Magnetite Nanoparticles for Stem Cell Labeling with High Efficiency

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Magnetite Nanoparticles for Stem Cell Labeling with High Efficiency and Long-Term in vivo Tracking Noelia Guldris, Barbara Argibay, Juan Gallo, Ramón Iglesias-Rey, Enrique Carbo-Argibay, Yury V. Kolen'ko, Francisco Campos, Tomás Sobrino, Laura M. Salonen, Manuel Bañobre-López, José Castillo, and Jose Rivas Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00522 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Bioconjugate Chemistry

Magnetite Nanoparticles for Stem Cell Labeling with High Efficiency and Long-Term in vivo Tracking Noelia Guldris,a,b Bárbara Argibay,c Juan Gallo,a Ramón Iglesias-Rey,c Enrique Carbó-Argibay,a Yury Kolenˊko,a Francisco Campos,c Tomás Sobrino,c Laura M. Salonen,a Manuel Bañobre-López,a José Castillo,c and José Rivas*a,b a

International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga,

Portugal b

Department of Applied Physics, Technological Research Institute, Nanotechnology and Magnetism

Lab, Universidade de Santiago de Compostela 15782, Spain c

Clinical Neurosciences Research Laboratory, Clinical University Hospital, Health Research Institute

of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela 15782, Spain E-mail: [email protected] Telephone: +34 881814021

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Abstract Superparamagnetic iron oxide nanoparticles (SPIO-PAA), ultrasmall iron oxide nanoparticles (USPIO-PAA), and glucosamine-modified iron oxide nanoparticles (USPIO-PAA-GlcN) were studied as mesenchymal stem cell (MSCs) labels for cell tracking applications by magnetic resonance imaging (MRI). Pronounced differences were found in the labeling performance of the three samples in terms of cellular dose and labeling efficiency. In combination with polylysine, SPIO-PAA showed nonhomogeneous cell internalization, while for USPIO-PAA no uptake was found. On the contrary, USPIO-PAA-GlcN featured high cellular uptake and biocompatibility, and sensitive detection in both in vitro and in vivo experiments was found by MRI, showing that glucosamine functionalization can be an efficient strategy to increase cell uptake of ultrasmall iron oxide nanoparticles by MSCs. Keywords:

cell

tracking,

glucosamine,

magnetite,

MRI,

stem

superparamagnetic iron oxide nanoparticles, ultrasmall iron oxide nanoparticles


cells,

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1 Introduction Stem cells show promise for the treatment of various diseases, such as glioma, ischemic brain injury, Alzheimer´s or Parkinson´s diseases,1 and cardiovascular disease,2–4 as demonstrated by their involvement in more than 360 clinical trials.5 Cell therapy is based on the administration of stem cells, relying on their ability to self-renew and differentiate into most cell types of the adult body. Therefore, the understanding of the needed dose, administration route, and biodistribution of these transplanted stem cells is of fundamental importance in order to develop safe therapies. Non-invasive cell tracking and specific imaging of labeled stem cells in vivo is an ideal methodology to follow the migration and grafting of transplanted cells, and evaluate their fate and therapeutic effect.6 Iron oxide nanoparticles (IONs) together with MRI are a common approach for cell tracking due to the biocompatibility and effectiveness of IONs, and the high spatial resolution, penetration depth, and the lack of ionizing radiations provided by MRI.7 Magnetic nanoparticles are known to shorten the transverse relaxation time or T2 in MRI, and therefore create dark contrast in T2-weighted MR images. Clinically approved IONs, such as Feridex or Ferumoxytol, provide low labeling efficiency.8 Although in their natural niches MSCs are phagocytic in nature, during ex vivo cell culture and expansion they lose their phagocytic ability, rendering their efficient labeling a challenge.2,9 A low labeling efficiency limits the use of IONs as magnetic labels to short-term detection, usually referred to 0−72 h after cell transplantation.10 Thus, significant efforts have been devoted to developing new ION formulations with different organic coatings to enhance cellular uptake and enable mid to long-term tracking.11–14 IONs can be classified as superparamagnetic iron oxide nanoparticles (SPIOs), if the hydrodynamic diameter (Dh) is above 50 nm, or as ultrasmall iron oxide nanoparticles (USPIOs), if it is below. USPIOs have often been studied as contrast agents because of their enhanced blood half-life when compared to SPIOs, but low cell uptake has hampered their use for cell tracking applications.15,16 The smaller size of USPIOs as compared to SPIOs could result in advantageous properties for cell tracking, such as higher stability in biological fluids,



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reproducible cell labeling procedures, and homogeneous labeling of the entire cell population.17 In this work, we have studied the cell labeling performance of SPIOs (SPIO-PAA), USPIOs (USPIO-PAA), and glucosamine-functionalized USPIOs (USPIO-PAAGlcN). The internalization of the samples by MSCs was significantly different. USPIO-PAA-GlcN showed promise as a labeling agent for cell tracking applications, displaying high biocompatibility, enhanced uptake by MSCs, and providing sensitive detection by MRI in both in vitro and in vivo experiments.

