Wireless Stimulation of Barium Titanate@PEDOT Nanoparticles Toward Bioelectrical Modulation in Cancer

Abstract

Cancer cells possess distinct bioelectrical properties, yet therapies leveraging these characteristics remain underexplored. Herein, we introduce an innovative nanobioelectronic system combining a piezoelectric barium titanate nanoparticle core with a conducting poly(3,4-ethylenedioxythiophene) shell (BTO@PEDOT NPs), designed to modulate cancer cell bioelectricity through noninvasive, wireless stimulation. Our hypothesis is that acting as nanoantennas, BTO@PEDOT NPs convert mechanical inputs provided by ultrasound (US) into electrical signals, capable of interfering with the bioelectronic circuitry of two human breast cancer cell lines, MCF-7 and MDA-MB-231. Upon US stimulation, the viability of MCF-7 and MDA-MB-231 cells treated with 200 μg mL^-1 BTO@PEDOT NPs and US reduced significantly to 31% and 24%, respectively, while healthy human mammary fibroblasts (HMF) were unaffected by the treatment. Subsequent assays shed light on how this approach could interact with cell's bioelectrical mechanisms, namely, by increasing intracellular reactive oxygen species (ROS) and calcium concentrations. Furthermore, this system was able to polarize cancer cell membranes, halting their cell cycle and potentially harnessing their tumorigenic characteristics. These findings underscore the crucial role of bioelectricity in cancer progression and highlight the potential of nanobioelectronic systems as an emerging and promising strategy for cancer intervention.

Keywords: breast cancer; cancer bioelectricity; multifunctional nanoparticles; nanobioelectronics; ultrasound stimulation; wireless stimulation.

Figure 1

Synthesis and characterization of BTO@PEDOT NPs. a) Schematic representation of the synthesis of the nanobioelectronic systems proposed in this work to obtain the BTO@PEDOT NPs. b) Hydrodynamic size and c) ζ-potential of the different intermediates of the synthesis of BTO@PEDOT NPs obtained by Dynamic-light scattering for the 200 and 500 nm BTO (n = 3). d) TEM images of the 500 nm BTO-PSS (orange) and BTO@PEDOT NPs (blue). e) Size distribution (n = 50) and f) elemental mapping of the 500 nm BTO@PEDOT NPs.

Figure 2

Piezoelectric and electrochemical analysis of BTO@PEDOT NPs. a) Topographical image of an individual nanoparticle and b) its representative hysteresis loop illustrating the piezoresponse behavior of the material. The amplitude and phase of the piezoresponse are plotted against the DC applied voltage. c) Cyclic voltammetry curves for BTO, and BTO@PEDOT NPs at 300 mV/s scan rate.

Figure 3

Treatment consisting of BTO@PEDOT NPs and US stimulation decreases the viability of human breast cancer cell lines. a) Representative fluorescence microscopy images of the MCF-7, MDA-MB-231 and HMF cells in the absence (controls) and presence of BTO@PEDOT NPs at a concentration of 200 μg mL-1 and ultrasound (US) stimulation (I = 0.4 W cm^–2, f = 1 MHz, DC = 50%) after live/dead staining. Scale bar 100 μm. Green fluorescence was acquired through cell-permeant dye calcein AM and red fluorescence was acquired through the high-affinity nucleic acid stain ethidium homodimer. b) Calculation of the percentage of viable cells at the previous conditions. Statistical analysis was performed using one-way ANOVA (***p < 0.001 and ****p < 0.0001). Data are shown as mean ± SD (N = 3, n = 2).

Figure 4

US stimulation in the presence of BTO@PEDOT NPs increases ROS production in MCF-7 and MDA-MB-231, activates caspase-3 in MDA-MB-231 cells and polarizes cell membranes. a) Fluorescence microscopy images of ROS generation and normalized semiquantitative analysis of b) ROS generation (N = 4) and c) caspase-3 activity (N = 3, n = 2) in MCF-7, MDA-MB-231 and HMF cells under different conditions. d) Normalized semiquantitative analysis of DiBAC₄(3) levels in MCF-7, MDA-MB-231 and HMF cells under different US application times and with BTO@PEDOT NPs (N = 4). e) Fluorescence microscopy images of control and US-stimulated MDA-MB-231 in the presence of BTO@PEDOT NPs after evaluation with DiBAC₄(3), which correlates higher cell membrane polarization with lower fluorescence. Scale bar 200 μm. . Statistical analysis was performed using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). Data are shown as mean ± SD.

Figure 5

US stimulation in the presence of BTO@PEDOT NPs increases intracellular Ca²⁺in MCF-7 and MDA-MB-231 cells, alters their cell cycle and upregulates CACNA1C and CACNA1H genes in MDA-MB-231 cells. a) Fluorescence microscopy images and b) normalized semiquantitative analysis of the intracellular Ca²⁺ content of MCF-7, MDA-MB-231 and HMF cells at increasing times of US stimulation in the presence of BTO@PEDOT NPs. Scale bar 200 μm. Statistical analysis was performed using two-way ANOVA (*p < 0.05, **p < 0.01 and ****p < 0.0001). Data are shown as mean ± SD (N = 4). c) Cell cycle analysis of MCF-7, MDA-MB-231 and HMF cells. Percentage of cells in each phase of the cell cycle as determined by fluorescence-activated cell sorting (FACS) analysis in control conditions and after US stimulation in the presence of BTO@PEDOT NPs. d) CACNA1C and e) CACNA1H gene expression of obtained by RT-qPCR analysis of MCF-7, MDA-MB-231 and HMF cells incubated with BTO@PEDOT NPs with and without US stimulation. Gene expressions are normalized against the housekeeping gene GAPDH and presented as fold-change levels relative to controls consisting on cells cultured on media (represented by dotted line) (N = 3, n = 3). Statistical analysis was performed using one-way ANOVA (***p < 0.001 and ****p < 0.0001). Data are shown as mean ± SD.