The impact of sphingosine kinase inhibitor-loaded nanoparticles on bioelectrical and biomechanical properties of cancer cells

Abstract

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to identify biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical properties in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, this study showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.

Fig. 1. A) Illustration of the fabricated microfluidic chip including delivery and constriction channels, cells inlet/outlet, and suction port. B) Process flow for fabrication of the microfluidic device. C) Image showing a single deformed cell travelling through the constriction channel. D) The impedance magnitude change at 1 kHz when a single cell passes through the constriction channel.

Fig. 2. Schematic image showing the operation of the microfluidic chip.

Fig. 3. A) Formula of the drugs loaded in NPs. B) Release profiles of drug-loaded NPs. C) TEM image of NPs uptake in one cell.

Fig. 4. Measured impedance changes at A) 1 kHz, B) 10 kHz, C) 100 kHz, and D) 1 MHz frequency recorded as single MDA-MB-231 cells after treatments pass through the constriction channel.

Fig. 5. A schematic illustration of the electric circuit model for the microchannel with an elongated cell used to characterize cell electrical parameters from the multi-frequency impedance measurements. Circuit element legend: C _dl1 and C _dl2 – double layer capacitance; C _par – parasitic capacitance; C _m – membrane capacitance; R _sp – spreading resistance; R _cyt – cytoplasm resistance; R _int – cell-channel wall interface resistance.

Fig. 6. Scatter plots of (A) interface resistance, (B) specific membrane capacitance, and (C) cytoplasm conductivity obtained for 48 h treated cells with free-drug and NP-packaged SphKIs in comparison to untreated and unloaded NPs-treated cells, respectively. **P ≤ 0.01, ****P ≤ 0.0001.

Fig. 7. Measured entry time and travel time changes recorded as single MDA-MB-231 cells before and after treatments with free-drug (in A and B) and NP-packaged SphKIs (in C and D) pass through the constriction channel.

Fig. 8. Immunofluorescence images showing difference in the actin organization of A) unloaded NP-treated, B) untreated, C) DuaLI treated, D) SphKI2 treated, and E) SphKI1 treated cells. F) Actin content intensity revealed treatments with SphKI, SphKI2, and DuaLI led to a decrease in the actin intensity, while the absorbed NPs increased the actin intensity. **P ≤ 0.01.

Fig. 9. A) Actin filaments protein WB of whole-cell extracts from untreated cells and cells treated with SphKI1, SphKI2, DuaLI, and unloaded NPs for 48 h. B) Normalized (to NPs) levels of actin protein bands in treated and untreated cells. **P ≤ 0.01.