Inhibition of tumor-cell invasion with chlorotoxin-bound superparamagnetic nanoparticles

Abstract

Nanoparticles have been investigated as drug delivery vehicles, contrast agents, and multifunctional devices for patient care. Current nanoparticle-based therapeutic strategies for cancer treatment are mainly based on delivery of chemotherapeutic agents to induce apoptosis or DNA/siRNA to regulate oncogene expression. Here, a nanoparticle system that demonstrates an alternative approach to the treatment of cancers through the inhibition of cell invasion, while serving as a magnetic resonance and optical imaging contrast agent, is presented. The nanoparticle comprises an iron oxide nanoparticle core conjugated with an amine-functionalized poly(ethylene glycol) silane and a small peptide, chlorotoxin (CTX), which enables the tumor cell-specific binding of the nanoparticle. It is shown that the nanoparticle exhibits substantially enhanced cellular uptake and an invasion inhibition rate of approximately 98% compared to unbound CTX ( approximately 45%). Significantly, the investigation from flow cytometry analysis, transmission electron microscopy, and fluorescent imaging reveals that the CTX-enabled nanoparticles deactivated the membrane-bound matrix metalloproteinase 2 (MMP-2) and induced increased internalization of lipid rafts that contain surface-expressed MMP-2 and volume-regulating ion channels through receptor-mediated endocytosis, leading to enhanced prohibitory effects. Since upregulation and activity of MMP-2 have been observed in tumors of neuroectodermal origin, and in cancers of the breast, colon, skin, lung, prostate, ovaries, and a host of others, this nanoparticle system can be potentially used for non-invasive diagnosis and treatment of a variety of cancer types.

Figure 1

Schematic representations of CTX-enabled nanoparticles (NPCs) inhibiting tumor cell invasion. (a) Surface chemistry of NPC conjugate. (b) NPC binding to lipid rafts of glioma cells containing MMP-2 and select ion channels. (c) NPC internalization and effect on cell morphology.

Figure 2

Viability of C6 glioma cells treated with NP, NPC, free CTX, or left untreated, as determined by percent reduction of Alamar blue.

Figure 3

Glioma cell binding and internalization of NPC in comparison with controls. (a) NPC uptake by C6 cells in comparison with nanoparticles with no CTX, quantified by iron content per cell. (b) C6 cells incubated with AF680 fluorescently-labeled NPC, NP or CTX and analyzed for total uptake by flow cytometry.

Figure 4

Intracellular localization of nanoparticles. C6 cells incubated with AF680 fluorescentlylabeled NPC, NP or CTX and analyzed by (a) Z-stacked 2-D projections and (b) 3-D reconstructions (DAPI nuclear stain in blue, WGA 594 membrane stain in green, and AF680 in red). (Scale bars in a, and b represent 10 μm).

Figure 5

TEM images showing increased membrane uptake subsequent to NPC binding. Scale bars represent 5 μm and 200 nm for whole cell (first row) and high magnification imaging (second row), respectively. White and black arrows identify NPC and endosomes, respectively.

Figure 6

Glioma cell invasion inhibition. (a) Functional inhibition of NPC, free CTX, and NP on MMP-2 in the presence of gelatin. Comparable inhibition by CTX and NPC indicates retention of catalytic activity of CTX bound to NPC. (b) Schematic of cell invasion assay. (c) Quantitative assessment matrigel cell invasion post-treatment. (d) Optical images of C6 cells (scale bar: 50 μm) and fluorescence images of EGFP-expressing C6 cells (scale bar: 20 μm) that have crossed through the pores of a matrigel invasion chamber, showing that NPC significantly limits cellular mobility compared to NP and CTX treatments.

Figure 7

Confocal fluorescence images highlighting (a) surface expression and (b) total expression of MMP-2 (green) by C6 cells (also, DAPI nuclear stain in blue, and WGA membrane stain in red). The scale bars represent 30 μm in a and b.

Figure 8

Confocal differential interference contrast (DIC) and confocal fluorescence imaging, showing the morphological changes of C6 cells exposed to NPC (scale bar: 20 μm).