2 Results and Discussion Synthesis and characterization of IONs In order to apply magnetic nanoparticles (NPs) as MR labeling agents in cell tracking, IONs were synthesized by a hydrothermal method18 using poly(acrylic acid) (PAA) as coating because of the high yield, magnetic response of the NPs, high stability in aqueous medium, and the possibilities the coating offers for further functionalization. Two sets of IONs were synthesized following the same synthetic procedure,18 but differing in purification protocols19 (for details, see Experimental Part 4.1), to render two different samples: SPIO-PAA and USPIO-PAA. The inorganic core composition was confirmed to be magnetite (Fe3O4) by X-ray diffraction (XRD). The diffractogram showed one single phase corresponding to an inverse spinel structure for both samples (Fig. S1). The nature of PAA in the organic coating of SPIO-PAA was confirmed by X-ray photoelectron spectroscopy (XPS) through the presence of aliphatic C−C and carboxylic O−C=O components in the spectrum (Fig. S2). On the other hand, the presence of PAA on USPIO-PAA was confirmed by infrared (IR) spectroscopy, as identified by the vibrational bands at 1633 cm−1 attributed to –CO2H, 1551 cm−1 for –CO2−, and 558 cm−1 for Fe−O (Fig. S3). The difference between SPIO-PAA and USPIO-PAA is related with the NP size and the organic matter content. On one hand, core size measured from transmission electron microscopy (TEM) images showed values more than double for SPIO-PAA compared to USPIO-PAA, 18 and 8.5 nm, respectively (Table 1 and Fig. S4). The same trend was observed with hydrodynamic diameter (Dh) measured by dynamic 4 ACS Paragon Plus Environment

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light scattering (DLS), where SPIO-PAA presented a size of 95 nm, more than twice to that of USPIO-PAA, 24 nm, the latter corresponding to ultrasmall NP size (Table 1).20 PAA content, calculated by thermogravimetric assay (TGA), was found to be 9.3 and 23 wt% for SPIO-PAA and USPIO-PAA, respectively (Table 1 and Fig. S5). As we showed recently,19 both Dh and the amount of organic coating of IONs play an important role in the colloidal stability of the nanoparticles and their interactions with biomolecules, both important features governing cell uptake. Regarding the magnetic properties, USPIO-PAA featured a saturation magnetization (Ms) of 65 emu g−1 and coercive forces (Hc) of 0.12 Oe by vibrating sample magnetometer (VSM) measurements (Fig. S6). As expected, these values are significantly below those of SPIO-PAA that featured Ms of 81 emu g−1 and Hc of 20 Oe, as a result of the larger core size. Table 1: TEM core size, Dh, organic coating calculated from TGA, and Ms, and Hc obtained by VSM a for SPIO-PAA and USPIO-PAA. Data for SPIO-PAA are from ref. 18.

Coating (wt%) Ms (emu g−1)

Core size (nm)

Dh (nm)

Hc (Oe)

SPIO-PAAa

18

95

9.3

81

20

USPIO-PAA

8.5

24

23

65

0.12

MRI performance of USPIO-PAA was then evaluated and compared to that exhibited by SPIOs by means of relaxivity (r), which represents the inverse of the amount of contrast agent needed to reduce water protons’ relaxation time by 1 s. Transverse r, r2, of USPIO-PAA was found to be 127 mM−1s−1 and longitudinal r, r1, 17.7 mM−1 s−1 at 1.4 T and 37 °C (Fig. S7), indicating USPIO-PAA as a three times more efficient contrast agent than the clinically approved SPIO Feridex, which has an r2 of 41 mM−1 s−1 under the same conditions.21 On the other hand, SPIO-PAA featured values of 19.3 and 202 mM−1 s−1 for r1 and r2, respectively. The higher r in comparison with USPIO-PAA is in agreement with the larger Ms as a consequence of the larger core size. However, the performance of IONs in cell labeling depends much more on their uptake than on their magnetic properties, since the latter are affected/modified by the cell internalization.22



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Glucosamine coating modification In general, USPIOs have been reported to be internalized through fluid-phase endocytosis, which results in a poor cell uptake.23 As an example, negligible amounts of commercial Ferumoxytol are internalized by MSCs in vitro even when incubated in the presence of protamine. To increase MSCs’ Ferumoxytol loading, the contrast agent has to be used in combination with protamine and heparine, known as the Ferumoxytol-heparin-protamine (HPF) nanocomplex methodology,24 and even then, MSCs only show an iron content of 2.12 ± 0.11 pg Fe/cell. Consequently, in an attempt to achieve receptor-mediated endocytosis, USPIO-PAA was functionalized with D-glucosamine (GlcN) by peptide coupling to yield USPIO-PAA-GlcN (Scheme 1, for experimental details, see Experimental Part 4.2 and Fig. S8).

