Engineering magnetic nanoparticles and their integration with microfluidics for cell isolation

https://doi.org/10.1016/j.jcis.2019.12.092Get rights and content

Abstract

Isolation of cancer cells, bacteria, and viruses from peripheral blood has important applications in cancer diagnosis, therapy monitoring, and drug development. Magnetic particles functionalized with antibodies that target receptors of cancer cells have been shown to isolate such entities using magnetic field gradients. Here, we report enhancement in capture efficiency and specificity by engineering magnetic nanoparticles and integrating them with microfluidics for the enumeration of tumor cells. Nanoparticles were made from iron oxide, coated with poly(ethylene glycol), and conjugated through avidin-biotin chemistry with antibody specifically against epithelial cell adhesion molecule (EpCAM). On exposure of targeted nanoparticles to tumor cells, specific uptake by EpCAM-expressing tumor cells (e.g., BxPC3, a pancreatic cancer cell) was observed, whereas there was negligible uptake by cells with low EpCAM expression (e.g., CCRF-CEM, a leukemia cell). Using an arrangement of magnets called a Halbach array, capture efficiency and specificity towards BxPC3 cells tagged with magnetic nanoparticles were enhanced, compared to conditions without the magnetic field gradient and/or without magnetic nanoparticles, either in buffer or in whole blood. These results illustrate that engineered magnetic nanoparticles and their integration with microfluidics have great potential for tumor cell enumeration and cancer prognosis.

Introduction

Magnetophoresis, a nondestructive method for collecting or separating magnetic particles, involves the motion of magnetic particles in a viscous medium under the influence of a magnetic field gradient [1]. The choice of magnetic particle, its surface functionalization, and the external field under which capture is performed are some of the critical factors in magnetophoresis [2]. Magnetic beads functionalized with targeting moieties are used in blood purification [3], removal of bacteria [4], [5] from body fluids, and in separation of cancer cells in batch [6], [7], [8] and continuous flow processes [9], [10], [11].

At the micro- (<1 μm) and nano-scale (<100 nm), various particle platforms have been explored to isolate and enrich biomarkers and cells [12], [13], [14]. Capture using particles at the micron scale [15] works efficiently in simple cell solutions as they rapidly separate due to the high magnetic moment of the microparticles, resulting in greater forces available for separation [16]. However magnetic microparticles are found to be less efficient in capture of cells under flow conditions [9], which has been attributed to poor binding capacity of microparticles for receptors on cells [17]. Furthermore, microparticles are often found to aggregate in biological fluids [18], [19], contributing to inefficient capture and recovery in those media. Commercial particles used for capture have also shown significant nonspecific binding [20], thereby affecting selectivity and capture efficiency.

In the ideal case of magnetophoretic capture of tumor cells under flow, one would use particles that are highly selective towards the tumor cells, with minimal interactions (surface binding or uptake) with other cells in the sample. Past studies of magnetophoretic capture of tumor cells have relied on commercial particles [7], [10] or particles that are coated with mono- and polysaccharides, all of which suffer from significant non-specific binding to cells [6], [8], potentially limiting specificity. To minimize non-specific interactions with non-targeted cells, here we use magnetic nanoparticles coated with a dense brush of poly(ethylene glycol) (PEG). PEG is a so-called “stealth” polymer that reduces protein binding to the nanoparticles and improves their colloidal stability even in whole blood [21], [22], [23]. To target the epithelial cell adhesion molecule (EpCAM), a commonly used diagnostic marker for cancer [24], we developed PEG coated magnetic nanoparticles that were functionalized with streptavidin, and then bound to biotinylated anti-EpCAM. The selectivity of these targeted particles to tumor cells was tested in a microfluidic capture system.

Microfluidic devices are often used to isolate and enumerate tumor cells from body fluids [25], [26]. They are designed to promote collisions between cells and antibody-functionalized walls (Fig. 1a) and/or features (e.g. pillars, nanoparticles) resulting in improved capture rates with minimal damage to cells [27], [28], [29], [30]. To improve throughput, sensitivity, and purity in capture of rare tumor cell populations from body fluids, various magnetophoresis assisted microfluidic capture platforms have been developed [31]. When combining microfluidics and magnetophoresis with targeted nanoparticles, the aim is to improve cross-stream migration of cells towards the antibody functionalized surfaces in the microfluidic device, improving contact between surface bound antibodies and their target epitopes on the cell surface. Here, we explore this approach by combining an antibody functionalized herringbone microfluidic capture device with a planar Halbach array and anti-EpCAM-targeted magnetic nanoparticles to capture EpCAM expressing cells from cell mixtures (Fig. 1b). With the magnetic field gradient generated by the Halbach array under the device, targeted magnetic nanoparticle-bound tumor cells can be forced onto the antibody-coated inner surfaces and captured. At high flow rates, the combined forces also allow for selective capture of tumor cells tagged with the particles, while the non-targeted cells are washed out due to the high flow rate.

Section snippets

Synthesis of magnetic nanoparticles via thermal decomposition synthesis

Magnetic nanoparticles were synthesized by the semi-batch thermal decomposition of iron oleate in the presence of molecular oxygen. The precursor iron oleate was synthesized by reacting iron acetylacetonate (>98% pure, Tokyo Chemical Industry, TCI America) and oleic acid (90% technical grade, Sigma-Aldrich) at 320 °C under Argon atmosphere [32]. Particle synthesis was performed as detailed in Unni et al. [33]. A mixture of iron oleate and octadecene (90% technical grade, Sigma-Aldrich) was

Preparation and characterization of anti-EpCAM conjugated magnetic nanoparticles

Magnetic nanoparticles, illustrated in Fig. 2a, were prepared in order to test their ability to enhance microfluidic device capture efficiency and specificity in cell mixtures under strong magnetic field gradients. The nanoparticles were synthesized via thermal decomposition [33], coated with polyethylene glycol [21], and further modified with streptavidin [41], to which biotinylated Anti-EpCAM antibody was attached as detailed in the methods section and in Section 1 of the Supporting

Conclusions

We developed targeted magnetic nanoparticles coated with polyethylene glycol and functionalized with biotinylated Anti-EpCAM. These particles exhibited high affinity towards EpCAM expressing tumor cells and negligible non-specific uptake by EpCAM negative cells. It was hypothesized these targeted magnetic nanoparticles would selectively bind EpCAM expressing pancreatic tumor cells from cell mixtures and, that in the presence of large magnetic field gradient they would facilitate enhanced tumor

Credit author statement

Mythreyi Unni, Jinling Zhang, Z. Hugh Fan and Carlos Rinaldi: Designed and conceptualized the experiments. Mythreyi Unni: Worked on development of the particle platform and understanding the interactions of particles with the cells. Jinling Zhang: Performed the microfluidic capture experiments and analysis. Mythreyi Unni and Carlos Rinaldi: Drafted the manuscript. Jinling Zhang, Z. Hugh Fan, Thomas J. George and Mark S. Segal: Contributed to review of the manuscript.

Declaration of Competing Interest

The authors declared that there is no conflict of interest.

Acknowledgements

We thank the University of Florida for providing financial support and infrastructure during the course of this research. This work was also partially supported by the National Institutes of Health, through awards K25CA149080 and RO1 AR068324. We thank Dr. Doty Andria for help performing flow cytometry at the ICBR. We are grateful to Dr. Sharon Matthews and Chao Chen, of the University of Florida College of Medicine Electron Microscopy Core Facility, for advice and technical assistance.

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