Programmable ultrasonic fields enhance intracellular delivery in cell clusters

TL;DR

Programmable Acoustic Standing-wave Transfection is established as a programmable, high-throughput, and non-invasive intracellular delivery platform, offering new opportunities for precision drug screening, gene editing, and mechanistic exploration of cellular membrane biophysics.

Abstract

Intracellular delivery of biomolecules remains a critical challenge in both basic cell biology and translational therapeutics. We introduce Programmable Acoustic Standing-wave Transfection (PAST), a microfluidic tool that leverages dynamically programmable ultrasonic fields to transiently permeabilize cell membranes and enhance biomolecular transport within cell clusters. By generating programmable acoustic potential landscapes, PAST drives cells through cycles of hydrodynamic and acoustic stresses that induce reversible pore formation, enabling diffusion-based delivery without chemical carriers or contrast agents. Experimental studies demonstrate controlled influx and efflux dynamics across multiple biomolecular species, with transport rates tunable via acoustic power, frequency modulation, and duty cycles. Theoretical scaling and numerical simulations reveal that membrane tension, pore energetics, and acoustic field distributions collectively govern transmembrane transport of biomolecules. Post-treatment assays confirm high cellular viability and sustained proliferation, underscoring the biocompatibility of the method. Remarkably, effective diffusivity estimates derived from model predictions closely match experimental transport timescales. Together, these findings establish PAST as a programmable, high-throughput, and non-invasive intracellular delivery platform, offering new opportunities for precision drug screening, gene editing, and mechanistic exploration of cellular membrane biophysics.

Figures (17)

• Figure 1: Programmable Acoustic Standing-wave (PAS) and dynamics of bioparticle aggregates. a. PAS setup with programmable frequency modulation generating reconfigurable acoustic potential landscapes. b. Experimental images showing cell trapping, aggregate nucleation, and growth into a desired cluster size. c. Aggregate motion under dynamic frequency modulation, illustrating translation, rotation, and morphological reconfiguration over a single frequency cycle (T). d. Measured peak-to-peak driving voltage across the acoustofluidic device as a function of actuation frequency for three acoustic powers.Scale bar represents 100 $\mu$m.
• Figure 1: Images of experimental setup. a) Image showing the inverted microscope setup along with high-speed camera used for experiments. The microscope is equipped with a epifluorescence lamp and different fluorescence emission filters that enable visualization of emitted fluorescence intensities from ultrasonically treated cells. The image on the right shows a close-up view of the mounted acoustofluidic device equipped with a piezoelectric actuation source. The device is driven by a signal generator and a power amplifier, b) Image showing the experimental setup of a confocal scanning microscope. The microscope is equipped with laser sources corresponding to different excitation wavelengths, and a resonant scanning system that permits visualization across the cross-section of the sample.
• Figure 2: Intracellular transport under Programmable Acoustic Standing-wave Transfection (PAST).a. Schematic of membrane permeabilization under external stresses: disruption of the lipid bilayer initiates hydrophobic pores, followed by lipid head reorientation into hydrophilic pores permitting biomolecular transport in aqueous media. b. Fluorescence images showing intracellular uptake of different cargos after multiple frequency cycles ($N_R$); Scale bar, 100 $\mu$m. c. Temporal evolution of normalized fluorescence intensity ratio ($\Delta I^*$) over experimental timescales ($N_R$=15), indicating continuous uptake/efflux of biomolecules. d. Confocal images of acoustically treated HeLa aggregates stained with live–dead assay: blue = nuclei of all cells, red = cells with permanently compromised membranes; Scale bar, 20 $\mu$m. e. Time evolution of normalized fluorescence intensity ratio ($\Delta I_{L/D}^*$) of treated vs control cells over 30 min post-exposure; Scale bar, 20 $\mu$m. f. Evolution of $\Delta I^*$ for calcein and doxorubicin uptake in aggregates subjected to two input powers. g. Evolution of $\Delta I^*$ for calcein and doxorubicin in HeLa vs MCF-7 aggregates.
• Figure 2: Control experiments with HeLa cells. Fluorescence emission recorded from HeLa cells exposed to different biomolecular agents and observed under corresponding fluorescence filters. Cells are trypsinized from cell culture flask, washed and resuspended in fresh cell culture medium supplemented with the biomolecular agent under investigation. These cells are then observed periodically over a period of 6 h. Permeation of biomolecular cargos is primarily through passive diffusion and is observed to relatively very slow compared to the ultrasonic-mediated transmembrane transport. Fluorescence signals under TRITC imaging filter, corresponding to propidium iodide and DOX, remains largely below detectable thresholds in the initial period till 30 mins. Scale bar indicates 50 $\mu$m.
• Figure 3: Biocompatibility and Long-Term Cell Viability Post-PAST a. Infrared imaging of temperature rise in the acoustofluidic device during frequency sweep cycles, figure shows average device temperature over one cycle at three input powers; background shading denotes actuation (30 s, 0.900–1.200 MHz) and resting (90 s) phases. b. Bright-field images of ultrasonically treated HeLa cells seeded on poly-L-lysine–coated dishes, monitored every 24 h; scale bar, 100 $\mu$m. c. Temporal evolution of normalized seeded cell area over 72 h compared with untreated controls. d. Confocal live/dead staining of proliferated cells after 72 h; scale bar, 100 $\mu$m. e. Normalized fluorescence intensity ratio for treated versus control cells, showing only marginal deviation and confirming sustained viability and biocompatibility. f. Confocal images of acoustically treated HeLa aggregates with calcein-AM and doxorubicin, demonstrating intracellular uptake and retention of doxorubicin with residual calcein signals after 72 h; scale bar, 20 $\mu$m. Inset arrows demarcate bleb formation on membrane surface.