Water-powered self-propelled magnetic nanobot for rapid and highly efficient capture of circulating tumor cells

Abstract

Nanosized robots with self-propelling and navigating capabilities have become an exciting field of research, attributable to their autonomous motion and specific biomolecular interaction ability for bio-analysis and diagnosis. Here, we report magnesium (Mg)-Fe₃O₄-based Magneto-Fluorescent Nanorobot ("MFN") that can self-propel in blood without any other additives and can selectively and rapidly isolate cancer cells. The nanobots viz; Mg-Fe₃O₄-GSH-G4-Cy5-Tf and Mg-Fe₃O₄-GSH-G4-Cy5-Ab have been designed and synthesized by simple surface modifications and conjugation chemistry to assemble multiple components viz; (i) EpCAM antibody/transferrin, (ii) cyanine 5 NHS (Cy5) dye, (iii) fourth generation (G4) dendrimers for multiple conjugation and (iv) glutathione (GSH) by chemical conjugation onto one side of Mg nanoparticle. The nanobots propelled efficiently not only in simulated biological media, but also in blood samples. With continuous motion upon exposure to water and the presence of Fe₃O₄ shell on Mg nanoparticle for magnetic guidance, the nanobot offers major improvements in sensitivity, efficiency and speed by greatly enhancing capture of cancer cells. The nanobots showed excellent cancer cell capture efficiency of almost 100% both in serum and whole blood, especially with MCF7 breast cancer cells.

Fig. 1. Schematic of the MFN fabrication and cancer cell capture strategy.

a Schematic illustration of the fabrication of Janus MFNs. b Schematic of the self-propelling MFN and HCT116 cell capture. Lower left side inset shows the upward moving nanobot due to the adhered O₂ bubble. Upper left side inset shows HCT116 cell captured by MFN and separated under magnetic field.

Fig. 2. Characterization study of MFN.

a TEM image of self-propelling Janus nanobot wherein the Mg nanoparticle forms the core covered by superparamagnetic shell of Fe₃O₄. The crystalline features and the identified lattice fringes co-related well to the structure of magnetite planes with a plane-to-plane separation of 0.249 nm (inset). b FTIR spectra of (i) Mg, (ii) Mg-Fe₃O₄, (iii) Mg-Fe₃O₄-GSH-G4 and (iv) Mg-Fe₃O₄-GSH-G4-Cy5, (v) Mg-Fe₃O₄-GSH-G4-Cy5-Tf, and (vi) Mg-Fe₃O₄-GSH-G4-Cy5-Ab. c Surface charge evolution upon conjugation of GSH-G4, Cy5, and Ab.

Fig. 3. Propulsion kinetics of MFN.

Analysis of upward and downward propulsion speed of the nanobot in: a PBS, b DMEM, c serum, d time-lapse images of nanobot in 0.5 M NaHCO₃ solution in PBS.

Fig. 4. Images of cancer cells captured by MFN from blood samples.

a HCT116 cell captured by MFN conjugated with Cy5. Cell visualized by nuclear staining with Hoechst reagent (blue) and Cy5 (green). b Schematic of the MFN conjugated with Tf/Ab. c Immunocytochemistry method based on Fluorescein Isothiocyanate-labeled anti-cytokeratin (green), and Hoechst (blue) nuclear staining was applied to identify and enumerate CTCs captured by MFNs. PE-labeled anti-CD45 (red) and Hoechst (blue) nuclear staining was used to identify leukocytes.

Fig. 5. Capture efficiencies of MFNs in the presence of NaHCO₃ compared to controls.

a Grouped plots of HCT 116 cell capture by the two MFNs under control conditions and in the presence of NaHCO₃, denoted by Δ. The MFNs are treated with four media, PBS, DMEM, Blood and Lysed Blood (simulated serum) as reported here. The MFNs are further exposed to incremental quantities of spiked HCT 116 cells in an identical manner across all treatments, as indicated by the addition bar (10, 25, 50, 75, 100 cells) depicted in the top left of the plot area. b Depicts a similar experimental outcome using MCF7 cells under identical conditions. Data is reported as mean ± SD cells with correlation of group data represented as R² above individual plots. c Linear regression treatment of the data shown in a and b; HCT116 cell capture trials displayed in the left panel depicts treatments with and without the added NaHCO₃ resulting in two grouped correlation with a very high degree of linearity (‘goodness of fit’, R² > 0.676). The panel for MCF7 cells (right) also depict a similar outcome with more pronounced linearity (R² > 0.929). The p values for statistical comparison were calculated as being less than 0.05 across the entire treatment panel for both cell groups.

Fig. 6. Fluorescent micrographs of CTCs captured from blood samples from a breast cancer patient.

Three-color immunocytochemistry method based on a FITC-labeled anti-Cytokeratin, and b Hoechst nuclear staining. c Composite image from FITC and Hoechst micrographs. d Plot of CTCs captured by MFN in 1 mL peripheral blood samples from epithelial cancer patients.