Designing Magnetically Responsive Biohybrids Composed of Cord

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Designing magnetically responsive biohybrids composed of cord blood-derived natural killer cells and iron oxide nanoparticles Rachel A. Burga, Daud H. Khan, Nitin Agrawal, Catherine M. Bollard, and Rohan Fernandes Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00048 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Bioconjugate Chemistry

Designing magnetically responsive biohybrids composed of cord blood-derived natural killer cells and iron oxide nanoparticles

Rachel A. Burga†‡, Daud H. Khan§, Nitin Agrawal§, Catherine M. Bollard†‡°, Rohan Fernandes†‡* †

Institute for Biomedical Sciences, George Washington University, Washington, DC 20037,

United States ‡

George Washington Cancer Center, George Washington University, Washington, DC 20052,

United States §

Department of Bioengineering, George Mason University, Fairfax, VA 22030, United States

° Center for Cancer and Immunology Research, Children’s National Health System, Washington, DC 20010, United States 

Department of Medicine, George Washington University, Washington, DC 20037, United States

*Corresponding Author [email protected] 800 22nd St NW GW Cancer Center – 8th floor Washington, DC 20052 Phone: 202-994-0899

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract We report the generation of magnetically-responsive, cord blood-derived natural killer (NK) cells using iron oxide nanoparticles (IONPs). NK cells are a promising immune cell population for cancer cell therapy as they can target and lyse target tumor cells without prior education. However, NK cells cannot home to disease sites based on antigen recognition, instead relying primarily on external stimuli and chemotactic gradients for transport. Hence, we hypothesized that conjugating IONPs onto the surface of NK cells provides an added feature of magnetic homing to the NK cells, improving their therapeutic function. We describe a robust design for conjugating the IONPs onto the surface of NK cells, which maintains their intrinsic phenotype and function. The conferred magnetic-responsiveness is utilized to improve the cytolytic function of the NK cells for target cells in 2D and 3D models. These findings demonstrate the feasibility of improving NK cell homing and therapeutic efficacy with our NK:IONP “biohybrid.”

Table of Contents Graphic

Keywords: natural killer (NK) cells, iron oxide nanoparticles, nanoimmunotherapy, biohybrid, magnetic-responsiveness, homing, avidin-biotin


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Natural killer (NK) cells, as the main effector cells of innate immunity, are built to quickly and specifically recognize tumor cells, and subsequently elicit an immune response through their own cytolytic activity as well as interactions with antigen presenting cells and dendritic cells.1 It is this unique ability to lyse target cells rapidly and without prior education that renders NK cells a promising immune cell population for adoptive cell therapy. We, and others, have demonstrated that NK cells demonstrate significant efficacy as a therapeutic.2-5 However, one of the challenges faced by NK cell-based cancer therapeutics is that, unlike their T cell counterparts, NK cells do not home to disease sites based on antigen recognition; rather, they rely primarily on chemokine gradients as a mode of transport to disease sites.6 Specifically, NK cells migrate out of the bone marrow through sinusoids, and travel in circulation in response to external stimuli,7 a process that is largely dependent on the CXCR4/CXCL12 and CXCR3/CXCL10 axes.8-10 A robust NK cell infiltrate in tumors is associated with improved clinical responses and antitumor effects;11 however, solid tumors are known to utilize various immune evasion strategies to alter their chemotactic environment, and thus prevent NK cell infiltration.12 Additionally, recent efforts using ex vivo-expanded NK cells as a cancer therapeutic have identified that these cell populations have altered expression of chemokine receptors, which can negatively impact cellular migration. As a result, there is very limited understanding of the nuances of the migration of activated NK cells towards solid tumors, which would be critical to developing enhanced NK cell-based cancer therapies. In order to overcome these NK cell limitations, specifically in terms of homing and migration to a desired disease site, we propose a “nanoimmunotherapy” approach to enhancing NK cell therapy. We utilize NK cells isolated from umbilical cord blood, which represents a readily available donor source for generating large banks of “off-the-shelf” therapeutics. Our approach combines the advantages of both nanomedicine and immunotherapy to engineer a robust and persistent therapeutic that integrates strengths from both the nano- and immuno- strategies, while supplementing their individual limitations. We hypothesize that by conjugating iron oxide nanoparticles (IONPs) onto the surface of NK cells using established and robust bioconjugation techniques, we will generate a “biohybrid” (NK:IONP) that provides the added functionality of magnetic guidance to the NK cells (Figure 1A). In addition to the potent inherent cytolytic ability of the NK cells, these engineered NK:IONP biohybrids will be capable of rapid and localized



