CHAPTER THREE

Recent Advances in Nanoparticle-Based Nuclear Imaging of Cancers Avinash Srivatsan, Xiaoyuan Chen1 Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Lipid-Based Nanoparticles 3. Dendrimers 4. Polymers 5. Quantum Dots 6. Iron Oxide Nanoparticles 7. Gold Nanoparticles 8. Carbon Nanotubes 9. Silica-Based Nanoparticles 10. Conclusion References

84 89 97 98 100 102 108 110 112 117 117

Abstract Nuclear imaging techniques that include positron emission tomography (PET) and single-photon computed tomography have found great success in the clinic because of their inherent high sensitivity. Radionuclide imaging is the most popular form of imaging to be used for molecular imaging in oncology. While many types of molecules have been used for radionuclide-based molecular imaging, there has been a great interest in developing newer nanomaterials for use in clinic, especially for cancer diagnosis and treatment. Nanomaterials have unique physical properties which allow them to be used as imaging probes to locate and identify cancerous lesions. Over the past decade, a great number of nanoparticles have been developed for radionuclide imaging of cancer. This chapter reviews the different kinds of nanomaterials, both organic and inorganic, which are currently being researched for as potential agents for nuclear imaging of variety of cancers. Several radiolabeled multifunctional nanocarriers have been extremely successful for the detection of cancer in preclinical models. So far, significant progress has been achieved in nanoparticle structure design, in vitro/in vivo trafficking, and in vivo fate mapping by using PET. There is a great need for the

Advances in Cancer Research, Volume 124 ISSN 0065-230X http://dx.doi.org/10.1016/B978-0-12-411638-2.00003-3

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2014 Elsevier Inc. All rights reserved.

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development of newer nanoparticles, which improve active targeting and quantify new biomarkers for early disease detection and possible prevention of cancer.

1. INTRODUCTION Noninvasive imaging techniques have shown great promise as agents for understanding diseases at cellular and molecular levels. As outlined by the society of nuclear medicine, molecular imaging is the ability to visualize, characterize, and understand cellular as well as molecular processes in humans and other living organisms. The agents that help accomplish this goal are identified as molecular imaging agents (Hoffman & Gambhir, 2007; Margolis et al., 2007). Molecular imaging is an area of imaging, which provides both anatomical and functional information. In a literal sense, molecular imaging agents provide images of molecules. The idea of imaging is to determine the characteristics of the tumor to develop a treatment regimen so as to get the best possible outcome for the patient. Imaging also allows visualizing the margins of the tumor, whether the tumor has spread to other sites in the body; it is also used for early detection of tumors, and finally, imaging is a powerful tool for real-time monitoring of the treatment. Imaging modalities include a variety of techniques using a number of different characteristics ranging from anatomical, physiological, and molecular properties of tumors. More recently, imaging modalities have been combined with each other to get the maximum information about the tumor so as to decide the best course of action. Based on the type, characteristics, location, stage, and grade of the tumor, clinicians can design the best treatment course for the patient so as to get the best possible outcome. Also, imaging is helpful in monitoring the efficacy of treatment to see if there is tumor shrinkage, and during treatment if the properties of the tumor change, a clinician can reevaluate the treatment paradigm for that patient. Also, once the treatment is complete, long-term monitoring of the patient is important to prevent the recurrence of tumor regrowth. A wide variety of molecular imaging techniques ranging from radionuclide-based positron emission tomography (PET) and single-photon emission computerized tomography (SPECT) and ionizing radiation-based computerized tomography (CT) and X-rays have been used individually or in conjunction with other techniques such as magnetic resonance imaging (MRI), fluorescence-based optical imaging, ultrasound imaging, and newly developed modalities such as photoacoustic imaging and optical coherence

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tomography ( Jokerst & Gambhir, 2011; Jokerst, Lobovkina, Zare, & Gambhir, 2011; Schober, Rahbar, & Riemann, 2009). Imaging systems have already been an integral part in screening, diagnosis, and staging of many cancers (Cai et al., 2006; Hoffman & Gambhir, 2007). Among them, MRI has been a popular tool in tumor detection because of its high depth penetration, spatial resolution, and high soft tissue contrast. Most MRI is based on the use of contrast enhancing agents such as Gadolinium (Gd3+)-based agents (Aime et al., 2002; Caravan, Ellison, McMurry, & Lauffer, 1999). But recent work has found the use of nanoparticles such as super paramagnetic iron oxide (SPIO) nanoparticles and regular iron oxide nanoparticles (IONPs) for improving contrast within tumors (Arbab et al., 2003; Das et al., 2009). The advantage of using such nanoparticles is that they allow for the delivery of other agents such as chemotherapeutic drugs or DNA or other imaging agents as well. Photosensitizers have also helped in the delivery of MRI agents. Porphyrin-based photosensitizers have found to be excellent candidates for the delivery of MRI contrast agents to tumors. Pandey et al. recently demonstrated the development of a photosensitizer (3-vinyl-3-[1-(hexyoxy)ethyl]pyropheophorbide a), which was conjugated to an MRI contrast agent (Gd(III)–aminobenzyl–diethylenetriamine pentaacetic acid (DTPA)) (Spernyak et al., 2010). This agent showed good therapeutic benefit as a photosensitizer as well as an excellent MRI contrast agent, providing a better contrast than Magnevist® at 10 times lower concentration. CT is another imaging method which has found to be very popular in the clinic. It is used primarily for screening, monitoring, and detecting tumors in the clinic. CT is predominantly done using iodine-based radionuclides. The main disadvantage of CT is that it uses X-ray for generation of contrast, which is risky for patients as there is a fear of generation of tumors from prolonged exposure to such type of radiation. But recent work in the field shows that low-dose CT has reduced the risk of secondary tumors and at the same time helped improve the early detection of tumors as well (Iyer, He, & Amiji, 2012). Among the currently available imaging modalities, PET has been a promising candidate in the field of molecular imaging. It is highly sensitive, requiring extremely low doses of imaging agent for detection and has good resolution. SPECT and PET are among the first techniques to be used for diagnosis of diseases in humans clinically (Townsend, Beyer, & Blodgett, 2003). The basic principle behind PET involves the use of radiolabeled contrast agents called as radiotracers, which emit a positron when the radiotracer

