Surface modification of iron oxide nanoparticles by biocompatible polymers for tissue imaging and targeting

    Surface modification of iron oxide nanoparticles by biocompatible polymers for tissue imaging and targeting Muthunarayanan Muthiah, In- Kyu Park, Chong-Su Cho PII: DOI: Reference:

S0734-9750(13)00064-5 doi: 10.1016/j.biotechadv.2013.03.005 JBA 6663

To appear in:

Biotechnology Advances

Received date: Revised date: Accepted date:

31 October 2012 19 February 2013 11 March 2013

Please cite this article as: Muthiah Muthunarayanan, Park In- Kyu, Cho Chong-Su, Surface modification of iron oxide nanoparticles by biocompatible polymers for tissue imaging and targeting, Biotechnology Advances (2013), doi: 10.1016/j.biotechadv.2013.03.005

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ACCEPTED MANUSCRIPT Surface modification of iron oxide nanoparticles by biocompatible polymers for tissue imaging and targeting Muthunarayanan Muthiaha, b, In- Kyu Parka, b,*, Chong-Su Choc,**

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Department of Biomedical Sciences and Center for Biomedical Human Resources (BK-21 project), Chonnam National University Medical School, Gwangju 501-757, South Korea b

Clinical Vaccine R&D Center, Chonnam National University Hwasun Hospital, Jeonnam 519- 763, South Korea; c

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Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea;

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*Corresponding author. Tel.: 82-61-379-8481 fax.: 82-61-379-8455; e-mail:[email protected] ** Corresponding author. Tel.: 82-2-880-4868; fax.:82-2-875-2494; e-mail:[email protected]

ACCEPTED MANUSCRIPT Abstract Superparamagnetic iron oxide nanoparticles (SPIONs) are excellent MR contrast agents when

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coated with biocompatible polymers such as hydrophilic synthetic polymers, proteins,

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polysaccharides, and lipids, which improve their stability and biocompatibility and reduce their aggregation. Various biocompatible materials, coated or conjugated with targeting

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moieties such as galactose, mannose, folic acid, antibodies and RGD, have been applied to

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SPION surfaces to provide tissue specificity to hepatocytes, macrophages, and tumor regions in order to reduce non-specific uptake and improve biocompatibility. This review discusses the recent progress in the development of biocompatible and hydrophilic polymers for improving stability of SPIONs and describes the carbohydrates based biocompatible

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materials that are providing SPIONs with cell/tissue specificity as ligands.

Keywords: Surface modification, iron oxide nanoparticles, biocompatible polymer, tissue

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imaging, targeting

ACCEPTED MANUSCRIPT 1. Introduction

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Superparamagnetic iron oxide nanoparticles (SPIONs), as their name implies, exhibit

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superparamagnetic behavior.(Iijima et al. , 1999) Paramagnetic materials exhibit magnetism only in the presence of an external magnetic field. They have a strong attraction towards

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magnetic fields and the attraction is lost in the absence of the magnetic field. These properties

magnetic field. (Scharf et al. , 1996)

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are due to the presence of unpaired electrons, which align in the direction of an external

SPIONs are iron oxide particles of a restricted size in the range of 1 to 100 nanometers. Two main forms of iron oxide nanoparticles are known: magnetite (Fe3O4) and

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its oxidized form maghemite (γ-Fe2O3).(Lee et al. , 2012a) These two types of iron oxide nanoparticles possess paramagnetic properties. Even though strong paramagnets like Cu, Co,

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and Ni are available, SPIONs are preferred as a MR contrast agent because Co and Ni

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materials are toxic and are easily oxidized, unlike the biocompatible iron oxides. In general, the applications of SPIONs include their use in terabit magnetic storage devices, catalysts,

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and sensors, as well as their use as high-sensitivity molecular resonance (MR) imaging contrast agents in the medical field.(Unger, 2003) Their biomedical applications, including MR imaging contrast enhancement, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery and in vitro cell separation, etc., make them important materials for research and diagnostic purposes.(Kuehnert et al. , 2012) This review covers the characteristics of SPIONs and the recent progress in SPION development aimed at improving their stability and targeting for use in tissue imaging through the use of biocompatible polymers.

ACCEPTED MANUSCRIPT 2. Characteristics of SPIONs SPIONs as MR contrast agents

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2-1.

SPIONs possess unique magnetic properties with strong shortening effects under

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longitudinal (or T1) relaxation and transverse (or T2) relaxation pathways. T1 relaxation is the result of energy exchange between the spins and the surrounding lattice (i.e., spin-lattice

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relaxation), which re-establishes the thermal equilibrium. The spins travel from a high energy to a low energy state, and during that period, the RF energy is released back into the surrounding lattice. T2 relaxation arises due to the spins getting out of phase. When the spins move together, an interaction of the magnetic fields is observed due to spin-spin interaction.

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Transverse relaxations are temporary and random and transverse magnetization decay is the

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result of cumulative loss in phase during the spin-spin relaxation. SPIONs shorten the T2 relaxation of the neighboring tissues, which results in a

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decrease in the signal intensity in MR images.(Le Renard et al. , 2011) SPIONs in the nano-

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dimension, in particular, help to create a better biodistribution than do the conventional chemical agents. When these SPIONs are conjugated with disease specific markers, they can serve as specific biomarkers with contrast capability for use in imaging with MRI, thereby opening the way to early diagnosis of a diseased condition. Moreover, when the SPIONs are delivered along with drugs, the estimations of drug distribution, release, tracking, and tissue level become easier when using MR imaging techniques.(Gandhi et al. , 2011) The in vivo drug response can be clearly monitored with MRI and the drug can also be specifically attracted in vivo to the region of interest with the help of external magnetic fields.

ACCEPTED MANUSCRIPT 2-3.

Method of SPION preparation SPIONs are prepared by co-precipitation, micro-emulsion, and thermal decomposition

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methods.(Muruganandham et al. , 2010) The advantages of the different methods of

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preparation are mentioned briefly in Table 1. Problems with naked SPIONs

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Naked SPIONs that lack a polymeric coating usually show aggregation in water,

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chemical instability in air, and a lack of biodegradability in the physiological environment.(Paul et al. , 2004) The most significant issues with naked nanoparticles are that they undergo non-specific interactions with the serum proteins and they tend to agglomerate in vivo due to hydrophobic-hydrophobic interactions between the particles when exposed to a

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physiological environment. SPIONs are also rapidly eliminated from the circulation due to this aggregation, following adsorption of serum proteins onto SPION surface. In addition,

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these particles circulate inside the body without any specific interaction with the organs or

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tissues. The surface of the nanoparticle needs be hydrophilic for in vivo applications, and it

2-5.