delivery to desired target tumor sites under magnetic guidance, effecting maximal NK cell-based tumor eradication. To realize the NK:IONP biohybrid, our bioconjugation scheme consists of first biotinylating the surface of the NK cells.13 The biotinylated NK cells are subsequently contacted with streptavidincoated IONPs to generate the NK:IONP biohybrid via robust streptavidin-biotin interactions, one of the strongest non-covalent interactions (Kd ~ 10-15 M).14-15 We have previously demonstrated this bioconjugation approach wherein antigen-specific T cells were coupled to ablative Prussian blue nanoparticles for improved combined cytolytic activity, and this immune cell-nanoparticle biohybrid demonstrated improved preclinical efficacy as a cancer therapy over either individual modality.16 In this study, we test our NK:IONP biohybrids in 2D and 3D models of neuroblastoma, a solid tumor which is known to evade immune detection and infiltration by generating an immunosuppressive microenvironment and altering immune cell phenotype, thus presenting a barrier to adoptive cell therapies.17-18 The combination of nanoparticles with different cell types (e.g. red blood cells, macrophages) in biohybrids has been explored in the literature.19 However the majority of these published reports focus on the use of the nanoparticles as imaging agents or for drug delivery.20-25 For instance, magnetic nanocomposites, frequently comprising superparamagnetic IONPs, cobalt ferrite kernel core-based structures, or other iron-based constituents, have readily been exploited as an important biomaterial for therapeutic and imaging applications.24 In the context of NK cells, there have been recent papers describing the uptake of IONPs - by the NK cell line NK-92MI for MR imaging26 and for magnetic targeting to tumors.27 Indeed, Jang et al. successfully intravasated silica-coated IONPs into immortalized NK cells, and were able to similarly demonstrate magnetic delivery of the cell line to subcutaneous tumors in response to an external magnet.28 Although rapid accumulation in response to external magnetization occurred, they were unable to demonstrate sustained accumulation or any therapeutic benefit in the long-term as compared to systemically injected non-magnetized NK cells. Our approach using primary NK cells derived from umbilical cord blood, as an “off-the-shelf” donor source, is distinct from these previous studies using commercial or immortalized cell lines.


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Umbilical cord blood units are an abundant source for generating primary NK cells, as over 500,000 validated banked units are available worldwide.29 Furthermore, reports propose that cord blood-derived NK cells can be more advantageous to other sources of primary NK cells and immortalized cell lines,30-33 as they represent a naïve population of NK cells that can be optimally selected for a killer-cell immunoglobulin-like receptor-mismatch between donors and recipients of the NK cells and is available “off the shelf” – an attribute that is critical for improved cytotoxicity and desirability of using this cell population as an agent for cancer therapy.34-35 A recently published study describes the loading of polydopamine-coated IONPs on peripheral blood mononuclear cell-derived NK cells. However, the study relies upon non-specific coincubation of IONPs with NK cells for twelve hours to allow for particle uptake and internalization into the NK cells.36 In contrast, we employed a robust and generalizable bioconjugation strategy, which enabled rapid loading (< one hour) of an optimized and quantifiable amount of IONPs specifically to the surface of primary NK cells. Additionally, we utilized a microfluidic platform to validate the magnetic-responsiveness of the NK:IONPs biohybrids under flow conditions.37 The NK:IONP biohybrid approach described here is customizable, and can be applied to equip any cell therapy with directed motion, and to develop cancer therapeutics that can be delivered directly and efficiently to target sites by simple external magnetic guidance.



