Superparamagnetic iron oxide nanoparticles as MRI contrast agents for non-invasive stem cell labeling and tracking.

Stem cells hold great promise for the treatment of multiple human diseases and disorders. Tracking and monitoring of stem cells in vivo after transplantation can supply important information for determining the efficacy of stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be the most effective and safest non-invasive technique for stem cell tracking in living bodies. Commercial superparamagnetic iron oxide nanoparticles (SPIONs) in the aid of transfection agents (TAs) have been applied to labeling stem cells. However, owing to the potential toxicity of TAs, more attentions have been paid to develop novel SPIONs with specific surface coating or functional moieties which facilitate effective cell internalization in the absence of TAs. This review aims to summarize the recent progress in the design and preparation of SPIONs as cellular MRI probes, to discuss their applications and current problems facing in stem cell labeling and tracking, and to offer perspectives and solutions for the future development of SPIONs in this field.

Introduction

Stem cells are biological cells found in all multicellular organisms, which possess the capability of self-renewal and differentiation into various cell lineages. Until now, stem cells have been applied to not only cell-based therapies, for example, the treatment of ischaemic 1, degenerative 2, immune 3 and genetic diseases 4, but also regenerative medicine, such as the repair or regeneration of damaged heart 5, cartilage 6, 7 and bone tissue 8. Accordingly, the tracking and monitoring of these stem cells after delivery into human body is very important for a comprehensive understanding of their proliferation dynamics, differentiation process and migration dynamics in vivo. Currently, several imaging methodologies have been applied for this purpose, including positron emission tomography (PET) 9-11, single photon emission computed tomography (SPECT) 12, bioluminescence imaging (BLI) 13, 14, fluorescence imaging 15-21, X-ray based computed tomography (CT) 22 and magnetic resonance imaging (MRI) 5, 23-27. Among them, MRI shows advantages over the others owing to its high spatial resolution (∼100 μm), long effective imaging window, rapid in vivo acquisition of images, and the absence of exposure to ionizing radiation 28-31. It has shown promising future in tracing cells in vivo. However, the sensitivity of MRI is generally lower as compared to SPECT and bioluminescence. Thus, the development of MRI contrast agents with high efficiency and sensitivity becomes essential to allowing successful bio-imaging at the cellular and molecular level.

The MRI contrast agents in clinic are divided into two parts, including T1 and T agents. Some of them in the commercial market are shown in Table 1. T agents, such as paramagnetic metal lanthanide, can alter the longitudinal (T) relaxation times of water protons to produce bright positive signal intensity in images and increase the conspicuousness of cells. Initially, chelate complexes of gadolinium (Gd3+), such as the clinical contrast agent gadolinium diethylene-trianmine pentaacetic acid (Gd-DTPA) with the aid of transfection agents (TAs), were employed to label stem cells 32-34. Recently, Gd3+ containing particles and macromolecules have been developed as a new generation of T contrast agents 31, 35-39. Gd3+-hexanedione NPs (GdH-NPs) produced stronger signal intensity than Gd-DTPA, probably because the larger Gd complexes with high molecular weight in GdH-NPs caused the slow tumbling rate of GdH-NPs 35. Gd3+-ion clusters within ultra-short single-walled carbon nanotubes (Gd3+n @US-tubes) exhibited a T relaxivity (r) 40-fold greater than that of Gd-DTPA. It shortened relaxation time of water in labeled pig mesenchymal stem cells (pMSCs) two-fold as compared to unlabeled MSCs 36. The Gd-DTPA bearing poly(ethyleneimine) (SiO2/Gd-DTPA-PEI) 31 and Gd3+ conjugated peptide dendrimers 37-39 provide great possibilities to efficiently label and monitor stem cell with high cell uptake efficiency and increased Trelaxivity.

Table 1

Some commercial MRI contrast agents 51, 52.

Brand name	Structure	Hydrodynamic size (nm)	Classification	Target	Company	Ref.
Magnevist®	Gd-DTPA		T1 agent	Extracellular	Bayer Schering (Germany)	32
Omniscan®	Gd-DTPA-BMA		T1 agent	Extracellular	GE-Healthcare (U.S.A) and Nycomed (Norway)	35, 40
Eovist®	Gd-EOB-DTPA, Gadoxetate		T1 agent	Extracellular	Bayer Schering (Germany)	51
Ferumoxides (Feridex IV®, Endorem ™)	Dextran-coated SPIOs	80-150 nm	T2 agent	Reticuloendothelial system , LiverStem cell labeling	Advanced Magnetics (U.S.A)	46, 53-55
Resovist®	Carboxydextran-coated USPIOsa	20 nm	T2 agent	Blood poolStem cell labeling	Bayer Schering (Germany)	45, 54
Sinerem® (AMI-227)	Dextran-coated USPIOs	15-30 nm	T2 agent	Blood pool	Guerbet (France)	56
Ferumoxytol®	Carboxylmethyl-dextran coated USPIOs	30 nm	T2 agent	MacrophageBlood pool	Advanced Magnetics (U.S.A)	57
Ferumoxsil ®	Silicon-coated SPIOs	300 nm	T2 agent	Liver	Guerbet, AdvancedMagnetics	58
Ferucarbotran®(SHU-555A)	Carboxydextran-coated USPIOs	60 nm	T2 agent	Liver	Bayer Schering (Germany)	59
Feruglose NC100150	Pegylated starch-coated USPIOs	20 nm	T2 agent	Blood pool	GE-Healthcare (U.S.A)	60
Banges®	SPIONs encapsulated inpolystyrene/divinylbenzene	0.69-1.73 µm	T2 agent	Cell labeling	Bangs Laboratories (India)	61

Note: a Ultrasmall superparamagnetic iron oxide NPs .

T agent is to alter the transverse (T) relaxation times of water protons. T agents provide dark negative signal intensity in images and can be used to visualize stem cells grafted in organs that appear as high signal intensity (e.g. kidney or lymphoid tissues). Compared to Tagents, superparamagnetic iron oxide NPs (SPIONs) based T agents appear to be the preferred MRI contrast agents for monitoring stem cells due to their high sensitivity and excellent biocompatibility 40. By far, the common labeling approach for stem cell labeling and imaging is based on combining commercially available SPIONs (e.g. Feridex® and Revosit®) with a commercially available transfection agent (TA) (e.g. Superfect™, poly(L-lysine)(PLL) 41-43, Lipofectamine ™ 44, 45, or protamine sulfate 43, 46-48). However, one of the crucial problems of this approach is the potential toxicity of TAs to living bodies 47. For example, PLL can cause significant cell death at the concentration of 10 μg/mL in media 49. Feridex®-PLL complexes have been reported to inhibit the chondrogenic differentiation capacity of MSCs 50. In addition, Feridex® and Resovist® are no longer available commercially since 2009 29. Therefore, extensive efforts have been devoted to the development of novel SPIONs (some of them are shown in Table 2) in the last decade, leading to a rapid progress in the field of stem cell labeling. The present review summarizes the recent information involving the design consideration and preparation of SPIONs, discusses the current status of their applications in sensitive stem cell labeling and detection, and points out the current problems and perspectives on future directions in this field.