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has to be stable for prolonged circulation inside the body.(Frank et al. , 2003)

Surface coating of SPIONs Coating materials play an important role in the stabilization of aqueous SPIONs

suspensions as well as in their further functionalization. Surface modification of SPIONs with various materials can effectively render them water soluble and improve their stability under physiological conditions.(Juang et al. , 2009a) Two approaches can be taken for coating the SPIONs: one is ligand addition and the other is ligand exchange.(Bautista et al. , 2004) With the ligand addition mechanism, polymers physically adsorb to the surfaces of SPIONs due to electrostatic and hydrophobic interactions and by hydrogen bonding. Polymers with functional groups (i.e., hydroxyl, amine, carboxyl, etc.) at the periphery of SPIONs readily

ACCEPTED MANUSCRIPT absorb to their surfaces.(Ahmad et al. , 2012). The ligand exchange as one of simple coating methods of SPION surfaces is to

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replace the original surfaces with functional groups such as diol, amine, carboxylic acid and

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thiol for providing a better way to tailor the surface properties of the SPIONs (Na et al. , 2009). The surface modification of prepared particles is an essential step for safe clinical

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applications of the SPIONs. Tailoring the surface of SPIONs with polymers and other

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materials helps to improve their stability, surface charge, functionality, and targeting capability.(Boguslavsky and Margel, 2008) The ideal polymer for a SPION coating should have a high affinity for iron oxides but no capability for stimulation of the immune system or for antigenicity. The biocompatibility of the coating is a very essential determinant when

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considering in vivo applications. Hydrophilic materials used for SPION coatings include proteins, polysaccharides, lipids, and synthetic polymers. The most commonly used

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biocompatible coating materials are poly (ethylene glycol) (PEG), dextran, poly (vinyl

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alcohol) (PVA), chitosan, pullulan, alginate, gelatin, and poly (vinyl pyrrolidone) (PVP).

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3. Polymer coating for enhancing stability When hydrophobic SPION particles are injected into the bloodstream, they are surrounded by plasma proteins (hydrophobic surface) in a process called opsonization.(Elfick et al. , 2004) Once the SPIONs enter the physiological environment, they interact with the hydrophobic surfaces, which results in aggregation due to hydrophobic-hydrophobic interactions. Hydrophobic SPIONs are therefore prone to opsonization and tend to be cleared immediately by the mononuclear phagocytic system (MPS). On the other hand, if hydrophobic SPIONs are coated with hydrophilic polymers, the interaction of the SPIONs with the plasma proteins can largely be avoided. A hydrophilic coating on the SPIONs will thus increase the in vivo circulation by reducing uptake by the MPS. A hydrophilic coating

ACCEPTED MANUSCRIPT will also give stability and functionalization to which further drug, gene, or imaging agents can be conjugated. This makes the SPIONS multifunctional, as they can serve both as a

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contrast agent and as a drug carrier with a particular specificity.(Saba and Di Luzio, 1965)

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(Moghimi et al. , 1993) The following section describes the commonly used biocompatible

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polymers, including chitosan, PEG, dextran, PVA, and PVP.

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3-1. Chitosan

Chitosan is a natural polymer available in abundance from sea sources. It is biocompatible, hydrophilic, bio-degradable, non-antigenic, and non-toxic.(Kim et al. , 2008) It has repeated hexosaminide residues consisting of one amino group and two hydroxyl groups

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per unit.(Kang et al. , 2009) The hydroxyl and amino functional groups present on the chitosan rapidly form complexes with iron oxide surfaces, making the SPIONs hydrophilic,

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biocompatible, and stable. The positively charged amino groups can interact with the

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negatively charged nucleic acids for therapeutic gene delivery coupled to MR imaging.(Zhou et al. , 2009),(Qiao et al. , 2011) Furthermore, chitosan is known to facilitate particle

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movement across cellular barriers and transiently opens the tight junctions between epithelial cells.(Kuroiwa et al. , 2008, Zhu et al. , 2009) Until now, this natural polymer has been utilized for coating SPIONs to provide better contrast agent for MR imaging.(Chen et al. , 2012, Juang, Shen, 2009a, Juang et al. , 2009b, Juang et al. , 2010, Kim et al. , 2009, Kuroiwa, Noguchi, 2008, Li et al. , 2010, Peng et al. , 2010) Hong et al. prepared chitosan-coated ferrite nanoparticles (CFNs) to be investigated as an MRI contrast agent.(Hong et al. , 2010) They coated chitosan simultaneously with the synthesis of the ferrite nanoparticles. The amino groups (–NH2) of chitosan were bonded to the particles; however, the hydroxyl groups (–OH) of chitosan remain unbonded. Consequently, these types of coated particles were slightly positively charged. Due to the

ACCEPTED MANUSCRIPT Coulomb repulsion between the positively charged particles, the aqueous solution of coated particles remained in a state of colloidal suspension when no organic solvent or surfactant

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was used. The CFNs showed an average diameter of 67.0 nm. The authors also confirmed the

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strong bonding of the chitosan molecules to the surfaces of the ferrite nanoparticles with Fourier transform infrared (FT-IR) measurements.

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Once the CFNs were injected into mice, the liver part of the MR image became

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darker than the image without the injection of the agent, because the T2 relaxation of nuclear spins in the liver is faster due to the ferrite nanoparticles that are taken up by Kupffer cells (liver macrophage cells). The researchers observed a signal reduction of 29.3% after injection when compared to control images acquired before injection. When compared to other organs,

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the liver appeared darker than surrounding tissues. The gallbladder has no macrophage cells; thus, it cannot uptake ferrite nanoparticles. Hence, the signal intensity (image brightness) of

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the gallbladder was not changed, even after injection of the agent. The researchers then

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injected the CFNs into a hepatoma model. They found that the signal intensity at the site of the hepatoma was not much changed, as there are no Kupffer cells or the number of Kupffer

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cells is reduced in the hepatoma. Consequently, the hepatoma site could be easily identified using their contrast agent of ferrite nanoparticles. Finally, from their animal experimentation, they showed that a 31.7% signal loss from the MR image was observed 20 min after the injection of the nanoparticle agent. These results confirmed that their particles can be used as a T2 contrast agent in MRI. Ge et al. prepared nanoparticles coated with a modified chitosan possessing a magnetic oxide core with a covalently attached fluorescent dye. (Ge et al. , 2009) Their chemical synthesis was based on covalent coupling with modified organic fluorophores onto the chitosan, which strongly interacted with the surface of the ferric oxide nanoparticles. They observed, by confocal laser scanning microscopy (CLSM), that the magnetic particles

ACCEPTED MANUSCRIPT were located inside the cells and also on the cell surfaces. Hence, the labeling of SPIONs with fluorescent chitosan enabled their direct imaging and localization in living cells. The

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authors used TEM analysis to provide an even higher resolution image, which was better than

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optical imaging results. The nanoparticles were found inside the cells, in late endosomes or lysosomes, and the particles were exclusively present in the form of agglomerates. Overall,

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85% of the cells were labeled after incubation with the FITC-labeled particles. The uptake of

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FITC-labeled particles began as early as 30 min after incubation with the nanoparticles, and was comparatively more rapid within the first 2 h of incubation. The labeled cells were observed in vitro with a 1.5-T MR imager with detectable cell numbers of about 104. These magnetic fluorescent nanoparticles therefore served both as MR contrast agents and optical

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probes for intravital fluorescence microscopy. A cytotoxicity test also demonstrated that the prepared FITC-labeled particles were biocompatible and that they possessed suitable

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properties for biomedical applications although the FITC-labeled chitosan-coated SPIONs

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showed aggregation in vitro and this poor colloidal stability might be a major safety concern for further clinical application.

