Elsevier

Biomaterials

Volume 54, June 2015, Pages 158-167
Biomaterials

A DNA hybridization system for labeling of neural stem cells with SPIO nanoparticles for MRI monitoring post-transplantation

https://doi.org/10.1016/j.biomaterials.2015.03.017Get rights and content

Abstract

Neural stem cells (NSCs) demonstrate encouraging results in cell replacement therapy for neurodegenerative disorders and traumatic injury in the central nervous system. Monitor the survival and migration of transplanted cells would provide us important information concerning the performance and integration of the graft during the therapy time course. Magnetic resonance imaging (MRI) allow us to monitor the transplanted cells in a non-invasive way. The only requirement is to use an appropriate contrast agent to label the transplanted cells. Superparamagnetic iron oxide (SPIO) nanoparticles are one of the most commonly used contrast agent for MRI detection of transplanted cells. SPIO nanoparticles demonstrated to be suitable for labeling several types of cells including NSCs. However, the current methods for SPIO labeling are non-specific, depending mostly on electrostatic interactions, demanding relatively high SPIO concentration, and long incubation time, which can affect the viability of cells. In this study, we propose a specific and relatively fast method to label NSCs with SPIO nanoparticles via DNA hybridization. Two short single stranded DNAs (ssDNAs), oligo[dT]20 and oligo[dA]20 were conjugated with a lipid molecule and SPIO nanoparticle respectively. The labeling process comprises two simple steps; first the cells are modified to present oligo[dT]20 ssDNA on the cell surface, then the oligo[dA]20 ssDNA conjugated with SPIO nanoparticles are presented to the modified cells to allow the oligo[dT]20-oligo[dA]20 hybridization. The method showed to be non-toxic at concentrations up to 50 μg/mL oligo[dA]20-SPIO nanoparticles. Presence of SPIO nanoparticles at cell surface and cell cytoplasm was verified by transmission electron microscopy (TEM). SPIO labeling via DNA hybridization demonstrated to not interfere on NSCs proliferation, aggregates formation, and differentiation. NSCs labeled with SPIO nanoparticles via DNA hybridization system were successfully detected by MRI in vitro as well in vivo. Cells transplanted into the rat brain striatum could be detected by MRI scanning up to 1 month post-transplantation.

Introduction

Great effort has been made with cell replacement therapy due to the limited regenerative capacity of the central nervous system. Cell replacement therapy is a promising method for the treatment of degenerative disorders of the central nervous system, such as Parkinson's and Alzheimer's disease [1], [2], [3], [4], as well as traumatic brain injury and spinal cord injury [5], [6], [7].

Neural stem cells (NSCs) play an important role in cell replacement therapy and have been shown to be suitable candidates for the treatment of neurodegenerative disorders and central nervous system injury with their ability to survive, integrate, and differentiate within the host tissue [8], [9]. Although cell transplantation is a promising technique and has been extensively studied, monitoring the cells post-transplantation is a challenge. A non-invasive method to monitor transplanted cells and verify how the cells are integrating/migrating from the transplanted site, as well as study graft survival during the treatment time course, would provide crucial information for the advancement of cell replacement therapy.

Superparamagnetic iron oxide (SPIO) nanoparticles have been widely employed to label cells for magnetic resonance imaging (MRI) [10], [11], [12], [13], [14], [15]. In most studies, SPIO nanoparticles are co-incubated with cells in the presence of transfection agents to improve nanoparticle intake by the cells [11], [16], [17], [18]. The majority of the proposed methods are non-specific and rely on electrostatic interactions between cells and the nanoparticles. For example, Feridex®, an FDA approved contrast agent for MRI, is a dextran-coated SPIO nanoparticle usually co-incubated with cells in the presence of poly-L-lysine or protamine sulfate to improve the passive nanoparticle uptake by cells [18], [19], [20]. The non-specific characteristics of these methods demand relatively high SPIO nanoparticle concentrations and long incubation periods for proper labeling, which can interfere with cell viability [17], [21] and restrict clinical applications.

In our previous studies [22], [23], [24], we prepared a conjugate composed of a short single stranded DNA (oligo ssDNA) and specific sequence-poly(ethylene glycol)-phospholipid (ssDNA-PEG-lipids) and investigated its ability to specifically pattern cells to complementary ssDNA'-modified surfaces and induce rapid heterotypic aggregation between cells or immobilize various molecules on the cell surface via DNA hybridization. In the present study, the ssDNA-PEG-lipid was used to label NSCs with SPIO nanoparticles. Oligo[dT]20 ssDNA was conjugated with a lipid molecule, 1,2-dipalmitoryl-sn-glycero-3-phosphoethanolamine (DPPE), using poly ethylene glycol (PEG) as a linker (oligo[dT]20-PEG-DPPE). The oligo[dT]20-PEG-DPPE was anchored to the cell membrane through its hydrophobic DPPE tail, presenting oligo[dT]20 on the cell surface (surface-modified NSCs). The complementary ssDNA (oligo[dA]20) was conjugated to the maleic acid-coated SPIO nanoparticles (MA-SPIO), generating the modified SPIO nanoparticles (oligo[dA]20-SPIO). Cell labeling occurs via DNA hybridization by simply presenting the modified SPIO nanoparticles to the surface-modified NSC suspension.

Successful immobilization of SPIO nanoparticles was examined by transmission electron microscopy and the effect on cell phenotype assessed by immunostaining and RT-PCR. Labeled cells were examined by MRI in vitro and in vivo.

Section snippets

Neural stem cell isolation and culture

NSCs were isolated from the striatum of both standard SD-rat and transgenic enhanced green fluorescent protein (EGFP)-expressing SD-rat embryos on day 16 (E16). The striata of E16 embryos were isolated and dissociated into single cells with 0.05% trypsin in PBS. Dissociated cells were allowed to form aggregates and expand as free floating neurospheres for 3 days in base medium (DMEM/F12-GlutaMAX™ cell culture medium; Gibco, Life Technologies, New York, USA) supplemented with 2% (v/v) B27

Preparation of oligo[dA]20-SPIO nanoparticles and cytotoxicity assay

The peak of absorbance was determined as shown in Fig. 1B. Absorbance at 720 nm was plotted against known concentrations of Fe2+/Fe3+ ions to obtain the standard curve in Fig. 1C. The amount of MA-SPIO in a reaction mixture was determined using the standard curve before reaction with [dA]20-SH ssDNA.

Surface-modified NSCs were incubated for 30 min at 37 °C in the presence of increasing concentrations of oligo[dA]20-SPIO suspension. No significant decrease in viability was observed with oligo[dA]

Discussion

Labeling cells with a contrast agent for MRI has shown to be a reliable technique to monitor transplanted cells in a non–invasive manner. Recent reports have demonstrated that SPIO-labeled NSCs can survive and differentiate into neurons and glial cells after transplantation into the central nervous systems of rodents and monkeys [15], [26], [27]. In order to induce SPIO nanoparticle intake by cells, some sort of modification or coating with a biocompatible macromolecule is usually necessary [28]

Conclusions

The synthesized polymeric macromolecule, oligo[dT]20-PEG-DPPE, bound the cell surface and the oligo[dT]20 end exposed to interact (hybridize) with its counterpart, oligo[dA]20-SPIO. The DNA hybridization system provided specific binding sites on the cell surface for fast SPIO nanoparticle binding and uptake by the NSCs. Cultured labeled cells formed aggregates similar to non-labeled cells and exhibited the same phenotype, indicating that the method proposed in this work for cell labeling does

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Nanomedicine Molecular Science’’ (No. 2306) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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