Jessica M.
Rosenholm
*abc,
Riikka M.
Korpi
de,
Eveliina
Lammentausta
d,
Siri
Lehtonen
f,
Petri
Lehenkari
g,
Rasmus
Niemi
hi,
Wangchuan
Xiao
b,
Jixi
Zhang
bj,
Desiré
Lindberg
c,
Hongchen
Gu
b,
Cecilia
Sahlgren‡
hik and
Roberto
Blanco Sequeiros‡
*dl
aPharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland. E-mail: jerosenh@abo.fi; Tel: +358-2-215 3255
bNano Biomedical Research Center, Med-X Research Institute and School for Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, P.R. China
cLaboratory for Physical Chemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland
dDepartment of Diagnostic Radiology, Oulu University Hospital, Oulu, Finland
eDepartment of Radiology, Helsinki University Hospital, Helsinki, Finland
fUniversity of Oulu, Department of Anatomy and Cell Biology and Oulu University Hospital, Medical Research Center and Respiratory Research Unit, Finland
gDepartment of Anatomy and Clinical Research Center, University of Oulu, Oulu, Finland
hCell Biology, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland
iTurku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland
jChongqing University, College of Bioengineering, Chongqing, China
kDepartment of Biomedical Engineering, Technical University of Eindhoven, The Netherlands. E-mail: C.M.Sahlgren@tue.nl
lSouth West Finland Imaging Centre, Turku University Hospital, Turku, Finland. E-mail: roberto.blanco@tyks.fi; Tel: +358-2 313 1975
First published on 11th May 2015
Magnetic resonance (MR) imaging, with its inherent good spatial resolution and without tissue penetration depth limitations associated with other cell tracking techniques, is considered a valuable tool to assess the effects of cellular therapy. However, in order to allow in vivo tracking of transplanted cells with MRI, the cells must be labeled with contrast agents, usually in the form of magnetic or paramagnetic nanoparticles. Typically these are iron oxides, which are associated with a number of drawbacks related e.g. to the generation of hypointensities of the MR image due to signal loss. In this study, two chemically distinct manganese oxide-based nanostructures were developed and their feasibility as labels for human mesenchymal stem cells (hMSCs) was investigated. The ability to monitor the produced particles alone or within the labeled cells in vitro using MR imaging was further evaluated. Two novel synthetic approaches, the polyol process and microwave digestion, were combined to yield a “green”, and extremely rapid means of producing water-dispersible crystalline (MnO) and amorphous (MnOx) manganese oxides. To increase their water dispersability, capping agents in the form of organic polymers, poly(vinyl pyrrolidone) (PVP) and poly(acrylic acid) (PAA), were added to the synthesis process. Crystalline MnO was not formed when PAA was used as the capping agent, since Mn ions (Mn2+) cannot be hydrolyzed to Mn(OH)2, which is an intermediate step in the formation of MnO, under the acidic conditions provided by PAA. PVP, on the other hand, served to induce a spherical shape to the formed nanocrystals. Remarkably, the relaxation times of the as-prepared amorphous MnOx were significantly shorter than those of their crystalline counterparts, and the biocompatibility was also higher for MnOx. To the best of our knowledge, this is the first report which describes the use of an amorphous MnOx as a cell label for MR imaging.