Scheme 1: Synthesis route used to produce the three different IONs. After hydrothermal synthesis, SPIO-PAA was obtained. Its purification by centrifugation gave access to USPIO-PAA. Functionalization through peptide coupling of USPIO-PAA with GlcN molecules yielded USPIO-PAA-GlcN.

Glucose-coated NPs are known to be biocompatible and have been shown to present enhanced cellular uptake.25–27 The coupling reaction showed high reproducibility in terms of the amount of glucosamine attached to the NP between different batches, with an average of 143 µg GlcN/mg NP. Additionally, at basic pH, the ζ-potential of USPIO-PAA was −114 ± 14 mV, which after glucosamine modification shifted to −86 ± 7 mV (Fig. S9). While USPIO-PAA-GlcN reaches its isoelectric point at around pH 6.0 (−0.4 ± 0.1 mV), USPIO-PAA still presents a highly 6 ACS Paragon Plus Environment

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negative ζ-potential at that pH (−22.2 ± 0.8 mV), confirming the neutralization of a significant part of the carboxylic acid moieties with GlcN molecules. TEM images were acquired after glucosamine modification to ensure that no morphological changes took place after the functionalization procedure (Fig. S10), and core size did not show noticeable changes before and after the modification, confirming that the functionalization did not affect the integrity of the NPs. Dh as measured by DLS showed an increase from the original 24 nm to 40 nm after GlcN coupling, and did not show signs of NP aggregation. Due to the small amount of GlcN molecules attached per NP, the organic content by TGA did not show any significant changes between USPIO-PAA-GlcN and USPIO-PAA as expected.

In vitro studies In order to determine the behavior of SPIO-PAA, USPIO-PAA, and USPIO-PAAGlcN as labeling agents for cellular therapy, the NPs were evaluated in in vitro experiments with MSCs. The requirements for cell labeling are high labeling efficiency (i.e. uniformity in label distribution within different cells), high cellular dose that enables MRI detection, and absence of toxicological effects.13 Two sets of experiments were carried out with all synthesized NPs: SPIO-PAA, USPIO-PAA, and USPIO-PAA-GlcN. In the first set, the NPs were co-cultured with MSCs for a long incubation time (24 h) with a NP concentration of 100 µg mL−1. In the second one, the conditions were maintained, but polylysine (PLL) was used to promote particle uptake (1.5 µg mL−1; for details, see Experimental Part 4.3). PLL is a nonviral transfection agent employed to interact via electrostatic forces with the NP coating conferring a positive charge, and thus promoting cell adhesion and NP uptake.28 MSC labeling efficiency of SPIO-PAA, USPIO-PAA, and USPIO-PAA-GlcN was first qualitatively evaluated by Prussian blue (PB) staining (Fig. 1). In the absence of PLL, SPIO-PAA showed a very low labeling efficiency with inhomogeneous labeling within different cells (Fig. 1A) and USPIO-PAA (Fig. 1B) showed little to no internalization by MSCs. Surprisingly, USPIO-PAA-GlcN (Fig. 1C) showed a very similar behavior to USPIO-PAA with no significant uptake by MSCs even with the presence of glucosamine moieties. The inhomogeneous labeling of SPIO-PAA may be the 7 ACS Paragon Plus Environment


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consequence of NP aggregation in the protein-rich medium, resulting in an increase of the local NP concentration within certain cells due to NP sedimentation.29 Inhomogeneous cell labeling is undesirable for cell tracking due to the loss of detection of unlabeled cells and lack of reproducibility and control over NP uptake.13 On the contrary, for USPIO-PAA and USPIO-PAA-GlcN, the presence of nanoparticle aggregates was not observed in optical microscopy images. However, the affinity between MSCs and the negatively charged PAA-coated USPIOs was, as expected, very low. Moreover, receptor-mediated endocytosis was not promoted by glucosamine moieties on USPIO-PAA-GlcN in MSCs. We hypothesize that the combination of negative surface charge on USPIO-PAA-GlcN even after GlcN modification and protein corona formation30 in the protein-rich stem cell growth medium hinders the interaction of the glucose moieties with specific membrane transporters, such as GLUT1 and GLUT2, thus failing to promote cellular uptake. When PLL was employed, cells showed significant internalization of SPIO-PAA (Fig. 1D), but again with a lack of labeling homogeneity within the cells. Additionally, the presence of precipitated material indicates once more NP sedimentation. On the contrary, USPIO-PAA (Fig. 1E) remained stable with the addition of PLL, but no significant internalization was detected. However, when USPIO-PAA-GlcN was incubated in the presence of PLL, the labeling efficiency of MSCs was extremely high and MSCs were uniformly labeled (Fig. 1F). Hence, USPIOs modified with GlcN with the aid of PLL constitutes a feasible strategy for the labeling of MSCs with both high efficiency and homogeneity.


