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Wiley Interdiscip Rev Nanomed Nanobiotechnol. Author manuscript; available in PMC 2017 November 01. Published in final edited form as:
Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2016 November ; 8(6): 814–841. doi:10.1002/wnan. 1400.
Functional Nanoparticles for Magnetic Resonance Imaging Xinpei Mao1,2, Jiadi Xu3,4, and Honggang Cui1,2,5,6,* 1Department
of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N Charles Street, Baltimore, MD 21218, USA
2Institute
for NanoBioTechnology, The Johns Hopkins University, 3400 N Charles Street, Baltimore, MD 21218, USA
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3Russell
H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA 4F.
M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Research Institute, Baltimore, Maryland 21205, USA
5Department
of Oncology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
6Center
for Nanomedicine, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 North Broadway, Baltimore, Maryland 21231, USA
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Nanoparticle-based magnetic resonance imaging (MRI) contrast agents have received much attention over the past decade. By virtue of a high payload of magnetic moieties, enhanced accumulation at disease sites, and a large surface area for additional modification with targeting ligands, nanoparticle-based contrast agents offer promising new platforms to further enhance the high resolution and sensitivity of MRI for various biomedical applications. T2* superparamagnetic iron oxide nanoparticles (SPIONs) first demonstrated superior improvement on MRI sensitivity. The prevailing SPION attracted growing interest in the development of refined nanoscale versions of MRI contrast agents. Afterwards, T1-based contrast agents were developed, and became the most studied subject in MRI due to the positive contrast they provide that avoids the susceptibility associated with MRI signal reduction. Recently, chemical exchange saturation transfer (CEST) contrast agents have emerged and rapidly gained popularity. The unique aspect of CEST contrast agents is that their contrast can be selectively turned “on” and “off” by radiofrequency (RF) saturation. Their performance can be further enhanced by incorporating a large number of exchangeable protons into well-defined nanostructure. Besides activatable CEST contrast agents, there is growing interest in developing nanoparticle-based activatable MRI contrast agents responsive to stimuli (pH, enzyme, etc.), which improves sensitivity and specificity. In this review, we summarize the recent development of various types of nanoparticle-based MRI contrast agents, and have focused our discussions on the key advantages of introducing nanoparticles in MRI.
*
Correspondence to: [email protected]].
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Introduction
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Magnetic resonance imaging (MRI), developed on the principle of nuclear magnetic resonance (NMR), is one of the most powerful tools extensively used for noninvasive molecular and cellular imaging1. With superb spatial resolution and tissue contrast, MRI provides anatomic images of soft tissues and is considered as one of the most important diagnostic modalities for imaging of the brain2, cartilage, heart, blood vessels and for tumor detection3. Unlike other imaging platforms, such as computerized axial tomography (CAT), positron emission tomography (PET) and single-photon-emission computed tomography (SPECT), MRI techniques do not require the use of radioactive agents and ionizing radiation. Although MRI itself is able to provide detailed images of soft tissues, its intrinsic low sensitivity makes it hard to differentiate normal tissues from lesions. The introduction of supplements, called contrast agents, can enhance the contrast effect at regions of interest by accelerating magnetic relaxation4, 5. These MRI contrast agents can be divided into three groups based on the mechanism by which contrast is generated. T1 agents provide positive contrast by shortening the longitudinal relaxation time of surrounding water molecules, whereas T2 agents shorten the transverse relaxation time of water protons (basic principles will be discussed in Section 1). The third group relies on chemical exchange saturation transfer (CEST) and represents a relatively new approach to enhance MRI contrast. CEST agents exchange their presaturated exchangeable protons with those of bulk water, with the MRI contrast capable of being switched “on” and “off” by irradiation with radiofrequency (RF) pulses.
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Among the various contrast agents, nanoparticle-based contrast agents (especially nanoparticles with diameters of 1–100 nm) have become extremely attractive due to their unique features. Firstly, nanoparticles can be loaded with up to hundreds of thousands of imaging moieties per structure, providing superb signal amplification that enables good imaging contrast at a low dose of contrast agent, and reduces the potential for any cytotoxicity associated with the contrast agent6. For example, a 150 nm dendrimer nanocluster possesses a loading capacity of ~300,000 gadolinium ions, giving a r1 relaxivity value of 12.3 mM−1sec−1 per gadolinium ion and 3,600,000 mM−1sec−1 per particle (1.41 T, 40 °C)7, while the r1 relaxivity value of small gadolinium chelates is only 3.5 mM−1sec−1. Secondly, compared to bulk counterparts, nanoparticles possess a relatively large surface area that offers improved reactivity and an ability to be tailored with additional surface moieties to either improve targeting8 or introduce additional functionality (such as therapeutic features9 or fluorescence10). Multimodal MRI contrast agents are even more prevalent since they can reveal several properties at the same site by applying a single contrast agent. Thirdly, nanoparticles tend to accumulate at tumor sites through the enhanced permeability and retention (EPR) effect11, thereby rendering a higher signal-to-noise ratio in tumors12. Researchers have developed various types of nanoparticle-based MRI contrast agents of T1, T2 and CEST modalities to fulfill different purposes. Among them, T1 nanoparticle-based contrast agents have proven the most popular due to the positive contrast provided. Gadolinium (GdIII) complexes like Gd-DTPA are widely used to detect the breakage of the blood brain barrier (BBB) and characterize changes in vascularity13. However, GdIII chelates
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have a short circulation time and the relaxivity exhibited is relatively low, requiring a large dose of ion chelates to reach a useful detection level. Moreover, the non-biocompatible gadolinium has the potential to introduce toxicity into cells upon dechelation from its complexes. Alternatively, nanostructures (like dendrimers14, liposomes15, quantum dots16, mesoporous silica17 and carbon nanotubes18) can be employed as carriers for GdIII chelates, which significantly improves relaxivity, and the nanoparticle surface can be facilely modified with additional functional groups to create multimodal contrast agents. Since the number of GdIII chelates in these type of systems is limited by the available anchoring sites on the surface, and the size of ionic nanoparticle-based contrast agents are usually too large to avoid rapid excretion by the reticuloendothelial system (RES)19, smaller-sized inorganic nanoparticles composed of gadolinium oxide (Gd2O3)10, gadolinium fluoride (GdF3)20, gadolinium phosphate (GdPO4)21 or manganese oxide (MnO)22 have become more attractive, and relevant research has been conducted on functionalized inorganic nanoparticles as MRI contrast agents. Unlike T1 contrast agents, the negative contrast provided by T2 or T2* contrast agents is insufficient to fully differentiate pathogenic tissues from normal tissues, and the susceptibility generated distorts surrounding normal tissues. The intrinsic high magnetization and biocompatible features still lead to intensive research on iron oxide nanoparticles (extensively used T2* contrast agents) as MRI contrast agents, although the image contrast has been compromised to some degree. Superparamagnetic iron oxide nanoparticles (SPION) and ultra-small superparamagnetic iron oxide nanoparticles (USPION) are representative commercial products of T2 contrast agents that are widely used as liver and lymph node imaging contrast agents. As both core and shell affect magnetic properties of contrast agents, improvement on each aspect has been studied. As an alternative to always “on” MRI contrast agents, MRI nanoprobes that are responsive to various stimuli (i.e. pH, enzyme, RF saturation, etc.) have become attractive to researchers, since they could provide better sensitivity and specificity. Numerous studies have been conducted and reported on CEST contrast agents, as they are easily controlled to be “on” and “off” by RF saturation. Since the sensitivity is strongly dependent on the number of exchangeable protons, the incorporation of CEST contrast agents into nanoparticles can greatly increase the amount of exchangeable protons. Herein, we summarize representative nanoparticle-based MRI contrast agents on the basis of the three operating mechanisms: T1, T2, and CEST. Advances in nanotechnology enables researchers to develop nanoparticle-based MRI contrast agents with higher magnetization and the desired surface characteristics to meet the various needs for effective biodistribution. In this review, we will also discuss improvements focusing on magnetic materials and nanocarriers used to load magnetic materials.
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1. Basic principles of MRI contrast agents As illustrated in Figure 1a, upon exposure to an external magnetic field (B0), a greater proportion of protons will prefer to align parallel to the magnetic field (lower energy state), while the remainder aligns antiparallel (higher energy state). Therefore, a net magnetization (Mz) is produced along the direction of magnetic field (z-axis), and protons spin around the z-axis at a precession rate named the Larmor frequency (ω0). During this period, protons precess separately (out of phase). When an RF pulse is applied to the nuclei with the same
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precession frequency, some protons in the lower energy state are flipped to the higher state by absorbing the RF energy (Figure 1b). Furthermore, the protons become synchronized and precess together (in phase). Consequently, a transverse magnetization (Mxy) perpendicular to the static magnetic field is generated.