To date, small/ultra-small superparamagnetic iron oxide nanoparticles (SPIO/USPIO) have been the most widely used as contrast agents for cellular MR imaging.3–5 Traditionally, these are prepared via the conventional coprecipitation method, which is difficult to control in terms of size distribution, crystallinity, saturation magnetization values as well as particle aggregation and dispersability in aqueous solutions.6,7 To overcome these drawbacks, new synthesis approaches have been developed, such as high-temperature decomposition of iron precursors in nonpolar solvents, which produces high-quality monodisperse iron oxide nanoparticles with a tightly controlled size distribution and high crystallinity.7–9 These particles are inherently hydrophobic, and need to be either coated by a hydrophilic layer via surface functionalization procedures and/or phase transferred into water before they are suitable for biomedical applications. The addition of appropriate capping agents, such as carboxylic acids, in the synthesis process – resulting in hydrophilic groups on the formed particles has been an important development.10,11 Another method for producing intrinsically hydrophilic metal or metal oxide nanostructures with controlled properties is the polyol process, i.e. the reduction of metal salts in polyalcohols.6–8,10–17 When combined with microwave digestion, which is a well-established route in organic synthesis,18 this synthesis strategy may provide a simple, rapid (within minutes), effective, low energy-consuming, and environmentally friendly “green” synthesis method for producing inorganic nanostructures with controllable characteristics as well.8,14,18,19
Gadolinium-based complexes, such as gadopentate dimeglumine, and manganese (Mn) both accelerate longitudinal T1 relaxation increasing signal intensity on MR images as opposed to iron oxides. However widely used in drug administration applications,20 gadopentate dimeglumine is associated with nephrogenic systemic fibrosis21,22 and is therefore a less favorable agent for clinical applications. The molecular size of gadopentate dimeglumine is also an issue. Whereas free manganese ions (Mn2+) are also toxic and thus are mainly applicable for animal manganese-enhanced MR imaging studies,23 manganese oxide (MnO) in its crystalline form has a tolerable cellular toxicity range and is considered a promising agent for longitudinal cell tracking, as it is easy to deliver and maintain good image quality.23–25 However, it is still controversial how the labeling with MnO affects cell function and cell differentiation capacity26 and thus, there are only few studies reporting the use of MnO as a contrast agent for cellular tracking in vivo. Still, the obvious advantage of Mn-based substances related to the positive T1 contrast, which generates a signal increase that is opposite to iron oxide particles, opens up a multitude of opportunities in anatomical and functional imaging that can be used to augment tissue imaging. The effect could be especially beneficial in detecting metabolic events and adverse or pathological anatomy in tissues that naturally have low signal in MRI.24 Various advantages thus justify developing manganese-based nanoparticles for future clinical applications within molecular and cellular imaging.
The purpose of this study is to produce and characterize two chemically distinct types of manganese oxide, crystalline and amorphous, using the combined advantages of the polyol process and the microwave technique. We demonstrate the production of novel, water-dispersible (hydrophilic), fast-processed manganese oxide nanoparticles and assess the feasibility of these nanoparticles for the labeling of human mesenchymal stem cells as well as MR imaging.
For MR imaging measurements of particle treated MSC cells, 125000 MSCs were incubated with 20 and 40 μg ml−1 particles for 2 h. Subsequently, the medium was replaced and the cells were allowed to grow for 24 h after which the cells were harvested and pelleted in a 3% agar in Eppendorf tubes.
T1 and T2 relaxation times of all the samples were measured using a 3T clinical MR scanner (Siemens Magnetom Skyra, Siemens Healthcare, Erlangen, Germany) and an 8-channel receive-only small animal coil (RAPID Biomedical GmbH, Rimpar, Germany). The T1 relaxation time was measured using a single slice inversion recovery fast spin echo sequence (TR 10000 ms, TE 8.6 ms, ten TIs between 50 and 9500 ms, FOV 12 cm, matrix 256 × 256 yielding an in-plane resolution of 0.47 mm, slice thickness 3 mm, ETL 8, NEX 1). The T2 relaxation time was measured using a multi-slice multi echo spin echo sequence (TR 1680 ms, 12 TEs between 11.5 and 138 ms, ETL 5; FOV, matrix and slice thickness unchanged). Slices were positioned along the cross-section of the test tubes. Circular ROIs were manually segmented into each test tube, and the relaxation times were calculated pixelwise with non-linear fitting and assuming monoexponential relaxation using the in-house MATLAB application (The MathWorks Inc., Natick, MA, USA). The mean value and standard deviation were calculated for each ROI.
Ac− + H2O = HAc + OH− | (1) |
Mn2+ + OH− = Mn(OH)2 | (2) |
Mn(OH)2 = MnO + H2O | (3) |
Sample (reaction conditions) | Hydrodynamic diameter in EtOH | Hydrodynamic diameter in H2O |
---|---|---|
PVP 10 min@240 °C | 129 ± 9 nm | 383 ± 84 nm |
PVP 20 min@240 °C | 204 ± 8 nm | 349 ± 42 nm |
PVP 30 min@240 °C | 156 ± 22 nm | 411 ± 191 nm |
PAA 10 min@240 °C | N/A | 105 ± 11 nm |
PAA 20 min@240 °C | N/A | 178 ± 42 nm |
PAA 30 min@240 °C | N/A | 113 ± 11 nm |
Therefore, MnO was not formed when PAA was used as the capping agent, since Mn2+ cannot be hydrolyzed to Mn(OH)2 under the acidic conditions provided by PAA (a polyacid). Moreover, the reaction of Mn2+ to MnO indicates that there is no oxidation or reduction reaction taking place in this system.
