Multifunctional Rare-Earth Element Nanocrystals for Cell Labeling and

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Imaging and Diagnostics

Multifunctional Rare-Earth Element Nanocrystals for Cell Labeling and Multimodal Imaging Bianca Grunert, Jessica Saatz, Katrin Hoffmann, Franziska Appler, Dominik Lubjuhn, Norbert Jakubowski, Ute Resch-Genger, Franziska Emmerling, and Andreas Briel ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00495 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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ACS Biomaterials Science & Engineering

ACS Biomaterials Science and Engineering

Multifunctional Rare-Earth Element Nanocrystals for Cell Labeling and Multimodal Imaging Bianca Grunert1*, Jessica Saatz2, Katrin Hoffmann2, Franziska Appler1, Dominik Lubjuhn2, Norbert Jakubowski2, Ute Resch-Genger2, Franziska Emmerling2, Andreas Briel1 1 nanoPET Pharma GmbH,

Berlin, Germany -prüfung (BAM), Richard-Willstätter-Straße 11, 12489 Berlin, Germany

2 Bundesanstalt für Materialforschung und

*Address correspondence to [email protected]. [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] Abstract In this work, we describe a simple solvothermal route for the synthesis of Eu3+-doped gadolinium orthovanadate nanocrystals (Eu:GdVO4-PAA) functionalized with poly(acrylic)acid (PAA), that are applicable as cell labeling probes for multimodal cellular imaging. The Eu3+ doping of the vanadate matrix provides optical functionality, due to red photoluminescence after illumination with UV light. The Gd3+ ions of the nanocrystals reduce the T1 relaxation time of surrounding water protons, allowing these nanocrystals to act as a positive MRI contrast agent with a r1 relaxivity of 1.97 mM-1 s-1. Low background levels of Eu3+, Gd3+ and V5+ in biological systems make them an excellent label for elemental microscopy by Laser Ablation (LA)-ICP-MS. Synthesis resulted in polycrystalline nanocrystals with a hydrodynamic diameter of 55 nm and a crystal size of 36.7 nm, which were further characterized by X-ray diffraction (XRD), photoluminescence spectroscopy (PL) and transmission electron microscopy (TEM). The multifunctional nanocrystals were subsequently used for intracellular labeling of both human adipose-derived stem cells (MSCs) and A549 (adenocarcinomic human alveolar basal epithelial) cells. Keywords rare-earth element nanocrystals • multimodal imaging • cell labeling probe • LA-ICP-MS • MRI

Introduction In recent years, in the biomedical field, considerable attention has been paid to the development of multifunctional nanoparticles suitable for multimodal imaging.1,2,3,4 Nanoparticles are of special interest in this field because of their high payload of contrast-generating material as well as the easy integration of multiple properties within one system.5,6 Most popular are multimodal imaging probes combining MRI and optical imaging. Examples of multimodal probes based on T2 MRI contrast agents are superparamagnetic iron oxide nanoparticles (SPIOs) conjugated to quantum dots (QDs) or SPIOs conjugated to fluorescent organic or NIR (near infrared) dyes.7,8,53 Multimodal T1 contrast agents were obtained by functionalization of QDs with Gd3+-chelates or by generation of Gd2O3 nanoparticles having a dye-doped silica shell.7 At the molecular level, PAMAM (polyamidoamine) dendrimers were used as a polymeric matrix for contrast-generating ligands. Their well-defined size together with the large number of accessible functional groups allowed the successful conjugation of Gd3+-chelates and NIR fluorescent dyes.7,9 Up-conversion nanoparticles (UCNPs) of different host materials, such as a metalfluoride (e.g., NaYF4, CaF2), -oxide (Y2O3) or -phosphate (YPO4) are another class of multimodal imaging probes,10,11,12 Besides utilizing their up-conversion luminescence (UCL), they can simultaneously possess radioactivity and/or magnetic properties depending on their elemental composition (e.g. 18F for positron emission tomography (PET) and/or Gd3+ doping for magnetic resonance imaging (MRI)).13 Xu et al. also reported on functionalized UCNPs that are effective for phototherapy in cancer treatment and offer the capability for trimodal imaging (UCL, computed tomography (CT), MRI).14 Mesoporous silica materials, known as efficient drug delivery systems, have been additionally functionalized to provide magnetic and/or luminescence properties.15 1