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Figure 1: PB staining of MSCs incubated with NPs at 100 µg Fe mL−1 over 24 h at 37 °C. NPs employed are A) SPIO-PAA; B) USPIO-PAA; C) USPIO-PAA-GlcN; D) SPIO-PAA and PLL; E) USPIO-PAA and PLL; F) USPIO-PAA-GlcN and PLL.

The different behavior of USPIO-PAA and USPIO-PAA-GlcN may be related with their different interaction modes with PLL. While USPIO-PAA interact with PLL only by electrostatic attractions that are highly dependent on the ionic strength (moderate ionic strengths such as cell culture medium might disrupt these interactions),31 USPIO-PAA-GlcN may react with PLL through glycosylation (Scheme S1). The glycosylation of amino acids residues, such as lysine in PLL, has been proven to happen during incubation between PLL and glucose at 37 °C.32,33 This kind of reactivity could lead to the formation of covalent bonds between USPIO-PAA-GlcN and PLL, and subsequently the cell adhesion and cell uptake will be favored by the positive charges of the PLL moieties. The interaction between the PLL and USPIOPAA-GlcN was tested after their incubation under the same conditions as for the labeling procedure of MSCs but in phosphate buffer to simplify the NP characterization. After incubation, the NPs were purified from unbounded PLL by centrifugation (for details see Experimental Part 4.4). The IR spectra of USPIO-PAAGlcN after incubation with PLL showed two new bands at 1384 and 1085 cm−1



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corresponding to the vibrational bands of the PLL (Fig. S11), which indicates the attachment of PLL to the NPs. The outstanding performance displayed by USPIO-PAA-GlcN in the PB staining images prompted us to probe them further in in vitro and in vivo studies. First, the quantification of Fe uptaken by MSCs was carried out to determine the cellular dose by inductively coupled plasma optical emission spectroscopy (ICP-OES) after cell digestion (for details, see Experimental Part 4.5). Fe quantification after the incubation of MSCs with USPIO-PAA-GlcN indicated an average of 56.5 pg Fe per cell (Fig. 2A). To the best of our knowledge, this is the highest Fe uptake achieved to date ex vivo with USPIOs in stem cells. USPIOs have been reported to show low uptake by non-phagocytic cells: recently Gangenahalli and coworkers34 showed values around 12 pg Fe/cell employing USPIOs and PLL. Eamegdool et al.35 synthesized USPIOs coated with methoxypoly(ethylene) and Rhodamine B, and achieved around 3 pg Fe/cell after 24 h incubation. Kiessling and coworkers36 reported USPIOs functionalized with Flavin moieties and showed 3.65 pg Fe/cell after 30 min incubation. The cell uptake of USPIO-PAA-GlcN is high even compared with SPIOs, which are known to present higher uptake than USPIOs.16 As a reference, Reddy et al.37 reported uptake values of 18 pg Fe/cell employing Resovist (contrast agent approved by Food and Drug Administration) and PLL. Fe content uptake by MSCs In order to foresee the in vivo detection window by MRI, we performed in vitro experiments over time to follow the decrease of internalized Fe as MSCs proliferate. Even though in vitro experiments do not represent an accurate scenario to extract in vivo conclusions due to an enhanced growth rate (with the associated underestimation in Fe loading), they can provide a first rough estimation of the timeframe available for in vivo imaging. The Fe content of a constant number of cells, 105 MSCs, was analyzed at different time points after the labeling procedure (for experimental details, see Experimental Part 4.5). At day 0 post-incubation, 56.5 pg Fe/cell were found in MSC cultures. From this value, an exponential decrease in Fe concentration was observed (Fig. 2A). The exponential decrease in Fe is consistent with the growth rate of MSCs (Fig. S12). Up to day 2, Fe decrease can be mainly attributed to an exponential cell growth. From day 2 on, Fe loss may come 10 ACS Paragon Plus Environment