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decays. This positron undergoes immediate annihilation with an electron releasing two 511-keV γ-rays 180 apart. A dedicated camera with multiple detectors is used for collecting this energy; this energy is then processed electronically to generate an image showing the localization of the radiotracer within the body (Phelps, Hoffman, Mullani, & Ter-Pogossian, 1975; Ter-Pogossian, Phelps, Hoffman, & Mullani, 1975). Compared to SPECT, PET has greater advantages with respect to sensitivity and resolution and has been gaining in clinical popularity. The number of PET-based studies is expected to reach 3.2 million by 2010. Additionally, PET imaging allows for quantitative mapping of a drug or biomarker in vivo, which can be extremely important in evaluating whether a certain therapeutic can be utilized to target a receptor (Petersen, Hansen, Gabizon, & Andresen, 2012). The ideal properties for a PET radionuclide involve having the maximal positron energy (633.5 keV), which allows for better resolution of the image (Lewis et al., 2008). The half-life of the PET radionuclide used or PET imaging studies is extremely important as well. An ideal radiotracer should have short half-life, allowing for easy chemical synthesis and fast delivery of the agent to the clinic. In the past, the supply of the radiotracers for PET and SPECT was restricted because of limited availability of the relevant isotopes, which had to be produced in nuclear reactors or particle accelerators. With the advent of small cyclotrons, the self-contained radionuclide generator, and the dedicated small animal or clinical SPECT and PET scanners, the demand for SPECT and PET isotopes has shown a significant increase in the last few years (Gambhir, 2002). Most positron emitters have very short half-lives, and the common radionuclides used for PET are nonmetals such as carbon-11 (11C), oxygen-15 (15O), nitrogen-13 (13N), and fluorine-18 (18F) (Louie, 2010). These are common elements that make up biological molecules and thus can be incorporated without disturbing the overall behavior of the molecule. PET is a modern tool in cancer diagnostics and in planning of cancer treatments. Today, the most widely used PET radiotracer is deoxyglucose labeled with radioactive 18F (18FDG) for imaging of tumors. Even though 18FDG has been, and still is, highly successful in diagnosing cancer and shows high imaging quality in many tumor types, there are several cancer forms where 18FDG is not successful, including highly differentiated neuroendocrine (NE) tumors, brain tumors, and prostatic tumors (Binderup, Knigge, Loft, Federspiel, & Kjaer, 2010; Miele et al., 2008). 18F-FDG-PET shows high avidity for the cerebral cortex substantially limiting its usefulness for identifying possible cerebral metastases developing as a result of many known primary

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neoplasms. 18F-FDG-PET has issues with high accumulation in infections or inflammatory tissues resulting in high amounts of false-negative and false-positive cases. This limitation of 18FDG has led to the demand for research into other radionuclides as well as newer radiotracers for clinical diagnosis and imaging. It is preferable to use radionuclides having shorter half-lives in order to reduce the exposure time to the radionuclide in a clinical setting. The short half-lives of the positron emitters used for PET imaging, therefore, require that the loading and labeling procedure be efficient. The biodistribution and pharmacokinetics of a potential PET imaging agent play an important role in influencing and determining its development for a clinical application. Newer PET agents have been designed to take advantage of tumor characteristics. Antibodies, peptides, and ligands have been conjugated to radioactive tracers to target overexpressed receptors, antigens on tumor cells, and tumor vasculature. But a majority of such tracer agents have poor penetration or low bioavailability. For some tracers, conjugation with a peptide or an antibody or a targeting ligand is not efficient, leading to low yield of the radiotracer. These limitations have allowed for renewed interest in researching the use of nanoparticles as potential delivery agents for PET imaging agents. Nanoparticle-based platforms offer new strategies to design specific probes to detect and diagnose multiple diseases. Various nanoparticles offer a unique size and physiochemical properties that allow for loading of a variety of radiotracers using different synthetic strategies. The unique surface chemistry of nanoparticles also allows for improved targeting of disease sites using a wide variety of probes (Welch, Hawker, & Wooley, 2009). This improves the contrast of the disease site relative to other normal tissues improving sensitivity as well as specificity of PET. The use of various probes in combination with imaging techniques can allow for imaging of various biological events at cellular or molecular level (Lee, Xie, & Chen, 2010). The most well-investigated nanomaterials include liposomal carriers, magnetic IONPs, quantum dots (QDs), polymeric nanoparticles, silica nanoparticles, dendrimers, inorganic metal-based nanoformulations, and carbon nanotubes (Bartholoma, Louie, Valliant, & Zubieta, 2010; Elsabahy & Wooley, 2012; Jokerst & Gambhir, 2011; Lee et al., 2010; Louie, 2010; Nahrendorf et al., 2008; Ng, Lovell, & Zheng, 2011; Petersen, Hansen, et al., 2012; Tassa, Shaw, & Weissleder, 2011). For obtaining good contrast, the radiotracer should have a half-life long enough to be loaded on nanoparticles and allow for sufficient accumulation in the tumor tissue of the patient or subject. Table 3.1 outlines the most popular nanoparticles which have been studied for PET applications

Table 3.1 Labeling strategies for radiolabeling of nanoparticles Nanoparticle Radionuclide Method of labeling