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Kievit et al. used PEI-PEG-grafted chitosan for coating SPIONs.(Kievit et al. , 2009) They first grafted PEI to chitosan to increase the transfection efficiency, since PEI has high positive charge and helps materials to escape the endosomes through the proton sponge effect.(Benjaminsen et al. , 2012) At the same time, PEIs having high positive charges can disrupt the cell membrane, resulting in toxicity to the cells. In order to reduce the toxicity, the authors grafted PEG to PEI, which provided colloidal stability, reduced the MPS uptake, and neutralized the toxic effects of the PEI. The PEI-PEG-grafted chitosan-coated SPIONs bound to the DNA and helped in transfection, while the PEG protected the cells from the adverse effects

of

PEI.

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nanoparticle-chitosan-g-poly

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glycol)-

polyethylenimine (NP-CP-PEI)/DNA particles showed low cytotoxicity when compared to

ACCEPTED MANUSCRIPT commercially available transfection agents and PEI. The NP-CP-PEI/DNA particles exhibited a higher gene transfection efficiency when compared to PolyMag, but a lower efficiency than

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Lipofectamine 2000 and PEI. However, the low toxicity observed with NP-CP-PEI/DNA

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particles confirmed this preparation to be a novel transfection agent with minimal toxic effects and enhanced transfection efficiency.

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The MR imaging results indicated that the magnetism of the complexes was readily

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datable. The addition of DNA to NP-CP-PEI did not affect its relaxivity. The DLS data also supported the authors’ inference that no clusters of NP-CP-PEI /DNA occurred. In an in vivo test, the NP-CP-PEI/DNA particles were injected intravenously into mice with C6 xenograft tumors. The expression of GFP was observed in the tumors in the NP-CP-PEI/DNA-treated

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mice after 48 h, which was the time needed for the uptake and expression of the EGFP encoding DNA.

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The chitosan coating of SPIONs, together with FITC-labeling, served the dual

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purpose of intravital fluorescence microscopy imaging along with MR imaging. In another work, PEI-PEG-grafted chitosan provided the advantage of gene delivery together with in

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vivo imaging. These studies confirm that a modified polymer coating of SPIONs, in contrast to a polymer coating alone, renders the nanoparticles multi-purpose. The summaries from these studies are presented in Table 2.

3-2. PEG PEG is a water-soluble polymer widely used to enhance the aqueous solubility of hydrophobic drugs.(Chawla et al. , 2009, Chu et al. , 2011, Inada et al. , 1995, Park et al. , 2008) A PEG coating minimizes the uptake by the MPS and increases the circulation time without causing any immune interaction.(Betancourt et al. , 2009, Choi et al. , 2011, Csaba et al. , 2009, Free et al. , 2009, He et al. , 2010, He et al. , 2011, Kotsokechagia et al. , 2012,

ACCEPTED MANUSCRIPT Kouchakzadeh et al. , 2010) It minimizes the non-specific uptake in the body, which further helps in tumor accumulation due to the enhanced permeability and retention (EPR)

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effect(Asyarie and Rachmawati, 2007, Ayen and Kumar, 2012, Bruckheimer et al. , 2004,

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Cheng et al. , 2010, Deglau et al. , 2012, Fleming et al. , 2004, Gjetting et al. , 2010, Jeong et al. , 2005). Once the SPIONs are coated with PEG, the PEG acts as a good spacer for the

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attachment of different biomolecules.(Mengersen and Bunjes, 2012, Miki et al. , 2010, Ozcan

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et al. , 2010, Suh et al. , 2007, Vasudev et al. , 2011, Zhang et al. , 2007) If the targeting ligands, such as a protein or an antibody, are attached, the SPION accumulation will be more specific to the region of interest, thereby sparing normal tissues.(Kim et al. , 2010, Malek et al. , 2009, Mitra and Bachhawat, 1997, Schadlich et al. , 2011, Shah et al. , 2012)

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A few problems need to be addressed for effective drug release at the target site. The drugs are usually physically attached to the nanoparticle surface, so they can be quickly

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released upon injection before reaching the target. Ultimately, only the small portion of the

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drug will reach its final destination. Another problem is that the coating on the particles might be digested under cytosolic conditions, thereby exposing the particles to the cells. These

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particles may aggregate and cause adverse conditions for cell survival. These two problems can be overcome by using iron oxide nanoparticles coated with cross-linked poly (ethylene glycol)-co-fumarate (PEGF), as synthesized by Mahmoudi et al.(Mahmoudi et al. , 2009) They used fumaric acid together with PEG for crosslinking, because fumaric acid-containing macromers are highly unsaturated and can be cross-linked with or without the use of a crosslinking agent to form their corresponding polymeric networks. The presence of fumarate group along with PEG chain in the backbone was confirmed by 1H NMR spectroscopy. The authors did not observe any precipitation in the suspension of PEGF-coated SPIONs for more than six months, whereas precipitation occurred within a short period when the SPIONs were coated with other polymers, such as PVA.

ACCEPTED MANUSCRIPT The enhanced stability of PEGF-coated SPIONs in aqueous media is very likely due to the hydrogel property of PEGF, which is able to absorb water and thereby decreases the

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density of the core-shell nanoparticles. In addition, the hydrogel coating is able to take up and

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release drugs, due to their response to changes in the physical and chemical environment. The cross-linked PEGF coating reduced the burst effect rate by 21% when compared with non-

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crosslinked tamoxifen nanoparticles.(Mahmoudi, Simchi, 2009) These experiments also

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demonstrated the greater biocompatibility of the SPIONs with PEGF coating than with PVA coating.

In another interesting study, Yu et al. found that bare nanoparticles were more toxic, even at low concentrations, when the particles were used to treat cells cultured within 3D gels.

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(Yu et al. , 2012) Whereas majority of nanotoxicity studies have been performed with cells in 2D cultures, these authors investigated cellular toxicity in a 3D gel formed from sodium

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alginate. In the presence of calcium, alginate forms a gel, but specific cell attachment to

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alginate is not observed, so cells do not proliferate in culture in alginate gels. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide

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nanoparticles coated with the polysaccharide, dextran, or with PEG. In both 2D and 3D cell culture tests, they found that bare nanoparticles decreased cell viability, whereas dextran- and PEG-coated nanoparticles caused no reduction in cell viability. In the same manner, bare 5nm and 30 nm particles induced significant formation of reactive oxygen species (ROS), but the 5 nm- and 30 nm-coated nanoparticles did not increase the ROS levels. Alternatively, the dextran coating decreased ROS fluorescence intensity by 35.2%, while the PEG coating decreased this intensity by 62.6%, when compared to bare nanoparticles. The authors also observed that cells treated with bare nanoparticles were more elongated and showed actin cytoskeleton disruption, when compared to cells without nanoparticle treatment. Dextran- and PEG-coated nanoparticles had no effect on cell length and did not cause actin cytoskeleton

ACCEPTED MANUSCRIPT disruption. Enhanced tumor imaging was found for SPIONS coated with starch-crosslinked PEG

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when compared to uncoated SPIONs. Toxicity studies in 3D cell culture also revealed that

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the PEG-coated SPIONS are not toxic, in agreement with findings from 2D cell culture

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experiments. (Cole et al. , 2011, Tong et al. , 2010) The studies are summarized in Table 3.

