Therapeutic Innovations in Nanomedicine: Exploring the Potential of Magnetotactic Bacteria and Bacterial Magnetosomes

Introduction

Over the last decade, nanoparticles (NPs) and nanotechnology have attained significant consideration because of their exceptional and distinctive features.1 The properties are acquired by considering their narrow-sized geometry and structural and surface modifications. Today, nanoparticles have potential importance in medicine, electronics, environmental clean-up, research, defences, aerospace, etc.2,3 Nanomedicine is the emerging branch of nanosciences that applies NPs in the biomedical field.4,5 Among all the types of NPs, magnetic nanoparticles (MNPs) have emerged as transformative tools in nanomedicine, leveraging their unique physicochemical properties for various biomedical applications.6,7 MNPs have emerged as potential candidates in nanomedicine due to their response to an external magnetic field (EMF), recycling nature, low cost and easy availability, etc. Recently, MNPs have shown emerging roles in medicine for treatment and diagnosis. For instance, MNPs are widely used for drug delivery8 and in magnetic resonance imaging (MRI) as a contrast agent.9,10

The different types of iron oxide nanoparticles (IONPs) are magnetite, maghemite, hematite, and greigite, which have different magnetic properties. Out of all these MNPs, magnetite has a stronger magnetic strength than the others, and it has gained huge attention in medicine and environmental clean-up.11 MNPs are extensively utilized in drug delivery, MRI, contrast agents, etc.12 due to their high responsiveness to EMF and their magnetic properties, which allow precise targeting and raise localized hyperthermia for cancer therapy.13 Depending on their size, composition, and environmental conditions, MNPs exhibit diverse magnetic properties, primarily categorized as paramagnetic, superparamagnetic, or ferromagnetic. Paramagnetic NPs possess unpaired electrons, leading to a weak attraction to EMFs. They do not retain magnetization once the external field is removed, making them less suitable for applications requiring persistent magnetism. Superparamagnetic NPs, such as superparamagnetic iron oxide nanoparticles (SPIONs), can be magnetized in the presence of an EMF but exhibit no remanence or coercivity when the field is removed.14 This property allows them to be used effectively in drug delivery and MRI contrast agents, as they can be manipulated without residual magnetism.15 SPIONs can be magnetized in an external field and return to a non-magnetic state, facilitating minimal side effects during treatments like hyperthermia.14 Ferromagnetic NPs retain their magnetization even after removing the EMF, exhibiting hysteresis in their magnetization curves.16 They are typically larger in size and can be utilized in applications requiring stable MNPs, such as data storage, magnetic separation, and biosensing.14,17 Their strong magnetic properties are advantageous in industrial settings where consistent magnetic behavior is crucial. Superparamagnetic NPs are usually chosen for biomedical applications due to their non-toxic and biocompatible properties, but ferromagnetic NPs may be more appropriate for industrial applications that require stable magnetization.

IONPs (MNPs) can be synthesized by all three methods, ie, chemical, physical, and biological (plants and microorganisms).18 All these synthesis methods have their own advantages and drawbacks. For instance, the chemical method uses harmful chemicals that threaten the environment. Moreover, the chemical route requires a capping agent (polyvinyl alcohol, polyethylene glycol, etc) and stabilizing agents (chitosan, amino acids, etc) to control the size of the MNPs. So, this further increases the cost of the synthesis method. Moreover, the chemically synthesized MNPs require strict conditions to obtain uniform MNP, which may reduce the effectiveness of such MNPs.19,20 The physical approach requires sophisticated instruments for the synthesis, which is expensive and energy-intensive, increasing their production cost. The biological method is eco-friendly as it requires the least chemicals, and also, if microorganisms are used for synthesis, uniformity in size will be maintained.21 So, by microbial method, the synthesized MNPs have uniform shapes and sizes and are well crystalline in nature.

The shortcomings of the chemically and physically synthesized MNPs could be overcome by using the magnetotactic bacteria (MTB) and their magnetosomes as the source of MNPs, which possess well-ordered MNPs. Magnetosomes are membrane-bound MNPs synthesized by MTB that exhibit biocompatibility, uniform size, and strong magnetic properties. The role of MTB and their magnetosomes in nanomedicine is increasingly recognized for their nano-dimension, recyclability, and response to an EMF.22 MTB and their magnetosomes can be natural nanocarriers for anticancer drugs, antibodies, and nucleic acids, enhancing therapeutics’ stability and targeted delivery to tumor sites.23,24 Due to their high importance in nanomedicine, investigators have termed the MTB and their magnetosomes as potential nanobots/nanorobots. These microscopic robots could be easily manipulated and guided externally inside human beings. These nanobots may navigate through the human body driven to particular sites, for instance, tumor locations, while retaining their therapeutic and imaging properties.25–27

Even though some of the MTB synthesizes magnetite and greigite28 but magnetosomes are primarily composed of magnetite (Fe3O4) and are produced by MTB such as Magnetospirillum gryphiswaldense.29 They possess a narrow size distribution and uniform morphology, which is crucial for their functionality in drug delivery and imaging.23 Studies have shown that magnetosomes can improve the efficacy of treatments like magnetic hyperthermia, where localized heating is applied to cancer cells.30 Magnetosomes are also being explored as contrast agents in magnetic resonance imaging (MRI), providing enhanced imaging capabilities due to their magnetic properties.23,24 Advanced imaging techniques, such as Magnetic Force Microscopy (MFM), have been developed to characterize individual magnetosome chains, facilitating their application in biological settings.31

Earlier, some of the investigators have used magnetosomes for precise drug delivery to cancerous cells through hyperthermia under EMFs, showing potential in progressing focused and efficient cancer therapies.32 Xie et al reviewed and concluded that MTB-based magnetite NPs have several advantages over synthetic chemically synthesized magnetite NPs.33