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from a combination of MSCs proliferation plus NPs degradation/dissolution in endosomes/lysosomes.38 After 8 days in culture, MSCs were found to still contain around 3% of initial Fe, 1.63 pg Fe/cell, and after 12 days no differences in Fe content were found when compared to control cells. This result ensures the long-term detection of labeled MSCs by MRI. The same trend was observed in PB staining images (Fig. S13), where the cells were followed for 12 days at regular intervals. A clear labeling was observed up to 8 days post incubation. These results guarantee that the values from the ICP measurements come from internalized Fe rather than from extracellular NP aggregates. From day 5 on, an inhomogeneous cell labeling can be observed in the PB images. This is the result of the asymmetric cargo distribution after cell division as previously reported in several works.39,40 Compared to SPIO-PAA, the use of USPIO-PAA-GlcN delays the emergence of effects derived from the asymmetric cell division in vitro until day 5, which brings added value to USPIO-PAA-GlcN as MR cell tracking probes. To correlate the amount of Fe per cell with detectability by MRI, T2* values were calculated from MR phantom images of MSCs incubated with USPIO-PAA-GlcN. MR imaging results are in good agreement with the cellular Fe content measured by atomic emission spectroscopy. MSCs incubated with USPIO-PAA-GlcN showed an initial reduction in T2* of 57% after the labeling procedure, and even after 8 days in culture a 23% reduction in T2* was observed (Fig. 2B), following an exponential signal recovery until day 12 post-incubation. This suggests that USPIO-PAA-GlcN offer long-term detectability of MSCs by MRI, enabling longitudinal in vivo studies on the stem cell fate. Viability and cell proliferation The cell proliferation and viability of MSCs labeled with USPIO-PAA-GlcN were studied by means of cell count assays and lactate dehydrogenase assay (LDH), respectively, at different time points after the labeling procedure (Fig. 2C and 2D). No differences in viability were detected between the control and USPIO-PAA-GlcNlabeled cells at any time point investigated (Fig. 2D), confirming that these NPs do not affect the viability of the cells and therefore could be used as safe labeling agents for MSC tracking. A slight reduction in cell number is observed at day 5 after incubation with USPIO-PAA-GlcN when compared to the control (Fig. 2C). However, 11 ACS Paragon Plus Environment


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the number of cells does not statistically differ from the control, and before and after this point no diminishment of cell number was detected. This, together with the viability data, confirms the lack of toxicity of USPIO-PAA-GlcN.

Figure 2: A) ICP results for Fe quantification in MSCs after labeling procedure; B) signal reduction in T2* of MSCs labeled with USPIO-PAA-GlcN; C) cell count assay after incubation with USPIO-PAAGlcN; D) LDH viability assay after incubation with USPIO-PAA-GlcN.

In vivo MRI studies After the promising performance of USPIO-PAA-GlcN in in vitro experiments, cell tracking studies were conducted with labeled MSCs in order to evaluate their in vivo ability to reach the brain, and engraft and decrease the MR signal for sustained periods of time (for details, see Experimental Part 4.9). Many studies have reported good stem cell therapy results using the intra-arterial delivery route in experimental animal models of neurological diseases. The most common method of intra-arterial administration is the use of catheterization to guide the cells into the carotid artery, which enables the delivery of large numbers of cells directly to the brain.41 On the other hand, intravenous cell delivery has been also proven preclinically to be an 12 ACS Paragon Plus Environment

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effective route for cell therapy against neurological diseases, while being less invasive than intra-arterial administration. However, using intravenous route, a part of the administered cells accumulates in peripheral organs, leading to low cell concentrations in the brain.41 Thus, in order to validate USPIO-PAA-GlcN for cerebral cell tracking, the labeled cells were transplanted in healthy rats via intraarterial perfusion to deliver them in large numbers to the brain. In this study, labeled MSCs were administered at day 0 and MR imaging was performed right after cell administration (1 h) and after 1, 5, and 8 days. Dark signal voids were found over a wide area in the ipsilateral hemisphere (IH) (with respect to the carotid administration), with a prevalence for the area directly fed by the external carotid artery, while only a few dark spots were visible in the contralateral hemisphere (CH). T2-weighted images were also taken to discard ischemic formation, and no significant hyperintensity areas were observed, confirming that no severe damage of the brain had occurred. From the MR images, the reduction of T2*weighted signal in the IH was estimated over a broad area delimited in red boxes (Fig. S14) with respect to the CH. The values of MRI signal were calculated as [100 x (IH /CH)] and as an average of the MRI signal in 7 brain slices with labeled MSCs. Right after the cell administration, labeled MSCs were easily detected in every animal as strong hypointense signals corresponding to a 55% signal reduction in T2*weighted images (Fig. 3 and Fig. S14). At day 1, the strong dark signals were still clearly visible with a 32% of signal reduction in T2*-weighted images, predominantly in the IH, confirming the engraftment of MSCs mainly during the initial pass without systemic circulation.42 From day 1 to day 8, brain signal was partially recovered from an average signal reduction of 27% (day 2), to 16% (day 5) to 10% (day 8) in T2*-weighted images (Fig. S14), but well-defined hypointense spots were still visible even after 8 days of the administration. These results highlight the potential of USPIO-PAA-GlcN as an efficient and long-term labeling agent of MSCs in MRI. As opposed to the results obtained in vitro, where 8 days after the labeling the differences in signal intensity between the control and labeled cells were small, in vivo MR images showed that labeled cells can be easily followed in a wide area of the right hemisphere of the animals beyond the 8 days mark.



