Magnetic layer-by-layer coated particles for efficient ...

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Magnetic layer-by-layer coated particles for efficient MRI of dendritic cells and mesenchymal stem cells Aim: Cell detection by MRI requires high doses of contrast agent for generating image contrast. Therefore, there is a constant need to develop improved systems that further increase sensitivity, and which could be used in clinical settings. In this study, we devised layer-by-layer particles and tested their potential for cell labeling. Materials & methods: The advantages of layer-by-layer technology were exploited to obtain magnetic particles of controllable size, surface chemistry and magnetic payload. Results: Flexibility in size and surface charge enabled efficient intracellular delivery of magnetic particles in mesenchymal stem cells and dendritic cells. Owing to the high magnetic payload of the particles, high MRI contrast was generated, even for very low cell numbers. Subcutaneous injection of the particles and subsequent uptake by dendritic cells enabled clear visualization of dendritic cells homing towards nearby lymph nodes in mice. Conclusion: The magnetic particles offer several possibilities as efficient cellular MRI contrast agents for direct in vitro or in vivo cell labeling. Original submitted 16 August 2012; Revised submitted 13 April 2013 KEYWORDS: cell labeling n dendritic cell n iron oxide nanoparticle n layer-by-layer n mesenchymal stem cell n MRI

In the biomedical field, the interest in iron oxide nanoparticles (IONPs) for various applications, including MR I, magnetic drug/gene delivery, magnetic cell separation and magnetic hyperthermia of cancer cells, is rapidly increasing [1–3]. For several applications, IONPs are currently used in clinical settings or are undergoing clinical trials [4,5]. With respect to MRI, special attention is paid to applying IONPs for noninvasive cell labeling and imaging, which enables efficient and long-term monitoring of stem cell migration or tumor development [6,7]. Although MRI ensures excellent soft-tissue contrast and allows a high spatial resolution [8], it suffers from an inherently low sensitivity and, therefore, requires the use of magnetic contrast agents to clearly delineate the cells of interest [8]. In order to detect low cell numbers and their migration, high cellular concentrations of contrast agents need to be accumulated. Single-cell detection by MRI using IONPlabeled cells has been achieved under specific conditions, using high magnetic field strengths or very high cellular uptake levels of IONPs [9,10]. However, current IONP formulations typically suffer from relatively poor cellular uptake levels, requiring the use of cationic transfection agents or modulation of the particles’ surfaces to facilitate their cellular uptake [11–14]. Although high cellular IONP levels have been described,

this has been associated with toxic side effects, and generally results in a very heterogeneous partitioning of IONPs among the total cell population [15–18]. Alternative strategies to enhance the magnetic contrast of the nanoprobes rely on the use of different types of particle (e.g., iron–platinum) with higher magnetic moments [19], or by facilitating intracellular clustering of the particles, resulting in localized magnetic susceptibility effects that are larger than that of single particles [20,21]. Another way to improve magnetic resonance (MR) contrast exploits the synergistic effects of magnetically-coupled domains, resulting in stronger magnetic susceptibility effects by making use of micron-sized iron oxide particles (MPIOs). Such microspheres are commercially available from Bangs Laboratories (IN, USA) and consist of one, or a few, large magnetite cores encapsulated by a poly-(styrenedivinylbenzene) matrix that can also contain fluorophores. Compared with smaller US FDAapproved dextran-coated IONP formulations (Endorem®; Guerbet, Villepinte, France) and Feridex® (Bayer HealthCare Pharmaceuticals, CA, USA), the MPIOs were found to have increased R 2 * relaxivities (longitudinal relaxation rate with influence of magnetic field inhomogenities) at equal iron contents [22,23].

doi:10.2217/NNM.13.88 © 2013 Future Medicine Ltd

Nanomedicine (Epub ahead of print)