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Rare-earth gadolinium orthovanadate (GdVO4) as a host matrix for lanthanides (Ln3+) also combines optical and magnetic properties for in vitro applications and in vivo MR imaging.1,16,17,18,19,20 Back in 1992, Zaguniennyi et al. studied Ln3+-doped gadolinium orthovanadates as suitable compounds for laser materials.47 Since then, these materials have attracted much attention due to their excellent photoluminescence, magnetic, and electrical properties.4,21 Important benefits of Ln:GdVO4 nanocrystals functionalized with poly(acrylic)acid (PAA) are their well-defined particle size, the increased local Gd3+ concentration in the region of interest (ROI) with MRI, the easy doping with luminescent Ln3+, their simple synthesis and the possibility of further bioconjugation via EDC/NHS chemistry.16,22 Specifically, doping of the GdVO4 matrix with Eu3+ yields an efficient and intense red emission due to the strong absorption of UV light by GdVO4, followed by an energy transfer from the vanadate anions to the Eu3+ ions.19 This makes GdVO4 particularly interesting from the optical point of view, since the excitation of the dopant through an energy transfer from the vanadate anions is much more efficient than the direct excitation of Eu3+, thereby providing a higher luminescence.16 Moreover, Ln3+ emitters can be spectroscopically well distinguished from other chromophores present in biological systems because of their sharp characteristic emission bands and long luminescent lifetimes. Correspondingly, Shen et al. suggested that Ln3+ nanoparticles could be a potential reinforcement for current organic dyes and QDs bioprobes.7,23 Since cells can actively incorporate the Eu:GdVO4-PAA nanocrystals through endocytosis, these nanocrystals are a promising reagent for cell labeling. Hereby, the main challenge is to efficiently incorporate the label into the cells such that the labeled cells can be imaged at high sensitivity for prolonged periods of time, without the labeling process affecting cell functionality.24 Cell labeling is of special interest for tracking and monitoring of stem cells because these cells in particular hold great promise for the treatment of multiple human diseases and disorders.25,26 Determining the efficacy of cell-based therapies in vivo is crucial and requires the combination of an extremely sensitive detection technique and a stable cell labeling agent having a relatively long lifetime.27 Contrast-enhanced MRI is believed to be the most effective and safest non-invasive technique to follow the fate of cells in vivo and to determine the efficacy of a selected stem cell therapy.25 It can be used to image cell delivery, homing and engraftment but suffers from limited sensitivity and resolution for imaging at the cellular level.28,29,30,31 Cell-tracking methods followed by bioimaging of tissue sections also plays an important role in cancer research for understanding and interpretation of diseases.32,33 It is well-known that non-targeted nanoparticles accumulate within malignant lesions due to the EPR (enhanced permeability and retention) effect.34 Tumour vessel leakiness enables the nanoparticles to accumulate within the tumor matrix.1,34,35 The application of multimodal Eu:GdVO4-PAA nanocrystals overcomes the low sensitivity of in vivo MRI by enabling in-depth investigations of labeled cells with in vitro confocal laser scanning microscopy (CLSM) and LA-ICP-MS measurements.25 In recent years, especially LA-ICP-MS has been established as a powerful technique for in vitro determination of metal distributions within biological systems, because it offers precise, spatially resolved (few µm) measurements at the trace and ultra-trace level.33,36,37,38 Spatial resolution, however, depends on the employed Laserablation instrument and the detection limits of the analyte. Currently, subcellular resolutions of up to 1 µm can be achieved by LA-ICP-TOF-MS in a multiparametric immuno-imaging study.39,40 Alternative imaging techniques, such as MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry), allow localization of proteins and peptides in biological samples at resolutions varying from 20-50 µm.41 Whereas MALDI-MS benefits from imaging of the spatial distribution of larger molecules, SIMS (secondary ion mass spectrometry) is characterized by its excellent sensitivity to determine the elemental distributions on solid sample surfaces at trace and ultra-trace levels as well.42 SIMS offers superior lateral resolution of 200 nm, but suffers from matrix effects.42 Compared to LA-ICP-MS, the sample throughput of SIMS is small since all components of the system are housed in an ultra-high vacuum chamber.43 SIMS and LA-ICP-MS are based on two different principles to generate analyte ions: SIMS works with simultaneous evaporation/atomization and ionization processes, while in the laser ablation process the evaporation/atomization step is separated in time and space from the ionization step. Therefore, in LA-ICP-MS the sampling and ionization processes can be optimized separately, thus significantly reducing matrix effects.43 With the intention of developing multifunctional nanoprobes for bioimaging applications, we focused on Eu0,10Gd0,90VO4PAA nanocrystals as cell-labeling probe for MRI, CLSM, and elemental microscopy using LA-ICP-MS. Poly(acrylic)acid (PAA) was applied to provide an electrostatic stabilization to the dispersed nanocrystals and to enable further bioconjugation. We have chosen two different cell lines to investigate cell labeling with multimodal imaging techniques. Human adiposederived stem cells (MSCs) were chosen to demonstrate the efficacy of the nanocrystals for stem cell labeling and A549 cells were used due to their repeated application in tumor research.

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Results and discussion Yield, Size, Zeta Potential, Lanthanide Content. Eu0.10Gd0.90VO4-PAA nanocrystals as cell labeling reagents were synthesized in one batch with yields of up to 85 % (final Eu3+ amount, determined by ICP-MS, related to the applied amount). The purified nanocrystals were present in aqueous solution at 10 mg mL-1. DLS measurements showed an average hydrodynamic diameter of 55±5 nm with a PDI of 0.1±0.01 and a zeta potential of -48 mV, indicating that the nanocrystals have a monodisperse distribution and do not aggregate in aqueous solution. FTIR spectra were recorded to give a proof on the presence of the functional shell of poly(acrylic) acid (PAA). The corresponding FTIR spectra are shown in the supporting information in Figure S1. The Eu3+-doping level was determined by inductively coupled plasma mass spectrometry (ICPMS) and resulted in an average Eu3+-content of 10.4 %±0.4. Eu:GdVO4-PAA nanocrystals were also synthesized with Eu3+ contents of 5.6%±0.2 and 15.1 %±0.6, resulting in consistent hydrodynamic diameters, PDIs and zeta potentials. The excellent size distribution with reproducible quality make them a promising reagent for bioimaging. Structural Analysis. XRD patterns (Figure 1) of the Eu:GdVO4-PAA nanocrystals revealed that the data matches that of bulk GdVO4 (ICSD#15607) and indicated that Eu:GdVO4-PAA crystallized in the tetragonal space group (I41). The average crystallite size (B) was calculated by using the Scherrer formula: B=(K·λ)/(L·cosθ), where L is the full width half maximum (FWHM) and θ the diffraction angle. The calculation was examined using a Scherrer constant of K=1 and an X-ray wavelength λ of 1.54056 Å, whereby the calculated value for the crystallite size resulted in 20.3 nm.