Results and Discussion

Figure 1: (A) Schematic of the bioconjugation strategy to generate the NK cell-iron oxide nanoparticle biohybrid (NK:IONP). (B) Quantification of IONP binding to NK cells in the NK:IONP biohybrid using a reaction with Perls Prussian blue, where NK:IONP biohybrids (NP pellet) or supernatants from NK:IONP biohybrids after conjugation (supernatant) were reacted with ferric chloride in the presence of hydrochloric acid and potassium ferrocyanide (SI for details). Left: IONP concentrations bound (in the NP pellet) vs. unbound (in the supernatant) (on a per cell basis; pg/cell) for various amounts of added IONPs (pg/cell). Right: Ratios of bound vs. unbound IONP was used to determine optimal IONP:NK cell loading ratio. (C) Scanning electron microscopy images at different magnifications (1000×, 10 000×, 20 000×, 80 000× going from top left, top right, bottom left, bottom right, with individual scale bars indicative of 100 µm, 10 µm, 5 µm, and 2 µm, respectively) of unconjugated NK cells and the NK:IONP biohybrids (n=3 individual donor lines, error bars = standard error of the mean).


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Umbilical cord blood mononuclear cells were harvested from fresh cord blood units by density gradient separation, and NK cells were isolated and expanded ex vivo,30,

35

following repeat

stimulations with irradiated feeder cells and supplemental human IL-2 and IL-15 (SI for details).3839

To generate the biohybrid, the surface of the NK cells was biotinylated with sulfo-NHS-biotin

(1 mg/mL in PBS at 4°C on an orbital shaker) as previously described.13 The biotinylated NK cells were incubated with streptavidin-coated IONPs to form the NK:IONP biohybrid (Figure 1A). We determined the optimal IONP loading capacity by varying the amounts of streptavidin-coated IONPs added to the biotinylated NK cells (0-50 pg/cell; Figure 1B). The amount of bound versus unbound IONP after biohybrid formation was quantified using a reaction with Perl’s Prussian blue. Briefly, the NK:IONP biohybrids or supernatants from the biohybrids containing unbound IONPs, were reacted with ferric chloride in the presence of hydrochloric acid and potassium ferrocyanide. The absorbance at 680 nm was measured and the bound vs. unbound IONP concentration quantified using a standard curve (SI for details). Our quantification studies demonstrated that IONP binding was maximum at a concentration of 50 pg of streptavidin-coated IONPs per NK cell (henceforth designated as a 1× dose of IONP:NK cell). This reaction conditions yielded slightly greater than 2% IONP loaded per NK cell on a gram/gram basis (> 20 pg/cell; Figure 1B left panel) and greater than 50% labeling efficiency (> 1 Bound vs. Unbound ratio; Figure 1B right panel). Higher concentrations (100 pg/cell) yielded visible aggregation and were therefore not investigated further in this study (data not shown). The resultant NK:IONP biohybrid, which was visualized by SEM, exhibited a porous shell of IONPs on the NK cells (Figure 1C). Importantly, the IONPs in the biohybrid did not negatively impact the viability of cultured NK cells over time. The NK cells and the NK:IONP biohybrids both exhibited similar autonomous growth kinetics in the absence of feeder cells over time (p>0.90, Figure 2A). By utilizing fluorescent (AlexaFluor488-labeled) avidin-coated IONPs in the conjugation, we were able quantify the percentage of NK cells with IONPs attached to their surface using flow cytometry. To generate the fluorescent avidin-coated IONPs, the amount of fluorescent avidin added to the IONPs was varied (0 – 300 µg fluorescent avidin/mg IONP). Our studies employed the maximum avidin:IONP concentration for conjugation with biotinylated NK cells while preventing fluorescence saturation (SI for details). NK:IONP biohybrids (as well as single-component controls) were stained with CD56, a surface receptor highly expressed by NK cells, and conjugated



biohybrids were identified by dual positivity for both CD56 and Alexafluor488. This dual positive population and the gating strategy is demonstrated in Figure 2C. Using these conditions, we observed robust IONP:NK conjugation, with IONPs persisting on the surface of the NK cells for 7 days in culture, (Figure 2B, C). Thus, using a conjugation scheme reliant on the formation of a high affinity avidin-biotin complex, we were able to robustly generate a biohybrid composed of IONPs conjugated to the surface of umbilical cord blood-derived NK cells.

Figure 2: (A) Representative autonomous growth of NK cells and NK:IONP biohybrids for 14 days following in vitro expansion. (B) Aggregate results from flow cytometry indicating the persistence of IONPs (Alexafluor488+) on NK cells (CD56+) in the biohybrids over 7 days at 1× (50 pg/cell), 0.5× (25 pg/cell), and 0.1× (5 pg/cell) theoretical nanoparticle loading doses; n.s. = not significant (p>0.05). (C) Representative flow cytometry for determining the persistence of IONPs on NK cells; from left to right: unstained NK cells, stained NK cells (CD56+), fluorescent IONPs (Alexafuor488+), and NK:IONP biohybrid using CD56 stained NK cells and fluorescent IONPs at a 1× loading dose (n=3 individual donor lines, error bars = standard error of the mean).