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Figure 3: Representative T2- and T2*-weighted MR images of a healthy rat injected intra-arterially with USPIO-PAA-GlcN-labeled MSCs at different time points at 9.4 T. Inset: red regions highlight the labeled cells in same slice of T2*-weighted images after injection and after 8 days.

3 Conclusions SPIOs (SPIO-PAA) and USPIOs (USPIO-PAA) coated with polyacrylic acid, as well as USPIOs functionalized with glucosamine (USPIO-PAA-GlcN), were studied as labeling agents for MSCs. Dramatic differences were found in the cell-labeling performance of these magnetic NPs. SPIO-PAA showed signs of aggregation in cell culture medium and as a result the uptake was localized and non-homogeneous within the cell population. On the other hand, USPIO-PAA showed good stability in vitro but no significant internalization was observed by MSCs. In combination with polylysine, USPIO-PAA-GlcN displayed biocompatibility, high cellular dose of 56.5 pg Fe/cell, and sensitive detection in both in vitro and in vivo experiments by MRI, showing promise as labeling agent for cell tracking applications in animal models of cerebral ischemia.

4 Experimental 4.1 IONs synthesis procedure SPIO-PAA

was

synthesized

using

our

previously

reported

methodology.18 USPIO-PAA was synthesized following

hydrothermal

a slightly modified

procedure. Briefly, 8 mmol of FeCl2•6H2O and 14 mmol of FeCl3•6H2O were 14 ACS Paragon Plus Environment

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dissolved in 10 mL of ultrapure water in a 40 mL poly(tetrafluoroethylene) (PTFE) vessel. Next, 15 mL of aq. 25−30% NH4OH and 0.4 mmol of poly(acrylic acid) sodium salt in 5 mL of water were added to the reaction mixture. The PTFE vessel was placed in a stainless steel autoclave and kept at 200 °C for 24 h under autogenous pressure. NPs were isolated by magnetic separation and redispersed in water by sonication (two times). The resultant NPs were centrifuged at 3000 rpm for 10 min, the supernatant was subjected to an additional centrifugation at 4000 rpm for 12 h, and the supernatant was kept to yield sample USPIO-PAA (1.5 g). 4.2 Glucose modification The quantification of free carboxylic acids in PAA coating of USPIO-PAA was performed with cyanine3 amine (Cy3-NH2) (Table S1). For the coupling with GlcN, USPIO-PAA was dispersed in water at a NP concentration of 18.6 mg mL−1, and GlcN

(2.8

mg,

1000

eq.

per

nanoparticle)

was

added.

Subsequently,

8-diazabicyclo[5.4.0]undec-7-ene (DBU, 2.5 µL) was added to ensure a basic pH during the reaction, and last, 100 µL of freshly prepared aq. EDC solution (5 mg mL−1) were added. The reaction was stirred overnight at room temperature, and then purification was carried out by centrifugation at 7500 rpm during 10 min with Amicon Ultra-0.5 Centrifugal Filter Devices to obtain USPIO-PAA-GlcN (11 mg mL−1, 500 µL). Three washing cycles were performed with water, one with aq. 0.1 M NaOH solution, and three more with water, to recover functionalized NPs. The supernatants of all centrifugal purifications were combined and analyzed by the o-phthalaldehyde (OPA) method43 to determine the quantity of unreacted GlcN. A calibration curve of GlcN was performed in triplicate at 12, 10, 8, 6, 4, 2 µg mL−1 (Fig. S8), and the fluorescence was measured at λexcitation = 340 nm and λemission = 450 nm. Three different batches of USPIO-PAA-GlcN were quantified in order to ensure the reproducibility of the coupling reaction (Fig. S8). 4.3 In vitro experiments MSC lines were cultured in IMDM (78%), fetal bovine serum (10%), horse serum (10%), penicillin-streptomycin (1%), and amphotericin-B (1%). Cell passage numbers between 19 and 25 were used.