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3-3. Dextran

Dextran, a polysaccharide, has been extensively and successfully used for various in vivo applications.(Anzai et al. , 1994, Hsieh et al. , 2012) Dextran-coated SPIONs are well established and commercially available clinical contrast agents for MR imaging and have

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been shown to possess cancer nodal staging capabilities. The dextran coating has been improved by introducing a carboxymethyl group for enhanced stability and functionality.(Li

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et al. , 2011) Even after introducing the carboxymethyl group, however, a stronger

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attachment was still required since this polymer was susceptible to detachment. Consequently, the dextran was cross-linked with epichlorohydrin to form cross-linked iron oxide

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nanoparticles (CLIOs). The CLIOs had better stability than previous nanoparticles, even in harsh conditions, without undergoing any changes in their size and or their half-lives in the blood circulation. Their functionality was improved by treating them with amine to provide a primary amine group, which can be covalently bound with peptides or proteins.(Tassa et al. , 2011) Functionalized ligand-conjugated CLIOs have been extensively evaluated for MRI applications.(Lacava et al. , 2001) Harshingani et al. administered the CLIO ferumoxytol to 10 prostate cancer patients before starting the treatment.(Harisinghani et al. , 2007) The administered ferumoxytol accumulated in the benign lymph nodes and changed the signal to noise ratio (SNR). In contrast, the malignant lymph nodes showed only a slight change in the SNR ratio although

ACCEPTED MANUSCRIPT the ferrumoxtran-10 has been withdrawn from the market due to insufficient lymph node specificity.

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Josephson et al. synthesized SPIONs that were TAT-crosslinked and dextran-coated

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(TAT-CLIO) and studied their cellular localization in human lymphocytes, natural killer cells, and HeLa cells. (Josephson et al. , 1999) The CLIOs were prepared by the epichlorohydrin

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treatment and the peptide was attached through a disulfide linkage. The TAT-CLIO was

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localized in the nucleus and cytoplasm after FITC-labeling of the particles. The possibility exists that the disulfide cross-linked peptide can be cleaved in the cytoplasm and only the FITC reaches the nucleus, leaving the CLIO in the cytoplasm. To remove this doubt, the authors treated the cells with an anti-dextran antibody. Immunohistochemical staining

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localized the CLIO in the nucleus and not in the cytoplasm. The CLIO-labeled cells were

separation columns.

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highly magnetic, were detectable by MR imaging, and could be retained on magnetic

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In one of the above mentioned study they have checked the ability of dextran-coated SPIONs to distinguish the nodal stages during cancer occurrence with the help of MRI. In

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another study, attempts were made to stabilize the dextran coating by crosslinking the dextran, after coating, with epichlorohydrin.(Josephson, Tung, 1999)

3-4 Poly (Vinyl Alcohol) Poly (vinyl alcohol) (PVA) has been successfully used for tendon repair, contact lenses, ophthalmic materials, drug delivery, and other various biomedical applications.(Chen and Kao, 2006, Dong et al. , 2011, Franke and Nuttall, 1997, Liu et al. , 2012) The advantages of PVA are that it can resist protein adsorption and cell adhesion and it also possesses high biocompatibility.(Caramori et al. , 2012) Therefore, PVA can serve as an excellent biocompatible and water-soluble material coating for SPIONs.(Mahmoudi et al. ,

ACCEPTED MANUSCRIPT 2008) Liong et al. modified the PVA used for coating SPIONs because unmodified PVA

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nanocrystals precipitated during ultrafiltration.(Liong et al. , 2010) Strong attachment to the

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metal oxide surface was provided by introducing carboxylate groups onto PVA structure. Initially, bromo-acetic acid was added to PVA under basic conditions to convert its hydroxyl

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groups to carboxyl groups. The water-insoluble SPION-coated nanocrystals were transferred

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into the aqueous phase by treatment with a dilute aqueous solution of tetramethylammonium hydroxide (TMAOH), along with a quaternary ammonium solution as a phase transfer catalyst, which displaced the oleylamine and oleic acid surfactants, and surrounded the particle surface. Subsequent TGA analysis revealed a coating of 65% carboxylated PVA

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(CM-PVA) in weight percent on the SPIONs, which provided high stability to the colloids, lowered nonspecific binding, and extended the blood circulation half-life.

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A heat stress test also showed that only 2% of the polymer dissociated from the

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carboxymethylated PVA nanocrystals (CM-PVA-NCs), while, at the same time, nearly 30% of the polymer dissociated from the non-cross-linked dextran-coated nanoparticles. Cell

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labeling efficiency was demonstrated by conjugating the CM-PVA-NCs with anti-HER2/neu antibodies and then using them to treat SK-BR-3 (HER2 positive) and MDA-MB-231 (HER2 negative) cells. The cellular uptake of CM-PVA-NCs labeled with Her2/neu antibodies was increased up to 15 fold in the SK-BR3 cells when compared to the MDA-MB-231 cells. Liong et al. have demonstrated that carboxmethyl functionalization of SPIONs improved the stability of SPIONs and they have also utilized the carboxyl groups for labeling with antibody to increase the specificity of the contrast agent.

Fink et al. prepared SPIONs coated with PVA, CM-PVA, thiol-functionalized PVA, and amino-functionalized PVA (amino-PVA)(Petri-Fink et al. , 2005) and tested these

ACCEPTED MANUSCRIPT different functionalized PVAs against human melanoma cells. The Prussian blue reaction was used to determine the interaction of the PVA-coated SPIONs with different human melanoma

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cell lines. They found that the iron content was always below the detection limit in cells not

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exposed to nanoparticles, and was minimally detectable in cells exposed to PVA-SPIONs or exposed to CM-PVA-SPIONs or to thiol PVA-SPIONs. However, cells exposed to amino-

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SPIONs showed an increase in the cellular iron content after 24 h of continuous exposure;

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this increase was dependent on the amount of SPIONs administered. The SPIONs in the culture medium also showed particle aggregation and adherence to plastic surfaces at high concentrations. Thus, the cells showed efficient and rapid interaction only with aminoSPIONs. However, all of the samples of PVA-SPIONs, carboxy-PVA-SPIONs, thiol-PVA-

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SPIONs, or amino-PVA-SPIONs showed no toxicity against melanoma cell lines. Studies on temperature dependent cellular uptake of different PVA-SPIONs revealed