Table 2

Some novel SPIONs as MRI contrast agents in stem cell labeling and tracking.

Name	Magnetic core	Surface	Core diameter (nm)	Overall size (nm)	Zeta Potential(mV)	Magnetization/emu g-1	Relaxivity/mM-1s-1 (γ2)	B0a/T	Fe/cell(pg)	In vitro	In vivo	Ref
Fe2O3-PLL	γ-Fe2O3	PLL		6.2 b	-42		213	1.5	41.5	Rat MSCsHuman MSCsHuman umbilical cord blood MSCs	Rat brain	62-64
N-dodecyl-PEI2k/SPIO	Fe3O4	N-dodecyl-grafted PEI 2K		54.7 b	+40		345	3	7.1	Mouse MSC	Mice subcutaneous	65
iron oxide-loaded cationic nanovesicle	Fe3O4	(1)PEI-SA(2)PEG-PGA	6	150d	+20		343.1	1.5	50.02	Rat MSCs	Rat brain	66
CMCS-SPIONs	Fe3O4	(Carboxymethyl)chitosan	6-10	55.4d	-21.4	41.6	160.5	1.5	26.7	Human MSCs		67
ED-Pullulan coating SPIO	Fe3O4	Ethylenediamine Pullulan		94d	+10				65	Rat MSCs		68
UFH-SPIOs	Fe3O4	Unfractionated heparin		50-150c					4.93	Human MSCs	Nude micekidney	69
IONP-6PEG-HA	Fe3O4	Amine-functionalizd six-armed PEG covalently linked to hyaluronic acid	10	75d	-9.1	79	454.5	3	145.9 ng Fe/105 cells	Human MSCs		70
TMA-SPIONs	Fe3O4	Polyacrylic acid modified by 2-aminoethyl-trimethyl ammonium		101d	+40	44.9	728.23	7		Human MSCs	Mice brain	71
PDMAAm-coated γ-Fe2O3 NPs	γ- Fe2O3	PDMAAm		77.8d			27.26	0.5	36.9	Human MSCs		72
FITC-PLMA-MNPs	Fe3O4	FITC-PLMA		100 d	-34.5		164.8		32.7	Human MSCs		20
Magnetic PLGA MPs	Fe3O4	PLGA(carboxyl end-group)	10	0.4-3 μm		40	316.7		80	Mouse MSCs	BALB/C mice back and ears	73
Citrate SPION	Fe3O4	Citrate	6-7	90.13 d	-27.3			7	69.6	Human MSC	Mice muscle	74
D-mannose coated SPIONs	γ -Fe2O3	D-mannose		6b			140.4	0.5	51.7	Rat MSCs		64, 75
SPIO@SiO2-NH2	Fe3O4	SiO2-NH2	6	8.5 b		52.5	43.5	3	68.7	Rabbit MSCs	Rabbits brain	76
MNPs@SiO2(RITC or FITC)	Fe3O4	SiO2 containing RITC or FITC	9	30-80 b				1.5		Human MSC	NOD-SCID mice	77-79
Mag-Dye@MSNs	Fe3O4	SiO2 and FITC- incorporated mesoporous silica						1.5		Human MSC	Nude mice brain	80
TAT-CLIO	Fe3O4	Tat peptide functionalized cross-linking dextran		65.2d			73.4	0.47	2.15	Human NSCs		53, 81
LMWP-SPIO	Fe3O4	LMWP		26.77d	16.97	85			2.3	Human MSCs		82

Notes: a magnetic field. b size determined by TEM method. c size determined by AFM method. d hydrodynamic size measured by dynamic light scattering.

Design considerations of SPIONs for stem cell labeling

For designing SPIONs based MRI probes, there are several important aspects that need to be considered: 1) stem cell uptake, this is a necessary prerequisite for the application of SPIONs for stem cell labeling; 2) Trelaxivity, this is directly related to the MRI probe sensitivity; 3) a long-term stay in cells, SPIONs-labeled cells should retain the label and remain viable by MR for weeks, even months because preclinical and clinical trials mostly will need a long-term follow-up of tissue function and the fate of labeled cells; and 4) biosafety, the formulations should be biocompatible to stem cells and the host without side effects on their biological properties and functions.

NPs are carried into non-phagocytosis cells such as stem cells mainly via endocytosis path. Four basic mechanisms have been proposed by previous reports, including: macropinocytosis, clathrin- or caveolae-mediated endocytosis, and pathways that independent of clathrin and caveolae 83-85. There are a few factors that strongly affect the endocytosis of NPs, including particle size, surface charge, surface chemistry, and cell lines 63, 86. Numerous reviews have described and discussed the internalization mechanism and influential factors 30, 85, 87. On the size side, particles with the size less than 100 nm (hydrodynamic diameter) are generally preferred for cell uptake 68. In the ranging from 2 to 100 nm, it was reported that the most efficient cells uptake of herceptin conjugated colloidal gold NPs (Her-GNPs) occurred within the 25-50 nm size range 88. In term of surface charge, positive surface charges are expected to facilitate the phagocytotic uptake as a result of electrostatic attraction between the positively charged particles and negatively charged cell membrane. Most of transfection agents which can effectively introduce exogenous gene into various cells are cationic compounds, such as cationic lipids 89, 90, polymers, dendrimers 91, 92 and NPs. Moreover, Shui group reported a clear positive correlation of surface charge of PEG-PGA/PEI-SA/SPIO NPs and labeling efficiency in rat MSCs 66. In regard to surface chemistry, it is critical for uptake efficiency and specific cell internalization. For example, (Carboxymethyl) chitosan coated SPIONs and citrate coated SPIONs showed high efficiency in stem cell internalization due to the specific surface chemistry, despite they were negatively charged 67. Furthermore, our group has designed and prepared hydrophilic SPIONs with glucosaminic acid (GA) coating and found that GA modified SPIONs internalized more quickly to cancer cells than to normal cells lines 93. In addition, the cell targeting moieties on the surface of NPs, such as peptides from the human immunodeficiency virus (HIV) TAT protein and Herpes simplex virus (HSV), may mediate reporter-ligand endocytosis and efficiently enhance the internalization. Stem cells with different types (e.g. MSCs and NSCs) and different donors (e.g. pig, rabbit, rat, mouse and human) may show different internalization efficiency with the same NPs 63, 76, 81.