To determine the behavior in solution of the formed materials, whereby electron microscopy only provides information in dry state, the water/buffer suspensions of the amorphous and crystalline manganese oxides were investigated. Their suspension behavior especially in aqueous solvent is crucial for successful applicability, as the utilization of the produced materials, such as cellular labeling, will naturally take place under aqueous conditions. Thus, their hydrodynamic size and net surface charge (zeta potential) at neutral pH were investigated with dynamic light scattering and electrokinetic measurements (Fig. 2). For the crystalline sample, a hydrodynamic diameter of 99.17 ± 0.17 nm (z-average) was found (Fig. 2a), with a low polydispersity index (PdI = 0.076), indicative of well-dispersed particles with a narrow (hydrodynamic) size distribution. For the amorphous sample, the particles were not as discrete and well-defined, as also evident from their structure from TEM (Fig. 1) but they could also be readily dispersed in an aqueous solvent and provided better hydrodynamic characteristics than their crystalline counterparts under the same conditions (Table 1). However, under ethanolic (parent/storage solvent) conditions, also the crystalline MnO exhibited narrow hydrodynamic size distributions (Table 1), indicative of discrete particulates. Whereas the amorphous MnOx net surface charge, as measured by the ζ-potential in HEPES buffer at pH 7.2 yielded a negative surface charge −25.7 ± 1.47 mV due to the carboxylic acid groups resulting from the PAA used as the capping agent, the crystalline sample exhibited a net neutral charge at neutral pH (Fig. 2c), the characteristic isoelectric point of Mn(OH)2;27 further corroborated by considering that PVP does not contain chargeable groups. The net neutral charge at neutral pH is also reflected in the “worse” hydrodynamic size results in ddH2O of these samples, due to the absence of strong repulsive forces as in the case of the negatively charged MnOx. The overall characteristics of the PVP and PAA capped suspensions have been summarized in Table 1.
Based on dynamic light scattering data (Table 1), 20 min was concluded to be sufficient for obtaining manganese oxide particles when stabilized by polymeric agents PVP or PAA. For confirmation of the crystalline phase (or absence thereof), selected samples were analyzed by XRD (Fig. 3).
The diffraction patterns of samples produced via microwave digestion for 20 or 30 min at 240 °C without any additional polymer, showed clearly resolved peaks that could be indexed to a crystalline MnO phase.28 The same pattern was observed for the PVP-stabilized sample digested for 20 min at 240 °C, whereas for the corresponding sample prepared with capping agent PAA, no distinctive XRD patterns could be found, indicative of an amorphous phase and in accordance with what was deduced from TEM characterization.
TEM analysis of the above samples (Fig. 4) revealed some differences in size and morphology, also suggesting that 30 min provides for more uniform morphology than 20 min, whereas the addition of PVP seems to facilitate some additional morphology/size control inducing a more uniform and spherical shape rather than crystal-like particulates; whereby 20 min microwave digestion is sufficient for the formation of ∼10 nm MnO nanoparticles.