Figure 1: XRD patterns of dry Eu:GdVO4-PAA nanocrystals and standard data for tetragonal GdVO4 (ICSD#15607).

Morphology and crystal sizes were also investigated by high resolution transmission electron microscopy (HRTEM). Figure 2A-C depicts HRTEM images of the nanocrystals with increasing magnification and shows that these nanocrystals have a rough surface. In addition, Figure 2A and B indicate contrast variations of the nanocrystals. These variations could arise from overlying nanocrystals or rather their specific position on the TEM grid, which result in different contrast enhancements due to their tetragonal structure. The size measurements of 270 crystals were used to create a histogram of the frequency distribution of particle diameters (Figure 2D). The evaluation of all particle diameters resulted in a calculated medium diameter of 36.7±7.2 nm. Consequently, it was found that the actual crystallite size is moderately higher than determined by Scherrer evaluation. The discrepancy between Scherrer evaluation and TEM analysis can be explained by the fact that the nanocrystals are a composition of several crystallites resulting in a larger particle size. Crystallites are coherent diffraction domains in X-ray diffraction, whereas TEM analysis indicated the polycrystalline character of the nanocrystals. The polycrystalline character was confirmed by selected area electron diffraction (SAED), which resulted in diffraction patterns of concentric rings (see supporting information Figure S2).

Figure 2: HRTEM images of Eu:GdVO4-PAA nanocrystals with increasing magnification (from A to C) and histogram (D) of the frequency distribution after measurement of 270 particle diameters. Contrast variations in Figure 2A-C arise from overlying nanocrystals or rather their specific position on the TEM grid, which result in different contrast enhancements due to the tetragonal structure of the nanocrystals.

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Luminescent Properties. The photoluminescence properties of Eu:GdVO4-PAA nanocrystals are characterized by their Stokes or down-converted emission (Figure 3). Down-conversion (DC) occurs after absorption of UV photons by the VO43groups of the host matrix followed by a nonradiative energy transfer to the Eu3+ ions, resulting in the emission of lower energy photons from the excited Eu3+ ions. Figure 3A shows the excitation-emission contour plot of the Eu:GdVO4-PAA nanocrystals, which visualizes the optical absorption and emission features of the material. Intense excitation/emission regions can be observed, with the centers being located at excitation wavelengths λexc ≤ 350 nm and in emission regions between 580 and around 700 nm, respectively.

Figure 3: Photoluminescence properties of Eu:GdVO4-PAA nanocrystals suspended in water at a concentration of 1 mg mL-1.(A) Excitation-emission contour plot. (B) Emission spectrum (background corrected) with λexc = 330 nm.

The characteristic intense photoluminescence of Eu3+ resulting from the 5D07F2 transition appears as a strong doublet at 618 nm (Figure 3B). The weaker emissions bands at 593 nm and 698 nm can be assigned to the 5D07F1 and 5D07F4 transition, respectively. The band at 643 nm resulting from the 5D07F3 transition is very weak. 16,18,22,44,45,46 Biocompatibility. Gadolinium as the free ion (Gd3+) is highly toxic because it can compete with Ca2+ in biological systems. Especially for Ca2+-binding enzymes, the kinetics of the catalyzed biological process is often altered by the replacement of Ca2+ with Gd3+.48 The biocompatibility of Eu:GdVO4-PAA nanocrystals were tested with U937 cells via the MTT assay. The viabilities of the U937 cells were determined upon treatment with formulated nanocrystals in Gd3+ concentrations between 0.06 and 4.13 mM to investigate the nanocrystals’ biocompatibility and potential toxicity (Figure 4). For Gd3+ concentrations below 1.59 mM, negligible toxicity effects with viabilities > 90 % were detected. Gd3+ concentrations greater then 1.59 mM have an increasing impact on cell viability, resulting in lower viabilities of 75 % for Gd3+ concentrations > 4.13 mM. In summary, the results of the MTT assay reveal that the nanocrystals can be utilised as probes for tracking and labeling of cells in appropriate concentrations.

Figure 4: Cytotoxicity studies of Eu:GdVO4-PAA nanocrystals with U937 cells via MTT assay. Percentage of viability of cells was determined after 24 h incubation with the nanocrystals relative to the control with culture media (n=4). Results are presented as mean ± standard deviations.

MRI contrast of nanocrystal-labeled cells. Suitable cell phantoms for measurements with a preclinical PET/MRI were prepared in 0.2 mL PCR tubes. All applied concentrations refer to Gd3+ and correspond to 2.5 mM, 5 mM and 12.5 mM Gd3+ / 106 cells, respectively. Magnevist® (gadopentetate dimeglumine, 0.5 mmol/mL solution for injection, Bayer Vital) was used as reference to compare the contrast properties with a clinical, well-characterized imaging agent. Control samples were run with D-Mannitol instead of the testing compounds to exclude artifacts. Magnevist® is based on a low molecular-weight Gd-DTPA complex and is termed as extracellular contrast agent, since it distributes itself quickly from the intravascular space to the interstitial space after intravenous application.49 Particularly in cell culture, however, low molecular weight contrast agents are able to penetrate cells via micropinocytosis, whereas uptake of nanoparticles by cells primarily occurs via 4