Even though numerous investigations have been done in this field where MTB have been used for the drug delivery review, none of them have in-depth studies of the formation of magnetosomes, mass production of magnetosomes, and descriptive biomedical applications. Here, emphasis was given to the magnetosome’s role in targeted drug delivery, advanced MRI contrast enhancement, and hyperthermia therapy for cancer treatment, where their precise magnetic control minimizes off-target effects. Moreover, the study explores the genetic mechanisms of magnetosome formation, particularly the role of the magnetosome genomic cluster (MGC), and discusses bioengineering advances for customized applications. An emphasis was given to the state-of-the-art extraction and functionalization methods alongside detailed characterization of the magnetosomes. Moreover, the article addresses the scalability, safety, and regulatory hurdles, calling for interdisciplinary efforts to advance magnetosome biomedical applications and highlighting their transformative potential in nanomedicine.

In the present review, investigators have emphasized the basic information of magnetotactic bacteria and their magnetosomes. Further investigators have emphasized the habitats and morphology of MTB. The authors have reviewed the detailed information about the extraction, purification, and characterization of MTB and magnetosomes has been provided. Further, current and emerging applications of MTB and magnetosomes in biomedical applications, especially contrast agents, imaging, and drug delivery, have been highlighted. Finally, the authors have reviewed the current clinical trials on MTB-based drug delivery.

Magnetotactic Bacteria (MTB)

MTB is mainly a Gram-negative, flagellated microorganism that swims and inactively orients on the influence of geomagnetic fields.34 MTB is usually categorized under different structures based on elemental and synthesized mineral nanocrystals, which involve such as Fe3O4, Fe3S4 (greigite), combined greigite with the pyrite (FeS2), and a mixture of magnetite and greigite. These distinct mineral compositions give rise to different magnetic properties and behaviors within MTB.35 MTB exhibit a remarkable morphological diversity crucial for their ecological roles and evolutionary adaptations. The morphological diversity is phylogenetic and functional, as different morphotypes are adapted to specific environmental niches.36 The primary morphological types identified include coccoid, rod, spirillum, and vibrio forms, with coccoid shapes being the most prevalent.37–39 The most abundant are identified as magnetotactic cocci, characterized by their spherical shape and significant species-specific magnetosome arrangements. For instance, a giant rod-shaped species (QR-1) dominated in certain environments, showcasing a unique arrangement of magnetosomes in chains.40 In addition to the above two, spirillum and vibrio shapes contribute to the morphological spectrum, indicating various adaptations to aquatic environments.40

The variation in the morphology of MTB has ecological implications. For instance, MTB are vital in biogeochemical cycles, influencing iron and sulfur dynamics in ecosystems.25 Their morphological adaptations allow them to thrive in diverse habitats, from freshwater to marine environments, enhancing their ecological significance.41 MTB are predominantly anaerobic or facultatively anaerobic.42 Scientists have efficiently cultivated at least twenty kinds of magnetite-synthesizing MTB in pure culture.

Discovery, Natural Habitats and Biological Characteristics of MTB

Numerous magnetosomes producing MTB have been identified over several decades from various parts of the globe. The first identification of MTB in 1975 was Magnetospirillum magnetotacticum,43 followed by Magnetococcus marinus in 1978.44 In 1981, M. magnetotacticum MS-1 was described;42 by 1988, Magnetovibrio MV-2 was identified,45 and in 1991, researchers recognized Magnetospirillum magneticum, M. magneticum AMB-1, and Magnetovibrio MV-1.46

The discovery of Magnetovibrio MV-4 was made in the year 1993,45 while Magnetotactic bacterium strain WD-1 and Magnetotactic bacterium strain HM-1 were identified in 1994 and 1996, respectively.25 In 1999, Magnetospirillum sp. MSM-4 was reported.47 By 2002, Desulfovibrio magneticus RS-1 was discovered,48 followed by Magnetotactic bacterium strain YN-1 in 2003.49 In 2007, M. gryphiswaldense was identified.50

A significant period of discoveries occurred in 2010 and 2016. In 2010, Magnetovibrio blakemorei was identified33 and in 2016, several new species were discovered, including Magnetogaba australis IT-1,51Magnetospirillum QH-2,52Gammaproteobacterium BW-2, Gammaproteobacterium SS-5, and Magnetospira thiophila MMS-1.53Candidatus Desulfamplus magnetomortis BW-1, Delta proteobacterium ML-1, Delta proteobacterium ZZ-1, and δ-proteobacterium AV-1 were documented.53

Further discoveries in 2016 included Magnetoovoid strain MO-1,54Magnetotactic bacterium strain TH-1,45 and Herbaspirillum sp. TK-2.55 Other identified strains were Magnetotactic bacterium strain MG-1,56Magnetotactic bacterium strain MG-2,57 and Magnetotactic bacterium strain YSC-1.58Table 1 shows the year-wise identification of MTB from various parts of the world.

Table 1 Year-Wise Identification of MTB from Various Parts of the World

The above milestones reflect the progression from initial observations to identifying specific species, showcasing the increasing understanding of MTB’s ecological roles and biological mechanisms.

