Electric Field-Responsive Gold Nanoantennas for the Induction of a Locoregional Tumor pH Change Using Electrolytic Ablation Therapy

Electrolytic ablation (EA) is a burgeoning treatment for solid tumors, in which electrical energy catalyzes a chemical reaction to generate reactive species that can eradicate cancer cells. However, the application of this technique has been constrained owing to the limited spatial effectiveness and complexity of the electrode designs. Therefore, the incorporation of nanotechnology into EA is anticipated to be a significant improvement. Herein, we present a therapeutic approach based on difructose dianhydride IV-conjugated polyethylenimine-polyethylene glycol-modified gold nanorods as electric nanoantennas and nanoelectrocatalysts for EA. We demonstrate that square-wave direct current (DC) fields trigger a reaction between water molecules and chloride ions on the gold nanorod surface, generating electrolytic products including hydrogen, oxygen, and chlorine gases near the electrodes, changing the pH, and inducing cell death. These electric nanoantennas showed significant efficacy in treating colorectal cancer both in vitro and in vivo after DC treatment. These findings clearly indicate that gold nanoantennas enhance the effectiveness of EA by creating a localized electric field and catalyzing electrolytic reactions for the induction of locoregional pH changes within the tumor. By overcoming the limitations of traditional EA and offering an enhanced level of tumor specificity and control, this nanotechnology-integrated approach advances further innovations in cancer therapies.


Figure S1 .
Figure S1.Synthetic route of Schiff base DFA IV.

Figure S7 .
Figure S7.(a-d) Merged images of confocal fluorescence images and bright field images of DFA IV-PEI-PEG-GNR-treated cells exposed to strongly acidic (pH 2.5 and 3.4) and alkaline (pH 10.3 and 11.4) DMEM for multiple time intervals without a DC field (60× magnification; scale bar: 10 μm).Cells were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma Membrane for 30 min to stain the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane.

Figure S9 .
Figure S9.Merged confocal fluorescence images of CT-26 cell apoptosis and mitochondrial damage induced by PBS (control) and DFA IV-PEI-PEG-GNRs at the cathode and anode with a square-wave DC field using caspase 3/7 and TMRM double staining (20× magnification; scale bar: 250 μm).

Figure S11 .
Figure S11.Bio-TEM images of CT-26 cells treated with DFA IV-PEI-PEG-GNRs at the cathode (a), middle (b,c), and anode (d-f) under DC treatment for 10 min.The images show several morphological changes.N: nucleus, M: mitochondria, Ly: lysosome, ER: endoplasmic reticulum, G: Golgi apparatus, *: microtubules, N*: fragments of the nucleus, MDV: mitochondria-derived vesicles, and AB: apoptotic bodies.The red arrows indicate the gold nanorods (GNRs); the green arrowhead indicates the fragmented mitochondria; the black arrow and black arrowheads indicate the apoptotic bodies and doublemembrane compartment; the single and double white arrows indicate the doublemembraned autophagosomes and single-membraned autolysosomes; the blue arrows indicate the endoplasmic reticulum; the yellow arrow and yellow arrowhead indicate the mitochondria and cristae; the orange arrow indicates the mitochondria-derived vesicles; and the purple arrow indicates the fragmented Golgi apparatus.

Figure S12 .
Figure S12.(a-d) Merged images of confocal fluorescence images and bright field images of Ca 2+ and ROS generated by PBS (control) and DFA IV-PEI-PEG-GNR-treated CT-26 cells at the cathode and anode with and without square-wave DC field for various time intervals using Fluo-4 AM (green) and CellROX Deep Red (ROS) (60× magnification; scale bar: 20 μm).

Figure S13 .
Figure S13.The fluorescence intensities of Ca 2+ and ROS generated by PBS (control) and DFA IV-PEI-PEG-GNR-treated CT-26 cells at the cathode (a) and anode (b) with/without square-wave DC field for various time intervals using Fluo-4 AM (green) and CellROX Deep Red (ROS).

Figure S14 .
Figure S14.Intratumor pH was measured using a pH electrode with a pH meter connected to two Pt electrodes in response to DFA IV PEI-PEG-GNRs.

Figure S18 .
Figure S18.Biodistribution of Au concentrations in the blood, tumors, and major organs of mice treated with various groups.

Figure S20 .
Figure S20.Chamlide EC perfusion-type electric stimulation magnetic chamber with a Pt electrode and suction needle was used to maintain cell culture.