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endocytosis.49,50 Depending on size and molecular structure, uptake of macromolecular substances is often favored compared to low-molecular-weight substances.51 Nevertheless, Magnevist® was chosen as reference to compare cell labeling with a clinically approved contrast agent that is commercially available since to date, no clinical nanoparticulate T1 contrast agent is available. Figure 5A and 5B show the color-coded T1-weighted images of A549 cells and MSCs after labeling with Eu:GdVO4-PAA nanocrystals and Magnevist®, respectively. The cell pellets are visible at the bottom of the tubes and appear brighter after labeling with nanocrystals compared to labeling with Magnevist®. The labeling of A549 cells with Magnevist® (Figure 5A) shows nearly no contrast enhancement, whereas labeling of MSCs (Figure 5B) reveals a slight contrast enhancement compared to the overlying agarose layer. As expected, the control cells show no improved contrast for both cell lines. The enhanced contrast of the cells labeled with the nanocrystals indicates a shortening of the T1 relaxation time due to the presence of the paramagnetic element gadolinium. In the case of the nanocrystals, even at the lowest Gd3+ concentration (2.5 mM) a sharp distinction between the cell pellets and the agarose layer is possible for both cell lines in contrast to Magnevist® at the same Gd3+ concentration. Direct comparison of both experiments (depicted in Figure 5) demonstrates a higher uptake of nanocrystals and Magnevist® in the case of the MSCs, since all cell pellets appear brighter than the pellets of A549 cells at the same Gd3+ concentration. Microscopic examination showed that MSCs are nearly 5-times larger than A549 cells, which has an impact on the ability of substance uptake and resulted in higher uptake of the nanocrystals by the MSCs.

Figure 5: In vitro T1-weighted MR images of the A549 cell pellets (A) and MSC pellets (B) labeled with Eu:GdVO4-PAA nanocrystals and Magnevist®, respectively.

MRI relaxation time. A series of nanocrystals differing in Gd3+ concentration from 0 to 1.5 mM were measured at 1 T with PET/MRI. Figure 6A shows the MR image of the nanocrystal series and demonstrates their contrast-enhancing properties with increasing Gd3+ concentration. Relaxation times (T1 and T2) were determined using a 0.94 T Bruker minispec to calculate the r1 and r2 relaxivity of the nanocrystals. The relationship between the reciprocal of relaxation time 1/T1 (s-1) and the applied Gd3+ concentration of the nanocrystals was evaluated in Figure 6B. The diagram illustrates a linear increase of 1/T1 (s-1) with increasing Gd3+ concentration. The slope of the graph corresponds directly to the r1 relaxivity with a value of 1.97 mM-1 s-1 at 0.94 T. The determination of the r2 relaxivity was analogously to the r1 relaxivity after measuring the T2 relaxation times and resulted in a value of r2=2,85 mM-1 s-1. The ratio of r2/r1 is 1.45 and indicates that the nanocrystals are applicable as positive MRI contrast agent. The relaxation times of the reference Magnevist® were also measured and resulted in a r1 relaxivity of 3.20 mM-1 s-1. The lower relaxivity of the nanocrystals compared to Magnevist® can be explained by their particulate structure, since the magnetic interactions with surrounding water protons are weaker when Gd3+ ions are on the inside of the nanocrystals. However, in contrast to Magnevist®, which rapidly extravasates through the vascular endothelium, the nanocrystals are expected to remain within the blood vessels for a prolonged period and to accumulate in tumor tissue due to the EPR-effect which results in better contrast properties.

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Figure 6: (A) T1-weighted images of Eu:GdVO4-PAA nanocrystals with Gd3+ concentrations between 0.1 and 1.5 mM measured at 1 T. (B) Determination of the r1 relaxivity of Eu:GdVO4-PAA nanocrystals at 0.94 T. The slope of the curve corresponds to a relaxivity value (r1) of 1.97 mM-1 s-1.

CSLM. Confocal laser scanning microscopy (CLSM) was performed after incubation of A549 cells and MSCs with the Eu:GdVO4-PAA nanocrystals at a concentration of 10 pmol Ln3+ per cell. CLSM imaging clearly demonstrates the uptake of the nanocrystals by both cell lines as exemplarily shown for A549 cells in Figure 7A-C and for MSCs in Figure 7D-F. Comparing both cell lines, the photoluminescence (PL) intensity recorded for MSCs was up to 40 % higher than that of A549 cells. This result is in accordance with the measurements of cell phantoms by PET/MRI. Here, a brighter contrast was observed after nanocrystal uptake by the MSCs, indicating a more pronounced uptake of the nanocrystals by MSCs than by A549 cells. The uptake of the nanocrystals as well as Magnevist® was also quantified after digestion of the labeled cells with concentrated nitric acid at 60 °C and analyzation of the lysates by ICP-MS. The determination of the Ln3+ contents confirmed the higher uptake of the nanocrystals by MSCs than by A549 cells and a greater uptake of the nanocrystals instead of Magnevist® by both cell lines. The supporting information shows in Figures S3 the corresponding graphics that illustrate the Ln3+ contents in the lysates as well as the relative uptake of the substances.

Figure 7: CLSM images of A549 cells (upper row) and MSCs (bottom row) labeled with Eu:GdVO4-PAA nanocrystals. Fluorescence signals (A and D) were recorded upon excitation with λexc = 355 nm; transmission images (B and E) and overlays (C and F) were taken using the 488 nm line of a multiline Ar+ excitation laser.