Natural Habitats of the MTB

MTB exhibit diverse physiological adaptations and occupies various ecological niches. These range from oxygen-rich aerobic environments to oxygen-deprived anaerobic habitats, spanning marine to freshwater ecosystems.59 Most MTB require an anaerobic environment; typically, the oxic and anoxic transition area near the nethermost residues (sediments) is the best zone for MTB growth. The MTB growing in oxygen-rich regions of water bodies, such as lakes, ponds, oceans, etc., have magnetite inside them. While going into deeper parts of the ocean and beyond this zone, there is an abundance of magnetite and greigite-producing MTB, mainly microaerophiles and anaerobic. Due to the anaerobic condition, there is a chemical change in magnetite into maghemite. Finally, there is a high concentration of sulphide users at the deepest part of the ocean or lake.60

From the physiology, ecology, and phylogeny aspects, MTB are found in water columns or sediments due to vertical chemical stratification. MTB have long been limited to environments with pH values close to neutral and at room temperature. Nevertheless, a moderately thermophilic MTB, capable of thriving up to a potential upper growth limit of 63°C, has been observed in hot springs, which requires a pH of 9.0 to grow well.22

Some theories claim that finding low-oxygen environments is a one-dimensional task rather than a three-dimensional one, with the latter typically connected to different cell taxi mechanisms. Freshwater bacteria from the genus Magnetospirillum are among the most extensively investigated MTB.

Mechanism of Magnetotaxis

The mechanism of magnetotaxis in MTB involves a combination of magnetic sensing and chemotactic responses, enabling these microorganisms to navigate their environments effectively. This navigation is primarily facilitated by magnetosomes that align the bacteria with the Earth’s magnetic field.61 Magnetosomes are made up of either magnetite or greigite crystals, organized in chains that function like a compass needle.62 The arrangement of these magnetosomes allows MTB to line up with geomagnetic fields, helping in their directional movement toward optimal habitats. MTB exhibit magneto-chemotaxis, responding to chemical gradients in their environment, such as oxygen and other repellents. This dual response allows MTB to navigate complex redox gradients, optimizing their position in stratified aquatic environments.63 Their ability to adapt to various ecological niches highlights the evolutionary significance of magnetotaxis in diverse habitats.25

MTB display remarkable magnetotaxis characteristics that permit them to orient themselves with the Earth’s geomagnetic field and navigate utilizing their flagella. There are six known magneto-aerotaxis variations, and research on the nature of magneto-aerotaxis is still ongoing.59 This unique ability allows MTB to harness the Earth’s MF as a natural compass, aiding it in precise orientation and movement.42 Two categories—polar and axial—can be used to categorize the magnetotaxis of MTB. Axial MTB demonstrate a relatively unrestrained, back-and-forth swimming pattern in multiple directions. Conversely, polar MTBs tend to swim preferably in a definite direction associated with the local geomagnetic field, showing a more directed and oriented movement.

Relying on the magnetic crystal formations within MTB, there are two main classifications of polar MTB: south-seeking MTB and north-seeking MTB. South-seeking MTB are principally observed in the southern hemisphere and tend to swim in the north pole line at a magnetic field.61 Conversely, north-seeking MTB are more frequently found in the northern hemisphere and prefer swimming towards the south magnetic zone in a magnetic field. The north-seeking and south-seeking MTB are widely obtained around the geographical zone (equator) with migration in opposite directions.61 In this area, there are two zones, north and south, seeking MTB with almost the same numbers. Intriguingly, a remarkable discovery has contested conventional expectations. While south-seeking MTB are generally associated with the southern hemisphere, a population of these bacteria has been determined in the northern hemisphere. Their exceptional swimming behavior sets them apart, contradicting the traditional patterns in formerly known MTB. This breakthrough introduces a breathtaking area of research and desires a re-evaluation of the factors affecting the magnetic orientation of these MTB in various geographic locations.22

Applications in Biomedical Research

MTB and their magnetosomes have significant potential in biomedical research, where they are commonly used in drug delivery systems (targeted drug delivery and as nanocarriers), imaging and diagnostics, and pharmaceutical applications.64 MTB can be engineered to deliver anticancer drugs directly to tumor sites, enhancing treatment efficacy while reducing the side effects.65 Magnetosomes act as natural nanocarriers for various therapeutic agents, including antibodies and siRNA, improving stability and targeted delivery.23 Besides this, magnetosomes are also used in advanced techniques like Magnetic Force Microscopy (MFM), which allow for the characterization of magnetosome chains, facilitating their use in imaging applications.31 One major feature of the application of magnetosomes in diagnostics is their good biocompatibility and low cytotoxicity.66 The establishment of pharmaceutical cell banks for MTB enables large-scale production of high-purity magnetosomes, which can be utilized in nanomedicine.29

Bacterial Magnetosomes

The bacterial magnetosome, a genuine prokaryotic organelle, displays a complexity equivalent to its eukaryotic counterparts. Magnetosomes of MTB are lipid bilayer membranes enclosing nanosized crystals of either greigite or magnetite, with most MTB comprising the mineral magnetite.61 These magnetosomes have a potential impact on the magnetic characteristics of the geometry of MTB (Fe3S4).59 Chemical, metabolic, and genetic controls particular to each species affect morphology and content, including the structural morphology of magnetic-based solid bio-minerals.56 Moreover, it also depends on the amount of O2 and food available, pH, redox potential, source of carbon, temperature, and mode of Fe absorption via MTB.