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After removal of the RF pulse, the protons relax to their equilibrium state via two relaxation pathways: longitudinal relaxation (T1 relaxation) and transverse relaxation (T2 relaxation). In T1 relaxation, antiparallel protons jump back to the parallel state and give up energy to molecules in the surrounding environment (lattice), so that T1 relaxation is known as spinlattice relaxation. The recovery of Mz is described in equation (1), where the T1 relaxation time is defined as the time taken to recover 63% of the original longitudinal magnetization. The T1 relaxation rate, R1, is given by the reciprocal of T1 (1/T1), which is a function of r1 (T1 relaxivity), an intrinsic property of T1 contrast agents, and contrast agent concentration (see equation (2)). The ideal T1 contrast agent should be able to effectively shorten the T1 relaxation time at low concentration. In terms of T2 relaxation (spin-spin relaxation), protons that are in phase begin to dephase, with the transverse magnetization (Mxy) decaying as a result. This decay process follows equation (3), where T2 refers to the time taken to decay to 37% of the original Mxy value. T2 relaxivity (r2), an intrinsic property of T2* contrast agents, affects the T2 relaxation rate, R2, as described in equation (4). Generally, spins decay faster than T2 due to the magnetic field inhomogeneity generated by T2 contrast agents. After taking this inhomogeneity into consideration, the effective relaxation time (T2*) is given by equation (5), where γ is the gyromagnetic ratio and ΔBi is the difference in the local magnetic field strength due to the inhomogeneity.
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(1)
(2)
(3)
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(4)
(5)
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2. T1 contrast agents
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The signal enhancing nature of T1 contrast agents generates a favorably positive contrast effect, making them the most popular contrast agents for clinical use, especially small molecular gadolinium (GdIII) chelates. The utility of GdIII lies in its electron configuration, possessing seven unpaired electrons in f orbitals that give a symmetric S electronic state. This combination bestows a large magnetic moment on the ion and generates a high relaxivity23. Since gadolinium is toxic as free ions, the chelation of multi-dentate ligands GdIII ions can help minimize toxicity, and GdIII complexes are divided into linear chelates (Gd-DTPA, Gd-BOPTA, and MS-325) and macrocyclic chelates (Gd-DOTA, etc.), where macrocyclic GdIII chelates exhibit stronger thermodynamic and kinetic stability than their linear counterparts24. Among them, Gd-DTPA (Magnevist®) and Gd-DOTA (Dotarem®) possess similar r1 relaxivities around 3.5 mM−1sec−1 (measured at 20 MHz and 37 °C), while protein binding contrast agents like Gd-BOPTA (Multihance®) and MS-325 (Ablacar®) have higher relaxivity of 9.2 mM−1sec−1 (measured at 0.7 T and 37 °C), attributed to the slower molecular rotation caused by non-covalent binding to albumin25. Although GdIII chelates exhibit invaluable utility in diagnosing cancer and sclerosis, they experience rapid renal clearance that limits the time window for MRI26. Unlike iron oxide nanoparticles (T2* contrast agents) that can remain at the imaging site for several months, 95% of GdIII chelates are excreted intact within 24 h of administration27. Regarding the intrinsic low sensitivity nature associated with MRI, the relatively small relaxivity of small molecular GdIII chelates need further improvement. Additionally, the nonspecific extravasating behavior of small molecular GdIII chelates in both normal tissue and at pathogenic sites is an obstacle to clinical application. Therefore, new platforms with specific targets should be incorporated to tackle these problems13. Intensive research has been devoted to developing nanoparticle-based T1 contrast agents, focusing on two methods in particular. The first involves either anchoring small molecular GdIII chelates onto the surface of the nanoparticles or loading GdIII chelates into the interior cavity of the nanostructure. The second method is the creation of paramagnetic inorganic nanoparticles composed of Gd2O3, Gd2F3 and GdPO4. Both methods enhance the potential relaxation by increasing the number of GdIII ions per unit volume, with the high payload of imaging moieties increasing the T1 relaxivity and lowering the dose of contrast agent required for proton relaxation. Furthermore, an additional benefit is derived from the preferential accumulation of nanoparticles at tumor sites due to the leaky vasculature of abnormal tissue, and the targeting efficiency can be further improved by modifying the nanoparticle surface with targeting ligands.
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Nano-sized carriers can be employed to load GdIII chelates, creating nanoparticle-based T1 contrast agents that not only incorporate advantages of nanoparticles for image diagnosis, but also demonstrate a positively enhanced contrast effect. Commonly used carriers include dendrimers28, liposomes, quantum dots, silica and supramolecular self-assembly29. 2.1 Soft nanoparticles—Dendrimers are highly branched synthetic spherical polymers, which are fabricated as the core in nanostructure-based GdIII contrast agent. Previous research revealed that polyamidoamine (PAMAM) dendrimer-DOTA- GdIII chelates could increase T1 relaxivity up to 45 mM−1sec−1 (for dendrimer generations (G) of 9 or 10),
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dramatically improving T1 sensitivity compared with small molecular GdIII chelates (r1 ~ 3.5 mM−1sec−1). Although dendrimer nanoclusters (DNCs) enhance r1 relaxivity, the slow and/or incomplete excretion of such large-size molecules hampers their potential for clinical translation as the accumulation of free GdIII ions from dechelation may cause nephrogenic systemic fibrosis (NSF) that impairs kidney function, especially those released from linear GdIII chelates (such as Gd-DTPA). Furthermore, gadolinium chelates can undergo transmetallation with copper and zinc ions in the body, followed by the onset of toxicity from the released GdIII ions30. Hence, it is important for researchers to focus on developing biodegradable DNCs with stable ligands. Recently, Tsourkas and co-workers integrated polydisulfide linkages between individual PAMAM (G-3) dendrimers (figure 2A), enabling DNCs to further degrade through thiol-disulfide exchange reactions into smaller molecules that were readily excreted through renal clearance26. Unlike the traditional method of labeling GdIII onto dendrimers with chelating ligands, Tsourkas and co-workers prepared [Gd-C-DOTA]−1first, and then reacted dendrimers with the pre-metallated ion complexes. By doing so, the potential toxicity stemming from free GdIII ions trapped within dendrimer cavities was reduced. The relaxivities of 59, 91 and 142 nm sized polydisulfide DNCs were 11.7, 18.4 and 22.4 mM−1sec−1 per GdIII ion, respectively, which were higher than PAMAM (G-3)–[Gd-C-DOTA] −1 without polydisulfide bonds (r1 = 10.9 mM−1sec−1)31 and other polydisulfide gadolinium chelates that have previously been reported as blood pool agents (r1 = 4.4–16.3 mM−1sec−1)32–34.
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Liposomes are biocompatible vesicles formed by either natural or synthetic amphiphilic lipids, with examples of commonly used materials being phosphatidyl choline (PC), phosphatidyl glycerol (PG), and cholesterol. Confining the liposomal diameter to between 60–500 nm, and the introduction of additional surface coatings has been shown to prolong circulation time and improve stability in vivo35. Intensive research has been devoted to investigate liposomes as possible carriers of Gd-chelates, and liposomal contrast agents could be synthesized through two approaches: core-encapsulated GdIII (CE-Gd) liposomes or surface-conjugated GdIII (SC-Gd) liposomes36. Ghaghada studied how liposome size and internal gadolinium concentration would affect T1 relaxivity, determining that the T1 relaxivity increased as the size of the liposomal nanoparticles decreased (highest r1 = 1.85 mM−1sec−1 at 2 T magnetic field strength), and T1 relaxivity did not change significantly with core GdIII concentration over the measured concentration range37. Annapragada et al. compared relaxivity among three liposomal formulations (Figure 2B): CE-Gd liposomes, SC-Gd-liposomes and dual-Gd liposomes (where GdIII chelates were encapsulated within the core and conjugated onto the surface)38. Since dual-Gd liposomes possessed the highest number of GdIII ions per liposome, they demonstrated the highest T1 relaxivity on an individual nanoparticle basis, which was more than 2000–8000 fold that of small molecular GdIII chelates. Furthermore, the dual-Gd liposomes demonstrated the highest signal-to-noise and contrast-to-noise ratios in CE-MRA studies. Very recently, supramolecular MRI contrast agents have quickly drawn researchers’ attention since their first introduction by Stupp and Meade39, where self-assembling peptide amphiphiles (PAs) were conjugated with macrocyclic GdIII chelates to furnish peptide amphiphile contrast agents (PACAs). Nanostructures self-assembled from PAs can emulate extracellular matrices, a biomimetic strategy widely applied in the field of regenerative Wiley Interdiscip Rev Nanomed Nanobiotechnol. Author manuscript; available in PMC 2017 November 01.
