Poly(N,N-dimethylacrylamide)-coated maghemite nanoparticles for labeling and tracking mesenchymal stem cells

PDMAAm-coated γ-Fe2O3-labeled MSCs

Shan L.

Publication Details



In vitro Rodents



Personalized diagnosis and treatment with allogenic or autologous cells are becoming a reality in the field of medicine (1, 2). Cytotoxic or engineered T-cells are under clinical trial for the treatment of hematopoietic or other malignant diseases (3). Contrast agent–tagged macrophages are used as cellular probes to image the early inflammatory processes in macrophage-rich conditions such as inflammation, atherosclerosis, and acute cardiac graft rejection (4). The roles of stem cells are under intensive investigation in therapeutic and regenerative medicine, such as regenerating cardiomyocytes, neurons, bone, and cartilage (1). Genetically modified cells are used to treat genetic disorders (5). With promising results from these studies, a critical issue is how to monitor the temporal and spatial migration and the homing of these cells, as well as the engraftment efficiency and functional capability of the transplanted cells in vivo (6, 7). Histopathological techniques have only been used to obtain information on the fate of implanted cells at the time of animal euthanization or via biopsy or surgery. To track the real-time changes of cell location, viability, and functional status, cell imaging techniques have been introduced during the last few years. Cells of interest are labeled with reporter genes, fluorescent dyes, or other contrast agents that transform the tagged cells into cellular probes or imaging agents (2, 6, 7).

The ability to monitor superparamagnetic iron oxide particles (SPIO) with magnetic resonance imaging (MRI) has been utilized in animal models as well as in a few clinical settings to investigate the fate of labeled cells (6-10). The advantages of using MRI for cell tracking include high spatial resolution with high anatomic background contrast, the lack of exposure to ionizing radiation, and the ability to follow the cells for months. In addition, cell labeling with SPIO nanoparticles is generally nontoxic and does not affect the cell proliferation and differentiation capacity, although a few studies have reported that the stem cells labeled with SPIO lose part of their differentiation capacity in a SPIO concentration–dependent manner. An important limitation of MRI is the fact that MRI signals cannot indicate whether cells are dead or alive. It is also unknown whether the MRI signal comes from targeted or labeled cells or from macrophages. Basically, SPIO particles are used to label the target cells by systemic application or by injection into the tissue area of interest to monitor target cell migration after phagocytosis. SPIO are more frequently used to label the cells in vitro by incorporating into the cells directly. Furthermore, SPIO are usually encapsulated by organic polymers to increase their stability and biocompatibility and allow the chemical modification of their surfaces. The fact is that the uptake of different particles varies largely between different cell types (6, 7).

Mesenchymal stem cells (MSCs) represent a heterogeneous subset of pluripotent stromal cells that can be isolated from different adult tissues including adipose tissue, liver, muscle, amniotic fluid, placenta, umbilical cord blood, and dental pulp, although the bone marrow remains the principal source for most preclinical and clinical studies (1, 11, 12). Although MSCs account for only 0.001–0.01% of the total nucleated cells within isolated bone marrow aspirates, they can easily be isolated and expanded in vitro through as many as 40 population doublings after 8–10 weeks of culture (1, 13). These cells exhibit the potential to differentiate into cells of diverse lineages such as adipocytes, chondrocytes, osteocytes, myoblasts, cardiomyocytes, neurons, and astrocytes. In addition, MSCs possess remarkable immunosuppressive properties and have been shown to be effective against tumor cell growth (14, 15). Babic et al. developed poly(N,N-dimethylacrylamide)-coated maghemite (PDMAAm-coated γ-Fe2O3) nanoparticles for labeling and MRI tracking of MSCs (16). Maghemite (γ-Fe2O3) nanoparticles are one of the most commonly used ferric oxide particles because of their simple synthesis and chemical stability. Because iron participates in human metabolism, ferric oxide particles are usually well tolerated by living organisms. The PDMAAm coating provides desired functional groups on the particle surface and increases the stability of the nanoparticles. PDMAAm-coated γ-Fe2O3 nanoparticles provide higher T2 relaxivity and better resolution than uncoated nanoprticles and Endorem. Endorem is a commercially available MRI contrast agent that is based on dextran-coated SPIO nanoparticles (a brand name for ferumoxide).



Babic et al. described the synthesis procedure of PDMAAm-coated maghemite nanoparticles and cell labeling in detail (16). In general, the nanoparticles were prepared in a two-step procedure. Colloidal Fe(OH)3 was first precipitated from FeCl3·6H2O added to less than an equimolar amount of ammonia, followed by the addition of FeCl2·4H2O (molar ratio, Fe(III)/Fe(II) = 2). The mixture was then poured into an excess of ammonia to form a magnetite (Fe3O4) coagulate. The pure magnetite was sonicated with sodium citrate solution and oxidized with sodium hypochlorite to become maghemite (Fe2O3). PDMAAm coating was achieved with a radical polymerization solution of N,N-dimethylacrylamide (DMAAm) in the presence of maghemite nanoparticles. 4,4'-Azobis(4-cyanovaleric acid) (ACVA) was preferred over 2,2'-azobisisobutyronitrile (AIBN) as an initiator for coating because of its much higher solubility in an aqueous monomer solution than that of AIBN, and because of its carboxyl group’s ability to interact with iron oxide.

