Development of Metal-Organic Framework-Based Dual Antibody Nanoparticles for the Highly Specific Capture and Gradual Release of Circulating Tumor Cells

Abstract

Circulating tumor cells (CTCs) have been well-established as promising biomarkers that can be leveraged to gauge the prognosis of patients with cancers and to guide patient treatment efforts. Although the scarcity of CTCs within peripheral circulation and the associated phenotypic changes that they exhibit owing to the epithelial-mesenchymal transition (EMT) process make the reliable isolation of these cells very challenging. Recently, several studies have discussed platforms capable of mediating the efficient and sensitive isolation of CTCs, but these approaches are nonetheless subject to certain limitations that preclude their clinical application. For example, these platforms are poorly-suited to minimizing damage in the context of cellular capture and release or the in vitro culture of captured cells for subsequent molecular analyses, which would better enable clinicians to select appropriate precision treatments on an individualized basis. In this study, we report the layer-by-layer assembly approach to synthesize a novel composite nanomaterial consisting of modified zirconium-based metal-organic-frameworks (MOFs) on the surface of magnetic beads with dual antibody surface modifications capable of capturing CTCs without being hampered by the state of cellular EMT process. Our analyses indicated that these dual antibody-modified nanomaterials exhibited greater capture efficiency than that observed for single antibody. Importantly, captured cells can be gradually released following capture and undergo subsequent in vitro proliferation following water molecule-induced MOF structural collapse. This release mechanism, which does not require operator intervention, may be effective as a means of minimizing damage and preserving cellular viability such that cells can be more reliably utilized for downstream molecular analyses and associated treatment planning. To further confirm the potential clinical applicability of the developed nanomaterial, it was successfully utilized for capturing CTCs from peripheral blood samples collected from cases diagnosed with gastrointestinal tumors.

Keywords: cell release; circulating tumor cells; isolation; layer-by-layer assembly method; metal organic frameworks.

FIGURE 1

(A) Nanoparticles with a core-shell structure were prepared via a layer-by-layer method. (B) Highly specific CTC capture approach using dual antibody-modified nanoparticles. (C) UIO-67 gradually collapses in aqueous solutions, thereby facilitating the gradual automated release of captured cells.

FIGURE 2

(A) Images of TEM for pure Fe₃O₄. (B) Images of TEM for Fe₃O₄@UIO-67. (C) Hydrodynamic diameter values for Fe₃O₄ and Fe₃O₄@UIO-67 preparations measured via DLS. (D) Zeta potential values for Fe₃O₄ and Fe₃O₄@UIO-67 preparations. (E) Fe₃O₄ and Fe₃O₄@UIO-67 FTIR spectra. (F) Representative MCF-7 cell images following capture using dual antibody-modified Fe₃O₄@UIO-67.

FIGURE 3

(A) Analysis of the nanoparticle modification status on MCF-7 cell capture efficiency. (B) Analysis of the effects of nanoparticle concentration on MCF-7 cell capture efficiency. (C) Analysis of the effects of time on MCF-7 cell capture efficiency.

FIGURE 4

(A) Representative confocal microscopy images of captured MCF-7 cells. (B) Representative confocal microscopy images of single captured MCF-7 cells.

FIGURE 5

Comparing the rates of capture efficiency when using anti-EpCAM-modified, anti-N-cadherin-modified, and dual antibody-modified nanoparticles to capture model target cell lines.

FIGURE 6

(A) Analysis of the capture efficiency for low numbers of MCF-7 cells in PBS or PBMC-containing solutions. (B) Analysis of the capture efficiency for low numbers of HeLa cells in PBS or PBMC-containing solutions.

FIGURE 7

(A) Brightfield and fluorescent images of HeLa cells following dual antibody-modified nanoparticle-mediated capture and subsequent culture for 24, 48, 72, or 96 h. (B) Absorbance (OD) at 450 nm for HeLa cell cultures following dual antibody-modified nanoparticle-mediated capture. (C) Fluorescent imaging of HeLa cell viability following nanoparticle-mediated capture. Scale bar: 100 μm.

FIGURE 8

(A) Representative fluorescent images of captured CTCs. (B) Quantification of the CTCs captured from the peripheral blood (5 ml) of patients with gastrointestinal tumors. Scale bar: 20 μm.