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medicine40. Depending on the design rationale and assembly environment, various onedimensional morphologies with different physical properties can be obtained upon selfassembly of PAs, which renders PACAs a versatile platform for imaging purposes41. Most importantly, it is well known that the relaxivity of contrast agents will be enhanced by conjugation to proteins and polymers with large molecular weight, or by preparation of micellar structures42. Similarly, the structure of self-assembled PAs allows the increase in rotational correlation time (τr) that subsequently enhances relaxivity. Bull et al. reported the relaxivity difference that arose from the varying morphology (see Figure 3A–B) of two selfassembled PAs, where the relaxivity of nanofibers was 14.7 mM−1sec−1, while the relaxivity of spherical micelles was 22.8 mM−1sec−1 before cross linking (pH = 7.41)43. Later, Bull et al. fabricated three molecules (1, 2, 3) with similar sequences, with PA 1 able to form selfsupporting hydrogels, while PACAs 2 and 3 possessed Gd-DOTA positioned at different distances from the hydrophobic alkyl tails (Figure 3C). Both PACAs self-assembled into nanofibers at pH greater than 7.0, though cannot form hydrogels by themselves39. Upon mixing PACA 2 or 3 with filler PA 1, a homogeneous hydrogel was formed that allowed MR images to be obtained. From the MR images of these phantom gels, the mixture of PACA 3 and PA 1 exhibited the greatest contrast, implying that positioning GdIII chelates closer to the hydrophobic end of PAs would result in higher relaxivity. The postulated reason behind this was due to the decreased internal flexibility and increased steric hindrance of GdIII chelates that occurs upon self-assembly. Since the highest contrast was generated with PACA 3, it was further mixed with various epitope-bearing PAs (such as the IKVAV or YIGSR epitopes for neuronal stem cell differentiation, and the RGD epitope for cell adhesion) to form hydrogels (doping with an equal amount of 3). The T1 values of each mixed hydrogel were found to be similar, proving the ability to use PACAs with various PA gels.
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The feasibility of employing supramolecular dual-modality nanoprobes (self-assembly of amphiphilic peptide conjugates containing the fluorophore 5-FAM and Gd-DOTA) as contrast agents for MR and fluorescence imaging was assessed by Cui and co-workers (Figure 4)44. The live-cell fluorescence imaging of two self-assembled nanoprobes (one with single hydrocarbon tail, and the other with two hydrocarbon tails) was studied in KB-3-1 human cervical cells to evaluate cell viability of PACAs. The dual-tailed PACA 2 (50 μM) showed 25-fold higher cellular uptake than PACA 1 (200 μM), which was due to membrane insertion through the two hydrophobic alkyl chains. Before self-assembly, the relaxivity, r1, of PACA 1 and PACA 2 were 4.3 mM−1sec−1 and 4.2 mM−1sec−1 (pH = 7.4, room temperature), respectively, which were similar to the relaxivity of small molecular GdDOTA contrast agents (3.5–4.8 mM−1sec−1)23. However, upon self-assembly, the nanospheres formed by PACA 1 and PACA 2 had relaxivities of 7.8 mM−1sec−1 and 14.3 mM−1sec−1 that were higher than the monomeric forms. The higher relaxivity of selfassembled PACA 2 stems from the denser packing of PACA 2 in its self-assembled state, which led to a higher aggregation number of molecules and effective molar mass than selfassembled PACA 1. As we described above, self-assembling PAs have the propensity to enhance T1 relaxivity, and one advantage associated with fibrous PA structures is their potential for biodegradation
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into natural building blocks, so that it is of importance to track the fate of biomaterials after implanted in vivo29. There are, however, limited studies that shed light on the degradation process in vivo. By incorporating MRI modality to image PACAs, we could rationalize using PACAs to enhance MRI contrast, and possibly allow us to track the fate of PACAs in vivo, thus laying a critical foundation for future development as therapies45. Stupp, Meade and coworkers have reported on the in vivo biomaterial localization with Gd(HPN3DO3A) labeled peptide nanofibers (Figure 5). These PAs were designed to have one chelate next to the Cterminal (PA1 and PA2), three chelates at the C-terminal end (PA3), or one chelate relatively far away from the C-terminus (PA4)29. Small-angle X-ray scattering (SAXS) experiments revealed that the bulky Gd (HPN3DO3A) conjugated to the outermost residue of the PAs exhibited β-sheet character and retained high-aspect-ratio structures. The in vivo degradation was evaluated with the mixture (gels) of PA (1 or 3) and filler C16V3A3E3-NH2, of which the PA1 gel produced positive contrast in T1-weighted MRI and PA3 gel produced negative contrast. Subsequent ICP-MS analysis was conducted to measure GdIII retention. The result showed that 62 ± 8% of PA1 and 54 ± 9% of PA3 remained in the mouse leg after 4 days, and the similar T1 relaxation time at day 0 and day 4 indicated the PA concentration did not change significantly, verifying that the approach of using MRI to track the fate of biomaterials is practicable.
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2.2 Inorganic nanoparticles—Quantum dots (diameters of 2–6 nm) are of interest as imaging modalities due to their bright fluorescence, phosphorescence, and narrow emission spectrum. Incorporating such semiconductor nanocrystals into the design of GdIII-chelated nanoparticles imparts multimodal functionality for both MRI and fluorescence microscopy. Mulder et al. fabricated quantum dots with GdIII chelates as well as a PEG-lipid coating (pQDs), which endowed these contrast agents as bimodal molecular imaging probes (see figure 2C–E)16. The PEGylated lipid coating protected the contrast agents from interacting with plasma proteins, so that they went through less rapid hepatic clearance. The T1 relaxivity of pQDs was more than 12 mM−1sec−1, three times higher than those of small molecular GdIII chelates. If pQDs were further functionalized with the RGD peptide sequence, a cell adhesion epitope, RGD-pQDs could be employed to detect angiogenesis (new blood vessel formation), an important pathological process in cancer and atherosclerosis46.
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Recently, researchers have been interested in investigating inorganic nanostructures like porous or mesoporous silica with high loading capacity. High relaxivities have been reported for GdIII chelates embedded in mesoporous silica. Lin and co-workers grafted GdIII chelates onto mesoporous silica nanospheres (MSN-Gd), which exhibited high T1 relaxivities of 28.8 mM−1sec−1 and T2 relaxivity of 65.5 mM−1sec−1 (determined with a magnetic field strength of 3.0 T)17. Considering its high r1 and r2 values, MSN-Gd is a promising candidate for intravascular MR imaging (T1) and depicting soft tissues (T2). Tsai et al. described multifunctional mesoporous silica nanorods loaded with Gd-DTPA and green fluorescing dye47. The magnetic nanorods exhibited no short-term cytotoxicity, and displayed high cellular uptake efficiency without the need for transfection agents. For the GdIII chelates grafted inside silica pores, however, the access of water molecules may be hindered. At the other extreme, the presentation of GdIII chelates on the surface of nonporous nanoparticles
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does not hinder the interaction of water molecules with each Gd site, thus allowing better contrast. For example, fluorescent quantum dots or colloidal gold nanocrystals were coated with a thin silica shell, onto which multiple Gd-DOTA molecules were covalently attached without negative influence on optical properties of the nanoparticle cores48. The particulate relaxivity was dependent on the number of gadolinium ions on the silica surface, and the number of Gd-DOTA molecules can be easily tuned from 20 to 320 GdIII ions per quantum dot. Moreover, due to the reduced rotational motion of Gd-DOTA molecules conjugated to the silica, both the r1 and r2 relaxivities were 6 to 15 times larger than free Gd-DOTA. One limitation associated with quantum dots or gold nanoparticles is that the light penetration depth in living tissue is just a few centimeters in an optimized scenario, which makes them uncompetitive as multimodal MRI contrast agents. The application of silica coatings and Gd-DOTA attachment endows high r1 and r2 relaxivities, and preliminary in vivo tests have verified the contrast enhancement provided by paramagnetic silica-coated nanoparticles.
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Although nanostructures labeled with ionic T1 contrast agents exhibit higher relaxivity than small molecular GdIII chelates, the number of paramagnetic ions attached to the nanostructure surface is limited by the total number of available anchoring sites. Since the number of paramagnetic ions intrinsically determines the magnetism of nanoparticles, a higher number of imaging moieties per nanoparticle is always preferred. Most significantly, the size of ionic T1 nanoparticles is usually larger than 100 nm, making them susceptible to excretion by the RES system19. Alternatively, transition metal and lanthanide metal based inorganic nanoparticles composed of gadolinium oxide (Gd2O3), gadolinium fluoride (GdF3)20, gadolinium phosphate (GdPO4)49 or manganese oxide (MnO) have drawn researcher’s attention as a second type of T1 nanostructure-based contrast agent, since they have increased magnetic moments stemming from the abundance of paramagnetic ions at the surface, and the overall size is smaller than 100 nm.