The investigators then characterized the coated nanoparticles with different techniques (16). Transmission electron microscopy (TEM) showed agglomerated maghemite particles in a dry state with an iron core diameter of 6.3 nm and a polydispersity index of 1.33. The coating process did not change the shape or size of the particles. X-ray diffraction measurements demonstrated the typical spinel structure of γ-Fe2O3. The molecular weight of PDMAAm prepared in the presence of maghemite was 717.9 kDa, lower than that of PDMAAm prepared in the absence of maghemite (965.2 kDa). This can be attributed to the growing polymer chains probably terminating on the nanoparticle surface. At γ-Fe2O3/DMAAm ratios of 0.5/0.375 (w/w) and lower, free PDMAAm was present in the mixture after polymerization. The colloidal stability decreased with increasing γ-Fe2O3/DMAAm ratios. The spectrum of the nanoparticles differed from that of the pure PDMAAm, indicating that the PDMAAm shell effectively formed on the surface of the iron oxide particles. Use of ACVA as the initiator produced a very stable colloid with particles typically in the hydrodynamic size range of 50−170 nm per dynamic light scattering. While TEM provides the number-average particle size, dynamic light scattering gives the z-average, which is sensitive to large-size particles. The particles were very stable in water due to the PDMAAm coating, in that there was no increase in the hydrodynamic size and the polydispersity was consistently low over several months. The uncoated nanoparticles were unstable, showing increased hydrodynamic diameter and polydispersity over time because of aggregation. For the reaction parameters, the more DMAAm in the polymerization feed, the more PDMAAm was bound to the particles. At high ACVA amounts, the stability of the colloidal system was lost, showing aggregated particles. Both the hydrodynamic particle size and the percentage of bound PDMAAm increased with increasing amounts of the initiator up to 15 mg ACVA. Particle aggregation occurred with amounts of 1.5 g or more DMAAm in the feed, probably because of the undesirable physical cross-linking of PDMAAm chains and the increased viscosity of the reaction mixture. At a constant amount of the initiator in the feed, the percentage of PDMAAm that bound to the particles did not change with increasing amounts of DMAAm. In the case of constant particle size, the amount of bound PDMAAm decreased with increasing amounts of γ-Fe2O3 in the feed.

To investigate the effect of the PDMAAm coating on the internalization of the maghemite nanoparticles by target cells, human bone marrow MSCs (hMSCs) and rat bone marrow MSCs (rMSCs) were incubated with PDMAAm-coated nanoparticles, Endorem, or uncoated maghemite nanoparticles. hMSCs were obtained from the bone marrow of healthy donors, and rMSCs were obtained from the tibia and femur of 4-week-old Wistar rats. The same iron concentration (15 µg Fe2O3/ml in medium) of all particles was used, and incubation was carried out for 72 h. rMSCs showed considerably higher uptake of the PDMAAm-coated maghemite nanoparticles (percentage of labeled cells, 59%) than Endorem (39%). A higher uptake of PDMAAm-coated nanoparticles was observed in hMSCs (82%), whereas Endorem uptake was 68%. More cells were intensely stained with PDMAAm-coated nanoparticles than with Endorem or uncoated maghemite. The labeling efficiency was stable and was not dependent on the number of cell passages (16).

In Vitro Studies: Testing in Cells and Tissues


Babic et al. first evaluated the acute toxicity of PDMAAm-coated maghemite nanoparticles after incubation of the rMSCs and hMSCs with the nanoparticles for 72 h at a concentration of 15 µg γ-Fe2O3/ml (16). The cell viability of both rat and human labeled MSCs did not markedly decrease compared with that of unlabeled MSCs (control). When the MSCs were labeled with Endorem, cell viability decreased by 32% and by 15% with uncoated maghemite nanoparticles. The TEM images showed that cells internalized the PDMAAm-coated nanoparticles in large numbers and accumulated them in endosomes. Organelles and cell structures were not affected. MSCs labeled with Endorem showed a heterogeneous distribution of the nanoparticles. Some of the cells contained numerous endosomes filled with nanoparticles; others contained only a few or none. The majority of the cells labeled with uncoated maghemite underwent programmed cell death, probably by autophagia.

Babic et al. then analyzed the relaxometry after dispersing the labeled cells in gelatin at concentrations of 50, 100, 200, 400, and 800 cells/µl, separately (16). Relaxation rates r1 and r2 were related to the number of cells/µl. PDMAAm-coated maghemite-labeled hMSCs provided significantly higher r1 and r2 rates than Endorem- and uncoated maghemite–labeled cells. Large differences were observed for the relaxation rates between PDMAAm-labeled human and rat cells, with r2 nearly 10 times higher for human cells. PDMAAm-coated maghemite-labeled rat cells provided higher relaxation rates than Endorem-labeled cells; however, they were quite comparable to uncoated maghemite–labeled cells. These results indicated that human cells preferred PDMAAm-coated nanoparticles to both Endorem and uncoated ones, whereas in the case of rat cells, uncoated iron particles were internalized at slightly higher rates than PDMAAm-coated ones. Similarly, the T2- and T2*-weighted MRI of gelatin showed that cells labeled with PDMAAm-coated maghemite nanoparticles had higher negative contrast enhancement than cells labeled with Endorem or unlabeled cells.

Animal Studies



In vivo MRI was performed on a 4.7-T imager after implantation of PDMAAm-coated maghemite-labeled cells and Endorem-labeled cells (5,000 cells suspended in 5 µl phosphate-buffered saline) into the left and right rat brain hemispheres, respectively (n = 5 rats). Unlabeled cells were injected as a control. Only cells labeled with PDMAAm-coated maghemite nanoparticles were detected (16).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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