We next examined the effect of the IONP conjugation on the phenotype and effector cell function of the NK cells in the NK:IONP biohybrid. Conjugation with IONPs at a dose of 50 pg/NK cell did not impact the viability of NK cells, nor did placement within a magnetic field for 10 min, as measured by acridine orange/propidium iodide live-dead staining (Figure 3A and SI). The presence of the IONPs in the biohybrid did not impair the phenotype of expanded NK cells, which expressed


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equal levels of activating receptors NKp44, NKG2D, NKp30, and CD69, and exhaustion marker PD1 regardless of whether they were coated with or without IONP (p>0.80, Figures 3B, C). Accordingly, we found similar dose-dependent cytotoxicity of NK cells and the NK:IONP biohybrid against nonspecific target cells (K562) and against neuroblastoma cells (SHSY5Y, Figure 3D). In this manner, we observed that NK cells in the NK:IONP biohybrid were able to maintain phenotype and function comparable with unconjugated NK cells.

Figure 3: (A) Viability based on acridine orange/propidium iodide live/dead counts of unconjugated NK cells, NK cells after 10 minutes of exposure to a magnetic field (5754 G), NK:IONP, and NK:IONP after 10 minutes of exposure to the same magnetic field. Flow cytometry demonstrated unaltered surface expression of NKp44, NKG2D, NKp30, CD69, and PD1 on unconjugated NK cells, and NK:IONP. Cells were gated as live based on their scatter profile and CD56+ expression: (B) aggregate results of mean fluorescence intensity (MFI) as well as (C) representative histograms are shown; (D) A flow cytometry-based cytotoxicity assay was used to assess the cytotoxicity of effectors (NK cells or NK:IONP biohybrids) against K562 (left) and SHSY5Y (right) target cells with varying effector to target (E:T) ratios (n=5 individual donor lines, error bars = standard error of the mean).



Paramount to the success of this biohybrid construct is its ability of the NK:IONP to respond to an externally applied magnetic field. To assess this, a simple transwell assay was used, in which NK:IONP biohybrids were placed atop a 5 µm pore insert submerged in media, and an external neodymium magnet (5754 G) was placed beneath designated wells for 15 minutes of magnetic field exposure. Following 15 minutes, the resultant cell flow-through in each well was quantified. When a magnetic field was applied, there were notably increased cell counts from the flow through of NK:IONP as compared to controls, which exhibited lower flow-through cell counts (Figure 4A). To examine the biohybrid migration in response to an externally applied magnetic field in the setting of fluid flow, we fabricated a microfluidic device mimicking tissue/tumor vasculature (top flow through channel diameter: 250 µm, connector channels: 15 µm; please refer to the SI for details of the device fabrication and rationale for the selected geometry and flow conditions utilized in our study). Fluorescently-labeled NK cells (unconjugated or in the NK:IONP biohybrid) were infused at a steady flow rate (0.1-1 µL/min) into the top chamber of the device, and the resultant migration was examined into the bottom chamber, in response to the placement of an external magnetic field (using a 5754 G neodymium magnet) perpendicular to the direction of fluid flow (Figure 4B). The flow was monitored for up to 30 minutes and no change was observed in the accumulation of unconjugated NK cells in the bottom chamber in response to the external magnet (Figure 4C, left panel). In contrast, in less than one minute’s time, we observed rapid migration and subsequent accumulation of NK:IONP biohybrids in the bottom chamber in response to the applied magnetic field (Figure 4C, right panel). Quantification of the percentage of cells in the top vs. bottom channels revealed increased accumulation of NK:IONP biohybrids as early as 20 seconds after application of the magnetic field (79% in bottom vs. 11% in bottom with unconjugated NKs, Figure 4D); this increased accumulation persisted over the study duration. Having established magnetic guidance of the NK:IONP as a viable method of targeted homing in a 2D setting with fluid flow, we sought to investigate this in a 3D tumor model. We established a solid tumor model of 3D neuroblastoma in agarose (by culturing SHSY5Y cells in 2% agarose gel), and visualized cell migration following the addition of a suspension of NK or NK:IONP to the right side of the tumor gel. After only 5 minutes of exposure to a 5754 G magnetic field placed laterally on the left side of the tumor gel to pull magnetically-responsive nanoparticles from right to left through the gel, fluorescent NK:IONP biohybrids (green) were seen infiltrating the tumor gel with fluorescent neuroblastoma cells (red) in the presence of the external magnetic field (Figure