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Early research led to the development of paramagnetic ultrasmall Gd2O3 nanoparticles with an optimal particle diameter of 1 nm and r1 of 9.9 mM−1sec−1, which was higher than small molecular GdIII chelates50. This may be due to the induced longitudinal relaxation of water protons by surface GdIII ions of the Gd2O3 nanoparticles. A biocompatible PEG coating can enhance the steric repulsion of nanocrystals to avoid undesired aggregation, but conventional procedures to graft PEG onto Gd2O3 are time consuming. Fortin and co-workers reported a new, fast and efficient one-pot technique to prepare PEGylated nanoparticles with small sizes of 1.3 nm51. The PEGylated nanoparticles were colloidally stable in aqueous media, ideal for T1-weighted MRI since the ratio of r2/r1 was small enough (r2/r1 = 1.20 at 60 MHz). Furthermore, these ultrasmall PEG-Gd2O3 nanoparticles allowed the visualization of labeled cells implanted in vivo. Labeled F98 brain cancer cells were implanted into animals with brain tumors, and positive contrast was observed up to at least 48 h after implantation. Multimodal contrast agents are attracting increasing attention because they combine several complementary properties into a single object. Bridot et al. encapsulated Gd2O3 within a polysiloxane shell that carried organic fluorophores, and the surface of the nanoparticles was further coated with PEG-COOH to avoid agglomeration and precipitation10. Designed Gd2O3 nanoparticles with 2.2 nm core exhibited higher r1 and r2 values than small molecular GdIII chelates (r1 = 8.8 mM−1sec−1, r2 = 11.4 mM−1sec−1), and the small r2/r1 ratio rendered the nanoparticle as ideal T1 contrast agents (r2/r1 = 1.3). Fluorescence reflectance imaging Wiley Interdiscip Rev Nanomed Nanobiotechnol. Author manuscript; available in PMC 2017 November 01.
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after 3 h injection revealed no undesired accumulation in lungs and liver, which was due to both the small size and PEG coating. T1-weighted MRI of rats 1 h after injection and ICPMS of some organs and urine confirmed that the uptake of Gd2O3 nanoparticles in lung, spleen and heart was insignificant.
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Since relaxivity is inversely proportional to the sixth power of the ion–nuclear distance, metal ions like GdIII with seven unpaired electrons are the most predominantly used paramagnetic contrast agents52. However, GdIII ions are not naturally present in the human body. Alternatives based on transition metals, like manganese (five unpaired electrons), have progressed since there are natural processes that utilize these ions and could potentially be exploited. For instance, MnII ions are natural cellular constituents that play an important role as cofactors of enzymes and receptors. Moreover, the uptake of manganese is highly dependent on mitochondrial density that makes MnII a very promising contrast agent for mitochondria-rich organs like the liver, pancreas and kidney53. It is this combination of unique features that make MnII an attractive study object. Cai et al. designed hollow mesoporous Prussian blue (HMPB) nanoparticles loaded with a Mn-containing Prussian blue analogue (MnPBA), which also acted as an excellent drug carrier (high loading efficiency of 97.5% and loading capacity of 62.3%) and a photothermal agent, and most importantly had a high relaxivity (r1 = 7.43 mM−1sec−1) at pH 5.0 (tumor site conditions)54. This pH-responsive theranostic nanoplatform could concurrently release MnII ions and doxorubicin (DOX) at the tumor site. The positive correlation between MRI intensity and DOX release allowed controlled drug release (Figure 6). Additionally, the combination of strong absorbance in the NIR and chemotherapy of DOX realized a synergistic chemothermal therapy that achieved enhanced tumor therapeutic efficacy.
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One drawback, however, is that MnII chelates can cause hepatic failure and cardiac toxicity, severely limiting their application in animal studies. Hence, studies on functionalized MnO/Mn3O4-based nanoparticles have been undertaken. Hyeon, Gilad and co-workers studied mesoporous silica-coated hollow manganese oxide nanoparticles (HMnO@mSiO2) with a relaxivity of 0.99 mM−1sec−1 at 11.7 T55, a value much higher than existing MnO nanoparticle-based T1 contrast agents (MnO encapsulated within PEG-phospholipids, MnO coated with dense silica, and non-etched mesoporous silica-coated MnO)56, 57, simply because the mesoporous structure allowed the rapid penetration of water into the manganese core, and hence greater exposure of the MnII ions to water molecules (Figure 7). Although the relaxivity of these MnO nanoparticle-based contrast agents was compromised compared to small molecular MnII compounds, they were specifically designed to label and track cells that MnII compounds could not. With electroporation, HMnO@mSiO2 nanoparticles exhibited high cellular uptake (more than 75% in 24 h) by mouse adipose-derived mesenchymal stem cells (MSCs), and the applied electroporation only minimally impacted cell viability. In mice transplanted with labelled MSCs, the feasibility of HMnO@mSiO2 nanoparticles for long-term (over 14 days) in vivo cell tracking was studied, and the results revealed a hyperintense region at the transplantation site. The sustained contrast in vivo over a prolonged time period rendered HMnO@mSiO2 nanoparticles as competitive candidates for noninvasively monitoring the fate of transplanted cells in vivo.
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3. T2 MRI contrast agents
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3.1 Superparamagnetic Iron Oxide Nanoparticles—Shortly after the introduction of GdIII chelates, increased investigations were conducted on iron oxide nanoparticles as T2weighted MRI contrast agents, whose characteristics cannot be replaced by bulk counterparts or atoms. Contrary to the T1 contrast effect, T2 contrast agents generate a negative contrast effect due to a signal reduction mechanism that at times can make it difficult to differentiate normal tissue from lesions. It is also known that T2 contrast agents induce high susceptibility that can induce local magnetic field inhomogeneities in normal tissues (blooming effect)6. As a result, the background contrast of tissues surrounding lesions would be distorted, giving obscured images. Superparamagnetic iron oxide nanoparticles (SPIONs), composed of magnetite (Fe3O4) and/or maghemite (γ-Fe2O3; an oxidized form of magnetite), have been popularly used as T2 MRI contrast agents. Although the limitations of the T2 mechanism compromise the image contrast provided by SPIONs, they still possess many advantages that attract researchers’ interest, such as an intrinsic high sensitivity and biological tolerance. Usually, SPIONs are composed of a core containing thousands of iron atoms and a surface coating to stabilize the magnetic nanoparticles. This feature of the core content endows higher sensitivity over gadolinium complexes in the micromolar and nanomolar ranges, rendering SPIONs as competitive MRI contrast agents, particularly for T2-weighted MR imaging. Moreover, iron, as one of the metallic elements in living organisms, is extensively involved in biological processes, with oxygen transport being one such example. After intravenous injection, iron oxide nanoparticles that contain biodegradable iron will be cleared by macrophages of the RES, and further degraded by lysosomes following recycling and red blood cell reproduction processes58. Additionally, the magnetic properties of SPIONs can be easily manipulated by controlling nanoparticle size and shell thickness, where the shell may be functionalized by biological and targeting probes to realize multifunctional MRI.
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Dextran, a biocompatible material, is commonly employed as a surface coating for SPIONs, with several dextran-coated commercial products approved for clinical use. Feridex (diameter of 120–180 nm) was the first commercial product approved by FDA for the detection of liver lesions, and was intravenously administrated through slow diffusion. It allowed the differentiation of normal hepatic cells from abnormal ones through their selective uptake by phagocytic Kupffer cells in the reticuloendothelial system (RES) and spleen. Since malignant liver cells lack these Kupffer cells, no Feridex would be taken up and there was no subsequent reduction in the MR signal intensity of this region6. In other words, the signal intensity of normal regions was lowered since iron oxide nanoparticles shorten T2 relaxation, and a contrast was thus generated between malignant cells and normal cells. Another in vivo application of Feridex was studied by Zhu et al. (Figure 8), showing the feasibility of Feridex I.V. to noninvasively track stem-cell engraftment and migration after implantation with the use of MRI59. The autologous cultured neural stem cells from humans were labelled with Feridex I.V. and Effectene (a lipofection reagent) one day before implantation, and then stereotactically implanted around the region of brain trauma. A hypointense signal was found at the injection sites on the first day after implantation, and faded thereafter. However, the signal intensified at the periphery region around the lesion, indicating the migration of neural stem cells from the site of injection to the border of
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damaged tissue. In this pilot case, superparamagnetic iron oxide nanoparticles were proven to successfully label and track neural stem cells in vivo in patients with brain trauma. There are, however, numerous side effects, like hypotension and body pains, associated with Feridex administration60. Resovist is a carboxydextran-coated SPION that replaced the use of Feridex, as Resovist can be injected in a bolus fashion and causes weaker side effects.