Liposomes

18

F

Encapsulation

68

Ga

DTPA

64

Cu

BAT

124

I

Tyrosine conjugation

Dendrimers

64

Cu/ Ga/

Polymeric nanoparticles

76

Br/

124

64

Cu

DOTA

18

F

[18F]FETos

18

F

Nucleophilic substitution

64

Cu

DOTA

68

Ga

NOTA

18

F

Click chemistry

64

Cu

DOTA/direct doping/ dithiocarbamate (dtc)

68

Ga

Direct labeling/NOTA

Quantum dots

Iron oxide nanoparticles

68

124

Gold nanoparticles

Carbon nanotubes

Silica nanoparticles

I

I

111

In DOTA Tyrosine conjugation

Tyrosine conjugation

89

Zr

DOTA - 1,4,7,10tetraazacyclododecane1,4,7,10-tetraacetic acid

64

Cu

DOTA/direct labeling

18

F

Peptide labeling

64

Cu/86Y

DOTA

89

Zr

Desferrioxamine B

124

I

Bolton–Hunter reagent

68

Ga

NOTA - 1,4,7triazacyclononane-triacetic acid

64

Cu

DOTA

Porphysomes

64

Cu

Chelator-free building blocks

Copper sulfide nanoparticles

64

Cu

Chelator-free building blocks

Upconversion nanoparticles

18

F

Inorganic interaction

124

Tyrosine conjugation

Graphene oxide nanoparticles

64

I

Cu

DOTA

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along with the radionuclide with the appropriate labeling strategies used for imaging of either cancer or other diseases.

2. LIPID-BASED NANOPARTICLES One of the most popular and widely used approaches for delivery of radiotracers has been liposomes. Lipid-based carriers were one of the first to be studied, and their usage as delivery agents for a variety of compounds has been summarized very well in recent years (Petersen, Hansen, et al., 2012). For delivery through the bloodstream, the lipid carrier system needs to have a hydrophilic surface and a combination of internal hydrophobic domains. The dual-phase character of these vehicles allows one to position contrast agent payloads in either hydrophobic or hydrophilic compartments, as part of the lipid framework or free in the aqueous core, respectively. The first liposomal-based radiotracer formulation was called Vescan™ ( Jensen & Bunch, 2007). It was developed and envisioned as a broadspectrum imaging agent. It was developed by loading an 111In-based ionophore within the micellar structure of the liposomes. Vescan has not received approval by the FDA to be used commercially but the clinical trials provided great understanding of the liposomal interactions within the body. It also allowed for understanding of clearance rate of liposomes from patients and was successful in imaging a variety of tumors during the clinical trials. The studies with Vescan allowed for the development of newer liposomal formulations such as Ambiosome and DaunoXome ( Jensen & Bunch, 2007; Proffitt et al., 1983). Radiolabeled liposomes have been the focus of many preclinical studies and have been found to offer good sensitivity as well as specificity. Liposomes can be used for concomitant delivery of both therapeutic and diagnostic agents, and such strategies termed “nanotheranostics” have gained a lot of interest in recent times. Radiolabeled liposomes could be used to determine the biodistribution of a liposome-delivered drug, which in addition could be important for other treatments strategies, including radiation therapy and antiangiogenic agents in cancer patients (Phillips, Goins, & Bao, 2009). A variety of loading strategies have been developed for radiolabeling of liposomes. Figure 3.1 shows a schematic for the four main loading strategies for the preparation of radiolabeled liposomes, namely, passive encapsulation (Gabizon et al., 1991; Holmberg et al., 1989; Marik et al., 2007; Oku et al., 2011), membrane labeling (Morgan et al., 1981; Richardson, Jeyasingh, Jewkes, Ryman, & Tattersall, 1977), surface chelation (Andreozzi, Seo,

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Figure 3.1 Schematic diagram of the remote loading, membrane labeling, passive encapsulation, and surface chelation methods for preparing radioactive liposomes. Radionuclides can be associated with the lipid membrane by hydrophobic interaction, through membrane conjugation, or surface chelation using chelator–lipid conjugates in preformed liposomes (blue (white in print version) radionuclides). Radionuclides can alternatively be encapsulated inside liposomes during lipid hydration or can be transported through the lipid membrane of preformed liposomes by ionophores or lipophilic chelators (yellow (dark gray in print version) radionuclides). In the latter case, the radionuclides are trapped inside the aqueous lumen by a hydrophilic chelator with high affinity for the radionuclide. Reprinted with permission from Elsevier 2012.

Ferrara, & Louie, 2011; Goto, Kubo, & Okada, 1989; Laverman et al., 1999; Petersen, Binderup, et al., 2012; Petersen, Hansen, et al., 2012; Seo et al., 2010, 2011; Seo, Zhang, Kukis, Meares, & Ferrara, 2008; Urakami et al., 2007; Wong et al., 2013), and remote loading (Awasthi, Garcia, Goins, &

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Phillips, 2003; Awasthi, Goins, Klipper, Loredo, et al., 1998; Awasthi, Goins, Klipper, & Phillips, 1998; Bao, Goins, Klipper, Negrete, Mahindaratne, et al., 2003; Bao, Goins, Klipper, Negrete, & Phillips, 2003; Glaus, Rossin, Welch, & Bao, 2010). The first method of passive encapsulation involves incorporation of the radiolabel within the liposome during its formation. Due to the low efficiency of loading of radiolabeled compounds into the liposomes (90%). But the main issue with the use of chelators for developing PET agents is the exposure of the radionuclides to proteins and other biomacromolecules in the bloodstream which