Marie-Luce De Temmerman‡1, Stefaan J Soenen‡1,2, Nathalie Symens1, Bart Lucas1, Roosmarijn E Vandenbroucke3, Claude Libert3, Jo Demeester1, Stefaan C De Smedt1, Uwe Himmelreich4 & Joanna Rejman*1 Laboratory of General Biochemistry & Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium 2 Centre for Nano- & Bio-photonics, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium 3 Molecular Mouse Genetics Unit, Department for Molecular Biomedical Research, Vlaams Instituut voor Biotechnologie, Ghent University, Technologiepark 927, 9052 Ghent, Belgium 4 Biomedical MRI Unit/MoSAIC, Katholieke Universiteit Leuven Campus Gasthuisberg, Department of Imaging & Pathology, Herestraat 49, B3000 Leuven, Belgium *Author for correspondence: Tel.: +32 9264 8078 Fax: +32 9264 8189 j [email protected] 1

Authors contributed equally

‡

part of

ISSN 1743-5889

Research Article

De Temmerman, Soenen, Symens et al.

Table 1. r2 values of different particles suspended in agar. Particle

r2 value (s ·mM )

Bangs particle (Bangs Laboratories, IN, USA)

498 ± 53

-1

-1

LbL (positive) 100 nm 200 nm 500 nm

349 ± 43 388 ± 32 450 ± 58

LbL (negative) 100 nm 200 nm 500 nm

332 ± 32 377 ± 31 465 ± 45

The values given are the mean ± standard deviation. LbL: Layer-by-layer; r2: Longitudinal relaxivity.

These particles enabled single-cell detection of mouse hepatocytes [24,25], peripheral T cells [26], hematopoietic and mesenchymal stem cells (MSCs) [22], and low numbers of endogenous migrating neural progenitor cells in rats [27]. The larger MPIOs ensure higher magnetic contrast than IONPs at the same iron content. Moreover, they allow cell tracking over longer time periods, even if cells undergo cell division. This is possible because cells that contain a single MPIO can still be detected [24], whereas small particles are usually diluted below detection limits in the process of cell proliferation, as has been demonstrated by several groups [28,29].

One should keep in mind, however, that the frequently used Bangs particles have, thus far, mostly been used for labeling of cells with distinct phagocytic capacities since their size exceeds 800 nm. Moreover, the iron content per particle and the size of individual particles is highly variable, which makes homogeneous cell labeling and imaging difficult. In order to overcome these problems, the use of magnetic layer-by-layer (LbL)-coated particles has been proposed [30]. The technique allows the formulation of particles of controllable size, surface chemistry and iron content. It has been demonstrated by several groups that the close compartmentalization of IONPs in a physically restricted area results in a coupling of the magnetic domains of the individual particles. This results in an enhanced MR contrast compared with the same number of IONPs that are freely distributed and the magnetic moments of which are not interacting with each other [31]. In view of this, we propose that encapsulation of the magnetic particles in a confined space (the layers of the LbL capsules) will result in a strong MR contrast. The LbL technique is based on the adsorption of charged polymers on a surface [32,33]. Multilayers can be deposited on regular and irregular substrates ranging in size PSS PAH Iron oxide nanoparticle

Fluorescent polystyrene bead

LbL assembly

Further LbL assembly + functionalization

Figure 1. Functionalization of fluorescent polystyrene beads with iron oxide nanoparticles by means of the layer-by-layer technique. Electrostatic adsorption of polyelectrolyte layers onto the surface of the polystyrene bead was performed first. Iron oxide nanoparticles were incorporated inbetween the polyelectrolyte multilayers. The beads were further coated with additional layers of oppositely charged polymers. Six different formulations were obtained: positively charged ironcontaining beads of 100, 200 and 500 nm; and negatively charged iron-containing beads of 100, 200 and 500 nm. LbL: Layer-by-layer; PAH: Poly(allyl)amine hydrochloride; PSS: Polystyrene sulfonate.