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On the other hand, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with diameters of
Wiley Interdiscip Rev Nanomed Nanobiotechnol. Author manuscript; available in PMC 2017 November 01. Published in final edited form as:
Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2016 November ; 8(6): 814–841. doi:10.1002/wnan. 1400.
Functional Nanoparticles for Magnetic Resonance Imaging Xinpei Mao1,2, Jiadi Xu3,4, and Honggang Cui1,2,5,6,* 1Department
of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N Charles Street, Baltimore, MD 21218, USA
2Institute
for NanoBioTechnology, The Johns Hopkins University, 3400 N Charles Street, Baltimore, MD 21218, USA
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3Russell
H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA 4F.
M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Research Institute, Baltimore, Maryland 21205, USA
5Department
of Oncology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
6Center
for Nanomedicine, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 North Broadway, Baltimore, Maryland 21231, USA
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Nanoparticle-based magnetic resonance imaging (MRI) contrast agents have received much attention over the past decade. By virtue of a high payload of magnetic moieties, enhanced accumulation at disease sites, and a large surface area for additional modification with targeting ligands, nanoparticle-based contrast agents offer promising new platforms to further enhance the high resolution and sensitivity of MRI for various biomedical applications. T2* superparamagnetic iron oxide nanoparticles (SPIONs) first demonstrated superior improvement on MRI sensitivity. The prevailing SPION attracted growing interest in the development of refined nanoscale versions of MRI contrast agents. Afterwards, T1-based contrast agents were developed, and became the most studied subject in MRI due to the positive contrast they provide that avoids the susceptibility associated with MRI signal reduction. Recently, chemical exchange saturation transfer (CEST) contrast agents have emerged and rapidly gained popularity. The unique aspect of CEST contrast agents is that their contrast can be selectively turned “on” and “off” by radiofrequency (RF) saturation. Their performance can be further enhanced by incorporating a large number of exchangeable protons into well-defined nanostructure. Besides activatable CEST contrast agents, there is growing interest in developing nanoparticle-based activatable MRI contrast agents responsive to stimuli (pH, enzyme, etc.), which improves sensitivity and specificity. In this review, we summarize the recent development of various types of nanoparticle-based MRI contrast agents, and have focused our discussions on the key advantages of introducing nanoparticles in MRI.
*
Correspondence to: [email protected]].
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Introduction
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Magnetic resonance imaging (MRI), developed on the principle of nuclear magnetic resonance (NMR), is one of the most powerful tools extensively used for noninvasive molecular and cellular imaging1. With superb spatial resolution and tissue contrast, MRI provides anatomic images of soft tissues and is considered as one of the most important diagnostic modalities for imaging of the brain2, cartilage, heart, blood vessels and for tumor detection3. Unlike other imaging platforms, such as computerized axial tomography (CAT), positron emission tomography (PET) and single-photon-emission computed tomography (SPECT), MRI techniques do not require the use of radioactive agents and ionizing radiation. Although MRI itself is able to provide detailed images of soft tissues, its intrinsic low sensitivity makes it hard to differentiate normal tissues from lesions. The introduction of supplements, called contrast agents, can enhance the contrast effect at regions of interest by accelerating magnetic relaxation4, 5. These MRI contrast agents can be divided into three groups based on the mechanism by which contrast is generated. T1 agents provide positive contrast by shortening the longitudinal relaxation time of surrounding water molecules, whereas T2 agents shorten the transverse relaxation time of water protons (basic principles will be discussed in Section 1). The third group relies on chemical exchange saturation transfer (CEST) and represents a relatively new approach to enhance MRI contrast. CEST agents exchange their presaturated exchangeable protons with those of bulk water, with the MRI contrast capable of being switched “on” and “off” by irradiation with radiofrequency (RF) pulses.
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Among the various contrast agents, nanoparticle-based contrast agents (especially nanoparticles with diameters of 1–100 nm) have become extremely attractive due to their unique features. Firstly, nanoparticles can be loaded with up to hundreds of thousands of imaging moieties per structure, providing superb signal amplification that enables good imaging contrast at a low dose of contrast agent, and reduces the potential for any cytotoxicity associated with the contrast agent6. For example, a 150 nm dendrimer nanocluster possesses a loading capacity of ~300,000 gadolinium ions, giving a r1 relaxivity value of 12.3 mM−1sec−1 per gadolinium ion and 3,600,000 mM−1sec−1 per particle (1.41 T, 40 °C)7, while the r1 relaxivity value of small gadolinium chelates is only 3.5 mM−1sec−1. Secondly, compared to bulk counterparts, nanoparticles possess a relatively large surface area that offers improved reactivity and an ability to be tailored with additional surface moieties to either improve targeting8 or introduce additional functionality (such as therapeutic features9 or fluorescence10). Multimodal MRI contrast agents are even more prevalent since they can reveal several properties at the same site by applying a single contrast agent. Thirdly, nanoparticles tend to accumulate at tumor sites through the enhanced permeability and retention (EPR) effect11, thereby rendering a higher signal-to-noise ratio in tumors12. Researchers have developed various types of nanoparticle-based MRI contrast agents of T1, T2 and CEST modalities to fulfill different purposes. Among them, T1 nanoparticle-based contrast agents have proven the most popular due to the positive contrast provided. Gadolinium (GdIII) complexes like Gd-DTPA are widely used to detect the breakage of the blood brain barrier (BBB) and characterize changes in vascularity13. However, GdIII chelates
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have a short circulation time and the relaxivity exhibited is relatively low, requiring a large dose of ion chelates to reach a useful detection level. Moreover, the non-biocompatible gadolinium has the potential to introduce toxicity into cells upon dechelation from its complexes. Alternatively, nanostructures (like dendrimers14, liposomes15, quantum dots16, mesoporous silica17 and carbon nanotubes18) can be employed as carriers for GdIII chelates, which significantly improves relaxivity, and the nanoparticle surface can be facilely modified with additional functional groups to create multimodal contrast agents. Since the number of GdIII chelates in these type of systems is limited by the available anchoring sites on the surface, and the size of ionic nanoparticle-based contrast agents are usually too large to avoid rapid excretion by the reticuloendothelial system (RES)19, smaller-sized inorganic nanoparticles composed of gadolinium oxide (Gd2O3)10, gadolinium fluoride (GdF3)20, gadolinium phosphate (GdPO4)21 or manganese oxide (MnO)22 have become more attractive, and relevant research has been conducted on functionalized inorganic nanoparticles as MRI contrast agents. Unlike T1 contrast agents, the negative contrast provided by T2 or T2* contrast agents is insufficient to fully differentiate pathogenic tissues from normal tissues, and the susceptibility generated distorts surrounding normal tissues. The intrinsic high magnetization and biocompatible features still lead to intensive research on iron oxide nanoparticles (extensively used T2* contrast agents) as MRI contrast agents, although the image contrast has been compromised to some degree. Superparamagnetic iron oxide nanoparticles (SPION) and ultra-small superparamagnetic iron oxide nanoparticles (USPION) are representative commercial products of T2 contrast agents that are widely used as liver and lymph node imaging contrast agents. As both core and shell affect magnetic properties of contrast agents, improvement on each aspect has been studied. As an alternative to always “on” MRI contrast agents, MRI nanoprobes that are responsive to various stimuli (i.e. pH, enzyme, RF saturation, etc.) have become attractive to researchers, since they could provide better sensitivity and specificity. Numerous studies have been conducted and reported on CEST contrast agents, as they are easily controlled to be “on” and “off” by RF saturation. Since the sensitivity is strongly dependent on the number of exchangeable protons, the incorporation of CEST contrast agents into nanoparticles can greatly increase the amount of exchangeable protons. Herein, we summarize representative nanoparticle-based MRI contrast agents on the basis of the three operating mechanisms: T1, T2, and CEST. Advances in nanotechnology enables researchers to develop nanoparticle-based MRI contrast agents with higher magnetization and the desired surface characteristics to meet the various needs for effective biodistribution. In this review, we will also discuss improvements focusing on magnetic materials and nanocarriers used to load magnetic materials.
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1. Basic principles of MRI contrast agents As illustrated in Figure 1a, upon exposure to an external magnetic field (B0), a greater proportion of protons will prefer to align parallel to the magnetic field (lower energy state), while the remainder aligns antiparallel (higher energy state). Therefore, a net magnetization (Mz) is produced along the direction of magnetic field (z-axis), and protons spin around the z-axis at a precession rate named the Larmor frequency (ω0). During this period, protons precess separately (out of phase). When an RF pulse is applied to the nuclei with the same
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precession frequency, some protons in the lower energy state are flipped to the higher state by absorbing the RF energy (Figure 1b). Furthermore, the protons become synchronized and precess together (in phase). Consequently, a transverse magnetization (Mxy) perpendicular to the static magnetic field is generated.