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that the internalization of amino PVA-SPIONs in human melanoma cells was not passive, but

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was dependent on an active, saturable, and energy-dependent mechanism. The observation that the presence of amino groups improves cell uptake of the SPIONs was not surprising,

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since the preferential uptake of cationic liposomes has been widely exploited in the field of basic molecular biology, mainly for transfection purposes. A very clear maximum efficiency of amino-SPIONs was observed for particles with a median diameter between 24 and 32 nm for Me237 cells, whereas a less clear behavior was found for Me275 cells. For these latter cells, a first maximal interaction was observed around 24 nm as for Me237 cells, but at 32 nm, a secondary maximum was observed. This indicates that each cell type has a specific particle size for optimal interaction. From this study it is clear that particle size and functionalization play a significant role in cellular interaction of those particles. Kayal et al. coated SPIONs with different percentages of PVA and utilized the drug doxorubicin for anti-cancer drug delivery.(Kayal and Ramanujan, 2010) PVA is known to

ACCEPTED MANUSCRIPT adsorb nonspecifically onto oxide surfaces through hydrogen bonding arising from the polar functional groups of PVA and the hydroxylated and protonated surface of the oxide, which

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these authors confirmed through FTIR studies. The FTIR results can be interpreted as

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showing that attachment of DOX to the PVA-coated iron oxide nanoparticles occurs via the interaction of the –NH2 and –OH groups of DOX with the –OH groups of PVA, through

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hydrogen bonding. DOX loading and release profiles for the PVA-coated iron oxide

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nanoparticles showed that up to 45% of adsorbed drug was released in 80 h and the drug release pattern indicated a Fickian diffusion-controlled process. The therapeutic drug is formulated along with imaging agent in this work, which can be utilized to non-invasively analyze the drug distribution and release in in vivo.

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One study that investigated carboxymethylation of PVA indicated that this step provided a more stable attachment of the polymer to the SPION surface and also allowed

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more space for conjugating the targeting agents (Liong, Shao, 2010). Among the

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functionalized PVAs examined, the amine functionalized PVA showed a better interaction with cells; this form could also be utilized for further conjugation with carboxyl

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functionalized agents. Lastly, studies of a drug-loaded PVA coating indicated that magnetic targeted drug release could be efficiently performed with polymer-coated SPIONs in a targeted and controlled manner. In summary, SPIONs coated with carboxy-PVA had an excellent stability even at high temperature due to negative charges of the carboxylic acids and SPIONs coated with amino-PVA had a higher cellular uptake due to positive charges of amine groups.

3-5 Poly (Vinyl Pyrrolidone) Poly (vinyl pyrrolidone) (PVP) has been used extensively in various biomedical applications due to its biocompatibility, aqueous solubility, and neutral charge.(Sheng et al. ,

ACCEPTED MANUSCRIPT 2011, Tan et al. , 2009) The PVP coating was achieved by covalent interaction in most of published work, and this increased SPION stability in physiological media. (Zhang et al. ,

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2010)

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Lee et al. synthesized biocompatible PVP-coated iron oxide nanoparticles (PVPSPIONs) to evaluate their efficacy as a magnetic resonance imaging contrast agent.(Lee et al. ,

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2008) They synthesized PVP-SPIONs by a one-step thermal decomposition process. In

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addition to its water solubility, PVP is also soluble in various buffer solutions including PBS, serum, and saline. The T2∗-weighted MRI results indicated that PVP-SPIONs were slightly better than Feridex as a T2∗ negative contrast agent for MRI. The PVP-coated SPIONs retained a high magnetic moment, resulting in high relaxivity. They compared the uptake of

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PVP-SPIONs by macrophages to that of Feridex, a current clinically used MRI contrast agent

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that is known to be taken up by macrophages. The macrophages took up greater amounts of the large core PVP-SPIONs than Feridex. Even at low concentrations of Fe, PVP-coated

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SPIONs exhibited a greater negative contrast enhancement than did Feridex. Due to the monodispersity and high solubility of the PVP-SPIONs in aqueous buffer, the authors were

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able to perform a bolus injection of PVP-SPIONs without the presence of a filter, whereas filtration is required when using Feridex. Feridex and large core PVP- SPIONs were both able to show the reduced signal intensity in the parenchyma of a rabbit liver model, but the T2∗ effect of PVP-SPIONs was more obvious than that of Feridex. Arsalani et al. modified the SPION surface with 3-(trimethoxysilyl) propyl methacrylate (silan A) to introduce reactive vinyl groups onto the particle surface, and PVP was then grafted onto these modified nanoparticles by surface-initiated radical polymerization.(Arsalani et al. , 2010) They confirmed, by FT-IR, that poly (N-vinyl pyrrolidone) chains were successfully grafted onto the Fe3O4 nanoparticle surface. The

ACCEPTED MANUSCRIPT surface-grafted magnetic nanoparticles could be easily dispersed in water to form a uniform suspension and remained stably preserved for several months. This suggested that the

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tendency toward aggregation of the particles was considerably weakened, whereas the bare

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magnetic nanoparticles in a similarly prepared suspension completely precipitated from the solvent. The r2/r1 of these SPIONs was calculated as 28.1, which is much larger than that of

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dextran-coated SPIONs, and demonstrated that PVP-grafted SPIONs should perform well as

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T2-contrast agents in MR imaging. The PVP grafting with SPIONs has also retained the stability and dispersity of the particles as same as the other mechanisms of PVP coating over SPIONs.

In summary, the PVP-SPIONs will be very promising for a clinical application due to

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monodispersity and solubility although the targeting with a specific ligand will make PVPSPIONs an even better contrast agent for clinical studies.

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The various advantages of the PVP-coated SPIONs, along with PVP- and dextran-

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coated SPIONs, are summarized in Table 4.

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4. Polymer coatings for tissue targeting Nanoparticles in the range of 200-300nm tend to be non-specifically accumulated in a tumor region due to the enhanced permeation and retention (EPR) effect.(Acharya and Sahoo, 2011) However, the possibility of accumulation in normal tissues still remains, resulting in minimized bioavailability and cytotoxicity. If these nanoparticles can be targeted to specific locations with the help of biomarkers unique to that region, this would enhance the bioavailability and reduce the toxicity to normal tissues. In the case of tumors, various genetic alterations and cellular abnormalities specific to that region are available that can be utilized as cancer-specific targets.(Orth et al. , 2008) For example, some tumor cells express unique cell surface receptors, which still need to be identified and targeted.(Choi et al. , 2007)

ACCEPTED MANUSCRIPT The pH and temperature are also different in the tumor region compared to the normal regions, which means that pH and temperature responsive polymers could be exploited for

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delivering anti-cancer drugs.(Alexander, 2006) Targeted SPIONs can be formed using

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targeting ligands like sugar, antibodies, peptides and small molecules, which can be attached to the polymers used to coat the SPIONs. The SPIONs coated with these polymers containing

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a target ligand can accumulate more efficiently in the tumor or diseased region, resulting in