Structure and Composition

MTB are unique microorganisms that produce magnetosomes, specialized organelles containing magnetic iron minerals. These magnetosomes are crucial for the bacteria’s navigation in geomagnetic fields.61 The structure and composition of magnetosomes involve several key components, including the magnetic crystals, associated proteins, and the surrounding membrane. Inside the bacteria, the magnetosomes are typically grouped in chains. Even following bacterial disruption for the extraction of magnetosomes, the structural integrity of the magnetosome configuration persists, displaying robust stability that permits preservation and further analysis. This configuration is desirable because it felicitates uniform distributions and enhanced internalization into human cells, characteristics which may typically preferred for medicinal purposes.31

Magnetic Crystals

The structure of magnetosomes mainly comprises magnetic crystals and membrane enclosure. Magnetosomes typically contain magnetite or greigite crystals, which are biomineralized within the bacteria.62 Each magnetosome is enclosed by a lipid bilayer membrane derived from the inner membrane (IM) of the bacteria, which helps maintain the integrity of the magnetic crystals.61 Although uncultured coccus bacteria can develop exceptional magnetite in bulk shapes and sizes (up to 250 nm), established solid magnetite structures usually come under the confined nanoscale around 35–120 nm. The magnetosome’s size range has physical implications mirrored in its magnetic pull. It has two cores: an organic membrane and an organized arrangement of inorganic magnetite. An examination of isolated magnetosomes through lipid studies has uncovered that the magnetosome membrane mainly comprises a combination of amino acids, lipids, or lipid polymers. On the contrary, vesicles formed by the IM are considered the organic inner core of magnetosomes, including phospholipids, acids, glycolipids, and sulfolipids. Active sites on the magnetosome surfaces were identified as carboxyl, -OH, and amino monomers. Using a microscope, magnetosomes of different shapes have been observed, varying from rectangular, cubooctahedral, bullet-shaped, and elongated prismatic morphologies.67 Magnetite, which can oxidize to maghemite (Fe2O3), typically makes up the core of magnetosomes. The magnetosome core often has high degrees of crystallinity and purity.

Proteins

In addition to this, several proteins are associated with the magnetosomes. For instance, proteins such as MamK and MamJ are critical in organizing magnetosomes into chains, facilitating their function as geomagnetic sensors.61,68 Nonetheless, “Mam” refers to the magnetosome membrane, and “Mms” denotes the membrane specific to magnetic particles. These protein structural units play an essential role in forming the magnetosome membrane. This biological covering produces negatively charged magnetosomes with good water dispersion. The magnetosome surface is decorated with several chemical groups, facilitating straightforward functionalization.69 Moreover, some of the novel proteins, like Mad28, have also been identified, which contribute to the structural integrity of magnetosome chains.68 Besides this, Magnetosome Gene Clusters (MGCs) regulate the formation of magnetosomes that encode proteins essential for biomineralization.62

Magnetotactic Bacteria’s Genetic Landscape

Concerning molecular biology, genes specific to MTB were discovered following the sequencing of the genomes of numerous MTB species, which regulate the formation of magnetosomes. These genes are located within the MGC region, which plays a valuable role in various functions encompassing magnetosome membranes’ development. Their responsibilities extend to developing iron transport into magnetosomes, contributing to the structural composition, core formation, and the controlled morphological growth of magnetite crystals. Molecular genetic research was originally conducted using M. magnetotacticum MS-1. According to a hypothesis, certain genes from M. magnetotacticum MS-1 could be found within Escherichia coli. This is feasible because both organisms’ transcription and translation machinery are compatible, assisting genetic manipulation at the molecular level. This organism’s recA gene was cloned and expressed in E. coli. M. magnetotacticum MS-1 strain cloned a 2 kb DNA fragment to complement the iron uptake deficits in Salmonella typhimurium and mutants of E. coli lacking a functioning gene aroD. The above investigation concluded that such a 2 kb DNA fragment could help regulate the uptake of Fe.70 It is now possible to test non-magnetic mutants that lack magnetosomes due to the advancements in MTB culture technique on agar plates. The chromosome of a non-magnetic mutant strain of M. magneticum AMB-1 bacterium grown on an agar plate contained Tn5 transposon. A gene called magA was discovered using transposon mutagenesis, expressed in E. coli. Cultivation of M. magneticum AMB-1 bacterial cells followed by Fe-deficient optimizations produced extra magnetosomes, and magA appeared higher. Recent research shows magA may be a contender for enhanced MRI impacts. The aor gene was discovered to be expressed within a cytoplasm of M. magneticum AMB-1 and restricted to microaerobic environments. The mam22 gene was successfully cloned in M. magnetotacticum via reverse genetics. A gene for the homologous protein mamA was discovered in M. gryphiswaldense MSR1, M. magneticum AMB-1, and Magnetococcus sp. MC-1. Because of a shorter magnetosome chain developed by M. magneticum AMB-1, when mamA was deleted, it was determined that mamA is necessary to generate functioning magnetosome vesicles.71,72

Four preserved gene clusters inside a sizable unsteady genomic area known as the magnetosome island contain the bulk genomic substantially involved in magnetosome development. Various forms of primary genes are carried to form magnetosomes where the putative iron transporter genes are mainly mamB and mamM among the discovered primary genes. The mamK gene for the bacteria encodes several protein units. In contrast, the mamJ gene produces an acidic protein crucial for assembling the magnetosome chain.71,72 A summary of all the genes involved in the MTB for producing magnetosomes and their role is provided in Table 2.

Table 2 Summary of the Genes Involved in the MTB for Producing Magnetosomes and Their Role

Biosynthesis of Magnetosomes

The formation of magnetosomes in Magnetospirillum species, such as M. magnetotacticum and M. gryphiswaldense, is a complex process extensively studied across various strains. The formation of magnetosomes varies from one MTB to another, but the overall process remains the same. The formation of magnetosomes involves a series of genetic, biochemical, and structural processes crucial for their function as geomagnetic sensors. This understanding is primarily derived from research on multiple strains of Magnetospirillum, which have provided insights into the genetic and molecular mechanisms underlying magnetosome formation.68,73 The key reason for this is that these microorganisms are more accessible to cultivate than the majority of other MTB, that have made it easier to analyze their physiological and metabolic processes.74 The following phases make up the magnetosome formation mechanism:

Intrusion into cytoplasmic membrane and creation of the magnetosome vesicle. Uptake of Fe by MTB and its transportation into the magnetosome membrane vesicle. A partial oxidative process catalyzed by an oxidation-reduction enzyme guides the development of low-density hydrous ferric oxides resembling the mineral ferrihydrite. Hydrous oxides have reduced a third of their Fe3+, and additional dehydration produces Fe3O4.