doi:10.2217/NNM.13.88

Nanomedicine (Epub ahead of print)

future science group

Magnetic layer-by-layer coated particles for efficient MRI of DCs & MSCs

40 30 20

IONPs + PSS

PSS

PAH

PAH

PAH

PAH PSS

IONPs + PSS

-10

IONPs + PSS

PSS

0

PAH

10 PAH

Zeta potential (mV)

from nano- to micro-meters. The inherent characteristic of the LbL technique is its versatility and f lexibility. It allows a high level of control over the composition and thickness of the layers. In addition to the plethora of polymers, which can be used for layer deposition in LbL particles, almost any charged material including nanoparticles can be incorporated inbetween the layers [34]. In this study, we tailored fluorescent polystyrene beads of different sizes with LbL multilayers bearing IONPs. These particles were further evaluated for their labeling potential both in vitro and in vivo.

Research Article

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Materials & methods „„ Materials Polystyrene sulfonate (PSS), poly(allyl)amine hydrochloride (PAH) and sodium chloride were purchased from Sigma-Aldrich (Bornem, Belgium). FluoroSpheres ® red f luorescence (excitation maximum: 580 nm, emission maximum: 605 nm) of 100-, 200- and 500-nm average diameter were obtained from Invitrogen (Merelbeke, Belgium). Uncoated IONPs (8-nm nominal diameter as assessed by electron microscopy) used for the synthesis of magnetic LbL capsules were purchased from PlasmaChem (Berlin, Germany). Bangs particles, consisting of iron oxide cores encapsulated in a polystyrene/ polydivinylbenzene matrix with a nominal diameter of 0.86 µm were purchased from Bangs Laboratories. „„ Preparation of LbL-modified polystyrene beads Polystyrene beads were coated with the polyelectrolytes PAH and PSS by means of the LbL technique as described earlier [32,33]. Briefly, the polystyrene beads were incubated in a PAH solution (1 mg/ml) containing sodium chloride (0.5 M) for 10 min. After adsorption of the first PAH layer, the beads were centrifuged and washed with water to remove nonadsorped PAH. The beads were then dispersed in a PSS solution (1 mg/ml) in sodium chloride (0.5 M) for 10 min, followed by centrifugation and washing. This procedure was repeated with PAH. In the next step, 40 µl IONPs (the uncoated 8-nm diameter particles) were added, which was followed by the addition of PSS solution. After 10 min of incubation, particles were centrifuged and washed again. Finally, additional PAH and PSS layers were deposited as described earlier until the desired number of layers was reached. A total of four layers were deposited on the polystyrene future science group

-30 -40

100 nm

200 nm

500 nm

Figure 2. Layer-by-layer coating of polystyrene beads. Zeta-potential values for different coating steps of differently sized beads are shown. Data are expressed as mean of three independent measurements ± standard deviation. IONP: Iron oxide nanoparticle; PAH: Poly(allyl)amine hydrochloride; PSS: Polystyrene sulfonate.

templates, resulting in a total thickness that was identical for all LbL particles. The final size of the particles was, thus, dominated by the size of the original polystyrene beads, ranging from 100 to 500 nm. „„ Zeta-potential measurements The surface charge of the LbL-coated beads was determined by zeta-potential measurement using a Zetasizer® Nano series (Malvern Instruments, Malvern, UK). The beads were dispersed in distilled water. The reported zeta-potential values are the average of three consecutive measurements plus or minus the standard deviation. „„ Mice Female C57BL/6 mice were purchased from Janvier (Le Genest Saint Isle, France) and housed in a specified pathogen-free facility. All experiments were performed according to the regulations of Belgian law and the local ethical committee. „„ Murine bone marrow-derived dendritic cells Dendritic cells (DCs) were isolated from the bone marrow of C57Bl/6 mice. Mice were sacrificed and bone marrow was flushed out of the femur and tibia. After red blood cell lysis www.futuremedicine.com

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De Temmerman, Soenen, Symens et al.