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After removal of the RF pulse, the protons relax to their equilibrium state via two relaxation pathways: longitudinal relaxation (T1 relaxation) and transverse relaxation (T2 relaxation). In T1 relaxation, antiparallel protons jump back to the parallel state and give up energy to molecules in the surrounding environment (lattice), so that T1 relaxation is known as spinlattice relaxation. The recovery of Mz is described in equation (1), where the T1 relaxation time is defined as the time taken to recover 63% of the original longitudinal magnetization. The T1 relaxation rate, R1, is given by the reciprocal of T1 (1/T1), which is a function of r1 (T1 relaxivity), an intrinsic property of T1 contrast agents, and contrast agent concentration (see equation (2)). The ideal T1 contrast agent should be able to effectively shorten the T1 relaxation time at low concentration. In terms of T2 relaxation (spin-spin relaxation), protons that are in phase begin to dephase, with the transverse magnetization (Mxy) decaying as a result. This decay process follows equation (3), where T2 refers to the time taken to decay to 37% of the original Mxy value. T2 relaxivity (r2), an intrinsic property of T2* contrast agents, affects the T2 relaxation rate, R2, as described in equation (4). Generally, spins decay faster than T2 due to the magnetic field inhomogeneity generated by T2 contrast agents. After taking this inhomogeneity into consideration, the effective relaxation time (T2*) is given by equation (5), where γ is the gyromagnetic ratio and ΔBi is the difference in the local magnetic field strength due to the inhomogeneity.
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(1)
(2)
(3)
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(4)
(5)
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2. T1 contrast agents
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The signal enhancing nature of T1 contrast agents generates a favorably positive contrast effect, making them the most popular contrast agents for clinical use, especially small molecular gadolinium (GdIII) chelates. The utility of GdIII lies in its electron configuration, possessing seven unpaired electrons in f orbitals that give a symmetric S electronic state. This combination bestows a large magnetic moment on the ion and generates a high relaxivity23. Since gadolinium is toxic as free ions, the chelation of multi-dentate ligands GdIII ions can help minimize toxicity, and GdIII complexes are divided into linear chelates (Gd-DTPA, Gd-BOPTA, and MS-325) and macrocyclic chelates (Gd-DOTA, etc.), where macrocyclic GdIII chelates exhibit stronger thermodynamic and kinetic stability than their linear counterparts24. Among them, Gd-DTPA (Magnevist®) and Gd-DOTA (Dotarem®) possess similar r1 relaxivities around 3.5 mM−1sec−1 (measured at 20 MHz and 37 °C), while protein binding contrast agents like Gd-BOPTA (Multihance®) and MS-325 (Ablacar®) have higher relaxivity of 9.2 mM−1sec−1 (measured at 0.7 T and 37 °C), attributed to the slower molecular rotation caused by non-covalent binding to albumin25. Although GdIII chelates exhibit invaluable utility in diagnosing cancer and sclerosis, they experience rapid renal clearance that limits the time window for MRI26. Unlike iron oxide nanoparticles (T2* contrast agents) that can remain at the imaging site for several months, 95% of GdIII chelates are excreted intact within 24 h of administration27. Regarding the intrinsic low sensitivity nature associated with MRI, the relatively small relaxivity of small molecular GdIII chelates need further improvement. Additionally, the nonspecific extravasating behavior of small molecular GdIII chelates in both normal tissue and at pathogenic sites is an obstacle to clinical application. Therefore, new platforms with specific targets should be incorporated to tackle these problems13. Intensive research has been devoted to developing nanoparticle-based T1 contrast agents, focusing on two methods in particular. The first involves either anchoring small molecular GdIII chelates onto the surface of the nanoparticles or loading GdIII chelates into the interior cavity of the nanostructure. The second method is the creation of paramagnetic inorganic nanoparticles composed of Gd2O3, Gd2F3 and GdPO4. Both methods enhance the potential relaxation by increasing the number of GdIII ions per unit volume, with the high payload of imaging moieties increasing the T1 relaxivity and lowering the dose of contrast agent required for proton relaxation. Furthermore, an additional benefit is derived from the preferential accumulation of nanoparticles at tumor sites due to the leaky vasculature of abnormal tissue, and the targeting efficiency can be further improved by modifying the nanoparticle surface with targeting ligands.
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Nano-sized carriers can be employed to load GdIII chelates, creating nanoparticle-based T1 contrast agents that not only incorporate advantages of nanoparticles for image diagnosis, but also demonstrate a positively enhanced contrast effect. Commonly used carriers include dendrimers28, liposomes, quantum dots, silica and supramolecular self-assembly29. 2.1 Soft nanoparticles—Dendrimers are highly branched synthetic spherical polymers, which are fabricated as the core in nanostructure-based GdIII contrast agent. Previous research revealed that polyamidoamine (PAMAM) dendrimer-DOTA- GdIII chelates could increase T1 relaxivity up to 45 mM−1sec−1 (for dendrimer generations (G) of 9 or 10),
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dramatically improving T1 sensitivity compared with small molecular GdIII chelates (r1 ~ 3.5 mM−1sec−1). Although dendrimer nanoclusters (DNCs) enhance r1 relaxivity, the slow and/or incomplete excretion of such large-size molecules hampers their potential for clinical translation as the accumulation of free GdIII ions from dechelation may cause nephrogenic systemic fibrosis (NSF) that impairs kidney function, especially those released from linear GdIII chelates (such as Gd-DTPA). Furthermore, gadolinium chelates can undergo transmetallation with copper and zinc ions in the body, followed by the onset of toxicity from the released GdIII ions30. Hence, it is important for researchers to focus on developing biodegradable DNCs with stable ligands. Recently, Tsourkas and co-workers integrated polydisulfide linkages between individual PAMAM (G-3) dendrimers (figure 2A), enabling DNCs to further degrade through thiol-disulfide exchange reactions into smaller molecules that were readily excreted through renal clearance26. Unlike the traditional method of labeling GdIII onto dendrimers with chelating ligands, Tsourkas and co-workers prepared [Gd-C-DOTA]−1first, and then reacted dendrimers with the pre-metallated ion complexes. By doing so, the potential toxicity stemming from free GdIII ions trapped within dendrimer cavities was reduced. The relaxivities of 59, 91 and 142 nm sized polydisulfide DNCs were 11.7, 18.4 and 22.4 mM−1sec−1 per GdIII ion, respectively, which were higher than PAMAM (G-3)–[Gd-C-DOTA] −1 without polydisulfide bonds (r1 = 10.9 mM−1sec−1)31 and other polydisulfide gadolinium chelates that have previously been reported as blood pool agents (r1 = 4.4–16.3 mM−1sec−1)32–34.
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Liposomes are biocompatible vesicles formed by either natural or synthetic amphiphilic lipids, with examples of commonly used materials being phosphatidyl choline (PC), phosphatidyl glycerol (PG), and cholesterol. Confining the liposomal diameter to between 60–500 nm, and the introduction of additional surface coatings has been shown to prolong circulation time and improve stability in vivo35. Intensive research has been devoted to investigate liposomes as possible carriers of Gd-chelates, and liposomal contrast agents could be synthesized through two approaches: core-encapsulated GdIII (CE-Gd) liposomes or surface-conjugated GdIII (SC-Gd) liposomes36. Ghaghada studied how liposome size and internal gadolinium concentration would affect T1 relaxivity, determining that the T1 relaxivity increased as the size of the liposomal nanoparticles decreased (highest r1 = 1.85 mM−1sec−1 at 2 T magnetic field strength), and T1 relaxivity did not change significantly with core GdIII concentration over the measured concentration range37. Annapragada et al. compared relaxivity among three liposomal formulations (Figure 2B): CE-Gd liposomes, SC-Gd-liposomes and dual-Gd liposomes (where GdIII chelates were encapsulated within the core and conjugated onto the surface)38. Since dual-Gd liposomes possessed the highest number of GdIII ions per liposome, they demonstrated the highest T1 relaxivity on an individual nanoparticle basis, which was more than 2000–8000 fold that of small molecular GdIII chelates. Furthermore, the dual-Gd liposomes demonstrated the highest signal-to-noise and contrast-to-noise ratios in CE-MRA studies. Very recently, supramolecular MRI contrast agents have quickly drawn researchers’ attention since their first introduction by Stupp and Meade39, where self-assembling peptide amphiphiles (PAs) were conjugated with macrocyclic GdIII chelates to furnish peptide amphiphile contrast agents (PACAs). Nanostructures self-assembled from PAs can emulate extracellular matrices, a biomimetic strategy widely applied in the field of regenerative Wiley Interdiscip Rev Nanomed Nanobiotechnol. Author manuscript; available in PMC 2017 November 01.
