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.

Tuning the Magnetic Response of Magnetospirillum magneticum by Changing the Culture Medium: A Straightforward Approach to Improve Their Hyperthermia Efficiency.

Magnetotactic bacteria Magnetospirillum magneticum AMB-1 have been cultured using three different media: magnetic spirillum growth medium with Wolfe's mineral solution (MSGM + W), magnetic spirillum growth medium without Wolfe's mineral solution (MSGM - W), and flask standard medium (FSM). The influence of the culture medium on the structural, morphological, and magnetic characteristics of the magnetosome chains biosynthesized by these bacteria has been investigated by using transmission electron microscopy, X-ray absorption spectroscopy, and X-ray magnetic circular dichroism. All bacteria exhibit similar average size for magnetosomes, 40-45 nm, but FSM bacteria present slightly longer subchains. In MSGM + W bacteria, Co2+ ions present in the medium substitute Fe2+ ions in octahedral positions with a total Co doping around 4-5%. In addition, the magnetic response of these bacteria has been thoroughly studied as functions of both the temperature and the applied magnetic field. While MSGM - W and FSM bacteria exhibit similar magnetic behavior, in the case of MSGM + W, the incorporation of the Co ions affects the magnetic response, in particular suppressing the Verwey (∼105 K) and low temperature (∼40 K) transitions and increasing the coercivity and remanence. Moreover, simulations based on a Stoner-Wolhfarth model have allowed us to reproduce the experimentally obtained magnetization versus magnetic field loops, revealing clear changes in different anisotropy contributions for these bacteria depending on the employed culture medium. Finally, we have related how these magnetic changes affect their heating efficiency by using AC magnetometric measurements. The obtained AC hysteresis loops, measured with an AC magnetic field amplitude of up to 90 mT and a frequency, f, of 149 kHz, reveal the influence of the culture medium on the heating properties of these bacteria: below 35 mT, MSGM - W bacteria are the best heating mediators, but above 60 mT, FSM and MSGM + W bacteria give the best heating results, reaching a maximum heating efficiency or specific absorption rate (SAR) of SAR/f ≈ 12 W g-1 kHz-1.

Magnetotactic bacteria AMB-1 with active deep tumor penetrability for magnetic hyperthermia of hypoxic tumors.

Tumor hypoxia is a great physiological barrier for tumor treatment. The development of efficient detection and treatment methods for tumor hypoxia has great scientific and clinical significance. In this work, we investigated the potential of magnetotactic bacteria AMB-1 for magnetic resonance imaging (MRI)-guided magnetic hyperthermia treatment of hypoxic tumors. Our investigations reveal that AMB-1 bacteria can selectively migrate to the hypoxic regions of solid tumors due to their anaerobic characteristics, showing active deep tumor penetrability. Moreover, AMB-1 bacteria exhibit high MRI contrast and magnetic heating performances because of the excellent magnetic performance of their magnetosomes. In vivo studies demonstrate that AMB-1 can not only generate T2-weighted contrast signals in tumor tissue, but also efficiently ablate hypoxic solid tumors through the magnetic hyperthermia effect. We believe that this novel microbial therapy can be a potential weapon for hypoxic tumor treatment.

Tuning of Magnetic Hyperthermia Response in the Systems Containing Magnetosomes.

A number of materials are studied in the field of magnetic hyperthermia. In general, the most promising ones appear to be iron oxide particle nanosystems. This is also indicated in some clinical trial studies where iron-based oxides were used. On the other hand, the type of material itself provides a number of variations on how to tune hyperthermia indicators. In this paper, magnetite nanoparticles in various forms were analyzed. The nanoparticles differed in the core size as well as in the form of their arrangement. The arrangement was determined by the nature of the surfactant. The individual particles were covered chemically by dextran; in the case of chain-like particles, they were encapsulated naturally in a lipid bilayer. It was shown that in the case of chain-like nanoparticles, except for relaxation, a contribution from magnetic hysteresis to the heating process also appears. The influence of the chosen methodology of magnetic field generation was also analyzed. In addition, the influence of the chosen methodology of magnetic field generation was analyzed. The application of a rotating magnetic field was shown to be more efficient in generating heat than the application of an alternating magnetic field. However, the degree of efficiency depended on the arrangement of the magnetite nanoparticles. The difference in the efficiency of the rotating magnetic field versus the alternating magnetic field was much more pronounced for individual nanoparticles (in the form of a magnetic fluid) than for systems containing chain nanoparticles (magnetosomes and a mix of magnetic fluid with magnetosomes in a ratio 1:1).

