Set-up of a pharmaceutical cell bank of Magnetospirillum gryphiswaldense MSR1 magnetotactic bacteria producing highly pure magnetosomes.

We report the successful fabrication of a pharmaceutical cellular bank (PCB) containing magnetotactic bacteria (MTB), which belong to the Magnetospirillum gryphiswaldense MSR1 species. To produce such PCB, we amplified MTB in a minimal growth medium essentially devoid of other heavy metals than iron and of CMR (Carcinogenic, mutagenic and reprotoxic) products. The PCB enabled to acclimate MTB to such minimal growth conditions and then to produce highly pure magnetosomes composed of more than 99.9% of iron. The qualification of the bank as a PCB relies first on a preserved identity of the MTB compared with the original strain, second on genetic bacterial stability observed over 100 generations or under cryo-preservation for 16 months, third on a high level of purity highlighted by an absence of contaminating microorganisms in the PCB. Furthermore, the PCB was prepared under high-cell load conditions (9.108 cells/mL), allowing large-scale bacterial amplification and magnetosome production. In the future, the PCB could therefore be considered for commercial as well as research orientated applications in nanomedicine. We describe for the first-time conditions for setting-up an effective pharmaceutical cellular bank preserving over time the ability of certain specific cells, i.e. Magnetospirillum gryphiswaldense MSR1 MTB, to produce nano-minerals, i.e. magnetosomes, within a pharmaceutical setting.

Non-pyrogenic highly pure magnetosomes for efficient hyperthermia treatment of prostate cancer.

We report the fabrication of highly pure magnetosomes that are synthesized by magnetotactic bacteria (MTB) using pharmaceutically compatible growth media, i.e., without compounds of animal origin (yeast extracts), carcinogenic, mutagenic, or toxic for reproduction (CMR) products, and other heavy metals than iron. To enable magnetosome medical applications, these growth media are reduced and amended compared with media commonly used to grow these bacteria. Furthermore, magnetosomes are made non-pyrogenic by being extracted from these micro-organisms and heated above 400 °C to remove and denature bacterial organic material and produce inorganic magnetosome minerals. To be stabilized, these minerals are further coated with citric acid to yield M-CA, leading to fully reconstructed chains of magnetosomes. The heating properties and anti-tumor activity of highly pure M-CA are then studied by bringing M-CA into contact with PC3-Luc tumor cells and by exposing such assembly to an alternating magnetic field (AMF) of 42 mT and 195 kHz during 30 min. While in the absence of AMF, M-CA are observed to be non-cytotoxic, they result in a 35% decrease in cell viability following AMF application. The treatment efficacy can be associated with a specific absorption rate (SAR) value of M-CA, which is relatively high in cellular environment, i.e., SARcell = 253 ± 11 W/gFe, while being lower than the M-CA SAR value measured in water, i.e., SARwater = 1025 ± 194 W/gFe, highlighting that a reduction in the Brownian contribution to the SAR value in cellular environment does not prevent efficient tumor cell destruction with these nanoparticles. KEY POINTS : • Highly pure magnetosomes were produced in pharmaceutically compatible growth media • Non-pyrogenic and stable magnetosomes were prepared for human injection • Magnetosomes efficiently destroyed prostate tumor cells in magnetic hyperthermia.

A Method for Producing Highly Pure Magnetosomes in Large Quantity for Medical Applications Using Magnetospirillum gryphiswaldense MSR-1 Magnetotactic Bacteria Amplified in Minimal Growth Media.

We report the synthesis in large quantity of highly pure magnetosomes for medical applications. For that, magnetosomes are produced by MSR-1 Magnetospirillum gryphiswaldense magnetotactic bacteria using minimal growth media devoid of uncharacterized and toxic products prohibited by pharmaceutical regulation, i.e., yeast extract, heavy metals different from iron, and carcinogenic, mutagenic and reprotoxic agents. This method follows two steps, during which bacteria are first pre-amplified without producing magnetosomes and are then fed with an iron source to synthesize magnetosomes, yielding, after 50 h of growth, an equivalent OD565 of ~8 and 10 mg of magnetosomes in iron per liter of growth media. Compared with magnetosomes produced in non-minimal growth media, those particles have lower concentrations in metals other than iron. Very significant reduction or disappearance in magnetosome composition of zinc, manganese, barium, and aluminum are observed. This new synthesis method paves the way towards the production of magnetosomes for medical applications.

Enhanced antitumor efficacy of biocompatible magnetosomes for the magnetic hyperthermia treatment of glioblastoma.