During the initial phase, the inner membrane of MTB undergoes swelling, forming a vesicle this is followed by the uptake of Fe from the surroundings. The MTB have various transporters in the magnetosome membrane, which pumps in Fe2+/Fe3+, leading to the elevated concentration of Fe ion in the vesicle. The membrane of the magnetosome comprises a set of magnetosome-associated proteins (MAPs) that regulate the biomineralization environment, encompassing Fe concentration, redox state, and pH levels.35 In the final step, ie, in biomineralization, there is a high concentration of Fe ion, which is further crystallized by the magnetosome protein, guiding the creation of a magnetite crystal.75

The chronological order of the Fe intake, transport, and crystal biomineralization processes is well understood. However, whether the formation of magnetosome vesicles occurs before or after crystal biomineralization or if both processes occur simultaneously is unclear. The Fe-deficient cells of M. magnetotactic and M. gryphiswaldense possessed unfilled along the moderately full magnetosome vesicles, according to reported morphological studies. These findings suggested that magnetosome vesicles were present before the biomineralization of the mineral phase.74

In the first step of forming new cells, extracellular Fe2+/Fe3+ ions are generally obtained from cells. Subsequently, it penetrates the external membrane and enters the periplasm, where there is a potential for the formation of magnetite crystals within invaginations, leading to magnetite magnetosomes.76 On the other hand, particular details and definite steps in magnetosome biomineralization are yet unclear and may differ according to the MTB species. Nothing is known regarding the production process of greigite biomineralization in MTB outside the identification of genes and chemical precursors. It has been shown that many genes and proteins contribute to magnetosome development. The genes and proteins concerned in the biomineralization procedure are yet unclear.62 The species of Magnetospirillum were the principal organisms used to study the synthesis of bacterial magnetosomes. Figure 1 displays the scheme of the hypothetical mechanism of magnetite biomineralization.42

Figure 1 Schematic representation of the proposed magnetite biomineralization mechanism in MTB.

Notes: Reproduced with permission from Yan L, Zhang S, Chen P, Liu H, Yin H, Li H. Magnetotactic bacteria, magnetosomes, and their application. Microbiol Res 0.2012; 167:507–19. Copyright © 2012 Elsevier GmbH. All rights reserved.42

Magnetosome Vesicle Formation

In this stage, membrane vesicles are created, and a cytoplasmic film obtains magnetosome membrane findings. Approximately 3–4 nm double-layered lipid film, composed of proteins, phospholipids, glycolipids, and fatty acids comparable to those found in cytoplasmic membranes, makes up the membrane of magnetosome in various Magnetospirillum species. This shows that the cytoplasmic membrane may be the source of the magnetosome vesicle. The creation of the magnetosome membrane in M. magneticum is brought about by the magnetosome proteins MamB, MamI, MamL, and MamQ. The magnetosome formation and the association of oligomeric units under a process of magnetosome biomineralization may be coordinated by the MamA gene, which functions as a scaffolding protein. For magnetite to maintain its thermodynamic stability, the magnetosome vesicle may be alkaline.77

Uptake of Iron in Magnetotactic Bacteria

In this process, siderophores and transport proteins internalize exogenous iron, accumulating through transmembrane iron transporters into the magnetosome vesicles. Magnetospirillum utilizes both Fe2+/Fe3+ to create ferrites.78 Since Fe2+ can dissolve by limiting 0.1 mol/L at around seven pH, MTB can absorb it using non-specific processes. Contrarily, because Fe (III) is insoluble, many bacteria rely on Fe chelators for holding, dispersing, and subsequently ingesting Fe3+. Low molecular weight siderophore produced by MTB has a strong propensity for iron complexation. Ligand- or proton-induced magnetic oxide dispersion processes can accelerate the dissolution of Fe (III). As a result, siderophore boosts the likelihood that MTB will come into touch with Fe (III), improving MTB’s ability to absorb iron. Compared to non-magnetotactic bacteria, MTB have more iron, about 3% of their dry weight. However, there is no proof that MTB have unique Fe uptake mechanisms. Although the specifics surrounding iron intake by MTB for magnetosome production are unknown, it appears that each magnetotactic bacterial species has several iron uptake systems in operation.75,79

Transportation of Fe Within a Membrane Vesicle

Irrespective of the timeline of vesicle formation, the commencement of nanocrystal formation begins upon the internalization of dissolved iron into the vesicle. For instance, M. magneticum AMB-1 cells had a 200-fold higher capacity to collect iron than conventional E. coli cells, up to 0.01 mol/L. To attain such high iron concentrations, MTB must have an effective iron transfer system, albeit its exact workings are still unknown. However, many membrane proteins control how iron gets into magnetosomes for biomineralization. For instance, proteins (MamB and MamM), the cationic distribution of the metal-based carriers, have been found in the identified MTB genomes.80–82 Only a few species have been reported to contain the protein MamV, which can carry iron to the membrane vesicle or invasion of the magnetosome.75,83,84

Controlled Biomineralization Within the Magnetosome Vesicle

Fe2+ and Fe3+ are liberated at the interface between the compartment and the magnetosome after being bound by organic substrates, where A remains unidentified, and B is identified as ferritin. Magnetite nanocrystals are formed in the final phase because of closely bound magnetosome proteins.85 The development of magnetite crystals in magnetosomes seems to be mediated by specific magnetosome proteins. Mms proteins control how nanocrystals form and become biomineralized in the vesicles of MTB. The development of magnetic nanocrystals is regulated by five Mms-proteins (Mms5, Mms6, Mms7, Mms12, and Mms13).

