Nanoscale Roughness and Surface Charge of Functionalized Halloysite Nanotubes Control Selectin-Mediated Adhesion of Malignant and Non-Malignant Cells Under Flow

Nanoscale Roughness and Surface Charge of Functionalized Halloysite Nanotubes Control Selectin-Mediated Adhesion of Malignant and Non-Malignant Cells Under Flow

Michael J. Mitchell¹, Carlos A. Castellanos¹, Michael R. King¹

¹Department of Biomedical Engineering, Cornell University, Ithaca, New York USA

Introduction: The separation of circulating tumor cells (CTCs) in large numbers and at high purity levels from patient blood could lead to the development of effective personalized medicine regimens for those with metastatic cancer. Our lab has recently developed microscale flow devices containing nanostructured surfaces of halloysite nanotubes (HNTs) and recombinant human E-selectin (ES) to both isolate CTCs from patient blood and deliver targeted chemotherapeutics to cancer cells (1-3). Improvement of current CTC isolation purity levels is challenged by the fact that both CTCs and leukocytes possess ligands for ES (4). Herein, we summarize our study of the role of nanoscale roughness and surface charge of functionalized HNTs in differentially recruiting and capturing CTCs and leukocytes from flow.

Materials and Methods: HNTs were functionalized by incubation with sodium dodecanoate (NaL) or decyltrimethylammonium bromide (DTAB). Untreated, NaL- and DTAB-functionalized HNTs were immobilized within microscale flow devices, followed by incubation with ES. HNTs and immobilized HNT surfaces were characterized using dynamic light scattering, AFM, SEM, fluorescence microscopy, and contact angle goniometry.  Cancer cell lines (COLO 205 and MCF7) and human neutrophils isolated from peripheral blood were perfused through flow devices via a motorized syringe pump and exposed to physiologically relevant wall shear stresses. Micrographs and videos of adhesion phenomena were acquired to quantify cell rolling velocity, rolling flux, and the number of cells captured.

Results and Discussion: Adsorption of NaL surfactant into the HNT inner lumen increased the net negative HNT charge, which enhanced the electrostatic repulsion and thus the colloidal stability of the HNT solution.  NaL treatment altered the HNT average negative zeta potential from -25.1 to -103.4 mV (Fig. 1A), compared to the average negative zeta potential for untreated HNTs. Conversely, the HNT average negative zeta potential was reduced to -4.0 mV upon treatment with DTAB. The number of cancer cells captured from flow increased on surfaces coated with NaL-functionalized HNTs and ES (Fig. 1B). Interestingly, the number of leukocytes captured from flow decreased on identical surfaces (Fig. 1B). Cancer cell capture significantly increased on nanostructured surfaces of increasing net negative charge, while leukocyte capture significantly decreased (Fig. 1C).

Conclusions:  We have shown that surfaces of nanoscale roughness and altered surface charge significantly affect the differential adhesion of cancer cells and leukocytes to ES under flow. While both cancer cells and leukocytes possess adhesion receptors for ES, an increase in the net negative HNT charge acts to repel leukocytes from interaction with ES, while simultaneously enhancing cancer cell ES-mediated adhesion. The combination of both nanoscale roughness and HNT surface charge can serve to increase both the number and purity of CTCs isolated from patient blood, which can enable the development of effective personalized cancer therapies.

Acknowledgements: The work described was supported by the Cornell Center on the Microenvironment and Metastasis through Award Number U54CA143876 from the National Cancer Institute.

Figure 1. (A) Schematic of HNT functionalization and immobilization within a microscale flow device. (B) Reduced adhesion of leukocytes and enhanced adhesion of cancer cells from flow using a microscale flow device coated with NaL-functionalized HNTs and ES. Scale bar = 150 um. (C) Number of leukocytes and COLO 205 cancer cells captured from flow per 180,000 um² using functionalized HNT surfaces coated with ES. ***P<0.001. NS: not significant.

References:

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4.     Mitchell MJ, Castellanos CA, King MR. Nanostructured Surfaces to Target and Kill Circulating Tumor Cells While Repelling Leukocytes. Journal of Nanomaterials. 2012;2012(3):1–10.