Smart Magnetotactic Bacteria Enable the Inhibition of Neuroblastoma under an Alternating Magnetic Field.

Magnetotactic bacteria are ubiquitous microorganisms in nature that synthesize intracellular magnetic nanoparticles called magnetosomes in a gene-controlled way and arrange them in chains. From in vitro to in vivo, we demonstrate that the intact body of Magnetospirillum magneticum AMB-1 has potential as a natural magnetic hyperthermia material for cancer therapy. Compared to chains of magnetosomes and individual magnetosomes, the entire AMB-1 cell exhibits superior heating capability under an alternating magnetic field. When incubating with tumor cells, the intact AMB-1 cells disperse better than the other two types of magnetosomes, decreasing cellular viability under the control of an alternating magnetic field. Furthermore, in vivo experiments in nude mice with neuroblastoma found that intact AMB-1 cells had the best antitumor activity with magnetic hyperthermia therapy compared to other treatment groups. These findings suggest that the intact body of magnetotactic bacteria has enormous promise as a natural material for tumor magnetic hyperthermia. In biomedical applications, intact and living magnetotactic bacteria play an increasingly essential function as a targeting robot due to their magnetotaxis.

Biosynthesis of iron oxide magnetic nanoparticles using clinically isolated Pseudomonas aeruginosa.

Magnetotactic bacteria are microscale complex natural systems that synthesize magnetic nanoparticles through biologically controlled mineralization. Nanoparticles produced by this process are biocompatible due to the presence of surrounding membranes. The mechanism controlling synthesis is cost-effective and is executed by complex genomes (operons). The results are monodispersed magnetic nanoparticles displaying advantages over polydispersed ones synthesized by physical and chemical methods. In this work, we isolated Pseudomonas aeruginosa from clinical samples and demonstrated its ability to biosynthesize magnetic nanoparticles. P. aeruginosa was thrived in a carbon-minimal medium supplemented with iron at low pH. The cells aligned parallel to a magnetic field, confirming their magnetic properties. The magnetic nanoparticles were extracted, purified, and characterized using electron microscopy, magnetometry, dynamic light scattering, and X-ray diffraction. This work represents the first isolation of a magnetotactic bacterium from clinical samples. The aerobic nature of these bacteria allows them to be easily cultured under laboratory conditions, unlike their well-known microaerophilic counterparts. The biosynthesized magnetic nanoparticles can be used in many applications, including magnetic resonance imaging, diagnostics, and therapeutics (i.e., magnetic hyperthermia).

Magnetosome mediated oral Insulin delivery and its possible use in diabetes management.

Our study investigates the effect of magnetosome mediated oral Insulin delivery on diabetic induced rat models. The study involves the development of Magnetosome-Insulin (MI) conjugates by direct and indirect (by means of PEG) coupling method and further characterized by microscopic and spectroscopic analysis. The in vivo oral delivery of magnetosome-Insulin conjugate against streptozotocin-induced rat models and its efficiency was investigated. The impact of MI showed a remarkable change in the reduction of FBG levels up to 65% than the standard (Insulin). Similarly, the serum parameters: triglycerides (43.81%), AST&ALT (39.4 and 57.2%), total cholesterol (43.8%) showed significant changes compared to the diabetic control. The histological results of MI treated rats were found similar to control rats. Thus, these significantly notable results on diabetic rats depicts that magnetosomes can be employed as a potential approach and a very promising alternative for the parenteral route of Insulin delivery.

Bio-synthesized iron oxide nanoparticles for cancer treatment.