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Cell were plated in 6 well-plates, 1.5 × 105 cells/plate and labeled with 100 µg mL−1 of IONs for 24 h (n = 6) following the protocol described elsewhere44 with slight modifications. Briefly, MSCs were incubated for 24 h in the presence of IONs (100 µg mL−1) or IONs (100 µg mL−1) premixed with PLL (1.5 µg mL−1, vigorously shaken at 1300 rpm for 1 h). After the cell incubation period, the medium was removed and cells were washed three times with 1.5 mL of phosphate buffered saline (PBS) (PBS without Mg2+ and Ca2+). After washing, the cells were left for 12 h in 1 mL of fresh label-free medium. Next, the cells were washed once with 1.5 mL of PBS, and 0.5 mL of EDTA-trypsin was added to detach the cells from the well. The detached cells were collected, and after a centrifugation (1000 rpm for 5 min) the supernatant was discarded and the cells were resuspended in fresh medium. Labeled and nonlabeled MSCs were plated sequentially to evaluate the possible cellular toxicity and labeling clearance. Characterization of MSCs after labeling was performed at different cellular points: 12 h, and 2, 5, 8 and 12 days after labeling. 4.4 Incubation between PLL and USPIO-PAA-GlcN The incubation between PLL (1.5 mg mL−1) and USPIO-PAA-GlcN (100 mg mL−1) was performed in phosphate buffer at 37 °C for 24 h. The purification of the NPs from unbound PLL was performed by centrifugation (30000 rpm for 30 min), and the pellet was redispersed in water and purified again by centrifugation. NPs from the pellet were dried and analyzed by IR spectroscopy. 4.5 ICP of labeled MSC A total of 1 x 105 cells were dissolved in 1 mL of 37% HCl solution and 4 mL of water was added. The Fe concentration was determined by ICP using an ICPE-9000 Multitype ICP Emission Spectrometer from Shimadzu. Measurements were performed in triplicate and the results are shown as the mean value with the standard deviation. 4.6 PB staining PB staining was performed to demonstrate the uptake of the IONs by the cells. Labeled cells were plated and after 8 h washed with PBS and incubated for 20 min with a mix of equal parts of aqueous solution of HCl (20%) and aqueous solution of 16 ACS Paragon Plus Environment

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potassium ferrocyanide trihydrate (10%). After incubation, cells were washed three times with PBS and images were taken using an inverted microscope. 4.7 LDH assay For assessing the viability of the cells after labeling, supernatants from the incubation between USPIO-PAA-GlcN and MSCs were collected, including a negative control of lysed cells. Cell viability was determined by means of LDH following the manufacturer’s protocol. In brief, supernatants were subjected to centrifugation at 1000 rpm for 5 min and further incubated with LDH reagents for 20 min. Next, the absorbance of the plate was read at 490 nm and the viability rate was calculated with respect to control and lysed values. 4.8 Cell count Total cell count was performed with Trypan blue staining and a Neubauer counting chamber. Samples were diluted 1:5 with PBS and 1:2 with Trypan blue. Cell count was performed using an inverted microscope. 4.9 In vivo experiments Experimental animals: all procedures involving the use of research animals were approved by the Research Committee of the University Clinical Hospital of Santiago de Compostela (Spain) and were performed according to the “Principles of Laboratory Animal Care” (NIH publication No. 86-23, revised 1985), as well as specific Spanish (RD 1201/2005 and RD 53/2013) and European Union (Directives 86/609/CEE, 2003/65/CE, 2010/63/EU) regulations. Male Wistar rats (Harlan Laboratories, Barcelona, Spain) weighing 290 ± 10 g were kept in a controlled environment at 22 ± 1 °C and 60 ± 5% humidity, with 12:12 h light: darkness cycles and were fed ad libitum with standard diet pellets and tap water. All surgical procedures and MRI studies were conducted under Sevofluorane anesthesia (3–4%) using a carrier 65:35 gas mixture of N2O: O2. Healthy animals (n = 3) were anesthetized and 2.5 × 105 USPIO-GlcN labeled MSCs suspended in 300 µL of PBS were administered into the brain intra-arterially (perfusion rate: 20 µL/min) through one of the external carotid and with the common carotid opened.