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might cause release of the radionuclide from the chelator. The in vivo stability of surface-labeled liposomes is therefore very dependent on the radionuclide–chelator binding constant. Remote loading is an active loading of the radionuclide into the aqueous phase of liposomes and also into the interior of the liposomes, where an ionophore or a lipophilic chelator transports the radionuclide over the membrane of preformed liposomes, and the radionuclide is delivered to a preencapsulated chelator. The remote loading approach concentrates radionuclides in the internal aqueous compartment of the liposomes. It provides high loading efficiencies (>90%) and displays high in vivo stability because of the protection offered to the radionuclide preventing its interaction with the surrounding biological environment. Remote loading of the liposomes can be accomplished in a number of ways; the first involves entrapment of the radionuclide in the interior of the lipid by use of encapsulated chelators. The radionuclide is transported to the interior of the liposome via lipid-soluble chelators on the surface of the liposome and gets entrapped in chelator already encapsulated in the interior of the liposome. This procedure has been demonstrated by utilizing 8-hydroxyquinoline (oxine) (Gabizon, Goren, & Barenholz, 1988; Gabizon & Papahadjopoulos, 1988), which is a lipophilic chelator ferrying radiotracers such as 67Ga and 111In through the lipid bilayer into the center of the liposomes for entrapment by other chelators such as nitrilotriacetic acid or deferoxamine (Beaumier & Hwang, 1982; Hwang, Merriam, Beaumier, & Luk, 1982), which are nonlipophilic metal chelators. In another procedure, a lipophilic chelator can be converted into a membrane impermeable form after interaction with an encapsulated chelator. Lipophilic chelators, N-N-bis (2-mercaptoethyl)-N0 ,N0 -diethylethylenediamine and diisopropyl iminodiacetic acid, have similar features as hexamethyl propylene amine oxime and have been used to load radionuclides such as 99mTc into preformed liposomes containing glutathione (GSH) where the radiotracer interacts with the GSH forming an membrane impermeable complex (Bao, Goins, Klipper, Negrete, Mahindaratne, et al., 2003; Bao, Goins, Klipper, Negrete, & Phillips, 2003; Phillips et al., 1992; Suresh & Cao, 1998). Other methods involve a use of a pH gradient to chelate radionuclides and prevent release into the biological environment and the use of hydrophilic ionophores in the liposomal membrane for transfer of radionuclide into the interior of the lipid where it is entrapped by other chelators (Bao, Goins, Klipper, Negrete, & Phillips, 2004). These methods are extremely efficient and allow for high loading of the liposomes with radionuclides. Recent developments in the field have shown the increase in popularity

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of solid lipid particles. The main advantage of solid lipid particles is that it has a solid (hydrophobic or hydrophilic) interior core unlike an aqueous core in most liposomes. As a result, they offer more protection against chemical degradation of the payload they carry and also facilitate sustained drug release due to the zero-order kinetic breakdown of the solid lipid matrix. Louie et al. have demonstrated an easy synthetic method for efficient loading of 64Cu on solid lipid particles for PET imaging. The radioactive 64Cu was attached to the solid lipid particles through BAT (Andreozzi et al., 2011). Among the current methods tested in humans, remote loading (Presant et al., 1990; Proffitt et al., 1983; Turner et al., 1988) and surface chelation (Brouwers et al., 2000; Dams et al., 2000; Laverman, Brouwers, et al., 2000; Laverman, Zalipsky, et al., 2000) appear to be the most successful. Liposomes passively accumulate in tumors due to the enhanced permeability and retention effect of leaky vasculature and dysfunctional lymphatics (Wong et al., 2013). This effect has been used to enhance drug delivery (Wong et al., 2013) but can also generate contrast for imaging and detection of tumors. Being smaller than red blood cells, liposomes can extravasate relatively easily through fenestrated cellular barriers. Furthermore, the liposomes (and nanoparticles in general) become trapped in the extracellular space due to the ineffective lymphatic drainage within tumor tissue and the difference in the pressure between the interstitial space and the blood stream. This effect is termed as enhanced perfusion and retention (EPR) effect and is also termed as passive targeting. For successful in vivo applications, it has been established that the outer bilayer of the liposome should ideally be coated with a neutral polyethylene glycol (PEG) polymer to minimize colloidal instability, reduce bioadhesion, and limit immunological responses (Mitchell et al., 2013). Most importantly, the PEG coating reduces uptake of the liposome within the RES and therefore slows the rate of removal of the liposomes from the blood (Klibanov, Maruyama, Torchilin, & Huang, 1990; Presant et al., 1990). This effectively increases the biological half-life of the liposome. In clinical studies, conventional liposomes have been shown to have a half-life of 20 min in body fluids, whereas PEG liposomes can have a half-life of up to 5 days. Harrington et al. demonstrated that 111In-labeled PEGylated liposomes show significantly higher tumor accumulation than unmodified liposomes in xenografted mice (Harrington, Lewanski, & Stewart, 2000a, 2000b; Harrington, Rowlinson-Busza, Syrigos, Abra, et al., 2000; Harrington, Rowlinson-Busza, Syrigos, Uster, et al., 2000). Further in a clinical trial using 111 In-labeled liposomes similar to previously demonstrated in mice studies, it