Various living organisms, such as bacteria, plants, and animals can synthesize iron oxide nanoparticles (IONP). The mechanism of nanoparticle (NP) formation is usually described as relying on the reduction of ferric/ferrous iron ions into crystallized nanoparticulate iron that is surrounded by an organic stabilizing layer. The properties of these NP are characterized by a composition made of different types of iron oxide whose most stable and purest one appears to be maghemite, by a size predominantly comprised between 5 and 380 nm, by a crystalline core, by a surface charge which depends on the nature of the material coating the iron oxide, and by certain other properties such as a sterility, stability, production in mass, absence of aggregation, that have apparently only been studied in details for IONP synthesized by magnetotactic bacteria, called magnetosomes. In the majority of studies, bio-synthesized IONP are described as being biocompatible and as not inducing cytotoxicity towards healthy cells. Anti-tumor activity of bio-synthesized IONP has mainly been demonstrated in vitro, where this type of NP displayed cytotoxicity towards certain tumor cells, e.g. through the anti-tumor activity of IONP coating or through IONP anti-oxidizing property. Concerning in vivo anti-tumor activity, it was essentially highlighted for magnetosomes administered in different types of glioblastoma tumors (U87-Luc and GL-261), which were exposed to a series of alternating magnetic field applications, resulting in mild hyperthermia treatments at typical temperatures of 41-45 °C, leading to the full disappearance of these tumors without any observable side effects.

Comparative ecotoxicity assessment of magnetosomes and magnetite nanoparticles.

Magnetite nanoparticles (MNPs) are gaining attention because of their biomedical, environmental and industrial applications. However, they have limited uses because of ecotoxicity. On contrast, bacterially synthesized MNPs such as magnetosomes are found to be biocompatible and less toxic due to the lipid bilayer membrane found around magnetite. In this context, this study compares the physio-chemical properties and toxicology effects of MNPs and magnetosomes in different models such as human red blood cells, macrophage cell lines (RAW 264.7), onion root tips (Allium cepa), Artemia salina (A. salina) and zebrafish embryo (Danio rerio). MNPs showed 38.59% hemolysis whereas the maximum hemolysis induced by magnetosomes was 7.03% for the same concentration (250 µg/ml). The cytotoxicity of MNPs and magnetosomes were 36.01% and 13.4%, respectively, at 250 µg/ml. Onion root tip assay revealed high toxicity when treated with MNPs than magnetosomes. The MNPs were further tested for its toxicity against A. salina and 50% mortality rate was observed. Similarly, notable malformation was seen in zebrafish embryo treated with MNPs. However, magnetosomes did not exhibit any mortality and malformation in A. salina and zebrafish embryo. The study revealed that magnetosomes are safe and do not cause any potential risk to environment compared to synthetic MNPs.Abbreviation: MNPs: Magnetic nanoparticles; ATCC: American Type Culture Collection; MTB: Magnetotactic bacteria; MSR-1: Magnetospirillum gryphiswaldense; DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen; MSGM: Magnetospirillum growth medium; D-PBS: Dulbecco phosphate buffer saline; RBC: Red blood cells; SEM: Scanning electron microscopy; HRTEM: High-resolution transition electron microscope; FTIR: Fourier transform infrared spectroscopy; XRD: X-ray powder diffraction; AFM: Atomic-force microscopy; ZP: Zeta Potential; PSD: Particle Size Distribution; EDX: Energy-dispersive X-ray spectroscopy; PBS: Phosphate buffer saline; DMEM: Dulbecco's modified eagle medium; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); MTT:3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; DMSO: Dimethyl sulfoxide; ROS: Reactive oxygen species.

Biodegraded magnetosomes with reduced size and heating power maintain a persistent activity against intracranial U87-Luc mouse GBM tumors.

BACKGROUND: An important but rarely addressed question in nano-therapy is to know whether bio-degraded nanoparticles with reduced sizes and weakened heating power are able to maintain sufficient anti-tumor activity to fully eradicate a tumor, hence preventing tumor re-growth. To answer it, we studied magnetosomes, which are nanoparticles synthesized by magnetotactic bacteria with sufficiently large sizes (~ 30 nm on average) to enable a follow-up of nanoparticle sizes/heating power variations under two different altering conditions that do not prevent anti-tumor activity, i.e. in vitro cellular internalization and in vivo intra-tumor stay for more than 30 days. RESULTS: When magnetosomes are internalized in U87-Luc cells by being incubated with these cells during 24 h in vitro, the dominant magnetosome sizes within the magnetosome size distribution (DMS) and specific absorption rate (SAR) strongly decrease from DMS ~ 40 nm and SAR ~ 1234 W/gFe before internalization to DMS ~ 11 nm and SAR ~ 57 W/gFe after internalization, a behavior that does not prevent internalized magnetosomes to efficiently destroy U87-Luc cell, i.e. the percentage of U87-Luc living cells incubated with magnetosomes decreases by 25% between before and after alternating magnetic field (AMF) application. When 2 µl of a suspension containing 40 µg of magnetosomes are administered to intracranial U87-Luc tumors of 2 mm3 and exposed (or not) to 15 magnetic sessions (MS), each one consisting in 30 min application of an AMF of 27 mT and 198 kHz, DMS and SAR decrease between before and after the 15 MS from ~ 40 nm and ~ 4 W/gFe down to ~ 29 nm and ~ 0 W/gFe. Although the magnetosome heating power is weakened in vivo, i.e. no measurable tumor temperature increase is observed after the sixth MS, anti-tumor activity remains persistent up to the 15th MS, resulting in full tumor disappearance among 50% of treated mice. CONCLUSION: Here, we report sustained magnetosome anti-tumor activity under conditions of significant magnetosome size reduction and complete loss of magnetosome heating power.