Three major factors govern the T relaxivity of SPION agents, including particle size, composition, and crystallinity 96, 97. T relaxivity is highly sensitive to particle size and larger SPIONs generally have higher Trelaxivity 61. However, superparamagnetic size limit in magnetic iron oxide is 20 nm. Iron oxide cores with diameter beyond this limit are usually no longer superparamagnetic. Two methods have been reported to effectively maintain the superparamagnetism and enhance T relaxivity of SPIONs. One is the controllable aggregation of NPs into clusters, which induces the magnetic relaxation switch effect 65, 98-101. For example, the T relaxivity of amphiphilic alkyl-PEI/SPIONs micelles (323 mM-1s-1) with multiple SPIONs were higher than that with single SPION (118 mM-1s-1) at the magnetic field of 1.5 T. The other method is to confine SPIONs in micrometer-sized polymer particles, for example PLGA, which can enhance molar relaxivity of the Fe and cellular internalization 73. Other parameters of critical importance to the performance of NPs are the composition and the crystallinity. Lee et al 102 found that MnFe2O4 NPs showed the highest magnetic susceptibility and thus the strongest T shortening effect among a series of metal doped iron oxide NPs of spinel MFe2O4 (M = Mn, Fe, Co or Ni) at similar size. As for iron oxide NPs, Basti et al showed that the magnetite (Fe3O4) provided a stronger T shortening effect than the maghemite ( γ-Fe2O3) 103.

Proliferation and exocytosis are two main factors that hamper the long-retention of SPIONs in cells. When a cell proliferates, SPIONs are divided evenly or unevenly into two daughter cells. After several cycles, the label can be diluted below detectable levels. Proliferation is very likely to occur for stem and progenitor cells due to their strong self-renewal ability. Therefore, magnetic particles with high T relaxivity and high iron loading are of importance for cellular MRI due to the time-dependent decrease of Fe content in cells. Furthermore, exocytosis process of stem cells also dilutes the Fe content per cell 104. It has been reported that exocytosis is size dependent and smaller particles are exocytosed at a faster rate. Xu et al showed that the internalization of SPIONs loaded PLGA microparticles (SPIONs-PLGA MPs) in MSCs enhanced residence time inside the cells (3-fold) compared to SPIONs alone 73. Degradability of SPIONs is another factor that affects their duration time. One successful example is the SiO2 coated SPIONs, as reported by Wang's group 76. It had stayed in stem cells and remained visible by MRI for 8-12 weeks, probably due to the stability of SiO2 under the cellular circumstance.

For biosafety consideration, understanding the properties of NPs and their effects on the host are crucial before clinical use can occur. It is generally accepted that iron oxide is non-toxic to cells, since it can be degraded and utilized by cells via physical iron metabolism pathway 106. However, several groups recently have reported that high Fe load in cells is toxic to cells and would interfere the normal function of stem cells 94, 95, 107, 108. SPION toxicity is influenced by many factors including size, charge, surface chemistry, dose, and agglomeration state of NPs, etc. 105. Given a particular type of iron oxide NPs, an appropriate coating contributes to make the SPIONs less toxic. Meanwhile, systematic studies have to be conducted to assess the potential long-term toxicity of SPIONs in vivo 97.

Design strategies of SPIONs for stem cell labeling

Hydrophilic SPIONs for biological application usually consist of an iron oxide core and a surface coating. On one hand, synthesis methods of the iron oxide core, stabilizer and reaction parameters have significant effects on the size and magnetic properties of SPIONs 52, 109-111. On the other hand, surface coating materials, functionalization materials and surface engineering methods significantly affect ultimate size in living fluid, biocompatibility, cell internalization and duration in cells 26, 30, 112, 113. Therefore, to prepare ideal SPIONs, major factors such as core synthesis, surface coating and functional materials, and surface engineering methods should be carefully considered.

Synthesis of cores

Chemical methods used to synthesize SPIONs mainly include coprecipitation 63, 69, 72, thermal decomposition 114, pyrolysis method 114, hydrothermal reactions 115, and sol-gel syntheses 116. Until now, the main challenges in the synthesis of iron oxide core are (i) mono-dispersibility with required size, (ii) good magnetic properties. The two most extensively used methods in preparation of SPIONs for stem cells are coprecipitation and thermal decomposition technique. The following section summarizes the properties and applicability of both methods and provides a novel strategy for the synthesis of iron oxide cores.

The coprecipitation technique is probably the simplest and efficient chemical pathway to obtain SPIONs (either Fe3O4 or γ-Fe2O3). Magnetic particles are usually prepared by controlling the precipitation of iron oxides in aqueous Fe2+/Fe3+ salt and stabilizers through the addition of an alkaline solution in a non-oxidizing oxygen environment. The main advantage of coprecipitation process is high production of SPIONs 52. However, the main drawback of this method is that the prepared particles tend to be poly-disperse with non-unique shape. In the coprecipitation process, two stages are involved: a short burst of nucleation and slow growth of the nuclei. Controlling size and distribution at the first stage has been reported to be very important for the preparation of monodispersed particles. The size and shape of the NPs can be controlled with relative success by selecting the type of salts (e.g. chlorides, sulfates and nitrates), and adjusting the Fe2+/Fe3+ concentration ratio, reaction temperature, pH value, ionic strength of the media and the addition of the chelating organic anions (such as citric, gluconic or oleic acid) or polymer coating materials (such as polyvinylpyrrolidone (PVP), dextran and starch) 74, 112. The other problem is that the yielding ferrous colloid is often the mixture of Fe3O4 and γ-Fe2O3, and the former is not stable and subjected to oxidation into the latter in the presence of oxygen or at high temperature. In term of stem cell labeling, much more work have been carried out by using Fe3O4, due to its superior magnetic properties 117. Occasionally, it was reported that the uncoated γ-Fe2O3, exhibited higher r relaxivity as compared to commercially available Endorem™, Sinerem® and Resovist® 64.

