Abstract
Nanoparticle research has greatly benefitted medical imaging platforms by generating new signals, enhancing detection sensitivity, and expanding both clinical and preclinical applications. For magnetic resonance imaging, the fabrication of superparamagnetic iron oxide nanoparticles has provided a means of detecting cells and has paved the way for magnetic particle imaging. As the field of molecular imaging grows and enables the tracking of cells and their molecular activities so does the possibility of tracking genetically programmed biomarkers. This chapter discusses the advantages and challenges of gene-based contrast, using the bacterial magnetosome model to highlight the requirements of in vivo iron biomineralization and reporter gene expression for magnetic resonance signal detection. New information about magnetosome protein interactions in non-magnetic mammalian cells is considered in the light of design and application(s) of a rudimentary magnetosome-like nanoparticle for molecular imaging. Central to this is the hypothesis that a magnetosome root structure is defined by essential magnetosome genes, whose expression positions the biomineral in a given membrane compartment, in any cell type. The use of synthetic biology for programming multi-component structures not only broadens the scope of reporter gene expression for molecular MRI but also facilitates the tracking of cell therapies.
Keywords
- magnetosome
- iron biomineral
- reporter gene expression
- iron contrast
- magnetic resonance imaging
1. Introduction
With over a 20-year history, the field of molecular imaging is now well-entrenched [1, 2, 3] and continuing to expand its influence over multiple imaging modalities, including optical [4], nuclear [5], magnetic resonance (MR) [6] and acoustic [7]. In all these platforms, the use of contrast agents is a central theme, to enhance tissue structure and differentiate between healthy and diseased cells. Image-guidance has been achieved with simple molecules like the fluorophore indocyanin green [8], with macromolecules like antibodies [9], and with synthetic particles like superparamagnetic iron oxides (SPIO) [10] or perfluorocarbon emulsions [11]. Moreover, by adding targeting groups to these contrast agents, additional tissue specificity and/or image resolution may be obtained.
Despite these attributes, there are challenges in biomarker development for medical imaging, such as longevity of the signal and intrinsic biological activity. Exogenous contrast agents that reach their cellular targets may still be lost during cell division, metabolized, or decay too rapidly for effective longitudinal study. In addition, their role as beacon does not necessarily provide a measure of inherent biological activity. One solution is to adopt a gene-based approach in which contrast is synthesized by the cell and thus remains with it throughout its life cycle. Not only does this type of endogenous contrast get passed to daughter cells, it also permits reporter gene expression in response to biological cues. In this way, using the tools of molecular biology, cellular contrast may be directly linked to the presence of proteins (
In this chapter, the development of gene-based contrast for MR detection will be described using the bacterial magnetosome as a model for biogenic iron biominerals. Integral to this discussion are the factors that regulate gene expression, determine protein localization, guide macromolecular assembly, and permit iron crystal formation without the need for exogenous contrast agent.
2. Magnetosome model
The magnetosome is a remarkable structure synthesized by magnetotactic bacteria (MTB) [13]. These micron size cells produce nanometer size iron crystals for magnetotaxis, responding to the earth’s magnetic field through the creation of a single magnetic dipole within each biomineral. Ingeniously, to avoid cytotoxicity associated with the oxidation and reduction of iron, crystallization proceeds within a protective compartment,
In MTB, magnetosome biosynthesis is thus a protein-directed process, genetically encoded by structural genes arranged in units, termed operons, and located largely in a gene cluster, termed the magnetosome genomic island. Of the approximately 30 genes involved in magnetosome formation, roughly one third are located elsewhere in the bacterial genome, possibly indicative of magnetosome protein interactions with common cellular components. In support of this, mammalian cation diffusion facilitator protein complements bacterial MamM function [19]. In addition, mammalian molecular motors appear to interact with MamL [20]. While more studies are required to fully elucidate magnetosome structure, and potentially reproduce it in other cell types, the following functional categorization may prove useful for dissecting the steps and partners involved in magnetosome formation.