Unlocking the Potential of Magnetotactic Bacteria as Magnetic Hyperthermia Agents.

Magnetotactic bacteria are aquatic microorganisms that internally biomineralize chains of magnetic nanoparticles (called magnetosomes) and use them as a compass. Here it is shown that magnetotactic bacteria of the strain Magnetospirillum gryphiswaldense present high potential as magnetic hyperthermia agents for cancer treatment. Their heating efficiency or specific absorption rate is determined using both calorimetric and AC magnetometry methods at different magnetic field amplitudes and frequencies. In addition, the effect of the alignment of the bacteria in the direction of the field during the hyperthermia experiments is also investigated. The experimental results demonstrate that the biological structure of the magnetosome chain of magnetotactic bacteria is perfect to enhance the hyperthermia efficiency. Furthermore, fluorescence and electron microscopy images show that these bacteria can be internalized by human lung carcinoma cells A549, and cytotoxicity studies reveal that they do not affect the viability or growth of the cancer cells. A preliminary in vitro hyperthermia study, working on clinical conditions, reveals that cancer cell proliferation is strongly affected by the hyperthermia treatment, making these bacteria promising candidates for biomedical applications.

Tuning properties of biomimetic magnetic nanoparticles by combining magnetosome associated proteins.

The role of magnetosome associated proteins on the in vitro synthesis of magnetite nanoparticles has gained interest, both to obtain a better understanding of the magnetosome biomineralization process and to be able to produce novel magnetosome-like biomimetic nanoparticles. Up to now, only one recombinant protein has been used at the time to in vitro form biomimetic magnetite precipitates, being that a scenario far enough from what probably occurs in the magnetosome. In the present study, both Mms6 and MamC from Magnetococcus marinus MC-1 have been used to in vitro form biomimetic magnetites. Our results show that MamC and Mms6 have different, but complementary, effects on in vitro magnetite nucleation and growth. MamC seems to control the kinetics of magnetite nucleation while Mms6 seems to preferably control the kinetics for crystal growth. Our results from the present study also indicate that it is possible to combine both proteins to tune the properties of the resulting biomimetic magnetites. In particular, by changing the relative ratio of these proteins, better faceted and/or larger magnetite crystals with, consequently, different magnetic moment per particle could be obtained. This study provides with tools to obtain new biomimetic nanoparticles with a potential utility for biotechnological applications.

Effects of Environmental Conditions on High-Yield Magnetosome Production by Magnetospirillum gryphiswaldense MSR-1

Background: Magnetotactic bacteria are a heterogeneous group of Gram-negative prokaryote cells that produce linear chains of magnetic particles called magnetosomes, intracellular organelles composed of magnetic iron particles. Many important applications have been defined for magnetic nanoparticles in biotechnology, such as cell separation applications, as well as acting as carriers of enzymes, antibodies, or anti-cancer drugs. Since the bacterial growth is difficult and the yield of magnetosome production is low, the application of magnetosome has not been developed on a commercial scale. Methods: Magnetospirillum gryphiswaldense strain MSR-1 was used in a modified current culture medium supplemented by different concentrations of oxygen, iron, carbon, and nitrogen, to increase the yield of magnetosomes. Results: Our improved MSR-1 culture medium increased magnetosome yield, magnetosome number per bacterial cell, magnetic response, and bacterial cell growth yield significantly. The yield of magnetosome increased approximately four times. The optimized culture medium containing 25 mM of Na-pyruvate, 40 mM of NaNO3, 200 µM of ferrous sulfate, and 5-10 ppm of dissolved oxygen (DO) resulted in 186.67 mg of magnetosome per liter of culture medium. The iron uptake increased significantly, and the magnetic response of the bacteria to the magnetic field was higher than threefold as compared to the previously reported procedures. Conclusion: This technique not only decreases the cultivation time but also reduces the production cost. In this modified method, the iron and DO are the major factors affecting the production of magnetosome by M. gryphiswaldense strain MSR-1. However, refining this technique will enable a further yield of magnetosome and cell density.