To confirm the uptake of Eu:GdVO4-PAA nanocrystals and to rule out artefacts caused by scattering from cells or cell components, spatially resolved PL spectra were recorded. Five regions of interests (ROIs) were selected both within and 6


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outside the cells (Figure 8B). Fluorescence signals were mainly detected in ROIs inside the cells (see ROI 1 to 4), and these cell-related PL spectra exhibited the typical red emission at about 612 nm, characteristic for the 5D07F2 transition of Eu3+, see Figure 8A. A second band at about 595 nm, which can be assigned to the 5D07F1 Eu3+ transition, was observed in regions with high accumulation of nanocrystals, especially in ROI 1. In regions outside the cells (ROI 5), almost no signals could be recorded, indicating an efficient uptake of nanocrystals by the cells.

Figure 8: Spatially resolved photoluminescence (PL) spectra of MSCs incubated with Eu:GdVO4-PAA nanocrystals recorded with λexc=355 nm (A), and the corresponding color-encoded ROIs in the CLSM image (B). ROI 5 (pink circle) is positioned in an area outside the cells and represents a blank measurement.

LA-ICP-MS. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was applied as an elemental microscope to detect metal ions at subcellular levels and to simultaneously measure several isotopes by ICP-MS (see Table 1). In general, image resolution by measurements with LA-ICP-MS is determined by the spot size and scan speed of the laser ablation system, which is limited by the concentration of the analyte. Since the lanthanides Gd3+ and Eu3+ and as well as the transition metal V5+ are administered in a nanoparticulate formulation, they are present in significant concentrations in single cells. This enabled us to use relatively low spot sizes of 6 µm (see supporting information Figure S4) and even 4 µm (see Figure 9) to generate the elemental distribution maps of labeled A549 and MSC single cells. Images of control samples; (treated only with D-mannitol instead of nanocrystals); are also shown in the supporting information in Figure S4. Control samples were measured at a spot size of 12 µm due to the low intensities of the lanthanides. As expected, the control samples showed only the 193Ir signal of the cell nuclei, whereas the other signals were negligibly small. The elemental distribution maps in Figure 9 confirm that the nanocrystals can be detected with favorable intensities in single cells and demonstrate that even with low spot sizes of 4 µm, intensities of 100,000 cps (counts per second) can be achieved for the isotope 158Gd. Measurement of the 193Ir signal enabled visualization of the nuclei with favorable S/N (signal/noise) ratios until a spot size of 6 µm.52 A clear differentiation of the nuclei from the rest of the cell was no longer visible at a spot size of 4 µm. Because of the spatial similarities between the 193Ir, 158Gd, 153Eu and 51V signals, it can be assumed that the nanocrystals were efficiently taken up by the cells and that none of the elements leached from the nanocrystals during the labeling procedure.

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Microscope Image (Spot Size)

193

158

Ir

Gd

153

Eu

51

V

A549 (4 µm)

MSCs (4 µm)

Figure 9: Optical microscope images (far left) and corresponding LA-ICP-MS images of Eu:GdVO4-PAA labeled A549 cells and MSCs at a resolution of 4 µm.

Conclusion In summary, we confirmed the potential of Eu:GdVO4-PAA nanocrystals as cell labeling probes for use in multimodal spectroscopy such as for MRI, CLSM and LA-ICP-MS. The solvothermal synthesis of Eu:GdVO4-PAA led to high yields of up to 85 % and resulted in nanocrystals having a size of 36.7 nm. The synthesized nanocrystals had a tetragonal structure and could be prepared reproducibly with doping levels of 5 %, 10 % and 15 % Eu3+, determined by ICP-MS. Toxicity testing via the MTT assay revealed a suitable biocompatibility. Fluorescence spectroscopy showed the characteristic red photoluminescence of Eu3+ at 618 nm after illumination with UV light at 330 nm. The A549 cells and MSCs showed similar behavior in the labeling procedure with the Eu0,10Gd0,90VO4-PAA nanocrystals, even though the selected cell lines are biologically very different. Various imaging techniques verified the efficient uptake of the nanocrystals by A549 cells and MSCs, making these nanocrystals a useful label in biomedical science and a powerful tool for studying the kinetics of nanoparticle cell uptake. Cell labeling with nanocrystals followed by MRI resulted in brighter images compared to the widespread contrast medium Magnevist®. The shortening of the T1 relaxation time is another advantage compared to SPIOs, which are usually used to provide negative MRI contrast. CLSM measurements revealed fluorescence signals of the red Eu3+ emission originating mainly from inside the cells and confirmed the effectiveness of cell labeling. LA-ICP-MS demonstrated the spatial similarities between the 158Gd-, 153Eu- and 51V-signals of the nanocrystals and the 193Ir-signal of the DNAintercalator and verified the effective labeling of both cell lines. In conclusion, our studies have shown the multimodal character of the nanocrystals making them a promising cell-labeling probe for long-term in vivo cell tracking. Corresponding in vitro measurements with LA-ICP-MS can usefully complete preclinical data after administration of labeled cells. We assume that the easy doping of the nanocrystals with different lanthanides in various concentrations will enable cell labelling with so-called “nanocrystal-bar-codes” that are distinguishable by quantification with LA-ICP-MS analytics.