2.1 Membrane designation
Mutations designed to delete individual magnetosome genes from MTB have exposed the absolute requirement of a select few genes for magnetosome production. When anyone of these essential genes is missing, there is either no magnetosome vesicle and/or no biomineral [21]. Among these genes are
With a view to forming a rudimentary magnetosome-like nanoparticle in any cell type, we have proposed that essential magnetosome genes constitute a common base upon which diverse biominerals are synthesized [22]. This notion is predicated on the specificity of certain protein–protein interactions, needed to establish the magnetosome as a distinct structure. Plausibility is evident based on genomic sequencing and the commonality of sequence across diverse classes of MTB [26]. Likewise, large scale magnetosome gene expression has been successfully tested in a non-magnetic bacterium [27]. In this work, magnetosome related operons from the magnetotactic bacterium
Toward understanding the genetic make-up of a rudimentary magnetosome-like nanoparticle, MamI-MamL interactions have recently been described in a mammalian cell system [28]. This work showed that (1) MamI and MamL are compatible with a mammalian cell expression system; (2) MamL specifically recruits MamI to the same intracellular location despite co-expression in the complex intracellular environment of the mammalian host; and (3) MamL particles, alone and in the presence of MamI, also interact with putative mammalian molecular motors. These findings suggest that MamL may have a role in anchoring magnetosome assembly within a given membrane and raises the possibility that MamL also forms previously unrecognized cytoskeletal connections in MTB. Such a dual function further implies that membrane localization and magnetosome assembly may be initiated simultaneously, accounting for the essential role of MamL in both vesicle formation and subsequent biomineralization.
2.2 Protein recruitment
There are numerous corollaries to be considered for optimal expression of magnetosome-like nanoparticles in foreign non-magnetic cells. If the role of MamL is indeed to designate the membrane compartment, then eukaryotic cells equipped with vesicles may yet form magnetosomes by drawing on only those genes that attract biomineralizing activities (Figure 2). This would simplify magnetosome biosynthesis in eukaryotic cells. This is not to say that genetic encoding of vesicle formation should be ignored. A fuller understanding of how magnetosome vesicles form may be useful for ultrasound technologies that would benefit from reporter gene expression (discussed below). If the role of MamL lies in recruitment of magnetosome proteins involved in iron crystallization, then perhaps vesicle formation is largely carried out by other magnetosome proteins that shape the vesicle and accommodate biominerals of varying dimensions and morphologies [13, 21]. To this point, seven
Interestingly, there may be a dual role for MamI in both iron crystal nucleation [30] and size of the magnetosome vesicle [31]. Using a mammalian expression system to substantiate this hypothesis, we showed that MamI-derived contrast significantly increases MRI transverse relaxivity over the parental control, when cells are cultured in the presence of an iron supplement [32]. In this work, cells were mounted in a spherical gelatin phantom and placed in a knee coil for scanning at 3 Tesla using previously described MR sequences [33]. With this experimental setup, measurements obtained from a compact layer of cells can be assessed in any cell type, expression system and treatment condition. Using the same expression system and human melanoma cell line, the motility of fluorescent MamL particles increased in the presence of MamI, influencing both directed and Brownian motion and suggesting that particle size may be more compact in the presence of MamI [20]. These unexpected findings, from two small but essential magnetosome genes, reflect at once the beauty and simplicity of the MTB genome in its capacity to streamline the formation of magnetosomes using a minimum of genetic encoding.
2.3 Rudimentary nanoparticle
Given these findings, we might expect that the distinction between magnetosome vesicle formation and iron biomineralization is not so clear-cut. A subset of magnetosome genes, perhaps the essential genes, may link the two fundamental processes that define the magnetosome,
There is still much to learn about magnetosome assembly. Ideally, its formation in any cell can be accomplished by adapting the needed set of instructions from MTB. Toward this goal, the emerging picture of magnetosome assembly indicates that bifunctional proteins link one magnetosome component to the next, progressively defining the magnetosome compartment and biomineral. Until we can properly define how each genetic feature fits together, a rudimentary magnetosome-like nanoparticle is likely to bridge the gap created by our partial understanding of magnetosome biology. Since different cell types have different abilities for building and tolerating membrane-enclosed vesicles, research in this area should continue to expose fundamental processes involved in both magnetosome vesicle formation and iron biomineralization.
3. Iron biomineralization
Virtually all cells regulate iron carefully to prevent cellular damage from free radical formation all the while retaining access to a pool of iron co-factor, needed to drive vital cellular processes [36]. The magnetosome is a fine example of iron sequestration for the purpose of magnetite formation. This iron oxide (Fe3O4) has the necessary superparamagnetic properties for effective MRI detection [37]. Indeed, theoretical calculations indicate that approximately 3000 cells/voxel could be detected on large animal/human scanners at 3 Tesla if mammalian cells could be engineered to express the same magnetosomes as found in MTB [22]. On small animal scanners, this improves to as few as 3 cells/voxel. Therefore, a fuller understanding of how to regulate magnetosome formation will ultimately provide MR platforms with a sensitive and versatile method for long-term tracking of cells and their molecular activities.