As can be deduced from these TEM images, especially when PVP is used as the capping agent, there seems to be a coexistence of nanoclusters together with independent nanoparticles, which may explain why the individual particle diameter is about 10 nm but the hydrodynamic size (by DLS) is exceeding 200 nm (Table 1). In this case, the PVP thus seems to function primarily as a morphology inducer and/or growth regulator rather than an effective dispersion agent. Clearly, separate and almost spherical nanoparticles with diameters less than 10 nm are formed (Fig. 4c and d), but to keep them properly separated also in the final suspension, either the PVP concentration would need to be optimized, or a second dispersion agent would need to be added, or possibly some other suspension media could be used. It should be noted, however, that particle aggregation would also readily take place upon drying on the grids used for electron microscopy, due to the large surface energy of such small particles. Upon application in biological systems, further organic coating of the nanoparticles would also commonly come in question.3
Fig. 5 Relaxivity measurements of as-prepared manganese oxides in water. (a) & (b) amorphous and (c) & (d) crystalline MnO. Measurements were performed on a minispec table-top NMR instrument. |
Remarkably, the relaxation times of the amorphous samples (Fig. 5a and b) were significantly shorter than those of the crystalline particles down to extremely low values (T1 below 20 ms and T2 below 10 ms at higher concentrations of Mn). The corresponding relaxivity plots (Fig. 6) revealed r1,2 values of r1 = 15.6, 26.7, 24.9 mM−1 s−1 and r2 = 30.8, 52.2, 39.1 mM−1 s−1 for the amorphous MnOx. The corresponding relaxivity values for the crystalline MnO (ESI Fig. 1a and b†) are r1 = 0.3, 0.09 mM−1 s−1 and r2 = 6.7, 1.4 mM−1 s−1, i.e. approximately an order of magnitude lower. These striking differences in relaxometric properties can be due to many reasons, related to different material characteristics such as saturation magnetization, particle size and surface coating as well as dispersability in aqueous solvent.29–33 For instance, when superparamagnetic particles are very small, their 1/T2 is shown to be proportional to particle size; whereas when the particles are large, 1/T2 is proportional to the inverse particle size.29 The slight deviations from linearity observed for most of the samples may also be well related to the aggregation in solution, which, in turn, may very likely also partly be a consequence of time waiting dependence.32,33 Time dependence in the T2 relaxation time has been investigated for iron oxides, and found to be due to large particles generally having a high saturation magnetization value and poor stability in water, which, in combination, may lead to aggregation under the 1.4T magnetic field used for relaxivity measurements of suspensions; whereby the relaxation time changes with time.32 We have also recently shown the importance of an optimized surface functionalization for preventing aggregation of magnetic particles under a magnetic field.34 These notions point to the fact that careful surface engineering could be facilitative also from a relaxometric point of view.
Fig. 6 Relaxivity plots for the as-prepared amorphous MnOx prepared at different reaction times and temperatures. |
Labeled cell samples at 3T | ||||
---|---|---|---|---|
T 2 [ms] | T 1 [ms] | |||
Mean | SD | Mean | SD | |
Am 20 μg ml−1 | 3506 | 208 | 2040 | 5508 |
Am 40 μg ml−1 | 3707 | 351 | 1986 | 4603 |
Cry 20 μg ml−1 | 3451 | 149 | 1598 | 5341 |
Cry 40 μg ml−1 | 3183 | 132 | 1257 | 4296 |
The polyol process has cleared the ground amongst inorganic nanoparticle syntheses especially when biomedical applications are aimed at, due to the inherently hydrophilic particles that are produced as a result of the hydroxyl (–OH) groups that are formed on the surface by a layer of polyol molecules.7 The polarity of the polyols moreover offer the ability to dissolve various inorganic salts, act as the reducing agent for many kinds of metal ions for the synthesis of metal and metal oxide nanoparticles, and furthermore, the high boiling point enables elevated temperature conditions which, in turn, may improve the reactivity of the used reactants.17 Synergistically, carrying out the synthesis with the aid of microwave irradiation offers a striking reduction of reaction times as opposed to conventional heating (oil bath or autoclave) from hours or days to minutes, allowing for a time- and energy-saving process.14 A superior colloidal stability under aqueous conditions could potentially still be achieved by the addition of a polyelectrolyte such as PAA, the chains of which can strongly coordinate to metal ions such as iron on a nanocrystal surface, while uncoordinated carboxylic acid groups can extend into the surrounding water.10 In the present case, a change in pH was exerted by the polyacid, inducing the formation of an amorphous phase over a crystalline one. Shifting PAA to the strong coordinating agent PVP, an amphiphilic, nonionic polymer, widely used to improve the colloidal stability of various particle types as it is known to adsorb well onto a broad range of different materials,36 produced crystalline materials as expected. Here, the PVP seemed to rather function as a morphology (shape) inducer and size regulator, leading to spherical nanocrystals of less than 10 nm in diameter. Further optimization of surface coating procedures will facilitate the application of the produced Mn-based nanostructures as cellular labels from a colloidal stability, cytocompatibility and thus, ultimately, also the relaxivity point of view.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5qi00033e |
‡ Equal contribution. |
This journal is © the Partner Organisations 2015 |