Paramagnetic NPs

SPIOs generate MRI contrast by the production of signal voids on T2- or T2-weighted images. Whereas new tailored imaging sequences can partially recover this loss in signal intensity, inducing "white marker" contrast by exploiting phase and frequency differences generated by the SPIO (Mani et al., 2006; Cunningham et al., 2005), an alternative would be to produce positive (T1) contrast by the use of paramagnetic contrast agents. This dependence on the selective interaction with cellular water may have additional advantages for molecular and cellular imaging. For instance, it opens up the possibility of imaging the cell directly, as well as the design of smart probes that are responsive to changes in water exchange due to gene expression (Louie et al., 2000). On the other hand, this dependence raises the possibility of changes in contrast due to cellular compartmentalization (Terreno et al., 2006) and increased toxicity if the unchelated paramagnetic agent is released outside of the endosome.

Gadolinum-based contrast agents have been used successfully for both molecular and cellular imaging. Artemov et al. (2004) demonstrated successful targeting to Her-2/neu receptors and fibrin using relatively low payloads of 12.5 Gd ions per ligand. Overall, this approach has had limited success due to the necessity to increase the payload of contrast agent per ligand or cell to overcome the much weaker relaxitivity of Gd compared to the SPIO. It has been estimated that the number of Gd-chelates needed to visualize cells is around 100-1000 chelates or 10-100 |M per cell (Arbab et al., 2006a). This payload can be problematic for stem cell labeling due to the small amount of noncarrier-mediated uptake into many types of progenitor cells (Zheng et al., 2005). Therefore, approaches must be used to concentrate Gd contrast agents within the cell to a larger degree necessary than that required for SPIO. This cellular uptake has been achieved using cell translocation peptides such as HIVtat (Bhorade et al., 2000) peptides, polyarginine peptides (Allen et al., 2004), or lipophilic complexes (Zheng et al., 2005). In addition, unchelated forms of lanthanides are toxic to the cell. Careful examination of cellular function and paramagnetic complex stability at low pH (i.e., inside a endosome/lysosome) need to be performed. However, there is little doubt that cell and molecular MR imaging will benefit from ongoing research focused on designing paramagnetic complexes endowed with improved relaxivity within the cellular milieu.

An alternative approach for increasing cellular paramagnetic contrast agent is to increase paramagnetic payload of the contrast agents through the generation of fullerenes (Anderson et al., 2006), nanoparticulates (Modo et al., 2002; Santra et al., 2005b; Sharma et al., 2006; Sitharaman et al., 2005; Tan and Zhang, 2005), or liposomes (Mulder et al., 2006a,b). Such complexes have large surface areas that can be functionalized to contain contrast agents, progenitor cell-specific ligands, and cell penetration peptides. With the capacity to carry 50,000-90,000 gadolinium ions per particle, ~250 nm liquid perfluorocarbon nanoparticle emulsions by Wickline and Lanza et al. have been functionalized with paramagnetic chelates and homing ligands (fibrin, integrins) incorporated within an outer phospholipid surfactant monolayer (Lanza et al., 2004a,b; Morawski et al., 2005; Schmieder et al., 2005). Payload increases boost the molecular relaxivity of the nanoparticle to many times greater than the relaxivity of the Gd chelate alone, resulting in the detection of as few as 100 pmol/L of nanoparticles with a contrast-to-noise ratio of 5.57 even at 1.5 T (Morawski et al., 2004).