Precise Assembly of Genetically Functionalized Magnetosomes and Tobacco Mosaic Virus Particles Generates a Magnetic Biocomposite.

Magnetosomes represent magnetic nanoparticles with unprecedented characteristics. Both their crystal morphology and the composition of the enveloping membrane can be manipulated by genetic means, allowing the display of functional moieties on the particle surface. In this study, we explore the generation of a new biomaterial assembly by coupling magnetosomes with tobacco mosaic virus (TMV) particles, both functionalized with complementary recognition sites. TMV consists of single-stranded RNA encapsidated by more than 2100 coat proteins, which enable chemical modification via functional groups. Incubation of EmGFP- or biotin-decorated TMV particles with magnetosomes genetically functionalized with GFP-binding nanobodies or streptavidin, respectively, results in the formation of magnetic, mesoscopic, strand-like biocomposites. TMV facilitates the agglomeration of magnetosomes by providing a scaffold. The size of the TMV-magnetosome mesostrands can be adjusted by varying the TMV-magnetosome particle ratios. The versatility of this novel material combination is furthermore demonstrated by coupling magnetosomes and terminal, 5'-functionalized TMV particles with high molecular precision, which results in "drumstick"-like TMV-magnetosome complexes. In summary, our approaches provide promising strategies for the generation of new biomaterial assemblies that could be used as scaffold for the introduction of further functionalities, and we foresee a broad application potential in the biomedical and biotechnological field.

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.

Development of non-pyrogenic magnetosome minerals coated with poly-l-lysine leading to full disappearance of intracranial U87-Luc glioblastoma in 100% of treated mice using magnetic hyperthermia.

Magnetic hyperthermia was reported to increase the survival of patients with recurrent glioblastoma by 7 months. This promising result may potentially be further improved by using iron oxide nanoparticles, called magnetosomes, which are synthesized by magnetotactic bacteria, extracted from these bacteria, purified to remove most endotoxins and organic material, and then coated with poly-l-lysine to yield a stable and non-pyrogenic nanoparticle suspension. Due to their ferrimagnetic behavior, high crystallinity and chain arrangement, these magnetosomes coated with poly-l-lysine (M-PLL) are characterized by a higher heating power than their chemically synthesized counterparts currently used in clinical trials. M-PLL-enhanced antitumor efficacy was demonstrated by administering 500-700 µg in iron of M-PLL to intracranial U87-Luc tumors of 1.5 mm3 and by exposing mice to 27 magnetic sessions each lasting 30 min, during which an alternating magnetic field of 202 kHz and 27 mT was applied. Treatment conditions were adjusted to reach a typical hyperthermia temperature of 42 °C during the first magnetic session. In 100% of treated mice, bioluminescence due to living glioblastoma cells fully disappeared 68 days following tumor cell implantation (D68). These mice were all still alive at D350. Histological analysis of their brain tissues revealed an absence of tumor cells, suggesting that they were fully cured. In comparison, antitumor efficacy was less pronounced in mice treated by the administration of IONP followed by 23 magnetic sessions, leading to full tumor bioluminescence disappearance in only 20% of the treated mice.

Influence of the bacterial growth phase on the magnetic properties of magnetosomes synthesized by Magnetospirillum gryphiswaldense.

BACKGROUND: The magnetosome biosynthesis is a genetically controlled process but the physical properties of the magnetosomes can be slightly tuned by modifying the bacterial growth conditions. METHODS: We designed two time-resolved experiments in which iron-starved bacteria at the mid-logarithmic phase are transferred to Fe-supplemented medium to induce the magnetosomes biogenesis along the exponential growth or at the stationary phase. We used flow cytometry to determine the cell concentration, transmission electron microscopy to image the magnetosomes, DC and AC magnetometry methods for the magnetic characterization, and X-ray absorption spectroscopy to analyze the magnetosome structure. RESULTS: When the magnetosomes synthesis occurs during the exponential growth phase, they reach larger sizes and higher monodispersity, displaying a stoichiometric magnetite structure, as fingerprinted by the well defined Verwey temperature. On the contrary, the magnetosomes synthesized at the stationary phase reach smaller sizes and display a smeared Verwey transition, that suggests that these magnetosomes may deviate slightly from the perfect stoichiometry. CONCLUSIONS: Magnetosomes magnetically closer to stoichiometric magnetite are obtained when bacteria start synthesizing them at the exponential growth phase rather than at the stationary phase. GENERAL SIGNIFICANCE: The growth conditions influence the final properties of the biosynthesized magnetosomes. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