Thermal decomposition of organometallic precursors, such as Fe(Cup)3, Fe(CO)5 or Fe(acac)3 in high-boiling organic solvents containing surfactants can produce monodispersed magnetic NPs. The most outstanding advantage of this method is that the prepared NPs have controllable size in a narrow distribution and high crystallinity. The ratios of the starting reagents including organometallic compounds, surfactants, and solvents are the decisive parameters for the control of the particle size. In addition, the reaction temperature, reaction time, and aging period are also important factors for the precise control of size 52. Hyeon's group 114 has synthesized high crystalline and monodisperse γ-Fe2O3 nanocrystallites with size varied from 4 nm to 16 nm by controlling the experimental parameters. Sun's group 118 also synthesized monodispersed MFe2O4 (M= Fe, Co, Mn) NPs by thermal decomposition (Figure 1). Particle diameter can be tuned from 3 to 20 nm by varying reaction conditions or seed-mediated growth. Our group synthesized ternary SPIONs doped with Mn and Zn elements via thermal decomposition 119. The yielding magnetic NPs had high saturation magnetization (increasing 23% as compared to that without dopants) and small particle size (8 nm) with a narrow size distribution. The SPIONs prepared by thermal decomposition were hydrophobic because of the coverage of hydrophobic surfactants and needed to be transformed into hydrophilic ones for further biological application.

Fig 1

TEM images of MFe2O4 (M= Fe, Co, Mn) prepared by thermal decomposition. (A) 6 nm Fe3O4, (B) 12 nm Fe3O4 (C) 14 nm CoFe2O4 and (D) 14 nm MnFe2O4; reprinted with permission from ref. 118. Copyright (2004) American Chemical Society.

Based on the advantages of both coprecipitation and thermal decomposition method, our group explored a novel one-pot method with sodium oleate as both the surfactant and precipitant to synthesize monodispersed SPIONs in the water/ethanol/toluene system 110, 111, 120, 121. The convenient and mild reaction condition, high yield and narrow size distribution of the obtained SPIONs indicate this method has great potential for industry production. As shown in Figure 2, our group parepared SPIONs with a size of 8 nm by this simple method. This system merely required iron chlorides and sodium oleate because sodium oleate served as both an anionic surfactant and a key reactant in buffering the concentration of OH- for homogeneous SPION nucleation and crystal growth, which was quite different from the traditional preparation methods 110. Furthermore, the composition of products from the mixture of magnetite and maghemite to pure magnetite could be adjusted by varying the iron concentration and feed ratio 111. Similar to the thermal decomposition, the obtained hydrophobic SPIONs in our method required hydrophilic modification for biological applications.

Fig 2

(A) TEM micrographs and the corresponding particle size histograms by DLS (insets) (8 nm) of SPIONs prepared by a novel one-pot method with sodium oleate as both the surfactant and precipitant; (B) the SAED pattern of SPIONs; reprinted with permission from ref. 110 Copyright (2010) Elsevier.

Surface modification

Although many synthetic routes have been developed for the preparation of iron oxide core with tunable shape, size and magnetization, several challenges remain for the naked SPIONs in terms of stem cell labeling, including: (i) poor water solubility and tendency of aggregation due to large surface/volume ratio; (ii) low cellular uptake efficiency; (iii) potential toxicity. To address these problems, the most straightforward and effective method seems to be coating the iron oxide core by a layer. The nature of the surface coatings and modification methods determine the physical and biologic properties such as the overall size, surface charge, coating density, toxicity and degradability, which finally affect the fate of SPIONPs in the cells 93, 112. This following section focuses on the currently used surface modification materials (e.g. PLL, PEI, chitosan, PEG, citric acid and so on) and methods (e.g. in situ coating, post-synthesis coatings including blending, polymerization, ligand exchange) for the SPIONs applied for stem cell labeling and tracking. The influence of these factors on labeling efficiency and biocompatibility is also discussed.

Polymers

Polycations

Polycations are extensively used as the intracellular delivery carriers, because their positive surface charges induce them to interact with negatively charged cell membrane and facilitate internalization. Meanwhile it can be easily combined to the negatively charged SPIONs due to the electrostatic interaction.

Poly(L-lysine) (PLL), a positively charged peptide, is widely used as transfection agent for the complexation and delivery of genes 122. It is also known to enhance the cell adhesion to the surface of culture dish during cell cultivation. PLL can coat negatively charged SPIONs such as Feridex® and chaperon them into stem cells via electrostatic interaction. The common preparation method is post-synthesis coating. Briefly, an aqueous solution of PLL is added into the fresh water-based magnetic fluid prepared through coprecipitation and the reaction is carried on with stirring to obtain PLL capped NPs 62-64. Horάk's group 63 had tested the influence of the molecular weight of PLL (ranging from 146 D to 579 kD) and PLL/Fe2O3 feed ratio (from 0 to 0.009) on the capability of PLL to increase the intracellular uptake of the NPs (PLL-Fe2O3). The maximum cell labeling efficiency (labeled rat MSCs 92.2%) was achieved with 0.02 mg PLL per mL of PLL-Fe2O3 colloid (PLL/γ-Fe2O3 mass ratio of 0.009 and PLL molecular weight of 388 kD). The coating of the naked iron oxide with different amount of PLL (Mw 388 kD) did not change the morphology or the size of the core (~6 nm) (Figure 3A and 3B). The coating force between PLL and γ-Fe2O3 was electrostatic interaction (Figure 3C). TEM examination of PLL-Fe2O3 showed the successful internalization into lysosomes (Figure 3D), and the mechanism of cellular uptake was supposed to be endocytosis and/or diffusion through the cell membranes. Cell labeling with PLL-Fe2O3 was more efficient and safer than that with a conventionally used agent (Endorem™). Horάk's group also found that PLL-Fe2O3 had higher relaxivity (r2) value than PLL-Endorem™ complex and uncoated Fe2O3 64. Ju and coworkers 62 coated Fe2O3 with PLL by the similar post-synthesis as Horάk's group described, but they washed the PLL-Fe2O3 several times to remove free PLL. Therefore, the amount of PLL adsorbed on particle surface was only 0.01% of the total Fe2O3 mass. Prussian blue staining results demonstrated that almost all of human umbilical cord blood mesenchymal stem cells (UCB-MSCs) had shown clear blue after the treatment with PLL-Fe2O3 under the optimal Fe concentration (20 μg/mL of Fe). Owing to the low PLL content, no significant toxicity of PLL-Fe2O3 was observed as compared to the unlabeled cells, even at a high co-incubating concentration up to 200 μg/mL of Fe. T2 weighted image (WI) and T2*WI demonstrated significant decrease of signal intensity in vials containing 1×106 (1 day and 8 days) labeled cells, in comparison with the unlabeled cells.

Fig 3

TEM micrographs of (A) uncoated γ-Fe2O3 NPs and (B) PLL-Fe2O3 (PLL/Fe2O3 = 0.009, mass ratio); (C) Schematic illustration of the interaction between PLL and a citrate-treated γ-Fe2O3; (D) TEM micrographs of rMSC cells labeled with PLL-Fe2O3 (PLL/ Fe2O3 = 0.005) and scale bar 1 μm; reprinted with permission from ref. 63. Copyright (2008) American Chemical Society.