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was found that the liposomes were able to accumulate in tumors in high quantities generating a good contrast for tumor detection, indicating their usefulness for detection of solid tumors (Harrington et al., 2001). A variety of lipsosmal formulations using a variety of radionuclides such as 111 In, 99mTc, 67Ga, 64Cu, and 18F have shown good tumor accumulation via passive targeting in a number of preclinical studies in mice and rats (Awasthi et al., 2003; Erdogan, Roby, & Torchilin, 2006; Harrington, Rowlinson-Busza, Syrigos, Abra, et al., 2000; Marik et al., 2007; Oku, Tokudome, Namba, et al., 1996; Oku, Tokudome, Tsukada, et al., 1996; Oku, Tokudome, Tsukada, & Okada, 1995; Oku et al., 2011; Petersen, Binderup, et al., 2012; Seo et al., 2011, 2008; Urakami et al., 2007). PET imaging with 64Cu-labeled liposomes is being explored frequently because of its relatively long half-life. This allows for sufficient uptake of the liposomes into the tumor prior to imaging with PET. Preclinical studies with 64 Cu-loaded liposomes have demonstrated that in the mouse tumor model, 64 Cu liposomes generate images with contrast and volume estimates comparable to 18F-FDG. These liposomes also demonstrated higher amount of heterogeneity in tumor accumulation relative to 18F-FDG. Recent reports have demonstrated the use of 89Zr for PET imaging of tumors. Abou et al. synthesized a multifunctional particle for PET–MRI dual-modal imaging of tumors. The 89Zr was incorporated into the membrane of the liposome using a chelation-free labeling method. The dual-imaging particle was able to image C6 glioma cells by both PET and MRI using Gadolinium (Gd) incorporated within the liposomes, showing high tumor accumulation with the tumor site (Abou, Ku, & Smith-Jones, 2011; Abou et al., 2013). Other radiolabels have been incorporated in liposomes. Glutathione-encapsulated liposomes have been radiolabeled with Rhenium-186 (186Re) and 188Re (Weeks et al., 2011) for both diagnostic and therapeutic purposes using a rhenium–SNS/S complex (Beaumier & Hwang, 1982; Gabizon et al., 1991, 2003; Rossin et al., 2011; Wiessner & Hwang, 1982). Liposomes have also been used for the delivery of SPECT agents for multimodal imaging. Tabor et al. have developed liposomes for multimodal imaging of tumor. The liposomes consisted of the macrocycle DOTA conjugated the lipid head group of the liposomes using short n-ethylene glycol (n-EG) spacers of varying length. These liposomes allowed for chelation of a variety of ligands such as Gd3+, 64Cu2+, and 111In3+ (Mitchell et al., 2013). These liposomes showed high uptake into the tumor cells under in vitro and in vivo conditions allowing for the possibility of future development of flexible trimodal imaging agents.

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Targeting strategies using ligands that are directed toward overexpressed receptors on tumor cells or highly expressed markers on the endothelium of tumor vasculature can potentially improve the disease-specific targeting of liposomes (Danhier, Feron, & Preat, 2010; Kievit & Zhang, 2011) and work additively with the EPR effect to increase tumor accumulation. A variety of targeting ligands have been used for delivering radionuclide contrast agents to the tumors. These range from antibodies, peptides, carbohydrates, small molecules, and oligonucleotides. The goal is to increase the concentration of the contrast agent at the tumor site relative to healthy tissues and allow for earlier detection. A variety of preclinical studies have been performed for studying the effect of active targeting of tumors using liposomes. The results have been very mixed as often attaching a targeting moiety improves delivery of cargo to the target cells, but the biodistribution and tumor accumulation mirror nontargeting nanoparticles. It is believed that the EPR effect tends to dominate the accumulation of liposomes at the tumor site. Targeting strategies for targeting of cancers and other diseases involves use of ligands to target different receptors. Targeting liposomes have been studied in great detail using a wide variety of ligands such as arginine– glycine–aspartic (RGD) peptides (Cai & Chen, 2008; Huang et al., 2008; Schiffelers et al., 2003), folate (Gabizon et al., 2003; Henriksen, Schoultz, Michaelsen, Bruland, & Larsen, 2004), transferrin (Miyata et al., 2011), epidermal growth factor (Beuttler, Rothdiener, Muller, Frejd, & Kontermann, 2009; Gao et al., 2011, 2012; Rodriguez-Porcel et al., 2008), somatostatin (Abou et al., 2013; Petersen, Binderup, et al., 2012), membrane matrix metalloprotease substrates (Elsabahy & Wooley, 2012; Huang et al., 2008; Kondo et al., 2004; Medina et al., 2005; Rossin et al., 2011), nucleosomespecific monoclonal antibody (mAb) 2C5 (Elbayoumi & Torchilin, 2006; Erdogan et al., 2006), and vasoactive intestinal peptide (Dagar, Krishnadas, Rubinstein, Blend, & Onyuksel, 2003; Dagar, Sekosan, Lee, Rubinstein, & Onyuksel, 2001; Dagar, Sekosan, Rubinstein, & Onyuksel, 2001; Onyuksel, Ashok, Dagar, Sethi, & Rubinstein, 2003; Refai et al., 1999). Anderson et al. demonstrated the development of 64Cu-labeled somatostatin targeted liposomes for the detection of NE tumors. The PEGylated 64 Cu liposomes with the targeting moiety showed higher tumor uptake in NET xenograft model (NCI-H727) (high tumor-to-muscle ratio) compared to untargeted liposomes (Fig. 3.2). The targeted liposomes showed faster accumulation times relative to the untargeted liposomes in NE tumors allowing for earlier detection (Anderson et al., 1992). Torchilin and

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Figure 3.2 Positron emission tomography (PET)/computed tomography (CT)-images of 64 Cu-TATE-liposome distribution in a mouse xenograft model. Axial PET/CT fusion images 1, 8, 24, and 48 h postinjection of 64Cu-TATE-liposomes into a mouse with tumors (neuroendocrine carcinoma NCI-H727; marked with arrows) on right and left flanks. Reprinted from Petersen, Binderup, et al. (2012). Copyright (2012) with permission from Elsevier.