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contrast enhancement against the surrounding region that can be detected by MR imaging. The carbohydrate moieties used for targeted imaging are listed in Table 2. Considerable literature exists for targeted SPIONs and these particles will be used in clinics in the near future.(Hieu et al. , 2011, Lee et al. , 2009, Selim et al. , 2007, Vu-Quang et al. , 2012a, Vu-

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Quang et al. , 2012b)

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4-1. Hepatocyte targeted imaging

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The galactose moieties present on the surface can interact with the asialoglycoprotein receptors (ASGP-R) found on the hepatocyte cell surfaces. When the galactose or N-

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acetylgalactosamine residues are attached to any material or polymer, that material will be taken up specifically by the hepatocytes by mechanisms that have been studied by various research groups.(Jiang et al. , 2010, Jiang et al. , 2008, Kim et al. , 2006a, Kim et al. , 2005) Cho and coworkers emphasized the targeting capabilities of galactose-modified polymers for liver specific imaging, which they used to coat a MR contrast agent for targeted imaging. As a SPION coating, they used poly (vinylbenzyl-O-β-D-galactopyranosyl-Dgluconamide) (PVLA), which has galactose moieties that can be recognized by ASGP-R on hepatocytes (Fig. 1).(Yoo et al. , 2007) Significant uptake of the PVLA-coated SPIONs was observed within an hour, due to their specificity. The PVLA-coated SPIONs were preferentially accumulated in the liver, resulting in a relative signal enhancement of the T2-

ACCEPTED MANUSCRIPT weighted MR image, with a signal drop of 75.4%, when injected in vivo. A signal drop of only 36% was observed for PVP-coated SPIONs. The PVLA-coated SPIONs, therefore, were

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more successfully targeted to the liver than were the PVP-coated SPIONs.

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Lactobionic acid (LA)-coated SPIONs have been used by Kamruzzaman et al. to target hepatocytes.(Selim, Ha, 2007) LA falls under the classification of an aldonic acid, and

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the galactose group that aids in hepatocyte specific targeting is linked to it, forming gluconic

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acid. The uptake of LA-coated SPIONs by hepatocytes was enhanced when compared to uptake of unmodified and maltotrionic acid-modified nanoparticles. The in vivo targeting capabilities of LA-coated SPIONs were also confirmed. Specifically, the injection of the LAcoated SPIONs resulted in intensity changes only in liver cells. The LA-coated SPIONs could

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therefore be used as a specific recognition marker for hepatocytes, which would further help in specific imaging of the liver for diagnostic purposes.

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Lee et al. modified the surface of SPIONs with a targeting moiety and a nuclear

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imaging agent to provide both specific targeting and dual imaging.(Lee, Jeong, 2009) They first modified the SPION surface with dopamine by a ligand replacement reaction method.

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LA was then conjugated to the free amine groups of dopamine. A radiolabel was attached by treating the LA-SPIONs with 99mTc, using DTPA as a chelator. The authors co-injected galactose with these 99mTc-labeled LA-SPIONs, and showed that the liver uptake was reduced by up to 40 and 70% at 85 and 115 mol of galactose, respectively. This indicated that the LA-SPIONs were not phagocytosed by Kupffer cells but were selectively accumulated in hepatocytes. The authors also used Bio-TEM to confirm the location of the SPIONs in the hepatocytes of liver tissues after in vivo injection of the LA-SPIONs, and they found the LASPIONs in the cytoplasm. In vivo studies also revealed a specific localization of LA-SPIONs in the liver, using either Nuclear imaging (NI) or MR imaging (Fig.2). The ASGP-Rmediated uptake of LA-SPIONs was clearly confirmed by a competition study where the

ACCEPTED MANUSCRIPT receptors were blocked with galactose. These hepatocyte-specific targeting agents are useful for evaluating hepatocyte function in certain clinical conditions, such as following partial

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liver transplantation or in hepatitis cases, and for monitoring disease progression using

Immune cell targeting

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4-2.

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images.

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Mannan is a cell wall component of microorganisms that consists of D-mannose residues. Mannan in Saccharomyces cerevisiae is water soluble and consists of sugar monomers linked by alpha-(1,6)-, alpha-(1,3)-, and alpha-(1,2)-linkages. Mannan is recognized by mannose receptors of antigen-presenting cells (APCs) and reticuloendothelial

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cells, which mainly reside in the normal lymph nodes (LN), while circulating APCs also opsonize the circulating antigen and then migrate to the lymph nodes. (Kim et al. , 2006b,

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Yoo et al. , 2008) Mannan was coated directly onto the surface of the SPIONs to allow

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recognition by the mannose receptors on the macrophages. The mannan-coated SPIONs were small sized, stable, and showed low cytotoxicity.

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The uptake of mannan-coated SPIONs into macrophages was enhanced when compared to the uptake of PVA-coated SPIONs. The in vivo results suggested that intravenously administered mannan-SPIONs were mainly taken up by the liver, when compared with PVA-SPIONs, and confirmed that the hepatic uptake of mannan-SPIONs was mediated by mannose receptors. Prussian blue staining showed that the uptake of mannanSPIONs mainly occurred near the vasculature of the liver and the SPIONs were selectively distributed into the Kupffer cells, when compared with the PVA-SPION distribution. These findings indicated that the mannan-SPIONs were mainly accumulated by macrophagetargeted uptake.

ACCEPTED MANUSCRIPT 4-3.

Lymph node targeting Mannan-coated SPIONs (mannan–SPIONs) were used for specific delivery by

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receptor-mediated endocytosis to immune cells in the lymph nodes (LNs).(Hieu, Yoo, 2011)

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(Muthiah et al. , 2013)Mannan-SPIONs were taken up by antigen-presenting cells such as macrophages and dendritic cells, which was confirmed by Prussian blue and fluorescence

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staining. As early as 2 h post-treatment, the mannan-SPION-treated macrophages could be

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clearly visualized by MR phantom imaging. The mannan-SPIONs rapidly accumulated in the LNs, as early as 1 h post-injection, and this uptake was sustained for up to 24 h without any significant change in signal intensity. Injection of the mannan-SPIONs into a metastatic lymph node mouse model showed a preferential accumulation of the mannan–SPIONs in the

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normal LNs, but not in the metastatic tumor cells, resulting in negative contrast in MR images. In contrast, in the tumor regions, the metastatic LNs did not internalize these SPIONs

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(Fig.3); this was confirmed by Prussian blue staining, which showed SPION uptake into the

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normal LNs and their absence in the tumor region. The mannan-SPIONs also exhibited enhanced in vivo delivery efficiency and

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targeting of the macrophages in the LN when compared with PVA-SPIONs. This specificity resulted in greater accumulation of mannan-SPIONs in the LNs within a few hours after the intravenous injection, which indicates that they can be used for MR imaging for early detection of metastatic LN (Fig.4). The mannan-SPIONs had an LD 50 value of 44mg/kg when systemically injected in vivo. The bio-compatibility was improved by introducing carboxyl groups onto the surface of the mannan (CM-Mannan). This CM-Mannan was coated onto SPIONs (CM-SPIONs) to allow specific targeting to the immune cells in the LNs.(Vu-Quang, Muthiah, 2012a) The LD 50 of the CM-SPIONs increased up to 80mg/kg due to the carboxylation, resulting in the enhanced bio-compatibility of the contrast agent. The surface charge of the CM-SPIONs was

ACCEPTED MANUSCRIPT negative due to the carboxylation of mannan. The surface carboxylation of mannan did not hinder the targeting ability of mannan. The specific uptake by macrophages was observed

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within 2 h through phantom tube imaging, and within 1 h, the popliteal LN was clearly

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visible after injection of the CM-SPIONs into the foot pad. The CM-SPIONs showed reduced

in the LNs after in vivo administration.