Polyethyleneimine (PEI), another cationic polymer with many primary amino groups, has shown relatively higher gene transfection efficiency, as compared to other non-viral vectors (e.g. PLL, DOTAP liposome), especially for PEI 25kD. It can facilitate cellular uptake and endosomal escape through an “a proton sponge effect” 123. However, PEI also exhibits cytotoxic effect including cell death, apoptosis or inhibition of cell differentiation. Recently, low molecular weight PEI as an alternative to PEI 25kD has drawn more and more attention, because of its improved biocompatibility. Liu and co-worker prepared amphiphilic N-dodecyl-grafted PEI 2k with a graft ratio of 11% 65, 124. The hydrophobic SPIONs obtained by thermal decomposition and N-dodecyl-grafted PEI 2k were dispersed in chloroform. The mixture was under shaking for overnight and the alkyl-PEI/SPIO complexes were obtained after evaporation of chloroform. The mass ratio of N-alkyl-PEI2k to SPIO was a key factor of the stability, biocompatibility and relaxivity of the complex. The complex micelle with mass ratio of 0.6 could hold several SPIONs with a clustering structure (see Figure 4A and 4B), leading to much higher cell labeling and T relaxivities (345 mM-1s-1) measured on 3T MR scanner, as compared to single SPIONs for example Endorem™ (about 176 mM-1s-1). The incubation of MSCs with alkyl-PEI/SPIO (7 μg/mL of Fe, mass ratio = 0.6) for 24 h achieved 7.1 Fe pg/cell and labeled mice MSCs were unaffected in their viability, proliferation, or differentiation capacity. Subcutaneous injection of the labeled MSCs into BALB/c mice showed strong signal contrast against unlabeled cells under a 3T MR scanner for 19 days post-transplantation (Figure 4C and 4D). Shuai's group 66 synthesized cationic stearic acid-grafted PEI (PEI Mw = 423Da) copolymers (PEI-SA) and anionic poly(ethylene glycol)-poly(γ-benzyl-L-glutamate) (PEG-PBLG) (Mn = 9.3 kDa). They firstly coated hydrophobic SPIONs prepared by thermal decomposition with PEI-SA through a blending method, as described in Liu's report 65. Subsequently, PEI-SA modified SPIONs were coated with PEG-PBLG via electrostatic interaction. The zeta potential of PEG-PBLG/PEI-SA/SPIONs could be controlled through adjusting the mass ratio of PEG-PBLG to PEI-SA. It was found that labeling efficiency increased linearly with the zeta potentials of PEG-PBLG/PEI-SA/SPIONs. Under optimal cell labeling conditions involved an iron concentration of 3.15 μg/mL with 20 mV positive charge and 1 h incubation time, a mean iron concentration in rat MSCs reached 50.02 pg/cell.

Fig 4

Characterization of SPIONs (polymer/SPIO mass ratio = 0.6) and in vivo MRI of SPIO-labeled MSCs. (A) AFM height image; (B) TEM image of SPIONs nanoparticle clusters; (C) T*-weighted gradient echo image shows a prominent hypointense area of labeled injection in the right frank (19 d after transplantation); (D) the ratios of signal intensities of the control and the labeled injection; reprinted with permission from ref. 65. Copyright (2011) Elsevier.

Polysaccharides

Polysaccharides, especially those from natural sources, exhibit many specific properties compared to other materials, including water-solubility, biocompatibility, biodegradability, biological activity and low cost 125. Polysaccharides enriched with hydroxyl groups have been reported to interact with iron oxide via hydrogen bonding 126. Chitosan, pullulan, and heparin are the currently used polysaccharides for coating SPIONs.

Chitosan [poly(1,4-β-D-glucopyranosamine)], a biodegradable natural polymer, is derived by the deacetylation of chitin obtained from the shells of crustaceans. It has many biological applications because of its biological activities, biocompatibility, high charge density, low toxicity toward mammalian cells, and ability to improve dissolution. Chitosan coated SPIO (Chitosan-SPIO) NPs had the core size of 6 nm and hydrodynamic size of 65 nm including the chitosan coat, which are similar to those of Resovist® (core iron size: 4.2 nm, total hydrodynamic size: 62 nm) 127. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) data revealed that similar amounts of iron (~18 pg/cell) was taken up by hMSC when labeled with Chitosan-SPIO or Resovist® iron particles in the presence of PLL (388 kD). However, Chitosan-SPIO alone could not efficiently label cells. The carboxymethylation of chitosan increases its water solubility, and enhances the dispersion of SPIONs in aqueous media 67. Furthermore, carboxymethyl chitosan (CMCS) has been shown to enhance interactions with the cell membrane 128. Shi and co-workers produced (carboxymethyl) chitosan-modified SPIONs (CMCS-SPIONs) by covalently binding CMCS to (3-aminopropyl) trimethoxysilane-treated SPIONs. The hMSCs have a preferential uptake of CMCS-coated SPIONs as compared to SPIONs likely due to the nonspecific adsorption 67, 128, although both show negative surface charge (CMCS-coated SPIONs: -21.4 mV vs. SPIONs: -13.6 mV). Less than 100 CMCS-SPIONs labeled cells in agarose can be clearly detected by 1.5T MR system.

Pullulan is a water-soluble polysaccharide with a repeated unit of maltotriose condensed through α-1, 6 linkage. Jo 68 developed two pullulan derivates, including cationized ethylenediamine modified pullulan (ED-pullulan) and anionized succinic anhydride modified pullulan (Suc-pullulan). The Pullulan and/or its derivate-coated SPIONs were synthesized by the conventional coprecipitation of ferric and ferrous ions in the presence of pullulan derivatives. This method was called in suit coating. Various pullulan or its derivate coated SPIONs with different sizes (62 nm ~ 161 nm) and zeta potentials (-6.9 mV ~ +12 mV) were obtained by altering the mixing molar ratios of pullulan hydroxyl groups to ferric ions and mixing percentages among the pullulan, ED-pullulan and Suc-pullulan. Prussian blue staining revealed that positively-charged pullulan-SPIONs with the size less than 100 nm were internalized into almost all cells by co-culture for 1 h. The internalized NPs retained in the cells without negative effects on the cells viability for 21 days. However, Fe content in MSCs dramatically decreased when MSCs treated by NPs with the particle size around 100 nm or the surface potential around 0 mV.