colleagues have developed nanosized liposomes for tumor imaging via SPECT combined with CT. The authors utilized DTPA–polylysyl–Nglutarylphosphatidylethanolamine for stable loading of the SPECT imaging agent 99mTc. The liposomes were designed to be tumor specific by conjugation of mAb 2C5 to the liposomes, and iopromide was encapsulated inside the liposomes to acquire CT contrast. These tumor-specific liposomes were found to be effective multimodal agents displaying almost six times more uptake in tumor cells compared to nontargeted liposomes (Elbayoumi & Torchilin, 2006; Erdogan et al., 2006; Silindir et al., 2013). Current studies of radiolabeled liposomes in various preclinical models have provided promising results from a diagnostic perspective, for both targeted and nontargeted liposomes. But the studies when comparing spontaneous human cancers versus xenograft models make the transitions difficult. The differences in the tumor microenvironment, angiogenesis, tumor growth, and metastasis between such models make direct translations difficult. The advantages of targeting liposomes over conventional liposomes are also not well understood and need more studies to present a clear picture. The EPR effect tends to dominate the delivery of radionuclides to the disease site, and it is not well understood how the preclinical studies with xenograft tumors in mice will translate in clinical patients. There is a strong need to quantify the EPR effect in clinically relevant tumor models and complemented with work in large animals such as dogs and monkeys. In order to test this, more clinical trials are needed to get a better idea of how targeted liposomes can be utilized as diagnostic as well as therapeutic agents. Targeted liposomes can find great success for personalized medicine where the

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particles can be tailored based on the expression of specific tumor markers from the patient tumor.

3. DENDRIMERS The medical community has made use of synthetic polymeric and dendrimeric nanoparticles for a wide variety of biological applications such as drug and gene delivery, tissue engineering, and in vivo imaging because their structure presents a potentially large number of modifiable groups. Dendrimers are highly branched macromolecules having high polydispersity that can be easily tuned into various sizes and properties by chemical synthesis (Almutairi et al., 2009; Chang et al., 2011; Kobayashi et al., 2000). The branched nature of dendrimers provides a scaffold for conjugation of a variety of targeting, imaging, and therapeutic agents. The aliphatic polyesterbased dendrimers show excellent biocompatibility and are biodegradable in animals. Fre´chet and colleagues reported the synthesis of novel dendritic nanoprobes for PET imaging of tumor angiogenesis. They synthesized a variety of different dendrimeric constructs with functionalized tyrosine groups for attachment of different radioactive halogens (122I, 123I, 124I, 125 131 75 I, I, Br, 76Br, and 77Br). To improve the target specificity of the constructs, cyclic RGD peptide was covalently attached to the dendrimer. The newly synthesized nanoprobe having a radioactive core and a protective shell consisting of PEG chains appended with multiple peptide ligands to target αvβ3 integrin overexpressed in angiogenesis (Almutairi et al., 2009). In vivo studies in a mouse ischemia model with 76Br-based PET showed high accumulation for the targeted radiolabeled dendrimer compared to the nontargeted dendrimer. The ischemic hind limb of the mice showed high levels of the targeted dendrimer when imaged in a PET scanner with both the transaxial and the sagittal images confirming this observation. In the case of the nontargeted dendrimer, there was no difference observed in the sagittal view between the ischemic and the nonischemic hind limbs of the mice. The 76Br-labeled dendrimer allowed for highly selective imaging of an important clinically relevant condition which demonstrates the potential of dendrimers as agents for imaging and therapy. Asparagine-based oligosaccharides are attractive candidates for developing imaging agents because of the presence of sialic acid clusters allowing for increased biostability under in vivo conditions. Fukase and colleagues recently reported the first PET and fluorescence imaging using dendrimer-type glycan molecules. The clusters were designed to have a terminal lysine ε-amino group so that

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they could be efficiently labeled by fluorescent groups or 68Ga-DOTA as the PET radiolabel in the presence of numerous hydroxy groups through the 6-azaelectrocyclization protocol under mild conditions (Tanaka & Fukase, 2008; Tanaka, Fukase, & Katsumura, 2010; Tanaka et al., 2008). The PET imaging studies demonstrated a remarkable difference in the clearance and stability of n-glycan clusters under in vivo conditions. The presence of different sialosides in the clusters showed marked changes in the dynamics of accumulation and biodistribution of the radiolabeled compounds in tumored and nontumored mice. Unfortunately, no tumor targeting was achieved by the newly synthesized clusters (Almutairi et al., 2009; Chang et al., 2011; Kobayashi et al., 2000). Wickstrom et al. reported the utility of polydiamidopropanoyl dendrimer (generation m), with increasing numbers (n) of DOTA chelators (radiolabeled with 111In), extended via an N-terminal AEEA from a mutant KRAS2 PNA (peptide nucleic acid) with a C-terminal AEEA and IGF1 analogue allowing for radionuclide imaging of pancreatic cancer xenografts that overexpress IGF1 receptor and mutant KRAS2 mRNA. Scintigraphic imaging showed a high accumulation of the targeted dendrimer complex relative to nontargeted dendrimer complex. The work allows for the use of PNA-based agents for preclinical imaging studies (Amirkhanov, Zhang, Aruva, Thakur, & Wickstrom, 2010; Cheng, Chakrabart, Aruva, Thakur, & Wickstrom, 2004).

