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 TZcontaining 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.

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. Graphical abstract

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.

Nitrate ion (NO3−) in wastewater is a major cause of pollution in aquatic environments worldwide. Magnetospirillum gryphiswaldense (MSR-1) has a complete dissimilatory denitrification pathway, converts NO3− in water into nitrogen (N2) and simultaneously removes ammonium ions (NH4+). We investigated and confirmed direct effects of regulatory protein factors Mg2046 and MgFnr on MSR-1 denitrification pathway by EMSAs and ChIP-qPCR assays. Corresponding mutant strains were constructed. Denitrification efficiency in synthetic wastewater medium during a 12-h cell growth period was significantly higher for mutant strain Δmgfnr (0.456 mmol·L−1·h−1) than for wild-type (0.362 mmol·L−1·h−1). Presence of magnetic particles (magnetosomes) in MSR-1 greatly facilitates collection and isolation of bacterial cells (and activated sludge) by addition of a magnetic field. The easy separation of magnetotactic bacteria, such as MSR-1 and Δmgfnr, from wastewater using magnetic fields is a unique feature that makes them promising candidates for practical application in wastewater treatment and sludge pretreatment.

Biosynthesis provides a critical way to deal with global sustainability issues and has recently drawn increased attention. However, modifying biosynthesized magnetic nanoparticles by extraction is challenging, limiting its applications. Magnetotactic bacteria (MTB) synthesize single-domain magnetite nanocrystals in their organelles, magnetosomes (BMPs), which are excellent biomaterials that can be biologically modified by genetic engineering. Therefore, this study successfully constructed in vivo biotinylated BMPs in the MTB Magnetospirillum gryphiswaldense by fusing biotin carboxyl carrier protein (BCCP) with membrane protein MamF of BMPs. The engineered strain (MSR−∆F−BF) grew well and synthesized small-sized (20 ± 4.5 nm) BMPs and were cultured in a 42 L fermenter; the yield (dry weight) of cells and BMPs reached 8.14 g/L and 134.44 mg/L, respectively, approximately three-fold more than previously reported engineered strains and BMPs. The genetically engineered BMPs (BMP−∆F−BF) were successfully linked with streptavidin or streptavidin-labelled horseradish peroxidase and displayed better storage stability compared with chemically constructed biotinylated BMPs. This study systematically demonstrated the biosynthesis of engineered magnetic nanoparticles, including its construction, characterization, and production and detection based on MTB. Our findings provide insights into biomanufacturing multiple functional magnetic nanomaterials.