In most cases, these paramagnetic complexes can be coupled to optical dyes (i.e., quantum dots), which allow for the ex vivo corroboration of the MRI results or coupling to in vivo optical measurements capable of single cell tracking (Stroh et al., 2005b). For instance, during the last decade, the water-in-oil (W/O) microemulsion technique has become a very powerful tool to synthesize monodisperse nanoparticles (NPs) of various kinds (Arriagada and Osseo-Asare, 1999; Lin et al., 2003; Qhobosheane et al., 2001; Santra et al., 2001a,b 2004; Xiao et al., 2003), including magnetic NPs (Santra et al., 2001b), semiconductor quantum dots (Xiao et al., 2003; Yang et al., 2004), dye-doped NPs (Qhobosheane et al., 2001; Santra et al., 2001a,b), etc. at room temperature (Figure 6.3) (Santra et al., 2005a). The beauty of the W/O microemulsion technique is its versatility. It does not require drastic experimental conditions such as high temperature or high pressure. A variety of "tailor-made" NPs, such as core-shell or doped NPs, can be readily synthesized. It is also possible to synthesize monodisperse NPs as small as a couple of nanometers to several hundreds of nanometers by controlling the synthesis parameters. Previous studies report the synthesis of bifunctional contrast agents for dual (fluorescence and magnetic) imaging in vivo (Daldrup-Link et al., 2004; Huber et al., 1998;

Figure 6.3. Diagram of a multifunctional nanoparticle (A) generation using W/O microemulsion technique that is both MR (B; top) and optically active (Rubpy; B; bottom). T1 weighted MRI of PBS, and equal concentrations of a clinical available contrast agent and the Gd-Rubpy NP. A is reprinted from Santra et al. (2005a). Reproduced with permission from WILEY-VCH Verlag.

Figure 6.3. Diagram of a multifunctional nanoparticle (A) generation using W/O microemulsion technique that is both MR (B; top) and optically active (Rubpy; B; bottom). T1 weighted MRI of PBS, and equal concentrations of a clinical available contrast agent and the Gd-Rubpy NP. A is reprinted from Santra et al. (2005a). Reproduced with permission from WILEY-VCH Verlag.

Kircher et al., 2003; Sosnovik et al., 2005; Veiseh et al., 2005). In this contrast agent design, organic fluorescent dyes were used, which may have limitations for real-time imaging as these dyes often undergo a rapid photobleaching process. Qdots, also known as nanocrystals, are a nontraditional type of semiconductor used for many biological-imaging techniques due to their photostability, brightness, and tuneability (Michalet et al., 2005). In vivo applications of Qdots have just begun to be explored (Gao et al., 2002, 2004, 2005; Gao, 2003; Jiang et al., 2004; Smith et al., 2004; Voura et al., 2004). John Frangioni (Kim et al., 2004; Michalet et al., 2005) and Shuming Nie's (Gao et al., 2004) research groups have successfully demonstrated in vivo bioimaging applications of Qdots for cancer detection (see also Chapter 22 in this book). Frangioni's group took the advantage of near-IR Qdots to perform deep tissue imaging (Kim et al., 2004; Lim et al., 2003; Parungo et al., 2005). Recently a bifunctional paramagnetic 7.5 nm Qdot nanoparticle was synthesized for stem cell tracking by both optical and (Figure 6.4) MR imaging (Yang et al., 2006).

Manganese (Mn), one of the first paramagnetic MR contrast agents, has also been used to label cells, resulting in a decreased intracellular T1 time. Mn enters cells in vivo through calcium channels in the cell membrane, and has been used systemically to image both neuronal and cardiac activity (Arbab et al., 2006a). In cell phantoms, Aoki et al. (2006) have shown that MnCl2 can act as a positive

VcMtmy t

Gd(nr) yi Vreden)

Quantum Dot

Silica

Surface functionalizing agent

Gd chelated to Silica surface x &

Qdot T,

2717ms

Qdot +Gd3+

Figure 6.4. Multimodal quantum dots that generate dual functionalities of yellow fluorescence and paramagnetism. Water-soluble silica-coated ZnS-passivated CdS:Mn (CdS:Mn/ZnS core/shell) quantum dots (A) are functionalized with GdIn ions to impart the paramagnetic property (C) and the Qdot can be used to track labeled cells. (B) Multimodal quantum dots were used to label muscle-derived stem cells and their uptake confirmed in vitro.

contrast agent in allogeneic natural killer cells at 11.7 T. Based on large T changes measured in cell pellets, they estimated that 50-100 cells in a 100 ^m3 voxel should be detectable. Work still needs to be performed on the amount of time that Mn will be retained within the cell and whether any of the toxic effects associated with Mn are observed.

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