4. POLYMERS Amphiphilic polymers are a distinct class of molecules finding use in biomedical application because of their tunable in vivo pharmacokinetics and versatile conjugation chemistry. They possess a hydrophobic core providing an environment for loading of hydrophobic drugs and a hydrophilic exterior (e.g., poly(acrylic acid co-acrylamide)) coating presenting multiple opportunities for covalent attachment of functional units such as imaging moieties. Additionally, the multivalency of such polymers empowers flexible radiochemistry (64Cu, 76Br, 124I, and 18F) for PET applications (Li et al., 2012; Shrestha, Shen, Pollack, Taylor, & Wooley, 2012; Zeng et al., 2012). Welch et al. reported the use of star-shaped cross-linked polymers containing DOTA synthesized by a nitroxide-mediated radical polymerization protected by an outer shell of PEG to improve circulation times in vivo. Biodistribution studies presented a correlation between the lengths of the PEG chain used for coating the polymer. It was observed that the longer PEG chain improved retention of the compound in vivo and reduced

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clearance by the MPS system (Fukukawa et al., 2008; Li et al., 2012; Pressly et al., 2007; Rossin et al., 2011; Shokeen et al., 2011; Welch et al., 2009). Other studies involving the synthesis of poly(methyl methacrylate-comethacryloxysuccinimide-graft-poly(ethylene glycol)) (PMMA-coPMASI-g-PEG) via living free radical polymerization provided a stable amphiphilic block copolymer, which could be easily functionalized with DOTA for 64Cu labeling for PET-based applications. Other polymeric nanoparticles such as the comb-type nanoparticles synthesized by Liu and colleagues were designed to target the natriuretic peptide clearance receptor in a mouse angiogenesis model. DOTA chelation allowed for loading of 64 Cu for PET imaging studies. Imaging studies showed high targeting capability of the c-type atrial natriurietic factor toward the receptor which is overexpressed under ischemic conditions. The amount of radiolabeled polymer was significantly higher demonstrated by both PET imaging and ex vivo biodistribution. More importantly, the multivalent polymeric nanoparticle had improved uptake at the disease sites compared to monovalent radiotracers and nontargeted radiotracers, showcasing the advantages of using a multivalent nanoparticle for radionuclide-based molecular imaging studies (Liu et al., 2010, 2011). Similar studies have been reported using shell cross-linked polymeric nanoparticles as agents for PET imaging. Copolymers based on N-(2-hydroxypropyl)-methacrylamide (HPMA) and active ester methacrylates have also been radiolabeled with a variety of radionuclides for use in SPECT or PET imaging studies. R€ osch et al. reported 18 the use of 2-[ F]fluoroethyl-1-tosylate for easy one-step conjugation with HPMA polymers for developing PET imaging agents. The synthesis procedure was fast and simple and provided high yields of radiolabeled HPMA polymers. The radiolabeled polymers were easily filtered out of body of healthy rats in the urine indicating renal clearance. This was confirmed by PET imaging studies showing high accumulation in the kidneys and in the bladder (Herth, Barz, Jahn, Zentel, & Rosch, 2010; Herth et al., 2009). SPECT imaging has also benefited from the advances in nanotechnology. Borbe´ly and colleagues have developed self-assembling chitosan and folated poly-γ-glutamic acid nanoparticles radiolabeled with 99mTc. These nanoparticles were targeted toward folate overexpressing cells and were internalized by tumor cells which allowed for detection through SPECT as well as SPECT/modalities. Tumors transplanted in the kidney resulted in considerably higher uptake of the radiolabeled nanoparticles compared to nontumorous organs. The SPECT studies allowed for imaging of the

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tumorous kidney distinctly showcasing the nanoparticles ability to target and image tumors (Polya´k et al., 2013).

5. QUANTUM DOTS Quantum dots (QDs) have been gaining a lot of interest as alternatives to organic fluorophores. They have been investigated as means to attach multimodal imaging agents such as PET and MRI. The most straightforward modification of QDs employs bifunctional chemical cross-linkers to conjugate two dissimilar molecules to each other, methods that are routinely used to fluorescently label proteins. A routine way of developing QDs as agents for PET imaging would involve attaching a radionuclide to the QD nanoparticle. Amine reactive probes are the most popular method to conjugate metal chelators to the surface of the QDs. These chelators can be later on labeled with metal radionuclides. Chen et al. described the development of a dual-PET/near-infrared (NIRF) probe for tumor detection (Cai, Chen, Li, Gambhir, & Chen, 2007). They used a heterobifunctional linker containing N-hydroxysuccinimide (NHS) ester for conjugating a DOTA molecule to the surface of the QD. The DOTA then chelates 64Cu for allowing the QD to be used as a PET imaging agent as displayed in Fig. 3.3A. It is noted by the authors that PET imaging required far less 64Cu-labeled QD than near infra-red fluorescence (NIRF) whole-body optical imaging. Only 22 pmol of the probe was used for animal imaging by PET compared to 200 pmol used for NIRF imaging. Further work by the group showed that improvement in the uptake of the QDs at the tumor site by attaching a targeting peptide such as vascular endothelial growth factor (VEGF) (targeting angiogenesis) and arginylglycylaspartic acid (RGD) (targeting vasculature as well as tumor cells) is shown in Fig. 3.3C. Although there was no significant difference in the liver uptake between the nontargeted and the targeted QD, the amount in the tumor differed significantly (less than 1% injected dose per gram for the free QD vs 4.16  0.5%ID for the VEGF targeting QD 24 h postinjection) in U87MG tumor-bearing mice (Chen, Li, Wang, Cai, & Chen, 2008) seen clearly in Fig. 3.3D. Recent work by Tavitian et al. shows the development of phospholipidcoated CdSe/ZnS core–shell QDs labeled with 18F. The phospholipid has a terminal –SH (sulfhydryl) group which is easily conjugated to the maleimido [18F] reagent, coded [18F]FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl] pyrrole-2,5-dione). The presence of the maleimido group on the radioactive reagent allows for easy attachment to the –SH group on the macromolecule-coated QD. The radiolabeled QDs showed good optical

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Figure 3.3 (A) Synthesis of dual-function PET/NIRF probe DOTA–QD–RGD. DOTA–QD was prepared in similar manner, except that no RGD peptide was used. Overall diameter of QD conjugate is about 20 nm. PEG, polyethylene glycol. (B) Whole-body coronal PET images of U87MG tumor-bearing mice at 1, 4, 16, and 24 h postinjection of about 300 μCi of 64Cu–DOTA–QD and 64Cu–DOTA–QD–VEGF. Arrows indicate the tumor. (C) U87MG tumor uptake of 64Cu–DOTA–QD and 64Cu–DOTA–QD–VEGF over time as quantified by microPET scans (n ¼ 3 per group). *P < 0.05, **P < 0.01. Reprinted with permission from Cai et al. (2007). Copyright by the Society of Nuclear Medicine and Molecular Imaging, Inc.