Use of the magnetosome for this purpose in mammalian cells requires that some iron be diverted from its usual pathways of distribution, namely iron uptake, storage and export [38]. Little is known about how iron uptake into a magnetosome-like vesicle will compete for the available cellular iron. Factors to consider include the cell’s labile iron pool and response to shifts in iron homeostasis. For example, the mouse, multi-potent P19 embryonic carcinoma cell line is an iron exporting cell type, with high iron import and export activities similar to alternatively activated macrophages [39]. This cell type is programmed to recycle iron and, as such, retains very low levels of iron storage. Furthermore, P19 iron export is hormonally regulated by hepcidin, which induces a transient decrease in iron export protein (ferroportin) and an increase in the relationship between MR transverse relaxation rates and total cellular iron. In addition to this endocrine response, P19 cells secrete hepcidin activity that effectively decreases ferroportin levels in human THP-1 monocytes, indicating the ability for paracrine and/or autocrine regulation of cellular iron content [40]. How will the formation of magnetosome-like particles affect iron homeostasis in multi-potent cells like P19?
Despite the complexity of P19 iron metabolism, we know the cell’s capacity for iron retention is increased by expression of the MTB gene
Early results with mammalian MamI-expressing cells indicate the same capacity for enhancing the iron-related MR transverse relaxation rates as MagA-expressing cells [32, 33]. In a direct comparison, using the same MDA-MB-435 host cell, both irreversible
4. Applications in molecular imaging
The use of reporter genes to track molecular activity, and therefore cellular activity, is well known in biology. Reporter genes have provided all sorts of signals that may be detected optically under a microscope in cells or histological sections, or by using luminometry or chromatography on tissue extracts. Adapting reporter genes for non-invasive molecular imaging is an enabling technology that adds spatial information in the context of a living subject as well as the possibility of repetitive imaging for longitudinal study of
Nevertheless, the magnetosome is an interesting nanoparticle with multiple possible applications in molecular imaging. Magnetosomes may serve as a gene-based, contrast agent for tracking cell therapies without the need for exogenous substrate. By sequestering iron, the magnetosome is ideal for MR signal detection on various modalities, including MRI, hybrid imaging with positron emission tomography (PET)/MRI and MPI. The nature of the magnetosome biomineral may also be used to amplify and manipulate MR signals, by varying iron content and form [39]. For instance, in cultured P19 cells the negative regulation of iron export by hepcidin does little to increase total cellular iron; however,
Adding gene-based contrast to this mix widens the scope of MR detection even further. Genetic regulation of nanoparticles [50] means that expression of the magnetosome can be tailored to include desirable features or exclude what is not needed. For example, magnetosomes with genetically programmed, cell surface modifications have been prepared for a variety of applications from magnetic separation [51] to cancer diagnosis [52] and therapy [53]. In these examples, modified magnetosomes are isolated from MTB, ensuring the biomineral is fully formed and has the expected magnetic properties. A breast cancer model was used to compare isolated magnetosomes with chemically synthesized SPIO coated with serum albumin [54]. Both types of nanoparticle were crosslinked with fluorescent-labelled antibody to the epidermal growth factor receptor and examined in cultured MDA-MB-231 cells and their tumour xenografts. The modified magnetosomes outperformed SPIO with respect to MR signal and tumour distribution. At high field strength, low doses of iron in purified magnetosomes gave higher
A compelling future strategy entails direct expression of rudimentary magnetosome-like nanoparticles in any cell type. Envisioning gene-based contrast of this nature for molecular imaging, using essential magnetosome genes to produce partially formed magnetosomes, is still under development (Figure 3). Clearly, MRI detects significant increases in mammalian cell contrast derived from single, MTB and magnetosome gene expression systems, including
The combination of magnetosome genes that further elevates the MR signal is anxiously anticipated. Can we use the rudimentary magnetosome-like particle, consisting of essential magnetosome genes, to fashion nanoparticles that are MR silent until complemented by the protein(s) that trigger assembly of the complex or activation of biomineralization? Will subcellular arrangement of nanoparticles be sufficient to alter the MR signal? To what degree will changes in iron form or content alter MR detection? What types of cellular activity could be programmed to modulate magnetosome-like nanoparticle expression in mammalian cells?