Experimental Methods Materials. Gadolinium(III) nitrate hexahydrate (99.99 %), europium(III) nitrate pentahydrate (99.9 %), sodium orthovanadate (99.98 %), and poly(acrylic)acid (PAA) with Mw=1.800 g/mol) were purchased from Sigma-Aldrich. Ethylene glycol (99 %), ethanol (96 %), paraformaldehyde and agarose were purchased from Carl Roth. Dulbecco's modified eagle medium (DMEM), penicillin/streptomycin, fetal bovine serum (FBS), and Trypsin/ EDTA were purchased from PAN Biotech GmbH. Phosphate-buffered saline (D-PBS) and Ir-Intercalator were purchased from Biowest and Fluidigm, respectively. Nanocrystal Synthesis. Eu0.10Gd0.90VO4-PAA nanocrystals were prepared through a simple and optimized synthesis according to Nunez et al.16 Firstly, 25 mL of two starting solutions were prepared: a 0.08 mol L-1 solution of (Eu,Gd) (NO3)3

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in ethylene glycol and a 0.2 mol L-1 sodium orthovanadate (Na3VO4) solution containing 2 mg mL-1 poly(acrylic)acid (PAA) in a 3 : 2 ethylene glycol : water mixture. The PAA was directly dissolved in the Na3VO4 starting solution. Both solutions were heated to 80°C to facilitate dissolution. After cooling to room temperature, the solutions were mixed together in a sealed vessel under magnetic stirring. This mixture was aged for 2 h at 120°C. After cooling to room temperature, the reaction mixture was centrifuged so as to wash the nanocrystals twice with ethanol and twice with Milli-Q water followed by dialysis against Milli-Q water with 10 kDa MWCO. Characterization. The hydrodynamic diameter of the nanocrystals and their zeta potential were determined using a Zetasizer nano ZS (Malvern). Inductively coupled plasma mass spectrometry (ICP-MS, iCAP, Thermo Fisher Scientific) was used for quantitative analysis of the lanthanides. The shape and crystal sizes were investigated by high resolution transmission electron microscopy (HRTEM) using a FEI Tecnai G² 20 S-TWIN X-ray diffraction (XRD) measurements were performed on a D8 ADVANCE (Bruker AXS) using Cu-Kα radiation with a LYNXEYE XE-T detector. Diffraction data was recorded at 40 kV and 40 mA with a step size of 0.02°. Labeling of A549 cells and MSCs. Human lung cancer cells (A549) and human adipose-derived mesenchymal stem cells (MSCs) were cultivated in Dulbecco's Modified Eagle's medium (DMEM) containing 10 % fetal calf serum (FCS) and 1 % Penicillin/Streptomycin at 37°C and 5 % CO2 (standard conditions). Both cell lines were labeled with Eu0,10Gd0,90VO4-PAA nanocrystals that were previously formulated with D-Mannitol to ensure isoosmolar conditions (250-350 mosmol/kg) and adjusted to a pH of 7.4. Studies on the stability of formulated nanocrystals were performed over a period of 12 weeks as well as in DMEM culture medium and at different pH values. The evaluations are shown in the supporting information (see Figure S5-S7) and indicate stable nanocrystal formulations for the use as labeling reagent. The cell labeling was investigated with MR imaging, CSLM and LA-ICP-MS. For the preparation of MR phantoms, 1x106 cells were seeded in 75 cm2 cell culture flasks and adhered for 24 h under standard conditions. Subsequently, the culture medium was removed, and 10 mL fresh culture medium followed by 2 mL of formulated nanocrystals at different Gd3+ concentrations (2.5 mM, 5.0 mM und 12.5 mM) were added. After another 24 h of incubation, the cells were washed several times with D-PBS to remove excess nanocrystals. Subsequently, the cells were detached by trypsin/EDTA treatment followed by fixation with 4% paraformaldehyde. The fixed cells were transferred to PCR tubes to pellet them by centrifugation. The supernatants were discarded and the cell pellets were coated with 1.5 % agarose. For the investigation of intracellular labeling with CSLM and LA-ICP-MS, 5x105 cells were incubated with the nanocrystals in a concentration of 10 pmol Ln3+ per cell. The cells were seeded on 22x22 mm coverslips and adhered for 24 h, respectively. After enrichment of the cells, the culture medium was removed and 1 mL fresh culture medium was added followed by 0.5 mL of the formulated nanocrystals. Each cell line was incubated for 6 h under standard conditions. Subsequent to the labeling procedure, the cells were washed several times with D-PBS before fixing them with 4 % paraformaldehyde. For CSLM, the coverslips were treated with a few drops of mounting medium and placed upside down on microscope slides. For LA-ICP-MS measurements the cells were additionally treated with an Ir-Intercalator after washing and fixing of the cells. The Ir-intercalator binds to cellular nucleic acid and enables imaging of the nucleus with LA-ICP-MS.52 After incubation with the Ir-Intercalator for 45 min on a horizontal swivel, the coverslips were immersed in solutions with an increasing alcohol concentration without using any mounting media. Biocompatibility. Biocompatibility testing was performed via the MTT-Assay using U937 cells cultured in RPMI (Roswell Park Memorial Institute) medium containing 10 % fetal calf serum (FCS) and 1 % Penicillin/Streptomycin.54 U937 cells are commonly used for biocompatibility testing because they are one of the few human cell lines with monocytic properties. The MTT-Assay is based on the reduction of the yellow MTT salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by metabolically active cells to the water-insoluble blue-colored formazan product. Each test was run with both positive and negative controls. Culture medium and PBS without using any test substances were used as negative control. 2 % Triton destroys cellular membranes and is therefore toxic to cells; therefore, it was used as positive control. After 24 h of incubation of 4x104 U937 cells with the nanocrystals and the control samples, the cells were treated with 0.04 M HCl in isopropanol to dissolve the formazan. The absorption of the cell lysates was measured on a plate reader at 570 nm, which enabled the quantification of living cells. Excitation-emission mapping. Excitation-emission maps (EEM) of fluorescent nanocrystal samples dispersed in water were recorded with an FSP920 (Edinburgh Instruments Ltd.) spectrometer using standard 1-cm quartz cuvettes (Hellma GmbH & Co. KG). The excitation wavelength was varied systematically (∆λ= 5 nm) in the range of 300-410 nm. For each excitation wavelength setting, the emission spectrum was recorded in the range of 500-700 nm (∆λ= 1 nm). Photoluminescence measurements. Photoluminescence (PL) spectra were measured in the range of 500-700 nm with the same instrument as employed for EEM using an L-geometry for excitation and emission light paths. The spectral bandpass for measuring emission spectra was usually fixed to 1 nm. A 495 nm cut-off filter was used to suppress contributions from scattered excitation light.