Heparin is a highly sulfated glycosaminoglycan molecule that interacts with various proteins containing heparin-binding domains within the extracellular matrix milieu. It has been widely used in drug delivery systems and tissue engineering to improve the biocompatibility and blood compatibility of biomaterials 129. Recently, Lee and coworkers 69 synthesized SPIONs coated with unfractionated heparin (UFH-SPIOs) by in suit coating (Figure 5). The uptake efficiency of UFH-SPIO without the aid of transfection agents was greater than that of dextran coated SPIO by approximately 3 folds when treated for 1 h. This was because the coating of heparin on the surface of NP increased its hydrophilicity, which promoted cell attachment to the NP surface. When the UFH-SPIO-labeled hMSCs were transplanted into the left renal subcapsular membranes of nude mice, they were successfully visualized and detected by T2 weighted MRI imaging after a month.

Fig 5

Schematic illustration of the preparation of UFH-SPIO; TEM images of non-coated and UFH coated SPIONs (non-coated SPIONs (left) and UFH-SPIONs (right), scale bar: 100 nm); AFM images of UFH-SPIONs (height image (upper) and 3D image (lower)), reprinted with permission from ref. 69. Copyright (2012) Elsevier.

PEG

Poly(ethylene glycol) (PEG) is a flexible and water-soluble polymer. The high hydrophilicity of PEG chains can render the iron oxide core soluble and stabilized in the aqueous media. Park's group 70 evaluated the potency of a library of commercial PEG derivatives (Laysan Bio, Inc, Arab, AL) as surface modifying agents for iron oxide NPs (IONPs) (Figure 6). The PEG library included PEG derivatives with variations in structural configurations (linear, four-arm and six-arm), molecular weights (1, 2, 3.4, 5, 10, 15 kDa, etc.). PEG derivatives modified IONPs were prepared by post-synthesis coating. With the increasing of the molecular weight, the number of branched chains and functionalities, higher stability and better dispersion could be attained. The result demonstrated that six-arm amine-functionalized PEG (6(PEG-NH2)) was a superior dispersion agent for the monodispersed SPIONs. However, PEG is well known for its “stealthy” effect and not favorable for most cells to uptake SPIONs with PEG shell protection 130. Therefore, SPIONP-6PEG-NH2 was modified by hyaluronic acid (HA), a targeting moiety, for stem cell uptake. IONP-6PEG-HA showed higher saturated magnetization and relaxivity as compared to Feridex®. The amount of Fe inside MSCs was much higher for IONP-6PEG-HA (145.9 ± 15.2 ng Fe/105 cells) than that for Feridex® (45.1 ± 3.9 ng Fe/105 cells), probably due to CD44-mediated endocytosis.

Fig 6

Schematic illustration of the procedure for dispersion of IONPs in an aqueous phase using a library of PEG derivatives followed by functionalization with bioactive molecules for cellular labeling and imaging. Inset shows oleic acid on IONP surface; reprinted with permission from ref 70. Copyright (2011) American Chemical Society.

Other polymers

Other synthetic polymer such as polyacrylic acid (PAA), poly(N,N-dimethylacrylamide) (PDMAAm), poly(DL-lactic acid-co-α,β-malic acid) copolymer (PLMA), poly(lactide-co-glycolide) (PLGA) and cellulose have been used to coat or encapsulate SPIONs.

PAA with many carboxylic acids on its backbone can strongly bind to the surface of SPION through multivalent anchoring points. The other free carboxylic groups of the PAA provide SPIONs with high hydrophobicity 131. Our group reported monodispersed superparamagnetic Fe3O4/polymethyl methacrylate (PMMA) composite NPs with high saturation magnetization (39 emu g-1, total mass) which were fabricated by a facile novel miniemulsion polymerization method 99. The ferrofluid, MMA monomer and surfactants were co-sonicated and emulsified to form stable miniemulsion for polymerization. Then Fe3O4/PAA composite NPs could be easily obtained by hydrolysis. Kim et al 71 prepared PAA coated SPIONs by exchanging the oleic acid on the surface of SPIONs with PAA. Subsequently, the free carboxylic acids of the PAA backbone were conjugated with 2-aminoethyl-trimethyl ammonium (TMA) by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) initiation to produce particles called as ''TMA-SPION''. The quaternary amine derivatives have a permanent positive charge (+ 40 mV), independent of the physical pH in vivo. Prussian blue staining results showed that hMSCs treated with TMA-SPION (25 μg/mL, 4 h) acquired much more bright blue color from the stain than that treated with Feridex®. TMA-SPION treatment had no effect on the differentiation of hMSCs. In vivo MRI study confirmed that TMA-SPION labeled cells were visible at least 7 d.

Horák's group produced PDMAAm-coated γ-Fe2O3 NPs by the solution radical polymerization of DMAAm in the presence of SPIONs which were obtained by coprecipitation method and subsequent oxidation with sodium hypochlorite 64, 72. In comparison to dextran coating (Feridex®), PDMAAm coated magnetic NPs exhibited good dispersibility and high cellular internalization. However, it reduced magnetic susceptibility of SPIONs. PDMAAm-coated γ-Fe2O3 labeled hMSCs provided significantly higher r at both 0.5 and 4.7 T fields than Feridex®- and uncoated-iron oxide-labeled cells did due to its higher iron internalization. After the implantation of the three groups of iron oxide-labeled rMSCs (5,000 cells in 5 μL of PBS) in rat brain, only PDMAAm-coated γ-Fe2O3 labeled cells were detected by MR.

PLMA, biodegradable polyester, is biocompatible and easy to be bifunctionalized through conjugating other imaging probes. Wang and coworkers 20 coated magnetic NPs with PLMA covalently bound fluorescein isothiocyanate (FITC) (FITC-PLMA-MNPs) by ligand exchange method (Figure 7). The FITC-PLMA-MNPs labeled hMSCs could be observed under confocal fluorescence microscopy and imaged by a 1.5 T MR scanner at a threshold cell number of 1200 without adverse effects on the osteogenic and adipogenic differentiation potentials of hMSCs.

Fig 7

Schematic diagram and reaction scheme for FITC-PLMA-MNPs; reprinted with permission from ref.20. Copyright (2010) Elsevier.

