Research Article: Uptake and persistence of bacterial magnetite magnetosomes in a mammalian cell line: Implications for medical and biotechnological applications

Date Published: April 23, 2019

Publisher: Public Library of Science

Author(s): Jefferson Cypriano, Jacques Werckmann, Gabriele Vargas, Adriana Lopes dos Santos, Karen T. Silva, Pedro Leão, Fernando P. Almeida, Dennis A. Bazylinski, Marcos Farina, Ulysses Lins, Fernanda Abreu, Yogendra Kumar Mishra.


Magnetotactic bacteria biomineralize intracellular magnetic nanocrystals surrounded by a lipid bilayer called magnetosomes. Due to their unique characteristics, magnetite magnetosomes are promising tools in Biomedicine. However, the uptake, persistence, and accumulation of magnetosomes within mammalian cells have not been well studied. Here, the endocytic pathway of magnetite magnetosomes and their effects on human cervix epithelial (HeLa) cells were studied by electron microscopy and high spatial resolution nano-analysis techniques. Transmission electron microscopy of HeLa cells after incubation with purified magnetosomes showed the presence of magnetic nanoparticles inside or outside endosomes within the cell, which suggests different modes of internalization, and that these structures persisted beyond 120 h after internalization. High-resolution transmission electron microscopy and electron energy loss spectra of internalized magnetosome crystals showed no structural or chemical changes in these structures. Although crystal morphology was preserved, iron oxide crystalline particles of approximately 5 nm near internalized magnetosomes suggests that minor degradation of the original mineral structures might occur. Cytotoxicity and microscopy analysis showed that magnetosomes did not result in any apparent effect on HeLa cells viability or morphology. Based on our results, magnetosomes have significant biocompatibility with mammalian cells and thus have great potential in medical, biotechnological applications.

Partial Text

Nano-sized magnetic particles that functionally integrate into cells and subcellular structures are versatile tools for monitoring cell position and function in vivo, as well as for the development of drug-delivery systems [1]. Magnetosomes biomineralized by magnetotactic bacteria (MTB) possess many attributes that make them suitable for biotechnological applications [1]. Among these attributes are the high chemical purity of the magnetic mineral, well-defined crystals morphologies, narrow size range in nanometric scale, dimensions consistent with single magnetic domains and the presence of a phospholipid bilayer surrounding each crystal. Therefore, studies involving cell-magnetosomes interactions are potentially relevant for nanobiotechnology and medicine [2].

TEM observation of purified magnetite magnetosomes from M. blakemorei strain MV-1 showed the presence of the magnetosome membrane surrounding each crystal (Fig 1A) confirming the homogeneity and potential reproducibility of the sample. After the endocytosis assay, phase contrast images of HeLa cells in cultures showed confluent growth on the glass surface on both control (not incubated with magnetosomes) and treated cultures even after 120 h of incubation with purified magnetosomes (Fig 1B and 1C). Treated cultures displayed dark areas corresponding to aggregates of magnetosomes; even in these areas, no inhibition of growth was detected. The cytotoxicity assay showed that magnetosomes did not have any toxic effect on cells in all incubation times analyzed (Fig 1D). Magnetosome cytotoxicity have already been evaluated against a few cell lines. In these studies, H22 (mouse hepatocarcinoma cells), HL60 (human acute promyelocytic leukemia cells), EMT-6 (mouse mammary cancer cells) and ARPE-19 cells (human retinal pigment epithelium cells) were treated with 9 μg/ml of magnetite magnetosomes for the three first cell lineages and 10, 50 and 100 μg/ml for ARPE-19 cells and no toxic effect was observed [11,12]. Qi et al. (2016) showed that when the same amounts of synthetic magnetic particles were added to ARPE-19 cells (10, 50 and 100 μg/ml), their viability decreased significantly after incubation [12]. This study also reported that cell viability rates were directly correlated to the concentration of the synthetic nanoparticles as well as the incubation period. Similar results were obtained for 661W cells (mouse transformed cell line) [12]. Therefore, cell death was directly related to an increase in the concentration of synthetic magnetite nanoparticles incubated with cell cultures. On the other hand, the use of the same concentrations of magnetosomes promoted the growth of ARPE-19 cells [12]. For 661W cells, 10 μg/ml magnetosomes promoted cell growth during all incubation periods used in the study (24, 48 and 72 h) [12]. Therefore, our findings are consistent with these previous studies, indicating high biocompatibility between magnetosomes and mammalian cells even when high concentrations of magnetosomes were used. In addition to the non-deleterious effect on cells, the presence of a biological membrane involving the magnetosome also make these magnetic nanoparticles excellent tools in nanomedicine, because of the possibility of associating molecules to proteins embedded on this outer phospholipidic bilayer.

In this study, we evaluated the cytotoxicity and possible degradation of magnetosomes using HeLa cells as a model lineage. Our results showed that magnetosomes are biocompatible and are not oxidized or suffer morphological changes after internalization by cells. In contrast, studies using synthetic magnetic particles showed toxic effects over cells after 24 h of incubation [12]. The presence of fine crystalline iron-containing structures of approximately 5 nm near internalized magnetosomes might be an indication of a slow degradation or the detachment of small parts of the nanoparticles due to original defects (see Fig 3G). If true, this process would be a great advantage for the use of magnetite magnetosomes in many medical and biotechnological applications because it would guarantee nontoxic effects on the cell due to the derisory release of low concentrations of soluble iron over a long period. Moreover, the presence of a biological membrane in isolated magnetosomes and after some period in endocytic vesicles enlarges the range of possible applications of magnetosomes in the bioengineering area. Overall, this suggests that magnetite magnetosomes remain as stable structures in biological systems for long periods, which is also desirable for prolonged treatments.