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Confocal laser scanning microscopy (CSLM). Fluorescence microscopy was performed with a Fluoview 1000 (Olympus Deutschland GmbH). The excitation wavelength was 355 nm (Cobolt ZoukTM 355 nm DPSS laser (10 mW) for fluorescence imaging, or the 488 nm laser line of a multiline argon-ion laser (30 mW) for transmission images. A beam splitter 20/80 was used to reflect the excitation light, which was focused onto the sample through an Olympus UPLSAPO 60xW (NA:1.20) water immersion objective. The resulting fluorescence emission was re-collected with the same objective and focused onto the photomultipliers. The sampling speed was 200 µs/pixel. Fluorescence imaging was performed at room temperature using standard fluorescence imaging conditions. Magnetic resonance imaging (MRI). MR images were obtained using a T1-weighted sequence on a 1 T nanoScan PET/MRI device (Mediso Medical Imaging Systems) dedicated to small animal imaging. Relaxivity: Relaxivity measurements were performed on a Bruker minispec mq40 (0.94 T, 39°C) by preparing a series of dispersions of the nanocrystal at varying Gd3+ concentrations. Laser ablation(LA)-ICP-MS. Elemental microscopy images were performed with a commercial laser ablation system (New Wave Research-ESI 213 nm laser unit, New Wave Research, Inc.) coupled to an ICP sector field MS (Element XR, Thermo Fisher Scientific). The ICP-MS was synchronized with the laser ablation system in an external triggering mode. The fixed cells were ablated line by line with Helium as carrier gas at a flow rate of 1 L min-1. Argon was added at a flow rate of 0.8 L min-1 before the samples reached the ICP torch. The ICP-MS was tuned daily during ablation of a microscope glass slide and thin agarose films spiked with the analytes Eu, Gd and V were used as standard before and after each sample measurement to correct for the instrument drift.55 The oxide ratio (ThO/Th) was kept below 1 % for the maximum of ion intensity. Laser spot size and scan speed were typically set at 6 µm with 6 µm s-1 and 4 µm with 4 µm s-1 for analysis of exposed cells and 12 µm with 12 µm s-1 for control cells. The laser parameters were adjusted to ensure complete ablation of the cells; and the isotopes were selected with regard to high-percentage abundance and minimal interferences, (see Table 1).52 Table 1: Working conditions for LA-ICP-MS analysis

Laser system Laser Wavelength Fluence Repetition frequency Laser spot size Laser scan speed Ablation gas flow Ablation mode

Nd:YAG 213 nm 0.5 J cm−2 20 Hz 6, 4 µm 6, 4 µm s−1 1 L min−1 He scanning line by line

ICP-MS RF plasma source Plasma gas flow Auxiliary gas flow Sample gas flow: Scan type Measured elements

1350 W 16 L min−1 Ar 1.1 L min−1 Ar 0.8 L min−1 Ar E Scan 51 V, 153Eu, 158Gd, 193Ir

Data Treatment: Currently, no commercial software is available to evaluate analytical data from LA-ICP-MS measurements. The data output from the mass spectrometer was organized for every element in a single ASCII file for further evaluation with Origin. Acknowledgements

This publication is dedicated to Dr. Norbert Jakubowski for his 65th birthday, with the best wishes for a prosperous future Thank you for everything! This work was supported by the BMWi (Federal Ministry of Economic Affairs and Energy) funding programme MNPQtransfer. Supporting Information • Figure S10: FTIR spectra of Eu:GdVO4-PAA nanocrystals (red line), Eu:GdVO4 nanocrystals without poly(acrylic)acid (green line) and pure poly(acrylic)acid, PAA (black line). The absorption bands at 1.550 cm-1 are resulting from C=O