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systemic toxicity compared to mannan-SPIONs, but they still showed enhanced accumulation

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The SPIONs were also coated with glucan (Glu-SPIONs) for metastatic liver imaging, because glucan is taken up primarily by Kupffer cells in the liver, where it is slowly degraded. Previously, glucan was reported to elicit immune responses through the activation of macrophages with an immune cell-specific (1,3)-beta-D-glucan receptor or dectin-1

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receptor. (Vu-Quang, Muthiah, 2012b) A significant drop in MR signal intensity after 3 h was observed in the phantom tube containing macrophages incubated with Glu-SPIONs. This

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indicated preferential uptake of Glu-SPIONs by these macrophages, when compared to

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uptake of PVA-SPIONs and Dex-SPIONs. This enhancement possibly resulted from the specific interaction between the beta-glucan on the Glu-SPIONs and specific receptors such

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as Dectin-1 on the macrophage cells. In an in vivo study, the signal intensity in the normal liver was drastically reduced at 1h post administration when compared to the controls. When Glu-SPIONs were injected into a metastatic mouse liver model, they were taken up by the Kupffer cells, which are specialized macrophages in the liver. Abnormal proliferation of tumor tissues causes a localized lack of Kupffer cells, which results in an unaltered MR signal intensity because of the poor accumulation of SPIONs. These Kupffer cell-deficient tumor regions can be discriminated from the surrounding SPION-rich normal tissues. The immunohistochemical analysis with H&E staining and Prussian blue staining confirmed Glu-SPION accumulation in the Kupffer cells, located in the normal immune cellrich regions of the liver, whereas no staining was found in the highly metastatic and immune

ACCEPTED MANUSCRIPT cell-deficient tumor regions in the liver. In our recent work (Muthiah et al. 2013), we have used PEG as a spacer for

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improving the blood circulation and prevention of RES uptake in the liver of the mannose-

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SPIONs because the PEGylation is known to restrict the nanoparticles interaction with RES system and their normal accumulation in the immune cells. The results showed that the

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targeting capability of the mannose after PEGylation had not been compromised irrespective

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of the increase in biocompatibility in vitro and in vivo, and mannose-PEG-SPIONs accumulation in the immune cell rich organ was enhanced when compared to the conventional PEG-SPIONs, indication of decrease of RES uptake of SPIONs in the liver by

4-4.

Tumor targeting

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PEGylation.

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Folate receptors (FARs) present on the cell membrane can be targeted for tumor

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imaging because these FARs are overexpressed in epithelial carcinomas. FARs provide the opportunity for selective imaging or treatment of the tumor, while preserving the normal

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tissues. Another fact is that FARs are expressed at undetectable or very low levels in normal cells compared to cancer cells. Folic acid-conjugated PEG (FA-PEG) was coated onto SPIONs for targeted tumor imaging.(Islam et al. , 2010) Pretreatment of KB (ubiquitous keratin forming tumor cell line) cells with free folic acid inhibited the intracellular uptake of Cy5.5 labeled and FA–PEG conjugated SPIONs (FA-PEG-SPIONs-Cy5.5), indicating that uptake occurred by receptor-mediated endocytosis. The fluorescence signals in a lung cancer mouse model were stronger with FA-PEG-SPIONs-Cy5.5 than with folate-free PEGSPIONs-Cy5.5, which confirmed the targeted accumulation of FA-PEG-SPIONs in the folate receptor expressing tumor cells. A degradable polymer backbone, carrying branched PEI and PEG as brush-like side

ACCEPTED MANUSCRIPT chains, was coated onto SPIONs (PHEA-PEG-PEI (PPP)-SPIONs), and these were utilized to achieve the dual purpose of gene delivery with simultaneous MR imaging (Fig. 5).(Lee et al. ,

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2011, Lee et al. , 2012b) The SPIONs in the core helped in tracking the associated gene when

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injected in vivo. The PEI in the PPP has positive charges that lead to complexation with the negatively charge plasmid DNA. The PEG in the PPP helped with the surface hydrophilicity

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and enhanced circulation due to its stealth properties. The transfection ability and

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compatibility were demonstrated with respective in vitro studies. When the PPP-SPIONs were injected in vivo, the signal intensity(SI) at the tumor decreased significantly at 24 h post-injection. The relative signal drop of the PPP-SPIONs/pDNA complexes was very high when compared to the control. The high tumor accumulation via the EPR effect is mainly

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attributed to the size of the PPP-SPIONs (150–200 nm), compared to smaller micelles, which would show higher extravasation and lower capture in tumors.

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The surface of SPIONs was modified with dextran and conjugated with herceptin

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(herceptin–nanoparticles) by Chen et al. for specific imaging of breast cancer.(Chen et al. , 2009) When they administered herceptin–nanoparticles as intravenous injection to mice

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bearing a breast tumor allograft, the tumor site was detected in T2-weighted magnetic resonance images as a 45% enhancement drop, which indicated a high level of accumulation of the contrast agent within the tumor sites. The enhancement of the SKBR-3 tumor was significantly lower than that of the KB tumor, suggesting that the SKBR-3 tumor cells specifically took up the herceptin-coated SPIONs and the intensity was reduced during MR imaging, making the breast tumor distinguishable from other tissues. Many researchers have used anti-EGFR monoclonal antibodies for cancer treatment in patients. These are relatively large in size when compared to single chain antibodies, which limits the number of targeting ligands that can be linked to the surface of the nanoparticles. For example, an immunoglobulin G (IgG) antibody has an average size of 14.5*8.5*4nm3

ACCEPTED MANUSCRIPT and a molecular weight of 160 kDa, whereas a single-chain anti-EGFR antibody (ScFvEGFR) is a much smaller targeting ligand. A single-chain Fv (scFv) fragment consists of an antibody

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having heavy- and light-chain variable domains connected with a flexible peptide linker. The

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resulting antibody fragment (25 to 28 kDa) is smaller than 20% of an intact antibody but maintains a high binding affinity and specificity. Yang et al. utilized the ScFvEGFR antibody

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with SPIONs for in vivo targeted MR imaging of EGFR over-expressing tumors(Yang et al. ,

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2009). An amphiphilic triblock polymer was used to stabilize and functionalize the surface of the SPIONs. Two cell lines, MIA PaCa-2 cancer cells that have a high level EGFR expression and human embryonic kidney cell line (HEK 293) that have a low level of EGFR expression, were chosen to study the specificity.