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glutamine (Invitrogen), 10% heat-inactivated fetal bovine serum (HyClone), 10% horse serum (Invitrogen) and 100 U/ml penicillin/ streptomycin (Invitrogen). The cells were grown at 37°C in a humidified atmosphere containing 5% CO2. For experiments, MSCs were seeded at 50,000 cells/well in 24-well plates. „„ Particle uptake by DCs & MSCs

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Figure 3. Confocal images of mesenchymal stem cells that internalized beads containing iron oxide nanoparticles. Positively charged (A) 100-, (B) 200- or (C) 500-nm beads were incubated with mesenchymal stem cells for 3 h. After extensive washings, the images were acquired with a confocal microscope. (A,i, B,i & C,i) Transmission, (A,ii, B,ii & C,ii) red fluorescence and (A,iii, B,iii & C,iii) overlay. Fluorescence signals come from the beads (FluoroSpheres® red fluorescence [excitation maximum: 580 nm, emission maximum: 605 nm]; Invitrogen, Merelbeke, Belgium).

Flow cytometry was used to assess the uptake of coated polystyrene beads by DCs and MSCs. In the case of DCs, at day 8 of the culture, the cells were incubated with various particle formulations for 3 h, followed by vigorous washing of the cells with phosphate-buffered saline (PBS) to remove noninternalized particles. The cells were then incubated with 5% goat serum (Invitrogen) in PBS on ice for 30 min. DCs were identified by positive CD11c-APC antibody staining (BD Pharmingen, Erembodegem, Belgium) for 1 h on ice, followed by washing. MSCs were incubated with the modified beads for 3 h, which was followed by extensive washing with PBS to remove noninternalized particles. Finally, the DCs or MSCs were resuspended in stain buffer (BD Pharmingen) and analyzed with a FACSCalibur™ flow cytometer (BD Pharmingen). Untreated cells were used as controls. Data were analyzed using the CellQuest™ software (BD Biosciences). Confocal microscopy

(Pharm Lyse™, BD Biosciences, Erembodegem, Belgium), cells were seeded at a density of 2 × 105 cells/ml and incubated at 37°C in 5% CO2 . The cell culture medium was RPMI1640 (Invitrogen) supplemented with 5% fetal calf serum (HyClone™, Thermo Fisher Scientific, IL, USA), 1% penicillin/streptomycin (Invitrogen), 1% l-glutamine (Invitrogen), 50 µM b-mercaptoethanol (Invitrogen), 10 ng/ml IL-4 (Peprotech, NJ, USA) and 10 ng/ml granulocyte–macrophage colonystimulating factor (Peprotech). At days 2 and 6 of culture, the nonadherent cells were centrifuged, resuspended in fresh medium and seeded in the same flask. On day 7 of culture, the nonadherent cells were harvested, counted and plated at a density of 1 × 105 cells/ml in 24-well plates. „„ Mesenchymal stem cells MSCs were cultured in Iscove’s Modified Dulbecco’s Medium (BioWhittaker™, Thermo Fisher Scientific) supplemented with 2 mM doi:10.2217/NNM.13.88

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Confocal microscopy images of cells that internalized iron-containing LbL-coated particles were recorded using a Nikon C1si confocal laser scanning module attached to a motorized Nikon TE2000-E inverted microscope (Nikon, Brussels, Belgium). Samples were analyzed using a water immersion objective lens (Plan Apo 60X; Nikon). MSCs were plated in LabTek ® Chamber Slides™ (Sigma-Aldrich) and incubated with the particles for 3 h. Samples were extensively washed to remove noninternalized particles. Quantification of cellular iron content

Cellular iron content was quantified by a colorimetric method described previously [32]. Briefly, following incubation with the ironcontaining particles, the cells were vigorously washed to remove noninternalized particles and digested in acidic solution (H 2O:HNO3:HCL, 5:1:3) for 1 h at 37°C. Aliquots of all samples and standard iron solutions were transferred to future science group

