Molecular extraction in single live cells by sneaking in and out magnetic nanomaterials

Abstract

Extraction of intracellular molecules is crucial to the study of cellular signal pathways. Disruption of the cellular membrane remains the established method to release intracellular contents, which inevitably terminates the time course of biological processes. Also, conventional laboratory extractions mostly use bulky materials that ignore the heterogeneity of each cell. In this work, we developed magnetized carbon nanotubes that can be sneaked into and out of cell bodies under a magnetic force. Using a testing model with overexpression of GFP, the nanotubes successfully transported the intracellular GFP out at the single-cell level. The confined nanoscale invasiveness did not change cell viability or proliferation. This study presents the proof of concept of a previously unidentified real-time and single-cell approach to investigate cellular biology, signal messengers, and therapeutic effects with nanomaterials.

Keywords: drug screening; real-time detection; single-cell method.

Fig. 1.

Molecular extraction by spearing cells. (A) An external magnetic field drives MCNTs toward a cell cultured on a polycarbonate filter. To indicate the molecular extraction, the cell is transfected for GFP overexpression beforehand. (B) MCNTs spear into the cell under magnetic force. (C) MCNTs spear through and out of the cell and extract GFP. GFP-carrying spears are collected in the pores of a polycarbonate filter. (D) GFP representing the intracellular signal molecules can be used for analysis of individual pores.

Fig. 2.

Surface modification and characterization of MCNTs. (A) Schematic illustration of surface modification of MCNTs: Ni-coated CNT array by e-beam evaporation of Ni on the aligned CNT array, and poly-l-tyrosine coating by electropolymerization. (B) CV recording of the electropolymerization of l-tyrosine on CNTs with CNTs and Ag/AgCl as the working and reference electrodes, respectively. (C) Deposition charge (Q) (by integration of each cycle of CV) vs. the cycles. (D) SEM image of Ni-coated CNTs. (E) TEM images of Ni-coated CNTs whose surfaces have modified by poly-l-tyrosine coating (red arrow). (E, Inset) Low-magnification image. (F) Magnetization measurement of Ni-coated CNTs. (G) The aqueous suspension of magnetized MCNTs.

Fig. 3.

MCNT response to magnetic force. (A) Force analysis of MCNTs in the magnetically guided spearing. The net pulling force (F) on the MCNTs is the summation of the magnetic force (F_mag) and the drag force (F_d) in liquid. In the equations that describe the forces, µ₀ is the magnetic permeability of free space, χ is the magnetic susceptibility of MCNTs, V is the volume of the nanotube, B is the magnetic field density, η is the viscosity of the liquid, r is the radius of the nanotube, v is the velocity of the MCNT in motion, and K′ is the shape factor. (B) Microscopy image of MCNTs aligning in the magnetic field. (C) Movement of MCNTs by magnetic force. Images were taken over 8 s; numbers in the upper left corner (1–8) index the time. The same MCNT is circled in red in each of the eight images to show its movement.

Fig. 4.

MCNTs speared into and out of a cell viewed by SEM in top and bottom views. Local membrane surfaces appearing in the two red boxes are magnifications. Dashed circles highlight MCNTs positioned across the cell membrane. (Scale bars: 1 µm.)

Fig. 5.

Extraction of intracellular GFP via MCNTs speared into cells. (A–C) Bright-field, dark-field, and overlapped images of GFP-transfected HEK293 cells on a polycarbonate filter, respectively. (D and E) Bright- and dark-field images of MCNTs speared through cells and collected in the pores of a polycarbonate filter, respectively; the appearance of green fluorescence on MCNTs indicates that intracellular GFP was carried out of the cells by the MCNTs when speared through cells.

Fig. 6.

Flow cytometry detection of cell viability and apoptosis in cells speared by MCNT. (A) Mag-only group with normal culture under magnetic field. (B) MCNT-incubation group with MCNTs but without magnetic field driving. (C) MCNT-spearing group with MCNTs speared out of cells by magnetic field driving. (D) Cells from the group in C but left in culture for 12 h after spearing. FL1, propidium iodide channel; FL3, Annexin V channel.

Fig. 7.

Cellular morphology. (Upper) Bright-field images of the three groups of cells cultured for 24 h after spearing. The black chunks appearing in the MCNT-incubation and MCNT-spearing groups are the solid debris from the sample preparation. (Lower) The morphology of the nucleus in dark field. All cells were fixed before propidium iodide (PI) staining. Images are 300 μm in width.