PLGA and cellulose are Food and Drug Administration (FDA) approved for a variety of uses in humans and commonly employed for drug delivery and oral formulations. Shapiro's group incorporated monodispersed oleic Fe3O4 (~10 nm) into micron-sized particles (MPs) and NPs of PLGA (fast degradation) and cellulose (slow degradation) using an oil-in-water single emulsion technique 132. For all particles studied, SPIONs-loaded PLGA (505 Fe mM-1s-1) and cellulose NPs (399 Fe mM-1s-1) displayed higher relaxivity values per millimole of Fe, while had a much lower relaxivity per particle compared to PLGA MPs. Relaxivity of PLGA particles decreased faster than that of the cellulose particles after the incubation in endosomal mimicking solution (citrate buffer pH5.5). Without the aid of TAs, magnetic PLGA MPs showed a much higher labeling efficiency in MSCs compared to Feridex®/PLL as indicated by higher iron content per cell. Xu and coworkers 73 also reported oleic acid stabilized SPIONs (10 nm core size) encapsulated in PLGA (with carboxyl end-groups) using a single emulsion method. The yielding SPION/PLGA-MPs (0.8-3 μm) had higher rrelaxivity (316.6 Fe mM-1s-1) compared to SPIONs (61.16 mM-1s-1) as a result of the SPION aggregation in PLGA. After transplantation of labeled MSCs into mouse, average 1/T (R) signal from SPION/PLGA-MPs was approximately twice that of the signal generated from SPIONs. Moreover, this MPs enhanced residence time inside MSCs (3-fold) compared to SPIONs. The fabrication and internalization of SPION/PLGA-MPs into MSCs are shown in Figure 8. Therefore, SPIONs loaded polymeric MPs are also promising contrast agents for cellular MRI.

Fig 8

SPIONs/PLGA-MPs preparation and internalization by MSCs. (A) Schematic illustration of the preparation of SPIONs/PLGA-MPs with single emulsion method. (B) SEM image of SPIONs /PLGA-MPs. (C) TEM image of a representative SPIONs/PLGA-MP. (D) TEM image of SPIONs/PLGA-MPs internalized in a MSC, reprinted with permission from ref. 73. Copyright (2012) American Chemical Society.

Small molecule ligands

Small molecule ligands such as citric acid and mannose have been exploited as a coating material for SPIONs, which provides stability to the NPs in solution and is also helpful for cellular uptake.

Andreas et al 74 prepared citrate-coated SPIONs by simply mixing citrate acid and SPIONs prepared through coprecipitation in one step. Citrate-coated SPIONs showed negatively charged and hydrophilic surface, due to the exposure of their terminal carboxylic acid groups to the solvent 52. Compared to the commercial NPs, the citrate-coated SPIONs were effectively internalized by MSCs at 25 μg/mL of Fe in the cultivation medium (citrate-coated SPIONs 69.6 ± 5.1 Fe pg/cell, Resovist® 1.3 ± 2.3 Fe pg/cell and Endorem™ 4.9 ± 2.7 Fe pg/cell). The high labeling efficiency may be explained by an altered endocytotic uptake mechanism due to the highly negative surface charge 77, 133 and/or large size after aggregation in cell cultivation medium 86. Transplanted citrate SPIONs-labeled MSCs in vivo showed a considerable larger volume and surface area of hypointensity compared to Endorem™ labeled-MSCs.

D-Mannose-specific and energy independent transporter has been found on the surface of the majority of mammalian cells 134. D-mannose-coated NPs are most likely transported into the cells through this mannose transporter. Horάk et al 64, 75 prepared D-mannose-coated SPIOs by two methods: in suit coating in the mannose solution and post-synthesis coating with mannose. FTIR results indicated that the surface of γ-Fe2O3 NPs was covered with D-mannose, which could be attached to iron oxide NPs by the hydroxyl group located on the C2 carbon in the axial position. They found that D-mannose-coated SPIONs by in situ coating had a smaller size (~ 2 nm) by TEM as compared with that by the post synthesis coating (~ 6 nm). This small size was owing to the presence of D-mannose during precipitation, which interfered with the nucleation step of iron oxide formation. D-mannose-coated SPIONs by post-synthesis coating had higher cellular internalization (about 80% labeled MSCs) and relaxivity r than those of Endorem™ and D-mannose-coated SPIONs obtained by in situ coating (about 50% labeled MSCs).

Silica

Silica (SiO2) with good biocompatibility is demonstrated to serve as a good coating material for magnetic NPs 76, 78, 130, 135. Silica coating on the surface of SPIONs prevents their aggregation in aqueous solution, improves their chemical and biological stability, and provides better biocompatibility 76. The hydroxyl groups exposed on the silica-coated SPIONs can be easily linked to different functional groups, such as amine and carboxylate. Moreover, silica provides a platform for multi-model probes by integrating SPIONs and other imaging probes such as fluorescent dye 136. Usually, two strategies are exploited to prepare silica coating: 1) silica is formed in situ through a sol-gel process using precursors such as tetraethyl orthosilicate (TEOS) ; 2) micelles or inverse micelles are used to form the coating of silica on the iron oxide core 76, 77.

Wang's group 76 synthesized novel polyhedral crystalline SPIONs coated with aminosilica (SPIO@SiO2-NH2). The polyhedral SPIO was prepared by solvothermal treatment of the crude SPIONs, which was synthesized by coprecipitation. Aminosilica coating was formed on the surface of the recrystallized polyhedral SPIONs (SPIO@SiO2-NH2) through the hydrolysis of aminopropyltriethoxysilane (APTES). TEM image of the SPIO@SiO2-NH2 revealed an iron oxide core (dark dots)-silica shell (thin white layer around the dark dots) structure. ICP-OES and in vitro MRI results demonstrated that surface amine modification enhanced MSC-labeling efficiency of SPIO@SiO2-NH2 NPs compared to SPIO@SiO2 NPs. Importantly, after being implanted in rabbit brain, the labeled MSCs would be visible in a 3T MR system for 8-12 weeks, which was longer compared to other reports due to the stability of SiO2 in cellular environment 62, 72.

The combination of MRI and optical imaging methods has the advantages of high spatial resolution from MRI and good sensitivity from optical imaging methods. Dye molecules can be easily incorporated into a silica shell. Yoon and coworkers 77 synthesized cobalt ferrite magnetic NPs coated with a shell of amorphous silica containing rhodamine B isothiocyanate (RITC) or fluorescein isothiocyanate (FITC) and PEG by a modified PVP method and the sol-gel process (Figure 9A). The thickness of the silica shell could be easily controlled by adjusting the MNPs/tetraethoxysilane (TEOS) ratio and dye-modified saline (Figure 9B). These silica-coated magnetic NPs incorporating RITC (MNPs@SiO2(RITC)), achieves efficient MSC labeling. The labeled cells were bright, photostable, and easy to track in live NODSCID mice 136 and in an experimental rat model of liver cirrhosis 79 (Figure 10). Similarly, FITC-incorporated silica-coated core-shell SPIO NPs (SPIO@SiO2(FITC)) with diameters of 50 nm, were synthesized by a water-in-oil reverse micelle method 78. It could efficiently label hMSCs, via clathrin- and actin-dependent endocytosis. Detection threshold of labeled cell number in vitro was about 1 × 104 cells. Furthermore, 1.2 × 105 labeled cells could also be detected by clinical 1.5T-MRI in vivo.