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4E bottom) with little biohybrid infiltrate observed in the absence of the magnetic field (Figure 4E top). The biohybrid infiltrate was quantified and found to be significantly greater in the presence of a magnetic field, supporting the enhanced accumulation (Figure 4F). In summary, we demonstrated that the NK:IONP biohybrid was magnetically responsive, and capable of directed homing in a transwell assay, in a 2D model with fluid flow, and in a 3D tumor-mimetic model.

Figure 4: (A) Absolute cell counts of NK cells accumulated in the bottom well after unconjugated NK cells, or NK:IONP were placed into the top of a 5 µm transwell insert, with and without exposure to a magnetic field. (B) Schematic of our vasculature-mimetic microfluidic device – the section highlighted in red corresponds to the region zoomed in and depicted in the fluorescent snapshots in panel C, with “top” and “bottom” chambers labeled accordingly (C) Snapshots from a time course depicting migration of fluorescent NK cells (green) from the “top” chamber to the “bottom chamber”, entering at a constant flow; scale bar = 200 µm. (D) Quantified relative percentages of



fluorescently labeled cells or biohybrids in each chamber at each time point (0-60 s). (E) Representative fluorescence microscopy of NK:IONP (labeled with carboxyfluorescein succinimidyl ester, green) and SH5YSY neuroblastoma tumors in 2% agarose (labeled with Cell Trace Far Red, red) after 5 min of exposure to a magnet placed laterally on the left, and (F) quantification of NK:IONP infiltrate within the tumor gel in the absence or presence of the external magnet (n>2 individual donor lines, error bars = standard error of the mean).

The goal of engineering a NK cell-based biohybrid is to allow for improved targeting and consequently enhanced antitumor activity of NK cells. As a result of the increased accumulation observed in response to external guidance in both 2D and 3D tumor models, we next sought to examine if the increased accumulation led to improved cytolytic function of the magnetically guided NK:IONP biohybrid. Unconjugated NK cells and NK:IONP biohybrids (as well as IONP alone as a control) were co-incubated with fluorescently labeled neuroblastoma cells, which were grown overnight on the wells to ensure adherence. Immediately following the addition of effector components (NK, NK:IONP, or IONP), magnets (5754 G) were placed below the designated wells and all wells were washed to remove the non-adherent suspension. NK cells in the NK:IONP biohybrids remained in contact with the adherent neuroblastoma cells as a result of the applied external magnetic field whereas unconjugated NK cells were removed with the wash. This increased retention of NK cells in the NK:IONP + Magnet sample corresponded with increased tumor killing by the biohybrid after 4 hours of co-culture (p=0.001; Figure 5A). Similarly, we performed a cytotoxicity assay as described above using transwell inserts as a mechanism to gauge cytolytic activity following magnetic guidance through a barrier. Neuroblastoma cells were similarly cultured in wells, and NK cells, NK:IONP biohybrids, or IONP at varying doses, were added into the top transwell inserts. Magnets were placed below designated wells to establish migration through the transwell pores (under similar conditions to the previous study), and after 4 hours of co-culture we found that magnetically guided NK:IONP demonstrated increased migration and correspondingly increased cytotoxic killing of K562 target cells (Figure 5B). NK cell-mediated killing of target cells using a 1× IONP dose in the biohybrid (50 pg/cell) was significantly increased with the additional accumulation due to the external magnet (mean %killing was 33.2% with 1× NK:IONP without magnet vs. 58.3% with magnet; p=0.008). Similarly, when using the 0.5× IONP dose (25 pg/cell), there was a significant enhancement in target cell killing with the presence of an external magnet (%killing was 34.6% with 0.5× NK-IONP without magnet vs. 58.1% with magnet; p=0.044). Interestingly, the ten-fold lower dose of IONP (5 pg/cell) did not elicit any significant impact on target cell killing (%killing was 38.6% with 0.1× NK-IONP


