Molecular Imaging with Genetically Programmed Nanoparticles

*Donna E. Goldhawk*

## **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 wellentrenched [1–3] 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 (*i.e.* transcription factors, TF) that regulate genetically programmed contrast gene expression [12]. This approach has been tremendously effective with fluorescent proteins and the optical detection of cells and tissues, where depth of penetration is low enough to avoid losses in sample resolution from the scatter of light. Addressing gene-based contrast for other types of non-invasive detection systems is, in general, still a work in progress.

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, *i.e.* a vesicle invaginated from the cell's innermost plasma membrane [14]. Arguably one of the earliest examples of a subcellular organelle [15, 16], magnetosomes are typically arranged in a defined pattern within the cell and connected to cytoskeletal protein (**Figure 1**) [17]. Importantly, various magnetosome membrane (Mam) proteins and magnetosome membrane specific (Mms) proteins enable the compartment to carry out its functions [18]: recruiting the necessary activities to define the vesicle, connecting the magnetosomes to cytoskeletal elements, concentrating iron, defining the crystal, and assembling individual magnetosomes into an effective magnet.

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 *Molecular Imaging with Genetically Programmed Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.96935*

#### **Figure 1.**

*Magnetosome crystal morphologies. Transmission electron microscopy of MTB shows three types of magnetite crystal: cubooctahedral (A), prismatic (B) and bullet-shaped (C). Size, shape, composition, and subcellular arrangement of magnetosomes is generally species-specific. Adapted from Vargas et al. [17].*

production. When anyone of these essential genes is missing, there is either no magnetosome vesicle and/or no biomineral [21]. Among these genes are *mamB*, *mamE*, *mamI* and *mamL*. Numerous other genes may be selectively deleted without damaging the entire magnetosome structure [13]. In this case, what results is a compromised biomineral with a less than perfect crystal, altered size or disruption in cellular location. As the genes responsible for various magnetosome attributes become clearer so does the opportunity for designing nanoparticles that are not only compatible with a given intracellular environment but also impart desirable magnetic properties [22, 23]. For magnetic resonance imaging (MRI), subcellular arrangement of magnetosomes through MamJ-MamK interactions [24] may be a dispensable feature. Likewise, in magnetic particle imaging (MPI) individual magnetosomes constitute an ideal tracer owing to their perfect crystal morphology [25].

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 *Magnetospirillum gryphiswaldense* were transferred to the non-magnetic bacterium *Rhodospirillum rubrum.* Characterization of newly imparted magnetic properties included the appearance of intracellular, electron dense particles by transmission electron microscopy, with Fourier transforms in high-resolution images displaying intensity maxima typical of magnetite. In addition, magnetically transformed *R. rubrum* continued to perform photosynthesis, indicating compatibility between magnetosome-like nanoparticles and normal cellular function. Nevertheless, the minimum number of magnetosome genes required to build the basic magnetosome unit has not been clearly defined. Moreover, this knowledge would greatly enable the rational use of synthetic biology aimed at tailoring magnetosome-like nanoparticles for multiple purposes, above and beyond magnetotaxis, and in a wider variety of cell types.

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 *mam* genes, including the essential ones (*mamB, mamE, mamI* and *mamL*) have been implicated in magnetosome membrane formation in MTB [29].

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 *Molecular Imaging with Genetically Programmed Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.96935*

#### **Figure 2.**

*Modelling magnetosome formation in prokaryotes and eukaryotes. In MTB, genetic encoding of magnetosomes begins with plasma membrane invagination to form an intracellular vesicle (A). Once formed, magnetosome membrane proteins located in this subcellular compartment initiate iron crystal formation (B). The full complement of magnetosome genes specifies the final composition, size, shape, and arrangement of mature biominerals (C). Unlike these prokaryotes, eukaryotic cells readily synthesize intracellular vesicles (denoted in A with a solid line). To designate a magnetosome-like compartment requires a subset of magnetosome genes, providing genetic information for the initiation of biomineralization (outlined in B with stipple). The transition from rudimentary magnetosome to mature nanoparticle (outlined in C with stipple) has not been fully elucidated in non-magnetic (e.g. mammalian) cells.*

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, *i.e.* vesicle and biomineral, by recruiting proteins to a designated site on the membrane and establishing the base structure upon which the magnetosome is elaborated. In cells where the vesicle is otherwise formed, the key challenge is deciphering biomineralization. To this point, the reported activity of MamE fits into this framework [34, 35]. Also provisionally defined as a bifunctional protein, in the absence of MamE there is no biomineral, although, vesicle formation proceeds [21].

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.