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4.10 MRI studies All MRI studies were conducted using a 9.4 T MR system with 440 mT/m gradients and a combination of a linear birdcage resonator (7 cm in diameter) for signal transmission and a 2 × 2 surface coil array for signal detection. A quadrature volume coil (7 cm in diameter) was also used in phantom studies. MRI post-processing was performed using ImageJ software. 4.10.1 Agar phantoms Agar phantoms were made following a previously described procedure44 with 1 x 105 cells per condition. T*2-weighted and T2-weighted images were acquired 0, 2, 5, 8 and 12 days after the cell plantation. T2*-weighted images were acquired using a multi-gradient echo (MGE) sequence with 4.44 ms echo time, 1.8 s repetition time, 16 echoes with 6.75 ms echo spacing, implemented with a flip angle (FA) of 30º, 14 slices, 2 averages, field of view (FOV) of 7.5 × 7.5 cm2, and a matrix size of 256 × 256. T2-weighted images were acquired using a multi-slice multi-spin-echo sequence (MSME) with 10.44 ms echo time, 3 s repetition time, 16 echoes with 10.4 ms echo spacing, implemented with a FA of 108º, 14 slices, 1 average, FOV of 7.5 × 7.5 cm2, and a matrix size of 256 × 256. 4.10.2 In vivo MRI In order to assess USPIO-GlcN labeled MSCs in vivo, animals were scanned following T2-weighted and T2*-weighted sequences in MRI to evaluate the presence and the distribution of injected cells at 1 h, and 1, 5 and 8 days after the injection. T*2-weighted images were acquired using a MGE sequence with a 2.9 ms echo time, 1.5 s repetition time, 16 echoes with 3.28 ms echo spacing, implemented with a FA of 30º, 14 slices, 2 averages and with a 19.2 × 19.2 mm2 FOV, a 192 × 192 image matrix (isotropic in-plane resolution of 100 µm/pixel × 100 µm/pixel). T2-weighted images were acquired using a MSME sequence with a 9 ms echo time, 3 s repetition time, 16 echoes with 9 ms echo spacing, implemented with a FA of 180º, 2 averages, 14 slices of 1 mm and 19.2 × 19.2 mm2 FOV, a 192 × 192 image matrix (isotropic in-plane resolution of 100 µm/pixel × 100 µm/pixel).

Acknowledgements 18 ACS Paragon Plus Environment

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This work was funded by POCTEP (Operational Program for Cross-border Cooperation Spain-Portugal) through ‘InveNNta’ project and co-financed by the ERDF (European Regional Development Fund) and by the P.O Norte CCDR-N/ON.2 program. It was also partially supported by grants from Instituto de Salud Carlos III (PI13/00292; PI14/01879), Spanish Research Network on Cerebrovascular Diseases RETICS-INVICTUS (RD12/0014), and Xunta de Galicia (Consellería Educación GRC2014/027), all co-financed by the European Union program ERDF, and “Promoting Active Ageing: Functional Nanostructures For Alzheimer’s Disease At Ultra-Early Stages” (PANA_686009) funded within the EU Horizon 2020 Program. JG acknowledges the financial support from the Marie Curie COFUND Program (NanoTRAINforGrowth). TS (CP12/03121) and FC (CP14/00154) are recipients of a research contract from Miguel Servet Program of Instituto de Salud Carlos III.

Supporting information Supporting Information is available with Material and Methods section and complementary figures, including XRD diffractogram, XPS and IR spectrum, TEM images, TGA and VSM data, and additional images of PB staining.

Abbreviations DBU: 8-diazabicyclo[5.4.0]undec-7-ene; Dh: hydrodynamic diameter; DLS: dynamic light scattering; CH: contralateral hemisphere; FA: flip angle; FOV: field of view; GlcN: D-glucosamine; HPF: Ferumoxytol-heparin-protamine; ICP-OES: inductively coupled plasma optical emission spectroscopy; IC: ipsilateral hemisphere; IMDM: Iscove's modified Dulbecco's medium; IR; infrared; ION: iron oxide nanoparticle; LDH: lactate dehydrogenase assay; MEG: multi gradient echo; MRI: magnetic resonance imaging; Ms: saturation magnetization; MSC: mesenchymal stem cell; MSME:

multi-slice

multi-spin-echo

sequence;

NP:

nanoparticle;

OPA:

o-

phthalaldehyde; PAA: poly(acrylic acid) ; PB: Prussian blue; PBS: phosphate saline buffer; PLL: poly-lysine; PTFE: poly(tetrafluoroethylene); r: relaxivity; SPIO: superparamagnetic iron oxide nanoparticle; TEM: transmission electron microscopy; TGA: thermogravimetric assay; USPIO: ultrasmall iron oxide nanoparticle; VSM: vibrating sample magnetometer; XRD: X-ray diffraction; XPS: X-ray photoelectron spectroscopy. 19 ACS Paragon Plus Environment


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Magnetite Nanoparticles for Stem Cell Labeling with High Efficiency

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