Research Article

Magnetic layer-by-layer coated particles for efficient MRI of DCs & MSCs

Cells were seeded on MatTek ® chamber slides (MatTek Corporation, MA, USA) and incubated with the iron-containing particles for 3 h. After extensive washing with PBS, fresh culture medium was added. After overnight incubation, cells were fixed with 2% paraformaldehyde (Sigma-Aldrich) and incubated with Prussian blue reagent (SigmaAldrich; 6% HCl: 2% KFe 6CN in 1:1 ratio) for 30 min at 50°C, followed by washing.

Uptake by MSCs (%)

Prussian blue staining of intracellular iron

B

future science group

***

50 25

100 nm +

200 nm +

500 nm +

100 nm -

200 nm -

500 nm -

100 nm -

200 nm -

500 nm -

40 30

***

***

20 10 0

MRI detection of magnetic LbL particles

**

75

0

„„ MRI

100 nm +

200 nm +

500 nm +

Figure 4. Uptake of iron oxide-modified particles by dendritic cells and mesenchymal stem cells. (A) MSCs and (B) DCs were incubated for 3 h with different bead formulations. The fractions of cells that internalized the beads were assessed by flow cytometry. DCs were identified and gated based on positive CD11c staining. Graphs represent means ± standard deviation (n = 4). **p < 0.01. ***p < 0.001. DC: Dendritic cell; MSC: Mesenchymal stem cell.

Biospin).

100

Uptake by MSCs (%)

To assess the MRI contrast, all particles were diluted, to obtain the same iron concentration for every type of particle (100 µg/ml), mixed and solidified in small Eppendorf Tubes ® containing 1.5% agarose gel (Invitrogen). These Eppendorf Tubes were then placed in a larger agar block, and after solidification, the agar blocks were scanned using a Bruker BioSpec ® 9.4 T small animal MRI scanner (Bruker Biospin, Ettlingen, Germany; horizontal bore: 20 cm) equipped with actively shielded gradients (600 mT m-1). A quadrature radiofrequency resonator (transmit/receive; inner diameter: 7 cm; Bruker Biospin) was used. 2D multislice–multiecho experiments were acquired for the calculation of longitudinal relaxation time (T2) maps (repetition time [TR]: 3000 ms; 16 echo time [TE] increments of 10 ms; 256 matrix [256 pixel resolution]; 275 × 275 µm in-plane resolution; field of view: 70 × 70 mm; 0.35-mm slice thickness). For high iron concentrations (Table 1), experiments were repeated using multislice–multiecho experiments with lower resolution but shorter TE increments (32 TE increments of 6 ms; 128 matrix; in-plane resolution of 500 µm). 3D, high-resolution, longitudinal relaxation time with influence of magnetic field inhomogenities (T2*)-weighted MR images were acquired using a gradient echo sequence (fast low angle shot MRI; TR: 200 ms; TE: 15 ms; flip angle: 30°; isotropic resolution 75 × 75 × 75 µm). The temperature was maintained at 37°C during the acquisition of the MR images. Images were processed using ParaVision 5.1 (Bruker

***

A 100

Uptake by DCs (%)

a 96-well plate and Tiron solution (in sodium hydroxide) was added. After the addition of phosphate buffer, the absorbance was measured at 490 nm using an EnVision® plate reader (Perkin Elmer, MA, USA).

75

*** **

*

*

***

*

*** ***

*

**

50

25

0

100 nm + 200 nm + 500 nm + 3h

100 nm -

24 h

200 nm -

500 nm 48 h

Figure 5. Uptake of iron oxide-modified particles by mesenchymal stem cells – time course. MSCs were incubated for 3 h with different bead formulations. The fractions of cells that internalized the beads were assessed at different time points by flow cytometry. Graphs represent means ± standard deviation (n = 4). *p < 0.05. **p < 0.01. ***p < 0.001. MSC: Mesenchymal stem cell.