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medicine40. Depending on the design rationale and assembly environment, various onedimensional morphologies with different physical properties can be obtained upon selfassembly of PAs, which renders PACAs a versatile platform for imaging purposes41. Most importantly, it is well known that the relaxivity of contrast agents will be enhanced by conjugation to proteins and polymers with large molecular weight, or by preparation of micellar structures42. Similarly, the structure of self-assembled PAs allows the increase in rotational correlation time (τr) that subsequently enhances relaxivity. Bull et al. reported the relaxivity difference that arose from the varying morphology (see Figure 3A–B) of two selfassembled PAs, where the relaxivity of nanofibers was 14.7 mM−1sec−1, while the relaxivity of spherical micelles was 22.8 mM−1sec−1 before cross linking (pH = 7.41)43. Later, Bull et al. fabricated three molecules (1, 2, 3) with similar sequences, with PA 1 able to form selfsupporting hydrogels, while PACAs 2 and 3 possessed Gd-DOTA positioned at different distances from the hydrophobic alkyl tails (Figure 3C). Both PACAs self-assembled into nanofibers at pH greater than 7.0, though cannot form hydrogels by themselves39. Upon mixing PACA 2 or 3 with filler PA 1, a homogeneous hydrogel was formed that allowed MR images to be obtained. From the MR images of these phantom gels, the mixture of PACA 3 and PA 1 exhibited the greatest contrast, implying that positioning GdIII chelates closer to the hydrophobic end of PAs would result in higher relaxivity. The postulated reason behind this was due to the decreased internal flexibility and increased steric hindrance of GdIII chelates that occurs upon self-assembly. Since the highest contrast was generated with PACA 3, it was further mixed with various epitope-bearing PAs (such as the IKVAV or YIGSR epitopes for neuronal stem cell differentiation, and the RGD epitope for cell adhesion) to form hydrogels (doping with an equal amount of 3). The T1 values of each mixed hydrogel were found to be similar, proving the ability to use PACAs with various PA gels.
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The feasibility of employing supramolecular dual-modality nanoprobes (self-assembly of amphiphilic peptide conjugates containing the fluorophore 5-FAM and Gd-DOTA) as contrast agents for MR and fluorescence imaging was assessed by Cui and co-workers (Figure 4)44. The live-cell fluorescence imaging of two self-assembled nanoprobes (one with single hydrocarbon tail, and the other with two hydrocarbon tails) was studied in KB-3-1 human cervical cells to evaluate cell viability of PACAs. The dual-tailed PACA 2 (50 μM) showed 25-fold higher cellular uptake than PACA 1 (200 μM), which was due to membrane insertion through the two hydrophobic alkyl chains. Before self-assembly, the relaxivity, r1, of PACA 1 and PACA 2 were 4.3 mM−1sec−1 and 4.2 mM−1sec−1 (pH = 7.4, room temperature), respectively, which were similar to the relaxivity of small molecular GdDOTA contrast agents (3.5–4.8 mM−1sec−1)23. However, upon self-assembly, the nanospheres formed by PACA 1 and PACA 2 had relaxivities of 7.8 mM−1sec−1 and 14.3 mM−1sec−1 that were higher than the monomeric forms. The higher relaxivity of selfassembled PACA 2 stems from the denser packing of PACA 2 in its self-assembled state, which led to a higher aggregation number of molecules and effective molar mass than selfassembled PACA 1. As we described above, self-assembling PAs have the propensity to enhance T1 relaxivity, and one advantage associated with fibrous PA structures is their potential for biodegradation
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into natural building blocks, so that it is of importance to track the fate of biomaterials after implanted in vivo29. There are, however, limited studies that shed light on the degradation process in vivo. By incorporating MRI modality to image PACAs, we could rationalize using PACAs to enhance MRI contrast, and possibly allow us to track the fate of PACAs in vivo, thus laying a critical foundation for future development as therapies45. Stupp, Meade and coworkers have reported on the in vivo biomaterial localization with Gd(HPN3DO3A) labeled peptide nanofibers (Figure 5). These PAs were designed to have one chelate next to the Cterminal (PA1 and PA2), three chelates at the C-terminal end (PA3), or one chelate relatively far away from the C-terminus (PA4)29. Small-angle X-ray scattering (SAXS) experiments revealed that the bulky Gd (HPN3DO3A) conjugated to the outermost residue of the PAs exhibited β-sheet character and retained high-aspect-ratio structures. The in vivo degradation was evaluated with the mixture (gels) of PA (1 or 3) and filler C16V3A3E3-NH2, of which the PA1 gel produced positive contrast in T1-weighted MRI and PA3 gel produced negative contrast. Subsequent ICP-MS analysis was conducted to measure GdIII retention. The result showed that 62 ± 8% of PA1 and 54 ± 9% of PA3 remained in the mouse leg after 4 days, and the similar T1 relaxation time at day 0 and day 4 indicated the PA concentration did not change significantly, verifying that the approach of using MRI to track the fate of biomaterials is practicable.
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2.2 Inorganic nanoparticles—Quantum dots (diameters of 2–6 nm) are of interest as imaging modalities due to their bright fluorescence, phosphorescence, and narrow emission spectrum. Incorporating such semiconductor nanocrystals into the design of GdIII-chelated nanoparticles imparts multimodal functionality for both MRI and fluorescence microscopy. Mulder et al. fabricated quantum dots with GdIII chelates as well as a PEG-lipid coating (pQDs), which endowed these contrast agents as bimodal molecular imaging probes (see figure 2C–E)16. The PEGylated lipid coating protected the contrast agents from interacting with plasma proteins, so that they went through less rapid hepatic clearance. The T1 relaxivity of pQDs was more than 12 mM−1sec−1, three times higher than those of small molecular GdIII chelates. If pQDs were further functionalized with the RGD peptide sequence, a cell adhesion epitope, RGD-pQDs could be employed to detect angiogenesis (new blood vessel formation), an important pathological process in cancer and atherosclerosis46.
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Recently, researchers have been interested in investigating inorganic nanostructures like porous or mesoporous silica with high loading capacity. High relaxivities have been reported for GdIII chelates embedded in mesoporous silica. Lin and co-workers grafted GdIII chelates onto mesoporous silica nanospheres (MSN-Gd), which exhibited high T1 relaxivities of 28.8 mM−1sec−1 and T2 relaxivity of 65.5 mM−1sec−1 (determined with a magnetic field strength of 3.0 T)17. Considering its high r1 and r2 values, MSN-Gd is a promising candidate for intravascular MR imaging (T1) and depicting soft tissues (T2). Tsai et al. described multifunctional mesoporous silica nanorods loaded with Gd-DTPA and green fluorescing dye47. The magnetic nanorods exhibited no short-term cytotoxicity, and displayed high cellular uptake efficiency without the need for transfection agents. For the GdIII chelates grafted inside silica pores, however, the access of water molecules may be hindered. At the other extreme, the presentation of GdIII chelates on the surface of nonporous nanoparticles
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does not hinder the interaction of water molecules with each Gd site, thus allowing better contrast. For example, fluorescent quantum dots or colloidal gold nanocrystals were coated with a thin silica shell, onto which multiple Gd-DOTA molecules were covalently attached without negative influence on optical properties of the nanoparticle cores48. The particulate relaxivity was dependent on the number of gadolinium ions on the silica surface, and the number of Gd-DOTA molecules can be easily tuned from 20 to 320 GdIII ions per quantum dot. Moreover, due to the reduced rotational motion of Gd-DOTA molecules conjugated to the silica, both the r1 and r2 relaxivities were 6 to 15 times larger than free Gd-DOTA. One limitation associated with quantum dots or gold nanoparticles is that the light penetration depth in living tissue is just a few centimeters in an optimized scenario, which makes them uncompetitive as multimodal MRI contrast agents. The application of silica coatings and Gd-DOTA attachment endows high r1 and r2 relaxivities, and preliminary in vivo tests have verified the contrast enhancement provided by paramagnetic silica-coated nanoparticles.
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Although nanostructures labeled with ionic T1 contrast agents exhibit higher relaxivity than small molecular GdIII chelates, the number of paramagnetic ions attached to the nanostructure surface is limited by the total number of available anchoring sites. Since the number of paramagnetic ions intrinsically determines the magnetism of nanoparticles, a higher number of imaging moieties per nanoparticle is always preferred. Most significantly, the size of ionic T1 nanoparticles is usually larger than 100 nm, making them susceptible to excretion by the RES system19. Alternatively, transition metal and lanthanide metal based inorganic nanoparticles composed of gadolinium oxide (Gd2O3), gadolinium fluoride (GdF3)20, gadolinium phosphate (GdPO4)49 or manganese oxide (MnO) have drawn researcher’s attention as a second type of T1 nanostructure-based contrast agent, since they have increased magnetic moments stemming from the abundance of paramagnetic ions at the surface, and the overall size is smaller than 100 nm.