Fig 9

(A) Schematic illustration of the overall synthetic procedure for MNP-SiO2(RITC)-PEG. (B) TEM images of Co ferrite-silica (core-shell) MNPs with controlled shell thicknesses. TEOS/MNP = 0.12 mg/4 mg, scale bar = 100 nm (left), TEOS/MNP = 0.06 mg/4 mg, scale bar = 50 nm (middle), TEOS/MNP = 0.03 mg/4 mg, scale bar = 50 nm (right). As the ratio of TEOS/MNP (w/w) decreases, the shell thickness decreases. Reprinted with permission from 77. Copyright (2005) Wiley.

Fig 10

Optical, magnetic resonance (MR), and paraffin-embedded tissue images of a mouse injected with nanoparticle-labeled human UCB-derived MSCs (hMSCs). (A, B) The MNPs@SiO2(RITC)-labeled cells were injected into four sites (I-IV) in the mouse's subcutaneous cavity (I, 1 × 106; II, 5 × 105; III, 1 ×105; IV, 5 × 104 cells). Optical imaging shows that the labeled hMSCs express the RITC signal (A-1). With MRI, the coronal (A-2) and axial (A-3, A-4) planes, T*-weighted gradient echo pulse images show very hypointense and distorted dark areas (white arrows) in the abdominal wall. Subsequently, 1 × 106 or 1 × 105 labeled hMSCs were injected into the liver via the portal vein, and 7 days after injection a liver MR image was obtained in the axial plane. Compared to the liver of control mice (B-1), the liver of the treated mice had punctuated dark contrast spots distributed in a very hypointensive area (B-2). Injection of 1 × 105 cells into the liver resulted in a dark spot, but the number and intensity of black contrast spots were lower than that in the liver injected with 1 × 106 cells (B-3). After MRI to confirm that nanoparticle-positive hMSCs were present in the mouse liver (infused with 1 × 105 nanoparticle-labeled hMSCs), the liver was harvested and fixed it in a 4% paraformaldehyde solution. The sectioned slides were observed with CLSM. In a representative histological finding, red fluorescence from nanoparticle-labeled hMSCs, is clearly visible (B-4). Reprinted with permission from 136. Copyright (2010) Elsevier.

Mesoporous silica NPs (MSNs), in the form of stable aqueous dispersions, are emerging as good agents for biomedical imaging and drug/gene therapy. Huang's group developed tumbler-like magnetic/FITC-labeled mesoporous silica NPs (Mag-Dye@MSNs) 137, which were composed of silica coated SPIO (SPIO@SiO2) NPs co-condensed with FITC-incorporated mesoporous silica (FITC-MSNs) 80. Mag-Dye@MSNs displayed much greater labeling efficiency compared to SPIO@SiO2. The Mag-Dye@MSNs labeled cells could be visualized in a clinical 1.5-T MRI system with a detectable threshold of about 1.2×104 labeled cell in vitro and 1×105 cells in vivo. Our group prepared novel SPIO-SiO2 NPs which were composed of a nanospherical Fe3O4/PMMA composite core and a periodic mesoporous silica shell by the combination of a modified sol-gel method and a dual-template strategy. The NPs exhibited homogeneity, superparamagnetism, high saturation magnetization and good magnetic responsiveness (Figure 11) 100. They are promising MRI probes for stem cell labeling and tracking.

Fig 11

(A) The schematic synthesis of the Fe3O4/PMMA/SiO2 nanorattles with periodic mesoporous shells; (B) TEM micrographs of the Fe3O4/PMMA/SiO2 nanorattles with periodic mesoporous shells; (C) N2 adsorption-desorption isotherm and corresponding pore size distribution (inset graph) of Fe3O4/PMMA/SiO2 nanorattles with periodic mesoporous shells. Reprinted with permission from 100. Reproduced by permission of The Royal Society of Chemistry.

Functional moieties

Several small regions of proteins termed protein transduction domains (PTDs), including peptides from the human immunodeficiency virus (HIV) TAT protein and Herpes simplex virus (HSV), have received significant and widespread attention in the pharmaceutical and medical fields. They have the superior ability to deliver macromolecules, even NPs into living cells.

TAT peptide (GRKKRRQRRRGYK) purified from HIV TAT protein contains both transmembrane and nuclear localization signal. TAT-cross linked iron oxide (TAT-CLIO) was prepared by covalently conjugating CLIO-NH2 to C-terminal cysteine side chain of tat-peptide 81. The hNSCs labeled with tat-CLIO contained 2.15±0.3 Fe pg/cell, which was 59-fold, 430-fold and 6-fold higher than those of the hNSCs labeled with ferumoxides, monocrystalline iron oxide (MION) or CLIO-NH2, respectively 53.

Low molecular weight protamine (LMWP, VSRRRRRRGGRRRR) possessed with high arginine content and significant sequence similarity to that of TAT, was derived directly from native protamine by enzymatic digestion with thermolysin. LMWP enables the delivery of many compounds into cells, even an impermeable gelonin across tumor cells 116. In addition, unlike other cationic proteins or peptides, the LMWP hardly activated complement systems and elicited negligible hypotensive or toxic responses in dogs 138. LMWP modified SPIO (LMWP-SPIO) exhibited high Ms values of 85 emu/g Fe 82, which was much higher than that of SPIO (approximately 65 emu/g Fe) 109. The hMSCs labeled with LMWP-SPIO presented the highest iron content compared to those labeled with Federix® and the complex of Federix® with poly-L-lysine.

Problems and perspectives

SPIONs have gained intensive research interest in stem cell labeling due to their high MR sensitivity and amenability to surface modification. Despite their rapid advancement in magnetic NPs and MRI technologies for stem cell biology, several problems remain to be solved: 1) How to detect not only the location but also the function of the labeled cells? SPIONs combined with other artificial reporter genes 139 as MRI contrast agents probably hold promise in this regard, since the fate of the labeled cells can be probed. Moreover, MRI in conjugation with other noninvasive imaging modalities, such as PET, SPECT, ultrasound, and optical imaging, yield a hybrid imaging platform which may overcome the disadvantage of MRI 97, 140, 141. 2) What is the potential toxicity of SPIONs in vivo? For instance, after the SPION labeled cells being transplanted into the host, SPIONs influence not only the labeled cells, but also the liver and spleen of the host 97. The process and effects are not completely clear. Systematic preclinical studies have to be conducted with standardized assays to assess the potential long-term toxicity of the in vivo use of SPIONs. 3) How to translate stem cell tracking from preclinical models to human? Until now, there have been very few clinical studies due to issues in technologies and ethics 24.

In conclusion, the newer “smart” SPIONs together with a more thorough understanding of label particle-cell-host interactions will greatly improve the applicability of SPIONs-based stem cell tracking and monitoring.