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• •

•

•

•

•

bond stretching vibrations of carbonyl groups and give a poof on the existence of the functional polymeric shell, which has been attached on the particle surface during the aging process. Figure S2: Selected area electron diffraction (SAED) on Eu:GdVO4-PAA nanocrystals with transmission electron microscopy (TEM). The diffraction pattern of concentric rings confirms the polycrystalline character of the nanocrystals. Figure S3: Investigation of the cellular uptake of formulated Eu:GdVO4-PAA nanocrystals and Magnevist® by A549 cells and MSCs depending on the Ln3+ content per cell. After incubation, the cells were thoroughly washed with PBS followed by digestion with concentrated nitric acid at 60 °C and analyzation of the lysates by ICP-MS. The solid lines show the Ln3+ contents of the lysates, while the dotted lines show the relative uptake of the substances. Determination of the Ln3+ contents indicate a higher uptake of the nanocrystals by MSCs than by A549 cells and both cell lines showed a higher uptake of the nanocrystals than of Magnevist® (two orders of magnitude). While the Ln3+ contents of the lysates increase in both cell lines and for both substances, the percentage of uptake decreases for concentrations greater than 5 pmol Ln3+ per cell. Instead of A549 cells, the MSCs showed at concentrations greater than 15 pmol Ln3+ per cell a renewed increase of the percentage of uptake. Figure S4: Optical microscope images (far left) and corresponding LA-ICP-MS measurements images of Eu:GdVO4PAA labeled A549 cells and MSCs at a resolution of 6 µm as well as controls (incubated with D-mannitol) at a resolution of 12 µm. Figure S5: Evaluation on the stability of nanocrystal formulations by measuring their hydrodynamic diameter over 12 weeks. Eu0,10Gd0,90VO4-PAA nanocrystals were formulated with D-Mannitol to isoosmolar conditions (288 mosmol/kg) and adjusted to a pH of 7,40 at 25 mM Gd3+. The initial hydrodynamic diameter of the autoclaved nanocrystal formulation was 59 nm. Figure S6: Investigation of the hydrodynamic diameter of formulated Eu0,10Gd0,90VO4-PAA nanocrystals after mixing them with 37°C tempered DMEM culture medium in a ratio of 1:10. The hydrodynamic diameter was observed over a period of 216 h at 37°C. Figure S7: Investigation of the zeta-potential of formulated Eu0,10Gd0,90VO4-PAA nanocrystals as well as of a mixture of formulated nanocrystals with DMEM culture media in a ratio of 1:10 after adjustment to different pH values from 4-8.

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Stärk, H.-J.; Wennrich, R. „A new approach for calibration of laser ablation inductively coupled plasma mass spectrometry using thin layers of spiked agarose gels as reference,“ Anal. Bioanal. Chem., 2011, 399, 2211-2217. (doi: 10.1007/s00216-010-4413-1)

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For Table of Contents Use Only Multifunctional Rare-Earth Nanocrystals for Cell Labeling and Multimodal Imaging Bianca Grunert1*, Jessica Saatz2, Katrin Hoffmann2, Franziska Appler1, Dominik Lubjuhn2, Norbert Jakubowski2, Ute Resch-Genger2, Franziska Emmerling2, Andreas Briel1

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TOC Graphic 49x28mm (300 x 300 DPI)


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XRD patterns of dried Eu:GdVO4-PAA nanocrystals and standard data for tetragonal GdVO4 (ICSD#15607). 273x209mm (299 x 299 DPI)



Cytotoxicity studies of Eu:GdVO4-PAA nanocrystals with U937 cells by MTT assay. Percentage of viability of cells was determined after 24 h of incubation with the nanocrystals relative to the control with culture media (n=4). Results are presented as mean ± standard deviations. 201x109mm (300 x 300 DPI)


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LA-ICP-MS measurements of Eu:GdVO4-PAA labeled A549 cells and MSCs in 4 µm resolution. 60x94mm (300 x 300 DPI)





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In vitro T1-weighted MR images of the pelleted A549 cells (left) and MSCs (right) labeled with Eu:GdVO4PAA nanocrystals and Magnevist®, respectively. 445x301mm (300 x 300 DPI)



(A) T1-weighted images of Eu:GdVO4-PAA nanocrystals with Gd3+-concentrations from 0.1 to 1.5 mM measured at 1 T. (B) Determination of the r1 relaxivity of Eu:GdVO4-PAA nanocrystals at 0.94 T. The slope of the curve corresponds to a relaxivity value (r1) of 1.97 mM-1 s-1. 314x292mm (300 x 300 DPI)


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CLSM images of A549 cells (upper row) and MSCs (bottom row) labeled with Eu:GdVO4-PAA. Fluorescence signals (A and D) were recorded upon excitation with λexc = 355 nm; transmission images (B and E) and overlays (C and F) were taken using the 488 nm line of a multiline Ar+ excitation laser. 214x142mm (299 x 299 DPI)



Spatially resolved photoluminescence spectra of MSCs incubated with Eu:GdVO4-PAA nanocrystals, recorded upon excitation with 355 nm (A), and the corresponding color-encoded ROIs in the CLSM image (B). ROI5 (pink) represents a blank measurement, which was placed outside of the cell. 288x201mm (300 x 300 DPI)


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Spatially resolved photoluminescence spectra of MSCs incubated with Eu:GdVO4-PAA nanocrystals, recorded upon excitation with 355 nm (A), and the corresponding color-encoded ROIs in the CLSM image (B). ROI5 (pink) represents a blank measurement, which was placed outside of the cell. 135x135mm (72 x 72 DPI)



Photoluminescence properties of Eu:GdVO4-PAA nanocrystals suspended in Milli-Q at 1 mg mL-1. 288x201mm (300 x 300 DPI)


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Photoluminescence properties of Eu:GdVO4-PAA nanocrystals suspended in Milli-Q at 1 mg mL-1. 288x201mm (300 x 300 DPI)



HRTEM image of Eu:GdVO4-PAA nanocrystals 295x295mm (96 x 96 DPI)


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HRTEM images of Eu:GdVO4-PAA nanocrystals 295x295mm (96 x 96 DPI)



HRTEM images of Eu:GdVO4-PAA nanocrystals 295x295mm (96 x 96 DPI)


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Figure 2_TEM D (Histogram) 34x33mm (300 x 300 DPI)


Multifunctional Rare-Earth Element Nanocrystals for Cell Labeling and

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