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De Temmerman, Soenen, Symens et al.

***

150

***

Fe/MSC (pg)

125 100 75 50 25 0 100 nm +

200 nm + 500 nm +

100 nm -

200 nm -

500 nm -

200 nm -

500 nm -

Fe/DC (pg)

100 ***

75 *** ***

50 25 0

100 nm +

200 nm + 500 nm +

100 nm -

Figure 6. Intracellular iron content. Cellular iron content in (A) MSCs and (B) DCs. The cells were incubated for 3 h with differently sized iron-containing beads (10 µg of iron). Graphs represent mean values of three independent experiments ± standard deviation. ***p < 0.001. DC: Dendritic cell; MSC: Mesenchymal stem cell.

MRI detection of magnetically-labeled MSCs & DCs

DCs (100,000 cells) and MSCs (50,000 cells) were incubated with iron oxide-functionalized beads (10 µg iron in 1 ml medium) for 3 h, followed by washing using PBS. Cells were fixed with 2% paraformaldehyde. Cell suspensions were prepared in PBS and aliquots were transferred to Eppendorf Tubes containing serial dilutions of 2000, 500, 200, 50, 20 or 10 cells/µl in 1.5% agarose gel. Embedding of cells in agarose was necessary to avoid sedimentation during the MRI experiments, which may subsequently result in misleading T2/T2* measurements. Unlabeled cells at a density of 2000 cells/µl served as a negative control. The Eppendorf Tubes were placed in a plastic cylinder that was filled with 1.5% agarose gel. Following solidification, the agar blocks were scanned using a Bruker Biospec 9.4 T small animal MRI scanner as described above. MRI experiments were the same as for the LbL particles. MRI of in situ-labeled DCs in mice

Particles were administered to C57BL/6 mice by subcutaneous injection of IONP-modified 500-nm LbL particles, in a total volume of 100 µl doi:10.2217/NNM.13.88

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(20 µg of iron/ml) of isotonic saline solution in the left limb. Bangs particles were used as control formulation. At 4 and 20 h postadministration, animals were sedated and scanned using a Bruker Biospec 9.4 T small animal MRI scanner. Acquisition parameters for the in vivo MRI were as following: mice were anesthetized using isofluorane (2.5% for induction and 1.5% in O2 for maintenance during scanning), and positioned on an animal bed with regulation of body temperature. Temperature and respiration were monitored and maintained during the acquisition at 37°C and 60–80 breaths/min, respectively. Respiration-triggered MR images were acquired in axial and coronal orientation using a rapid acquisition with refocused echoes (R ARE) sequence, consisting of 56 (axial) or 24 (coronal) continuous slices of 0.6-mm thickness in interlaced mode. Other parameters were: TR: 4500 ms; TE: 15.9 ms; in-plane resolution: 100 µm; two dummy scans and two averages. In addition, respirationtriggered T2 maps (multislice–multiecho sequence) were acquired similarly to the phantom studies (TR: 3000 ms; 16 TE increments of 10 ms; 256 matrix; 275 × 275 µm in-plane resolution; and 0.35-mm slice thickness). For radiofrequency irradiation and detection, a 7-cm quadrature resonator (Bruker Biospin) was used. Following the last MRI scan (20 h after particle injection), the animals were sacrificed and the draining lymph nodes of the injection spot were collected. As a negative control, the lymph nodes of the right hind limbs were isolated. The lymph nodes were transferred to an agar block and analyzed by ex vivo MRI similar to the agar phantom containing magnetically-labeled cells. Alternatively, lymph nodes were analyzed for total iron content (as described previously). „„ Statistical analysis The uptake of the particles by the DCs and MSCs is expressed as mean ± standard deviation. To determine whether data groups differed significantly, statistical analysis was performed using SPSS® software (IBM, Antwerp, Belgium). Differences between means were assessed by Student’s t-test, with p-values