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Early research led to the development of paramagnetic ultrasmall Gd2O3 nanoparticles with an optimal particle diameter of 1 nm and r1 of 9.9 mM−1sec−1, which was higher than small molecular GdIII chelates50. This may be due to the induced longitudinal relaxation of water protons by surface GdIII ions of the Gd2O3 nanoparticles. A biocompatible PEG coating can enhance the steric repulsion of nanocrystals to avoid undesired aggregation, but conventional procedures to graft PEG onto Gd2O3 are time consuming. Fortin and co-workers reported a new, fast and efficient one-pot technique to prepare PEGylated nanoparticles with small sizes of 1.3 nm51. The PEGylated nanoparticles were colloidally stable in aqueous media, ideal for T1-weighted MRI since the ratio of r2/r1 was small enough (r2/r1 = 1.20 at 60 MHz). Furthermore, these ultrasmall PEG-Gd2O3 nanoparticles allowed the visualization of labeled cells implanted in vivo. Labeled F98 brain cancer cells were implanted into animals with brain tumors, and positive contrast was observed up to at least 48 h after implantation. Multimodal contrast agents are attracting increasing attention because they combine several complementary properties into a single object. Bridot et al. encapsulated Gd2O3 within a polysiloxane shell that carried organic fluorophores, and the surface of the nanoparticles was further coated with PEG-COOH to avoid agglomeration and precipitation10. Designed Gd2O3 nanoparticles with 2.2 nm core exhibited higher r1 and r2 values than small molecular GdIII chelates (r1 = 8.8 mM−1sec−1, r2 = 11.4 mM−1sec−1), and the small r2/r1 ratio rendered the nanoparticle as ideal T1 contrast agents (r2/r1 = 1.3). Fluorescence reflectance imaging Wiley Interdiscip Rev Nanomed Nanobiotechnol. Author manuscript; available in PMC 2017 November 01.
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after 3 h injection revealed no undesired accumulation in lungs and liver, which was due to both the small size and PEG coating. T1-weighted MRI of rats 1 h after injection and ICPMS of some organs and urine confirmed that the uptake of Gd2O3 nanoparticles in lung, spleen and heart was insignificant.
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Since relaxivity is inversely proportional to the sixth power of the ion–nuclear distance, metal ions like GdIII with seven unpaired electrons are the most predominantly used paramagnetic contrast agents52. However, GdIII ions are not naturally present in the human body. Alternatives based on transition metals, like manganese (five unpaired electrons), have progressed since there are natural processes that utilize these ions and could potentially be exploited. For instance, MnII ions are natural cellular constituents that play an important role as cofactors of enzymes and receptors. Moreover, the uptake of manganese is highly dependent on mitochondrial density that makes MnII a very promising contrast agent for mitochondria-rich organs like the liver, pancreas and kidney53. It is this combination of unique features that make MnII an attractive study object. Cai et al. designed hollow mesoporous Prussian blue (HMPB) nanoparticles loaded with a Mn-containing Prussian blue analogue (MnPBA), which also acted as an excellent drug carrier (high loading efficiency of 97.5% and loading capacity of 62.3%) and a photothermal agent, and most importantly had a high relaxivity (r1 = 7.43 mM−1sec−1) at pH 5.0 (tumor site conditions)54. This pH-responsive theranostic nanoplatform could concurrently release MnII ions and doxorubicin (DOX) at the tumor site. The positive correlation between MRI intensity and DOX release allowed controlled drug release (Figure 6). Additionally, the combination of strong absorbance in the NIR and chemotherapy of DOX realized a synergistic chemothermal therapy that achieved enhanced tumor therapeutic efficacy.
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One drawback, however, is that MnII chelates can cause hepatic failure and cardiac toxicity, severely limiting their application in animal studies. Hence, studies on functionalized MnO/Mn3O4-based nanoparticles have been undertaken. Hyeon, Gilad and co-workers studied mesoporous silica-coated hollow manganese oxide nanoparticles (HMnO@mSiO2) with a relaxivity of 0.99 mM−1sec−1 at 11.7 T55, a value much higher than existing MnO nanoparticle-based T1 contrast agents (MnO encapsulated within PEG-phospholipids, MnO coated with dense silica, and non-etched mesoporous silica-coated MnO)56, 57, simply because the mesoporous structure allowed the rapid penetration of water into the manganese core, and hence greater exposure of the MnII ions to water molecules (Figure 7). Although the relaxivity of these MnO nanoparticle-based contrast agents was compromised compared to small molecular MnII compounds, they were specifically designed to label and track cells that MnII compounds could not. With electroporation, HMnO@mSiO2 nanoparticles exhibited high cellular uptake (more than 75% in 24 h) by mouse adipose-derived mesenchymal stem cells (MSCs), and the applied electroporation only minimally impacted cell viability. In mice transplanted with labelled MSCs, the feasibility of HMnO@mSiO2 nanoparticles for long-term (over 14 days) in vivo cell tracking was studied, and the results revealed a hyperintense region at the transplantation site. The sustained contrast in vivo over a prolonged time period rendered HMnO@mSiO2 nanoparticles as competitive candidates for noninvasively monitoring the fate of transplanted cells in vivo.
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3. T2 MRI contrast agents
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3.1 Superparamagnetic Iron Oxide Nanoparticles—Shortly after the introduction of GdIII chelates, increased investigations were conducted on iron oxide nanoparticles as T2weighted MRI contrast agents, whose characteristics cannot be replaced by bulk counterparts or atoms. Contrary to the T1 contrast effect, T2 contrast agents generate a negative contrast effect due to a signal reduction mechanism that at times can make it difficult to differentiate normal tissue from lesions. It is also known that T2 contrast agents induce high susceptibility that can induce local magnetic field inhomogeneities in normal tissues (blooming effect)6. As a result, the background contrast of tissues surrounding lesions would be distorted, giving obscured images. Superparamagnetic iron oxide nanoparticles (SPIONs), composed of magnetite (Fe3O4) and/or maghemite (γ-Fe2O3; an oxidized form of magnetite), have been popularly used as T2 MRI contrast agents. Although the limitations of the T2 mechanism compromise the image contrast provided by SPIONs, they still possess many advantages that attract researchers’ interest, such as an intrinsic high sensitivity and biological tolerance. Usually, SPIONs are composed of a core containing thousands of iron atoms and a surface coating to stabilize the magnetic nanoparticles. This feature of the core content endows higher sensitivity over gadolinium complexes in the micromolar and nanomolar ranges, rendering SPIONs as competitive MRI contrast agents, particularly for T2-weighted MR imaging. Moreover, iron, as one of the metallic elements in living organisms, is extensively involved in biological processes, with oxygen transport being one such example. After intravenous injection, iron oxide nanoparticles that contain biodegradable iron will be cleared by macrophages of the RES, and further degraded by lysosomes following recycling and red blood cell reproduction processes58. Additionally, the magnetic properties of SPIONs can be easily manipulated by controlling nanoparticle size and shell thickness, where the shell may be functionalized by biological and targeting probes to realize multifunctional MRI.
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Dextran, a biocompatible material, is commonly employed as a surface coating for SPIONs, with several dextran-coated commercial products approved for clinical use. Feridex (diameter of 120–180 nm) was the first commercial product approved by FDA for the detection of liver lesions, and was intravenously administrated through slow diffusion. It allowed the differentiation of normal hepatic cells from abnormal ones through their selective uptake by phagocytic Kupffer cells in the reticuloendothelial system (RES) and spleen. Since malignant liver cells lack these Kupffer cells, no Feridex would be taken up and there was no subsequent reduction in the MR signal intensity of this region6. In other words, the signal intensity of normal regions was lowered since iron oxide nanoparticles shorten T2 relaxation, and a contrast was thus generated between malignant cells and normal cells. Another in vivo application of Feridex was studied by Zhu et al. (Figure 8), showing the feasibility of Feridex I.V. to noninvasively track stem-cell engraftment and migration after implantation with the use of MRI59. The autologous cultured neural stem cells from humans were labelled with Feridex I.V. and Effectene (a lipofection reagent) one day before implantation, and then stereotactically implanted around the region of brain trauma. A hypointense signal was found at the injection sites on the first day after implantation, and faded thereafter. However, the signal intensified at the periphery region around the lesion, indicating the migration of neural stem cells from the site of injection to the border of
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damaged tissue. In this pilot case, superparamagnetic iron oxide nanoparticles were proven to successfully label and track neural stem cells in vivo in patients with brain trauma. There are, however, numerous side effects, like hypotension and body pains, associated with Feridex administration60. Resovist is a carboxydextran-coated SPION that replaced the use of Feridex, as Resovist can be injected in a bolus fashion and causes weaker side effects.
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On the other hand, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with diameters of