as well as PET imaging capabilities. The presence of the phospholipids allowed for longer circulation times as reported by the authors (T½—2 h). The nanoparticles also avoided renal clearance which is attributed to be their larger size (>20 nm) (Duconge et al., 2008). Similarly, Lee et al. utilized a similar method to coat QDs with specific ligand-conjugated amphiphiles such as RGD acid-C18, mannose-C18, lactose-C18, and 2-(p-isothiocyanato-benzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acidC18. These targeted QDs could be easily radiolabeled with 68Ga using NOTA as a chelator. The newly synthesized nanoparticles were found to be very stable and showed high target specificity under both in vitro and in vivo conditions (Lee et al., 2012).

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Louie et al. recently developed new silicon-based QDs for multimodal imaging of tumors. The silicon QDs were synthesized from the precursor sodium silicide through a solution-phase reduction, where SiMn QDs (SiMn QDs ¼ 1% manganese-doped Si QDs) were coated with neutral dextran. The presence of dextran avoids the recognition of the QDs by macrophages under in vivo conditions. Using DOTA as a chelator, 64Cu was loaded on the newly synthesized QDs allowing for PET imaging. Biodistribution studies indicate that the highest amount of nanoparticles was found in the liver followed by high amounts in the kidney as well. This indicates that these nanoparticles could be eliminated in the body through renal clearance (Tu, Ma, House, Kauzlarich, & Louie, 2011; Wang, Jarrett, Kauzlarich, & Louie, 2007). Mareque-Rivas and colleagues have utilized QDs for generation of bimodal SPECT-optical imaging probes. The core–shell CdSe–ZnS QDs were made SPECT visible by simple addition of [99mTc(OH2)3(CO)3]+, an emerging radioprecursor. These bimodal nanoparticles allowed for light-mediated generation of cisplatin from an inert Pt(IV) prodrug under in vitro conditions (Maldonado et al., 2013). Recent work by Chen et al. demonstrates the construction of selfilluminating semiconducting QDS. The CdSe/ZnS QDs were doped with 64 Cu via a cation exchange reaction and exhibited Cerenkov resonance energy transfer. The presence of 64Cu allowed for PET imaging and measuring the biodistribution of the nanoparticles within the body of the mice with xenografted tumors. The newly synthesized radiolabeled nanoparticles showed high tumor uptake in a human glioblastoma model in mice and ability to self-illuminate in the absence of excitation light (Sun et al., 2014). QDs have a promising future in biomedical applications, but the main issue with QDs is their toxicity within cells. Literature has shown that proper coating of QDs can limit their toxicity as well as degradation but most studies agree that cadmium-based QDs are toxic to cells regardless of their surface coating. A current major focus is to develop new, cadmium-free QDs as safe and nontoxic probes for biological use. These QDs are nontoxic to cells, and their optical properties can be tailored by composition and size. Future studies would involve attachment of radiolabels to such QDs for safer multimodal imaging of tumors.

6. IRON OXIDE NANOPARTICLES IONP derivatives are clinically available, relatively benign contrast agents for MRI. They have super magnetic properties, are biocompatible

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exhibiting little toxicity, and are easy to synthesize in clinically relevant quantities. IONPs are primarily synthesized using a combination of Fe(II) and Fe(III) precursors (Quan et al., 2011; Xie, Chen, et al., 2010). To improve its biocompatibility, a number of coatings have been investigated in the literature ranging from hydrophilic polymers, normally added during the particle formation process, and protect against particle aggregation. Other ligands include polyvinylpyrrolidone, dendrimer, polyaniline, and dextran, with dextran and its derivatives being the most studied. To utilize IONPs as PET agents, Chen et al. proposed coating IONPs with dopamine to increase its hydrophilicity for easier attachment of human serum albumin (HSA). These HSA-coated IONPs were then labeled with Cy5.5 dye (Chen, Xie, & Chen, 2009) and 64Cu-DOTA chelates for use as optical as well as PET imaging agents. In vivo imaging and biodistribution studies were done in U87MG tumor-bearing mice. The PET imaging showed much higher tumor/muscle ratios of 4.55  0.42, 5.36  0.61, and 8.28  0.90 at 1, 4, and 18 h, respectively. Ex vivo biodistribution studies confirmed the imaging data with high amount of radioactivity found in the tumor relative to other organs such as spleen, kidneys, heart, and lung (Lee, Lee, et al., 2008; Lee, Li, et al., 2008; Quan et al., 2011; Xie, Chen, et al., 2010). To improve the target specificity of IONPs, Chen et al. reported the synthesis of integrin targeting dual MRI/PET agent. Polyaspartic acid-coated IONPs were conjugated with cyclic RGD peptides that bind to overexpressed integrin (αvβ3) receptors in neovasculture and tumor cells. The authors also conjugated DOTA for 64Cu labeling. In vivo PET studies done on U87MG tumor mice showcased the targeting capabilities of the newly synthesized IONPs. The U87MG tumor was clearly visualized with high contrast relative to the contralateral background from 1 to 21 h after injection of 64Cu–DOTA–IO–RGD. The targeted IONP showed higher tumor uptake (10.1  2.1%ID/g) compared to the nontargeted IONPs (

Recent advances in nanoparticle-based nuclear imaging of cancers.

Nuclear imaging techniques that include positron emission tomography (PET) and single-photon computed tomography have found great success in the clini...
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