Magnetized solid structures with comparable characteristics to those generated in MTB cells are noticed when M. gryphiswaldense MSR-1 is exposed to Mms6. In the absence of Mms6, the nanocrystals displayed irregular size and shape. In M. gryphiswaldense, MamX, MamZ, and MamH are a factor in governing the biomineralization of magnetite via redox control, which is believed to impact the size and maturation of magnetosome Fe3O4 crystals. MamK and MamJ, two proteins found in M. gryphiswaldense, were involved in regulating the development of the magnetosome chain. A recent discovery uncovered that MamY acts as a membrane-anchored mechanical scaffold, performing a vital role in positioning the motility axis of the Magnetotactic spirilla. This alignment helped reconcile the procedure of magnetoreception on moving orientations, furnishing significant insights into how these microorganisms navigate employing the earth’s magnetic field.74

Gene Cluster Involved in Magnetosome Formation

In MTB, a gene cluster is involved in the magnetosome formation called Magnetosome Island (MAI). These are highly conserved in MTB, which have five operons, namely, mamAB, mamGFDC, mms6/mms36-48, mamXY/mag123, and feoAB operons.86 The mamAB operon plays a crucial role in the biogenesis of magnetosomes in MTB, particularly in M. gryphiswaldense. This operon consists of several genes essential for forming and maturing magnetite crystals. Table 3 provides a summary of the role of various genes within the mamAB operon.

Table 3 A Summary of Roles of Various Genes Within the mamAB Operon in M. gryphiswaldense

Kolinko et al inserted these 5 operons from M. gryphiswaldense into Rhodospirillum rubrum (photosynthetic bacteria). R. rubrum is phylogenetically nearest to Magnetospirillum sp. but does not produce magnetosomes. Once these genes were introduced, small vesicles were formed in R. rubrum. Further, investigators also revealed that it was possible to recollect vesicles containing R. rubrum using a strong permanent magnet. This step proved that this vesicle may act as a magnetosome.89

Schüler and their group showed the function of genes in MAI by knocking out each one of them. If any of the genes, ie, mamL, mamQ, or mamB, coming under the mamAB operon, is defeated, there will be no magnetosome formation in MTB. It is believed that the proteins of these three genes are associated with an internal membrane and trigger vesicle construction. Moreover, the exact function of these three genes is not yet known.90Figure 2 illustrates the role of various genes of the mamAB operon in M. gryphiswaldense and the steps involved in forming bacterial magnetite.

Figure 2 Role of various genes of mamAB operon in M. gryphiswaldense.

Notes: Reproduced from IGEM Kyoto. Magnetosome formation. IGEM. Published 2014. https://2014.igem.org/Team:Kyoto/Project/Magnetosome_Formation. Accessed December 20, 2024. Creative Commons.91

Factors That Affect Magnetite Formation

Magnetosome composition in MTB is influenced by various environmental factors, including oxygen levels, nutrient availability, pH, redox potential, carbon sources, temperature, and iron absorption methods. These factors collectively impact the biomineralization method and the quality of magnetosomes produced. The formation of magnetosomes is a genetically controlled phenomenon, but environmental conditions play a significant role in modulating this method. Oxygen and redox potential play an important role in the composition of the magnetosomes of MTB. Magnetosome formation is favored in low-oxygen environments, as high oxygen levels can repress biomineralization. Redox modulation is critical, with specific redox proteins and pathways influencing magnetosome synthesis.92 The redox potential within magnetosomes is typically between −0.25 and −0.6 V, supporting magnetite formation.93 In addition to this, nutrient carbon sources also guide the composition of the magnetosomes. For instance, the availability of nutrients, particularly Fe, is essential for magnetosome formation. MTB can accumulate large pools of iron, which are distinct from the magnetite crystals, indicating efficient iron uptake and storage systems.93 Carbon sources such as Na-pyruvate have been shown to enhance magnetosome yield, with optimized culture conditions significantly increasing production.94 Some studies have shown that magnetosome synthesis is sensitive to pH and temperature. Optimal conditions for M. gryphiswaldense include a pH of 7 and a temp. of 28°C. Deviations from these conditions can limit magnetosome formation.95 High-resolution imaging has shown that variations in pH and temperature can lead to differences in magnetosome morphology and size.95 Some studies have proven that iron absorption and biomineralization directly impact magnetosome formation and composition. Iron uptake is a critical step in magnetosome formation, involving specific proteins and pathways. The process includes iron transport into magnetosomes, biomineralized into magnetite or greigite.71 The mode of iron absorption and the dissolved iron concentration significantly affect the efficiency of magnetosome production.94

No doubt environmental factors significantly influence magnetosome composition, but the genetic control of biomineralization remains a dominant factor. The interplay between genetic and environmental influences ensures magnetosomes’ high structural and compositional perfection, which are crucial for their applications in biotechnology and medicine.56,95 Understanding these dynamics can lead to improved methods for magnetosome production and potential applications in various fields.

Properties: Physicochemical and Magnetic and Their Relevance

MTB exhibit unique physicochemical and magnetic properties due to their ability to synthesize magnetosomes, which are intracellular MNPs. These properties facilitate magnetotaxis, allowing bacteria to navigate geomagnetic fields and hold significant potential for various applications, particularly in biotechnology and medicine.31 These magnetosomes, primarily composed of magnetite (Fe₃O₄) or greigite (Fe3S4), are arranged in chains that enhance the bacteria’s magnetic orientation and mobility. The magnetosomes have magnetite, which has ferromagnetic behavior.86 Besides this, it has a stable magnetic moment and adds physiological temperature. The MNPs formed inside the MTB are present in uniform chains coated with various biological molecules, which prevent their aggregation.