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without magnet vs. 31.6% with magnet; p=0.417), which we hypothesize may be attributed to a sub-optimal IONP loading dose failing to achieve improved enhancement in the accumulation of NK cells in response to the magnet. We next sought to examine if these findings of increase NK cell cytotoxicity were replicable in the 3D setting. In the 3D tumor model of neuroblastoma in agarose described earlier, we observed that infiltration of the NK:IONP in response to the external magnetic field corresponded to significant increases in perforin and granzyme B in the tumor digest, two proteins indicative of cytolytic killing of neuroblastoma cells by NK cells, in NK:IONP biohybrid + magnet samples compared with controls (perforin p=0.031, granzyme B p=0.045; Figure 5C), as measured by qPCR (SI for details). Taken together, these results highlighted that not only was the NK:IONP capable of localization in response to an external magnet, but also that this increased homing lead to enhanced antitumor cytolytic activity of the NK:IONP biohybrid, as compared to unconjugated NK cells alone and other controls.

Figure 5: (A) The killing of fluorescently labeled neuroblastoma target cells after contact and co-culture with effector cells or particles alone (NK cells, IONP, or NK:IONP) with and without exposure to a magnetic field, as assessed by flow cytometry. “Max” refers to a positive control of target cells incubated with 5% Triton X-100 to simulate maximal cell death, and “Spontaneous” refers to a background control of target cells incubated alone in media, to account for any spontaneous target cell death occurring over the length of the assay. NK:IONP biohybrids were established at 1× (50 pg/cell), 0.5× (25 pg/cell), and 0.1× (5 pg/cell) nanoparticle loading doses. (B) The percent killing of fluorescently labeled neuroblastoma target cells after co-culture with effector cells (NK cells, IONP, or NK:IONP at 1×, 0.5×, and 0.1× nanoparticle doses) that were added into transwell inserts, with and without exposure to a magnetic field, as



assessed by flow cytometry. (C) Perforin and granzyme B expression levels as quantified by qPCR of tumor digested RNA, obtained after 3D neuroblastoma tumors were harvested following 5 hours of co-culture with NK cells or NK:IONP (with and without exposure to a magnetic field) (n=3 individual donor lines, error bars = standard error of the mean).

Prior reports coupling magnetic nanoparticles and immune cells have relied on the internalization of magnetic particles, and exploit these cell-nanoparticle composites for tracking, and mechanical manipulation, among other applications.28, 40-45 Our findings build upon these models; specifically, our bioconjugation strategy leverages IONPs to increase the homing of NK cells to a target site that would otherwise be limited. In addition, our unique conjugation strategy that focuses on cell surface attachment rather than nanoparticle internalization represents an opportunity to confer this added functionality without affecting the intrinsic composition of NK cells. Our findings represent a new strategy for generating a nanoimmunotherapy that augments NK cell homing to a target site, and consequently, improves its therapeutic efficacy. Conclusion We have demonstrated a robust and generalizable schema for generating an immune cellnanoparticle biohybrid that (1) can be directed to a given location based on placement of an external magnet, and (2) is absent of genetic modification and allows the primary immune cell to retain all vital effector cell characteristics. By conjugating IONPs on to the surface of primary umbilical cord blood-derived NK cells, we have generated a therapeutic, which is capable of rapidly homing in response to magnetic guidance and achieving improved antitumor efficacy compared to unmodified NK cells. Supporting Information Detailed experimental procedures can be found in the Supporting Information which is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.xxx. Acknowledgements This work was supported the George Washington University Cancer Center and the Center for Cancer and Immunology Research at the Children’s National Health System. The authors would like to gratefully acknowledge the Institute for Biomedical Sciences at The George Washington University, where RAB is a doctoral candidate. The authors would also like to acknowledge the


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George Washington University Nanofabrication and Imaging Center, who were core resources for performing the scanning electron microscopy. The authors have no competing interests to disclose. Abbreviations HLA – human leukocyte antigen IONP – iron oxide nanoparticle K562 – chronic myelogenous leukemia cell line MFI – mean fluorescence intensity NK cell – natural killer cell NK:IONP – the natural killer cell-iron oxide nanoparticle biohybrid RNA – ribonucleic acid SEM – scanning electron microscopy SHSY5Y – neuroblastoma cell line STR – short tandem repeat



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