(7f) Nanoscale Roughness and Surface Charge of Functionalized Halloysite Nanotubes Control Selectin-Mediated Adhesion of Malignant and Non-Malignant Cells Under Flow

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:

1.    
Hughes AD, Mattison J,
Western LT, Powderly JD, Greene BT, King MR. Microtube
Device for Selectin-Mediated Capture of Viable Circulating Tumor Cells from
Blood. Clinical Chemistry. 2012 Feb 16;58:846–53.

2.     Mitchell
MJ, Chen CS, Ponmudi V, Hughes AD, King MR. E-selectin liposomal and
nanotube-targeted delivery of doxorubicin to circulating tumor cells. Journal of Controlled Release. 2012 Jun;160:609–17.

3.     Hughes
AD, King MR. Use of Naturally Occurring Halloysite
Nanotubes for Enhanced Capture of Flowing Cells. Langmuir. 2010 Jul 20;26(14):12155–64.

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.