Physiochemical Properties

The concept of a chain of spheres is employed to demonstrate the magnetostatic interactions that underlie the magnetic features of MTB and isolated magnetosomes, principally composed of magnetite crystals. Magnetosomes are mainly 30–120 nm in size and are enclosed in a lipid bilayer, forming linear chains that behave like magnetic dipoles. Magnetosomes primarily comprise magnetite or greigite NPs, with high purity levels exceeding 99.9% in some strains.29

The magnetostatic interactions between grains organized in chains block the magnetic transitions, even avoiding the external magnetic field for crystals as small as 30 nm. The arrangement of magnetosomes into chains appears essential for their maturation, but its impact on MTB cell division is minimal, according to experiments and computational models. Early in the biomineralization process, researchers looked at alterations in the state of iron oxidation and magnetosome crystal size distribution as a function of time. Controlled magnetosome solids, including MGC genes, can be impacted by budding MTB in a magnetic field weaker than the geomagnetic field.71

Magnetic Properties of Magnetosomes

The magnetosomes are typically large nanoparticles with a single magnetic domain. Under physiological temperatures, this guides the development of a magnetic moment that remains stable against thermal fluctuations.86 Consequently, it produces a magnetism surpassing chemically induced magnetic NPs, which are generally superparamagnetic and hold a temperature-based magnetic moment. This results in a larger coercivity (Hc 20–40 mT) and the remanent to saturation magnetization ratio (Mr/Ms 0.4–0.5). These magnetic qualities result in superior MRI causes, including temperature-induced capabilities toward the magnetosomes under specific circumstances.30

Magnetosomes are organized in chains, functioning like a compass needle, which enhances the bacteria’s orientation in magnetic fields.29 Different crystal morphologies of magnetite within magnetosomes, such as cuboctahedron and bullet shapes, exhibit distinct magnetic behaviors, influencing their coercivity and magnetic moments. The magnetic features of MTB are influenced by the arrangement of magnetosomes into chains, which behave like compass needles. For instance, the coercivity of these chains can vary significantly, with some exhibiting low coercivity (<30 mT) and others high coercivity (>50 mT). Pie et al calculated that larger MTB cells with bullet-shaped magnetosome chains have higher magnetic moments, enhancing magnetic navigation by overcoming viscous resistance.96

Studies have shown that magnetosomes exhibit distinct hysteresis loops, with coercivity values ranging from 9.8 mT to over 50 mT, depending on the crystal morphology.96,97 Exposure to static magnetic fields can enhance cell growth but may inhibit magnetosome formation, indicating a complex interaction between environmental magnetic fields and bacterial metabolism.98 The magnetic parameters of various Magnetospirillum strains have been characterized, revealing stable single-domain states and superparamagnetic properties crucial for their functionality.99 Studies have shown that magnetic hysteresis and effective magnetic anisotropy are critical for the magnetotactic behaviour of these bacteria.29

Growth, Isolation, and Purification of Bacterial Magnetosomes Growth of MTB in the Laboratory

MTB from their natural habitats could be isolated by various means, and the most widely accepted method is microfluidic system-based isolation, where the ferrofluids and the machinery are arranged so that MTB can be isolated with different amounts of magnetism.100

Despite numerous research attempts, only a few instances of this specific species of bacterium have been found since its discovery. MTB are extremely common and are found in considerable quantities in the sediments of various freshwater and marine ecosystems; however, their meticulous lifestyle makes MTB challenging to isolate and cultivate. Only a few MTB strains, primarily of the genus Magnetospirillum were purified and identified.25 Currently, γ-proteobacteria, δ-proteobacteria, Nitrospirae, and candidate phylum Omnitrophica are home to most MTB found. Magnetospirillum magnetotacticum strain MS-1, Magnetospirillium gryphiswaldense strain MSR-1, Magnetospirillum magneticum strain AMB-1, Magnetococcus marinus strain MC-1, and D. magneticus strain RS-1 were among the MTB strains that were successfully separated and cultured in the laboratory conditions.101

Isolation of Magnetosomes

Isolated magnetosomes resemble synthesized magnetic particles during the development of chain structures and variation in the rheological features in a suspension.56 The impact of the filament joining them in MTB and the power limitations of the magnetosome magnetostatic contact on particular sequences have been considered in mathematical models that detail the mechanical strength of magnetosome sequences. Immobilizing MTB engaged by the semisolid substrate of hydrated silica impacts the response of the magnetosome chain to shifts to align a particular magnetic zone, guiding sequencing units reorientation and structural disorder. However, applying MF (1 T) did not result in the disintegration of the chain for freeze-dried immobilized MTB.56

The first step involved in the isolation of magnetosomes from MTB is exposure to air, ultrasonic cell lysis or using a French press in a laboratory, agitation in a shaker, magnetic isolation of the magnetosomes or the elimination of the cellular debris by centrifugation followed by multiple times washing with distilled water and buffered solutions. The magnetic properties of magnetite can be controlled in a programmed MTB culture method by varying oxygen amounts to tailor them for the intended use. One mg of lyophilized MTB yields a mass of dry particles of only 20 g or less biomass. Under optimum conditions, the maximum daily dry biomass production can reach approximately 150 mg/L of culture.102

Purification of Bacterial Magnetosomes

It is well-studied that the magnetosomes made in the MTB have been isolated and purified by various techniques. Of all such purification techniques, one approach involves the growth of MTB under optimized conditions inside a bioreactor. Once the cells were grown completely, it was lysed by different methods like ultrasonication and organic solvent treatment. Once the cells were lysed, the magnetosomes were collected using an external magnet.103