Co-delivery of doxorubicin and recombinant plasmid pHSP70-Plk1-shRNA by bacterial magnetosomes for osteosarcoma therapy.

To explore a novel combination of chemotherapy, gene therapy, and thermotherapy for osteosarcoma, a targeted heat-sensitive co-delivery system based on bacterial magnetosomes (BMs) was developed. The optimal culture conditions of magnetotactic bacteria (MTB) AMB-1 and characterization of BMs were achieved. A recombinant eukaryotic plasmid heat shock protein 70-polo-like kinase 1-short hairpin RNA (pHSP70-Plk1-shRNA) under transcriptional control of a thermosensitive promoter (human HSP70 promoter) was constructed for gene therapy. Doxorubicin (DOX) and pHSP70-Plk1-shRNA were included in the targeted thermosensitive co-delivery system, and in vitro DOX release activity, targeted gene silencing efficiency and in vitro antitumor efficacy were investigated. The results showed that the optimal culture conditions of MTB AMB-1 are an oxygen concentration of 4.0%, a pH value of 7.0, 20 µmol/L of ferrous sulfate, 800 mg/L of sodium nitrate, and 200 mg/L of succinic acid. The temperature of BMs reached 43°C within 3 minutes and could be maintained for 30 minutes by adjusting the magnitude of the alternating magnetic field (AMF). The diameters of BMs, BM-DOX, BM-recombinant eukaryotic plasmid pHSP70-Plk1-shRNA (shPlk1), and BM-DOX-shPlk1 were 43.7±4.6, 79.2±5.4, 88.9±7.8, and 133.5±11.4 nm, respectively. The zeta potentials of BMs, BM-DOX, BM-shPlk1, and BM-DOX-shPlk1 were -29.4±6.9, -9.5±5.6, -16.7±4.8, and -10.3±3.1 mV, respectively. Besides, the system exhibited good release behavior. DOX release rate from BM-DOX-shPlk1 was 54% after incubation with phosphate-buffered saline at 43°C and 37% after incubation with 50% fetal bovine serum, which was significantly higher than that at 37°C (P<0.05). In addition, the expressions of Plk1 mRNA and protein were significantly suppressed in cells treated with BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF compared to other groups (P<0.05). Furthermore, evaluation of the effect of in vitro antitumor revealed that BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF was significantly more effective than others in tumor inhibition. In conclusion, the new heat-sensitive co-delivery system represents a promising approach for the treatment of cancer.

Transcriptome analysis reveals physiological characteristics required for magnetosome formation in Magnetospirillum gryphiswaldense MSR-1.

Magnetosome synthesis ability of Magnetospirillum gryphiswaldense MSR-1 in an autofermentor can be precisely controlled through strict control of dissolved oxygen concentration. In this study, using transcriptome data we discovered gene transcriptional differences and compared physiological characteristics of MSR-1 cells cultured under aerobic (high-oxygen) and micro-aerobic (low-oxygen) conditions. The results showed that 77 genes were up-regulated and 95 genes were down-regulated significantly under micro-aerobic situation. These genes were involved primarily in the categories of cell metabolism, transport, regulation and unknown-function proteins. The nutrient transport and physiological metabolism were slowed down under micro-aerobic condition, whereas dissimilatory denitrification pathways were activated and it may supplemental energy was made available for magnetosome synthesis. The result suggested that the genes of magnetosome membrane proteins (Mam and Mms) are not directly regulated by oxygen level, or are constitutively expressed. A proposed regulatory network of differentially expressed genes reflects the complexity of physiological metabolism in MSR-1, and suggests that some yet-unknown functional proteins play important roles such as ferric iron uptake and transport during magnetosome synthesis. The transcriptome data provides a holistic view of the responses of MSR-1 cells to differing oxygen levels. This approach will give new insights into general principles of magnetosome formation.