Video S11 .
Merged images of confocal fluorescence images and bright field images of CT-26 cells treated with DFA IV-PEI-PEG-GNRs at the anode under the squarewave DC field for various time intervals where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S12.Merged images of confocal fluorescence images of CT-26 cells treated with DFA IV-PEI-PEG-GNRs at the anode under the square-wave DC field for various time intervals where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S13.Merged images of confocal fluorescence images and bright field images of DFA IV-PEI-PEG-GNRs-treated CT-26 cells exposed to the strong acidic (pH 2.5) of DMEM for various time intervals without DC treatments where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S14.Merged images of confocal fluorescence images and bright field images of DFA IV-PEI-PEG-GNRs-treated CT-26 cells exposed to the strong acidic (pH 3.4) of DMEM for various time intervals without DC treatments where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S15.Merged images of confocal fluorescence images and bright field images of DFA IV-PEI-PEG-GNRs-treated CT-26 cells exposed to the strong alkaline (pH 10.3) of DMEM for various time intervals without DC treatments where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S16.Merged images of confocal fluorescence images and bright field images of DFA IV-PEI-PEG-GNRs-treated CT-26 cells exposed to the strong alkaline (pH 11.4) of DMEM for various time intervals without DC treatments where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S17.Merged images of confocal fluorescence images and bright field images of CT-26 cells apoptosis induced by PBS (control) at the cathode with square-wave DC field for various time intervals using the Annexin V-FITC/PI (60× magnification; scale bar: 20 μm).Video S18.Merged images of confocal fluorescence images and bright field images of CT-26 cells apoptosis induced by DFA IV-PEI-PEG-GNRs at the cathode with square-wave DC field for various time intervals using the Annexin V-FITC/PI (60× magnification; scale bar: 20 μm).Video S19.Merged images of confocal fluorescence images and bright field images of CT-26 cells apoptosis induced by DFA IV-PEI-PEG-GNRs at the anode with square-wave DC field for various time intervals using the Annexin V-FITC/PI (60× magnification; scale bar: 20 μm).Video S20.Merged images of confocal fluorescence images and bright field images of CT-26 cells apoptosis induced by PBS (control) at the anode with square-wave DC field for various time intervals using the Annexin V-FITC/PI (60× magnification; scale bar: 20 μm).Video S21.Merged images of confocal fluorescence images and bright field images of Ca 2+ generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the cathode with square-wave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S22.Merged images of confocal fluorescence images of Ca 2+ generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the cathode with square-wave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S23.Merged images of confocal fluorescence images and bright field images of Ca 2+ generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the anode with square-wave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S24.Merged images of confocal fluorescence images of Ca 2+ generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the anode with square-wave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S25.Merged images of confocal fluorescence images and bright field images of Ca 2+ generated by PBS (control)-treated CT-26 cells at the cathode with squarewave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S26.Merged images of confocal fluorescence images of Ca 2+ generated by PBS (control)-treated CT-26 cells at the cathode with square-wave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S27.Merged images of confocal fluorescence images and bright field images of Ca 2+ generated by PBS (control)-treated CT-26 cells at the anode with squarewave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S28.Merged images of confocal fluorescence images of Ca 2+ generated by PBS (control)-treated CT-26 cells at the anode with square-wave DC field for various time intervals using Fluo-4 AM (green) (60× magnification; scale bar: 20 μm).Video S29.Merged images of confocal fluorescence images and bright field images of ROS generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the cathode with square-wave DC field for various time intervals using CellROX Deep Red (60× magnification; scale bar: 20 μm).Video S30.Merged images of confocal fluorescence images of ROS generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the cathode with square-wave DC field for various time intervals using CellROX Deep Red (60× magnification; scale bar: 20 μm).Video S31.Merged images of confocal fluorescence images and bright field images of ROS generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the anode with square-wave DC field for various time intervals using CellROX Deep Red (60× magnification; scale bar: 20 μm).Video S32.Merged images of confocal fluorescence images of ROS generated by DFA IV-PEI-PEG-GNRs-treated CT-26 cells at the anode with square-wave DC field for various time intervals using CellROX Deep Red (60× magnification; scale bar: 20 μm).Video S33.Merged images of confocal fluorescence images and bright field images of ROS generated by PBS (control)-treated CT-26 cells at the cathode with squarewave DC field for various time intervals using CellROX Deep Red (60× magnification; scale bar: 20 μm).Video S34.Merged images of confocal fluorescence images of ROS generated by PBS (control)-treated CT-26 cells at the cathode with square-wave DC field for various time intervals using CellROX Deep Red (60× magnification; scale bar: 20 μm).

Table S1 . Comparative capabilities of recently published cancer treatment approaches based on EA.
Merged images of confocal fluorescence images and bright field images of CT-26 cells treated with PBS (control) at the middle region under the square-wave DC field for various time intervals where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).
Video S3.Merged images of confocal fluorescence images and bright field images of CT-26 cells treated with DFA IV-PEI-PEG-GNRs at the cathode under the squarewave DC field for various time intervals where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S4.Merged images of confocal fluorescence images of CT-26 cells treated with DFA IV-PEI-PEG-GNRs at the cathode under the square-wave DC field for various time intervals where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).Video S7.Merged images of confocal fluorescence images and bright field images of CT-26 cells treated with DFA IV-PEI-PEG-GNRs at the middle region under the square-wave DC field for various time intervals where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, Video S10.Merged images of confocal fluorescence images of CT-26 cells treated with PBS (control) at the anode under the square-wave DC field for various time intervals where the nuclei, mitochondria, endoplasmic reticulum, and plasma membrane were stained with Hoechst 33342, MitoTracker Green, endoplasmic reticulum (ER)-Tracker Red, and CellMask Deep Red Plasma membrane (60× magnification; scale bar: 10 µm).