help explain how MSR-1 cells initiate dissimilatory denitrification pathway and overcome energy deficiency under microaerobic conditions, and have broader implications regarding bacterial survival and energy metabolism strategies under hypoxia.

Regioselective C-H Amidation of (Alkyl)arenes by Iron(II) Catalysis.

A nondirected amidation reaction of aromatic C-H bond was developed under iron(II) catalysis, using sulfonyl azides as the nitrogen source. The reaction displayed a broad substrate scope and good regioselectivities in the aspects of aromatic ring vs alkyl chain and different aromatic position of (alkyl)arenes. This method provided a new protocol for the synthesis of some aromatic amines, which were hard to achieve in a previous report.

Strong and oriented conjugation of nanobodies onto magnetosomes for the development of a rapid immunomagnetic assay for the environmental detection of tetrabromobisphenol-A.

Variable domain of heavy chain antibody (nanobody, Nb) derived from camelids is an efficient reagent in monitoring environmental contaminants. Oriented conjugates of Nbs and bacterial magnetic particles (BMPs) provide new tools for the high-throughput immunoassay techniques. An anti-tetrabromobisphenol-A (TBBPA) Nb genetically integrated with an extra cysteine residue at the C terminus was immobilized onto BMPs enclosed within the protein membrane, using a heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithiol) propionate, to form a solid BMP-Nb complex. A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) based on the combination of BMP-Nb and T5-horseradish peroxidase was developed for the analysis of TBBPA, with a total assay time of 30 min and a half-maximum signal inhibition concentration (IC50) of 1.04 ng/mL in PBS (pH 10, 10% methanol and 0.137 moL/L NaCl). This assay can even be performed in 100% methanol, with an IC50 value of 44.3 ng/mL. This assay showed quantitative recoveries of TBBPA from spiked canal water (114-124%) and sediment (109-113%) samples at 1.0-10 ng/mL (or ng/g (dw)). TBBPA residues determined by this assay in real canal water samples were below the limit of detection (LOD) and in real sediments were between

Physiological characteristics of Magnetospirillum gryphiswaldense MSR-1 that control cell growth under high-iron and low-oxygen conditions.

Magnetosome formation by Magnetospirillum gryphiswaldense MSR-1 is dependent on iron and oxygen levels. We used transcriptome to evaluate transcriptional profiles of magnetic and non-magnetic MSR-1 cells cultured under high-iron and low-iron conditions. A total of 80 differentially expressed genes (DEGs) were identified, including 53 upregulated and 27 downregulated under high-iron condition. These DEGs belonged to the functional categories of biological regulation, oxidation-reduction process, and ion binding and transport, and were involved in sulfur metabolism and cysteine/methionine metabolism. Comparison with our previous results from transcriptome data under oxygen-controlled conditions indicated that transcription of mam or mms was not regulated by oxygen or iron signals. 17 common DEGs in iron- and oxygen-transcriptomes were involved in energy production, iron transport, and iron metabolism. Some unknown-function DEGs participate in iron transport and metabolism, and some are potential biomarkers for identification of Magnetospirillum strains. IrrA and IrrB regulate iron transport in response to low-oxygen and high-iron signals, respectively. Six transcription factors were predicted to regulate DEGs. Fur and Crp particularly co-regulate DEGs in response to changes in iron or oxygen levels, in a proposed joint regulatory network of DEGs. Our findings provide new insights into biomineralization processes under high- vs. low-iron conditions in magnetotactic bacteria.

Tumor detection using magnetosome nanoparticles functionalized with a newly screened EGFR/HER2 targeting peptide.

A novel peptide (P75) targeting EGFR and HER2 is successfully screened from a one-bead-one-compound (OBOC) library containing approximately 2 × 105 peptides built with the aid of computational simulation. In vitro and in vivo analyses show that P75 binds to human epithelial growth factor receptor (EGFR) with nanomolar affinity and to epithelial growth factor receptor-2 (HER2) with a lower affinity but comparable to other reported peptides. The peptide is used to modify the surface of magnetosome nanoparticles (NPs) for targeted magnetic resonance imaging (MRI). In vitro and in vivo fluorescence imaging results suggest peptide P75 modified magnetosomes (Mag-P75) specifically bind to MDA-MB-468 and SKBR3 cells as well as xenograft tumors with surprisingly low accumulation in other organs including liver and kidney. In vivo T2-weighted MR imaging studies of the xenograft tumors from SKBR3 and MDA-MB-468 cells show obviously negative contrast enhancement. The high affinity and specificity of P75 to EGFR and HER2 positive tumors, together with the success of peptide functionalized magnetosome NPs for targeted MRI demonstrate the potential of this peptide being used in the EGFR and HER2 positive tumors diagnosis and therapy.

Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria (MTB) synthesize unique organelles termed "magnetosomes," which are membrane-enclosed structures containing crystals of magnetite or greigite. Magnetosomes form a chain around MamK cytoskeletal filaments and provide the basis for the ability of MTB to navigate along geomagnetic field lines in order to find optimal microaerobic habitats. Genomes of species of the MTB genus Magnetospirillum, in addition to a gene encoding the tubulin-like FtsZ protein (involved in cell division), contain a second gene termed "ftsZ-like," whose function is unknown. In the present study, we found that the ftsZ-like gene of Magnetospirillum gryphiswaldense strain MSR-1 belongs to a 4.9-kb mamXY polycistronic transcription unit. We then purified the recombinant FtsZ-like protein to homogeneity. The FtsZ-like protein efficiently hydrolyzed ATP and GTP, with ATPase and GTPase activity levels of 2.17 and 5.56 mumol phosphorus per mol protein per min, respectively. The FtsZ-like protein underwent GTP-dependent polymerization into long filamentous bundles in vitro. To determine the role of the ftsZ-like gene, we constructed a ftsZ-like mutant (DeltaftsZ-like mutant) and its complementation strain (DeltaftsZ-like_C strain). Growth of DeltaftsZ-like cells was similar to that of the wild type, indicating that the DeltaftsZ-like gene is not involved in cell division. Transmission electron microscopic observations indicated that the DeltaftsZ-like cells, in comparison to wild-type cells, produced smaller magnetosomes, with poorly defined morphology and irregular alignment, including large gaps. Magnetic analyses showed that DeltaftsZ-like produced mainly superparamagnetic (SP) magnetite particles, whereas wild-type and DeltaftsZ-like_C cells produced mainly single-domain (SD) particles. Our findings suggest that the FtsZ-like protein is required for synthesis of SD particles and magnetosomes in M. gryphiswaldense.

A mutation upstream of an ATPase gene significantly increases magnetosome production in Magnetospirillum gryphiswaldense.

A mutant of Magnetospirillum gryphiswaldense, NPHB, was obtained from a conjugation experiment. An aberrant recombination occurred between a putative elongation factor-G gene (fus-like) of the bacterial chromosome and the chloramphenicol resistant gene (cat) of a suicide vector, pSUP202. Complementary experiments and transcription analysis of genes around the recombinant site showed that the cat promoter enhanced the expression of adenosine triphosphatase gene downstream. Adenosine triphosphate hydrolyzing activity in NPHB was 35% higher than in the wild-type strain (M. gryphiswaldense MSR-1). NPHB accumulated 71% less poly-beta-hydroxybutyrate and consumed 56% more oxygen and 40% more lactate than MSR-1. The magnetosome content of NPHB was 69% higher than MSR-1 in flask culture. NPHB cultured in a 7.5-L bioreactor gave a maximum yield of 58.4 +/- 6.4 mg magnetosomes per liter.

In vitro and in vivo antitumor effects of doxorubicin loaded with bacterial magnetosomes (DBMs) on H22 cells: the magnetic bio-nanoparticles as drug carriers.

Hepatocellular carcinoma (HCC) is the most common form of cancer although effective therapeutic strategy especially targeted therapy is lacking. We recently employed bacterial magnetosomes (BMs) as the magnetic-targeted drug carrier and found an antitumor effect of doxorubicin (DOX)-loaded BMs (DBMs) in EMT-6 and HL60 cell lines. The aim of this study was to evaluate the in vitro and in vivo anti-neoplastic effects of DBMs on hepatic cancer. DBMs, DOX and BMs displayed tumor suppression rates of 86.8%, 78.6% and 4.3%, respectively, in H22 cell-bearing mice. The mortality rates following administration of DBMs, DOX and BMs were 20%, 80% and 0%, respectively. Pathological examination of hearts and tumors revealed that both DBMs and DOX effectively inhibited tumor growth although DBMs displayed a much lower cardiac toxicity compared with DOX. The DBMs were cytotoxic to H22 cells manifested as inhibition of cell proliferation and c-myc expression, consistent with DOX. The IC(50) of DOX, DBMs and BMs in target cells were 5.309 +/- 0.010, 4.652 +/- 0.256 and 22.106 +/- 3.330 microg/ml, respectively. Our data revealed both in vitro and in vivo antitumor property of DBMs similar to that of DOX. More importantly, the adverse cardiac toxicity was significantly reduced in DBMs compared with DOX. Collectively, our study suggests the therapeutic potential of DBMs in target-therapy against liver cancer.