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A large number of iron-positive MIA PaCa-2 cells and very few positively stained HEK 293 cells were present when analyzed with Prussian blue staining. The T2 relaxometry

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measurement in phantom tube imaging also showed a significant decrease in the T2 value for

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the MDA-MB-231 cells treated with the ScFvEGFR-SPIONs compared to those treated with GFP-SPIONs, confirming the Prussian blue staining results. As the iron concentration

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increased, the T2 value decreased proportionally, indicating the specific binding of ScFvEGFR-SPIONs to the EGFR-expressing cells and resulting in the observed changes in the T2 weighted MRI contrast images. When this targeted SPIONs were injected into a mouse tumor model and MR was imaged, the signal intensity reduction was specific to the pancreatic tumor region due to the EGFR expression in those regions. When the targeted SPIONs and non-targeted SPIONs were compared, a 4.8 fold change in the MRI signal was observed in the pancreatic tumor mice that received ScFvEGFR-SPIONs when compared to the mice that received non-targeted SPIONs. Nanoparticles without targeting agents are usually taken up into the liver and spleen after an in vivo injection, but this study showed MRI signal reductions in the animals receiving targeted SPIONs that were 25% lower in liver

ACCEPTED MANUSCRIPT and 52% lower in spleen when compared to the animals that received non-targeted SPIONs. This finding suggested that uptake of the nanoparticles into the liver and spleen was reduced

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by the use of targeted SPIONs.

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Sawant et al. prepared PEG–phosphatidylethanolamine (PEG–PE) micelle-coated SPIONs(Sawant et al. , 2009) with a surface that was modified with the cancer cell-specific

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anti-nucleosome monoclonal antibody 2C5 (mAb 2C5). This antibody recognizes the surface

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of various tumor cells but not normal cells via tumor cell surface-bound nucleosomes. This made the resulting immune-micelles efficient for specifically targeting various tumors. ELISA assays confirmed the activity of the mAb 2C5-immunomicelle-SPIONs toward the specific antigen nucleosomes. The activity was maintained almost at the level of native

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unmodified mAb 2C5. At similar concentrations of SPIONs, the micelle-SPIONs had a significantly better magnetic resonance imaging T2 relaxation signal compared to the parent

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SPIONs. A significant increase in cell-associated T2 relaxation rate (1/T2) was seen for cells

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treated with mAb 2C5 immunomicelle-SPIONs when compared to non-targeted micelleSPIONs. The mAb 2C5 immunomicelle-SPIONs showed specific binding to cancer cells in

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vitro and more SPIONs were brought to the cancer cells, demonstrating this carrier’s potential as a targeted MRI contrast agents for tumor diagnosis.(Peng et al. , 2008) The work on antibody-coated SPIONs is summarized in Table 6. The small size and low immunogenicity of the peptides are making them more attractive for the purpose of targeting when compared to the antibody-based targeting. Many peptides have been identified by phage display technique and utilized for targeting studies. Among them, arginine-glycine-aspartic acid (RGD) peptide is very specific for targeting the integrin receptors which are over expressed on angiogenic endothelium. Moreover the expression of the receptors on tumor vessels also correlates with the tumor progression. Yang et al. have used this peptide for targeting the dual contrast agent nanocarrier

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Cu-labeled

ACCEPTED MANUSCRIPT SPIONs for PET/MR imaging (Yang et al. , 2011). They have also conjugated the drug doxorubicin (DOX) with nanocarrier using a pH-sensitive hydrazone bond to acquire pH-

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triggered release of the drug. The RGD-functionalized nanocarrier showed much higher level

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of tumor accumulation compared to the RGD-free nanocarrier. The RGD specificity enhanced the quantification and non-invasive detection of the drug at the target site with PET

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imaging and subsequently helped in monitoring the anti-cancer effects of the drug with MRI, 64

Cu-labeled SPIONs are perfect

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suggesting that RGD peptide and DOX drug conjugated

examples for tumor targeting drug delivery and PET/MR imaging with single delivery agent.

Conclusion and Perspectives

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The SPIONs can be coated by various biocompatible polymers for stability, enhanced contrast imaging, and tissue targeting. When the coating materials consist of targeting

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moieties, the imaging becomes more specific by reducing the non-specific interaction and

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increasing the availability in the targeted region. Biocompatible polymers such as chitosan, PEG, dextran, PVA, and PVP for enhancing stability and targeting moieties such as galactose,

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mannose, and folic acid, which target hepatocytes, immune cells, and cancer cells, were discussed in this review. In future, SPIONs will be utilized to target diseased tissue more specifically with the help of cell penetrating peptides, aptamers, and their fusion aptides (aptamer-peptide). SPIONs can also be loaded along with quantum dots for dual imaging, which will help in optical detection and tracking in a better way than by using SPIONs alone. Polymer-coated SPIONs can also be labeled with

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In or similar materials to obtain

functional and anatomical imaging through SPECT and MRI.

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Table 1 Method of preparation of SPIONs

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Table 2 Chitosan coated SPIONs.

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Table 3 PEG-coated SPIONs.

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Table 4 Dextran-, PVA-, and PVP-coated SPIONs

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Table 5 Carbohydrate moiety-coated SPIONs for targeted imaging

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Table 6 Antibody-coated SPIONs for tumor-specific imaging.

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bind with ASPG-R for hepatocyte-specific imaging.

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Fig. 2 In vivo micro-SPECT/CT dual imaging of mice in a three-dimensional view (A, left side), as coronal (b) and sagittal (c) sections at 1 h after injection of 99mTc-labeled LBA-

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SPIONs. The organs are labeled in short form as follows: H, heart; Lu, lung; Li, liver; B,

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bladder. T2-weighted images of the middle part of the liver before (a) and after (b) injection of LBA-SPION (B, right side). At 1 h post injection of the contrast agent, the signal of the liver tissue became noticeably darker. GB, gallbladder; R, right. Fig. 3 The mannan-SPIONs interact with the mannose receptors present on the surface of

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immune cells. These immune cells are present in the region surrounding the tumor in the lymph nodes, while the tumor itself is devoid of immune cells. The mannan-SPIONs

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accumulate in the immune cells surrounding the tumor, making that region appear dark, while

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the tumor region appears bright in the MR image. Fig. 4 MRI of partially metastatic lymph node (LN) and pathological specimens. (a) MR

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images before and after injection. Blue indicates the negatively enhanced region in the LN due to the accumulated SPIONs and white arrows indicate the metastatic LN area. (b) Gross image of a Prussian blue stained lymph node. (c) Magnified images of normal LN tissue that had accumulated mannan–SPIONs, stained with Prussian blue (blue arrows) in residual normal LN region. (d) Proliferating tumor cells (white arrows), stained with HE. Fig. 5 A scheme showing SPION encapsulation in a PPP polymersome. The PEI with a positive charge at the surface of the polymersome helps in binding with the negatively charged pDNA.

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