Recently, Martinez et al extracted and purified the magnetosomes from M. gryphiswaldense MSR-1 by three approaches, ie, enzymatic treatment, probe sonication, and high-pressure homogenization. Further, the investigators studied a systemic comparison between all these three methods. In this experiment, the focus was to study the effect of the extraction techniques on the chain length, integrity, and aggregation state of magnetosomes isolated from M. gryphiswaldense MSR-1 cells. The cell disruption yield from all three methods was about (>89%).104

Mass Production of Magnetosomes

The mass production of magnetosomes from MTB is a promising area of research due to its exceptional features and potential applications in nanomedicine. Various approaches have been developed to enhance the yield and purity of magnetosomes, which focus on optimizing bacterial cultivation, cell disruption, and purification processes. These advancements aim to overcome the hurdles associated with the low natural yield of magnetosomes and the complexity of their extraction and purification. Earlier, some of the studies achieved a significant number of magnetosomes using a specific strain where the yield has reached up to 170 mg/L/day. The magnetosomes have good biocompatibility and low toxicity when manufactured under appropriate circumstances. Finally, magnetosomes can be produced by growing MTB in a non-toxic growth medium (for instance, ATCC medium 1653 for the AMB-1 species).69

In some of the cases, cultivation strategies have been applied, like high cell density fermentation. In this technique, a scalable platform using M. gryphiswaldense MSR-1 has been developed, achieving high cell density and magnetosome yield through a two-stage continuous fermentation process. This method allows for efficient cell disruption and magnetosome recovery, with the potential for continuous production.97 Chades et al also developed and established PCB for M. gryphiswaldense MSR1, enabling large-scale bacterial amplification under minimal growth conditions, producing highly pure magnetosomes with over 99.9% iron content.29 Corrêa et al, 2022 used a fed-batch strategy for M. blakemorei, supplemented with iron and nitrous oxide, followed by continuous culture. There was improved magnetosome production and productivity.105

In some of the cases, cell disruption and purification methods were applied to achieve efficient magnetosomes. For instance, Masó-Martínez et al, 2023 applied high-pressure homogenization as an effective method for preserving magnetosome chain integrity during cell disruption, compared to enzymatic treatment and probe sonication.104 Fernández-Castané et al, 2024 used high-gradient magnetic separation and magnetically enhanced density separation to purify magnetosomes, achieving high purity and yield without damaging the crystal structure.97

Some of the studies applied a genetic approach to increase the yield and purity of the magnetosomes. For instance, Dziuba et al, 2023 transferred the magnetosome biosynthesis pathways into non-magnetic bacteria. Further, the transformed bacteria have shown potential, although challenges remain in achieving high yields in new hosts.106 Some of the investigators used synthetic bacteria for the production of magnetosomes. For instance, Mickoleit et al, 2019 genetically modified R. rubrum to produce magnetosomes, with cultivation conditions optimized for high yield and potential for functionalization.66

Characterization of Magnetosomes

Characterization techniques for the magnetosomes of MTB involve a variety of advanced methodologies that enhance the understanding of their magnetic properties and structural integrity. These techniques are crucial for fundamental research and potential applications in biotechnology and medicine. The detailed investigation of the magnetosomes by various analytical techniques will help in their specific applications in nanomedicine.31 Various investigators have reported the electrical and physicochemical aspects of the bacterial magnets; for instance, it has been analyzed by spectral techniques like X-ray diffraction (XRD), Fourier transform infrared (FTIR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), Mossbauer spectroscopy, vibrating sample magnetometry (VSM), microscopy such as Magnetic Force Microscopy (MFM), scanning electron microscope (SEM), transmission electron microscope (TEM), etc.107 All these instrumentation techniques revealed detailed morphological and elemental information about the bacterial magnetosomes. All these microscopic techniques helped confirm the shapes, spectroscopic techniques helped identify several functional groups on the surface of these magnetosomes, and the XRD technique helped determine the crystalline nature of the MTB and their magnetosomes and magnetite. The most crucial role of these instruments is Raman and Mossbauer spectroscopy, which helps identify and confirm iron oxides’ phases (magnetite, maghemite, or hematite) in the magnetosomes. Electron diffraction spectroscopy (EDS) is another technique that helps reveal various elements in the bacterial magnets, ensuring their purity. Besides this, since a biological membrane encloses bacterial magnets, their analysis could also be done using a carbon-hydrogen nitrogen and sulfur (CHNS) analyzer to detect carbon-hydrogen composition.108

The ultrastructure of MTB has been extensively examined through a range of electron microscopy procedures, with a specific emphasis on methods utilizing signals from inelastically scattered electrons. It is possible to map the scattered elements, their compositional ratios, and the binding energies of chemical bonds inside MTB using EDS and XRD techniques. New advancements in TEM enable high-resolution direct observation of the biomineralization procedure utilizing a liquid graphene cell with an encapsulated 1 µL sample of MTB.

Marqués-Marchán et al, 2024, have used MFM to characterize individual magnetosome chains, overcoming challenges posed by their low magnetic signal and the larger size of the MTB. Custom probes enhance sensitivity in various environments, allowing for quantitative data collection under in-situ magnetic fields.31 Some studies have used ultrasensitive torque magnetometry (UTM) to measure individual bacteria’s magnetic hysteresis, revealing magnetosome configurations and magnetic moments. This is complemented by transmission electron microscopy (TEM) to visualize the magnetosome structures. In some of the studies, dynamic light scattering (DLS) and nano-flow cytometry (nFCM) were used to provide insights into magnetosome size and quality.104

Crystallographic measurement

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