4.1 Reporter gene expression
A special application of gene-based contrast is referred to as reporter gene expression. Basically, this is the difference between constant expression of the reporter gene versus its selective expression. Regions on DNA that promote gene expression (
Historically, most reporters are single gene expression systems that encode any protein for which there is a suitable means of detection. Of course, how the reporter signal is detected is intrinsically connected to the type of sample used for measurement and the available equipment. Luminometry using the reporter gene firefly luciferase, for example, began as a routine tool for the analysis of cell extracts but expanded to include small animal bioluminescence imaging once these scanners became available. For MR applications, however, single iron-handling reporter genes do not afford a large enough signal to be competitive with chemically synthesized SPIO. Since the evidence in MTB indicates that iron biomineralization
The unique protein–protein interactions found in single-cell organisms like prokaryotes offer a unique opportunity to build reporter gene expression systems in eukaryotes that faithfully reproduce complex structures for non-invasive imaging modalities. The magnetosome is easily such a candidate, well-suited to MR signal detection platforms by virtue of its iron biomineral. Just what facsimile of this nanoparticle is required for a given application still needs to be properly defined. For MPI, uniform, well-formed iron crystals are required; however, genetically programming variations in biomineral size would provide distinct signals for reporter gene expression [22]. For MRI, there is a great deal of latitude in magnetosome-like particle detection, given the sensitivity of transverse relaxation rates to both the quantity and form of iron. Building reporter gene expression around multiple TF signals that successively add desirable features to the magnetosome-like particle, enhancing MR detection at each step, opens a new frontier in non-invasive imaging. This vision begins with the understanding of magnetosome root structure.
5. Conclusions
Medical imaging has transformed medical care: guiding diagnosis and the timely delivery of therapy, monitoring treatment success and avoiding unnecessary procedures. MRI, with its superior soft tissue resolution and depth of penetration in a non-ionizing form of radiation, is continually expanding its reach. To keep up with inroads in pre- and post-natal care [67, 68], pediatric MRI [69], specialty coils for the brain and cardiac imaging [46], as well as inserts for hybrid PET/MRI [70], there is a continuing need to foster technological developments in MR-sensitive contrast agents. Cellular imaging is enabled by magnetic nanoparticles. Furthermore, molecular imaging successes achieved with exogenous SPIO [47] indicate that future imaging with gene-based contrast is a realistic expectation. To this end, the magnetosome offers the necessary blueprint for patterning iron biomineralization in a safe and effective way.
Gene-based contrast permits greater understanding of a given disease process because it can be tied to the gene expression responsible for the cell’s behaviour, be this oncogenic, inflammatory, fibrotic, infectious, apoptotic, or the lack of appropriate signal transduction. While genetic regulation of contrast gene expression will initially pertain to preclinical research in animal models, many learnings will benefit clinically useful cell therapies either directly or indirectly. Microbiome research, for example, has already led to widely accepted probiotic supplements and experimental procedures like fecal microbiota transplantation [71]. Stem cell therapies are likewise destined to become mainstream. Developing the methods to visualize these therapies, deep within the body, is of paramount importance [72]. Holding back both microbial and mammalian cell therapies is an understanding of where these cells disseminate once introduced, how long they remain in the body, and how well they function. Molecular imaging of cellular activity holds the answer to many of these questions.
The introduction of multi-component assemblies as contrast agents for molecular imaging is an exciting new direction in nanoparticle research. Compared to single gene expression with injected contrast agent as substrate [73], multi-gene complexes offer a wider variety of imaging opportunities. For example, there may be no need for exogenous substrate, as assembly of the structure itself provides the imaging signal. In addition, there may be multiple levels of regulation, permitting finer control of assembly, disassembly and perhaps reassembly under the correct circumstances. This opens the possibility of creating suboptimal structures that are imaging silent until complemented by gene expression that switches on a detectable signal. Developing such structures could involve a role for constitutive and reporter gene expression. Further, by augmenting contrast incrementally, different stages of development could be monitored in (stem) cells that fulfill their therapeutic mission by reaching a terminally differentiated phenotype. This would also permit troubleshooting cell therapies that fall short, including (re)programming the timing of signal detection to validate stages where therapeutic function was successfully delivered.
The magnetosome is formed in a multi-step process that is regulated by a cohort of essential and auxiliary proteins. The genes that encode this process sequester iron in a membrane-enclosed compartment, shaping the biomineral while protecting the cell from iron toxicity. Can other cells be taught how to synthesize a magnetosome-like nanoparticle? Research is steadily showing this is the case. What then are the essential components required in any cell to reproduce the main structure? The notion that a minimal root structure underlies magnetosome formation has been advanced. Are all features of the bacterial magnetosome necessary? The MR evidence indicates that select magnetosome genes provide a measure of contrast enhancement when individually expressed in mammalian cells. What then are the protein(s) required for biosynthesis of the most desirable MR signal(s)? As outlined in this chapter, the magnetosome genes that define this compartment are steadily being elucidated, demonstrating that iron biomineralization can be programmed in all types of cells.
Acknowledgments
DEG is supported by a grant from the Ontario Research Fund Research Excellence program, ORF-RE07-021, in partnership with Multi-Magnetics Inc.
Conflict of interest
The use of magnetosome genes in mammalian systems is patented technology assigned to Multi-Magnetics Inc.
Appendices and nomenclature
magnetic particle imaging magnetic particle membrane specific magnetic resonance magnetosome membrane magnetotactic bacteria superparamagnetic iron oxides
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