In this study, biologically synthesized iron oxide nanoparticles, called magnetosomes, are made fully biocompatible by removing potentially toxic organic bacterial residues such as endotoxins at magnetosome mineral core surfaces and by coating such surface with poly-L-lysine, leading to magnetosomes-poly-L-lysine (M-PLL). M-PLL antitumor efficacy is compared with that of chemically synthesized iron oxide nanoparticles (IONPs) currently used for magnetic hyperthermia. M-PLL and IONPs are tested for the treatment of glioblastoma, a dreadful cancer, in which intratumor nanoparticle administration is clinically relevant, using a mouse allograft model of murine glioma (GL-261 cell line). A magnetic hyperthermia treatment protocol is proposed, in which 25 µg in iron of nanoparticles per mm3 of tumor are administered and exposed to 11 to 15 magnetic sessions during which an alternating magnetic field of 198 kHz and 11 to 31 mT is applied for 30 minutes to attempt reaching temperatures of 43-46 °C. M-PLL are characterized by a larger specific absorption rate (SAR of 40 W/gFe compared to 26 W/gFe for IONPs as measured during the first magnetic session), a lower strength of the applied magnetic field required for reaching a target temperature of 43-46 °C (11 to 27 mT compared with 22 to 31 mT for IONPs), a lower number of mice re-administered (4 compared to 6 for IONPs), a longer residence time within tumours (5 days compared to 1 day for IONPs), and a less scattered distribution in the tumour. M-PLL lead to higher antitumor efficacy with full tumor disappearances achieved in 50% of mice compared to 20% for IONPs. This is ascribed to better ability of M-PLL, at equal iron concentrations, to maintain tumor temperatures at 43-46°C over a longer period of times.

Biocompatible coated magnetosome minerals with various organization and cellular interaction properties induce cytotoxicity towards RG-2 and GL-261 glioma cells in the presence of an alternating magnetic field.

BACKGROUND: Biologics magnetics nanoparticles, magnetosomes, attract attention because of their magnetic characteristics and potential applications. The aim of the present study was to develop and characterize novel magnetosomes, which were extracted from magnetotactic bacteria, purified to produce apyrogen magnetosome minerals, and then coated with Chitosan, Neridronate, or Polyethyleneimine. It yielded stable magnetosomes designated as M-Chi, M-Neri, and M-PEI, respectively. Nanoparticle biocompatibility was evaluated on mouse fibroblast cells (3T3), mouse glioblastoma cells (GL-261) and rat glioblastoma cells (RG-2). We also tested these nanoparticles for magnetic hyperthermia treatment of tumor in vitro on two tumor cell lines GL-261 and RG-2 under the application of an alternating magnetic field. Heating, efficacy and internalization properties were then evaluated. RESULTS: Nanoparticles coated with chitosan, polyethyleneimine and neridronate are apyrogen, biocompatible and stable in aqueous suspension. The presence of a thin coating in M-Chi and M-PEI favors an arrangement in chains of the magnetosomes, similar to that observed in magnetosomes directly extracted from magnetotactic bacteria, while the thick matrix embedding M-Neri leads to structures with an average thickness of 3.5 µm2 per magnetosome mineral. In the presence of GL-261 cells and upon the application of an alternating magnetic field, M-PEI and M-Chi lead to the highest specific absorption rates of 120-125 W/gFe. Furthermore, while M-Chi lead to rather low rates of cellular internalization, M-PEI strongly associate to cells, a property modulated by the application of an alternating magnetic field. CONCLUSIONS: Coating of purified magnetosome minerals can therefore be chosen to control the interactions of nanoparticles with cells, organization of the minerals, as well as heating and cytotoxicity properties, which are important parameters to be considered in the design of a magnetic hyperthermia treatment of tumor.

Biocompatible and stable magnetosome minerals coated with poly-l-lysine, citric acid, oleic acid, and carboxy-methyl-dextran for application in the magnetic hyperthermia treatment of tumors.

Magnetic hyperthermia, in which magnetic nanoparticles are introduced into tumors and exposed to an alternating magnetic field (AMF), appears to be promising since it can lead to increased life expectancy in patients. Its efficacy can be further improved by using biocompatible iron oxide magnetosome minerals with better crystallinity and magnetic properties compared with chemically synthesized nanoparticles (IONP - Iron Oxide Nanoparticles). To fabricate such minerals, magnetosomes are first isolated from MSR-1 magnetotactic bacteria, purified to remove potentially toxic organic bacterial residues and stabilized with poly-l-lysine (N-PLL), citric acid (N-CA), oleic acid (N-OA), or carboxy-methyl-dextran (N-CMD). The different coated nanoparticles appear to be composed of a cubo-octahedral mineral core surrounded by a coating of different thickness, composition, and charge, and to be organized in chains of various lengths. The in vitro anti-tumor and heating efficacies of these nanoparticles were examined by bringing them into contact with GL-261 glioblastoma cells and by applying an AMF. This led to a specific absorption rate of 89-196 W gFe -1, measured using an AMF of 198 kHz and 34-47 mT, and to percentages of tumor cell destruction due to the exposure of the nanoparticles to the AMF of 10 ± 3% to 43 ± 3% depending on the coating agent. These results show the potential of this protocol for the tumor treatment by magnetic hyperthermia.