Acoustofluidics-Based Intracellular Nanoparticle Delivery

Abstract

Controlled intracellular delivery of biomolecular cargo is critical for developing targeted therapeutics and cell reprogramming. Conventional delivery approaches (e.g., endocytosis of nano-vectors, microinjection, and electroporation) usually require time-consuming uptake processes, labor-intensive operations, and/or costly specialized equipment. Here, we present an acoustofluidics-based intracellular delivery approach capable of effectively delivering various functional nanomaterials to multiple cell types (e.g., adherent and suspension cancer cells). By tuning the standing acoustic waves in a glass capillary, our approach can push cells in flow to the capillary wall and enhance membrane permeability by increasing membrane stress to deform cells via acoustic radiation forces. Moreover, by coating the capillary with cargo-encapsulated nanoparticles, our approach can achieve controllable cell-nanoparticle contact and facilitate nanomaterial delivery beyond Brownian movement. Based on these mechanisms, we have successfully delivered nanoparticles loaded with small molecules or protein-based cargo to U937 and HeLa cells. Our results demonstrate enhanced delivery efficiency compared to attempts made without the use of acoustofluidics. Moreover, compared to conventional sonoporation methods, our approach does not require special contrast agents with microbubbles. This acoustofluidics-based approach creates exciting opportunities to achieve controllable intracellular delivery of various biomolecular cargoes to diverse cell types for potential therapeutic applications and biophysical studies.

Keywords: Acoustofluidics; Metal-organic frameworks; Nanocarriers; Sonoporation.

Fig. 1.

Schematics illustrating the mechanism of our acoustofluidics-based intracellular delivery device. (a) Device schematic. (b) Cross-sectional view of the device for illustrating the generation of standing bulk acoustic waves with a pressure node at the top capillary wall, which can push a cell loaded in the capillary to the top wall. (c) Illustration of the cell movement in a capillary coated with cargo-encapsulated nanoparticles under continuous flow and acoustic waves. The acoustic waves can push cells to the top wall, enabling controllable contact between cells and nanoparticles when the cells flow through the capillary along its top wall. (d) A schematic illustrating the intracellular cargo delivery process.

Fig. 2.

Characterization of cargo-encapsulated nanoparticles and nanoparticle-coated capillary walls. (a, b) TEM images of FBSA@ZIF-8 and DOX@ZIF-8 nanoparticles (scale bars: 250 nm). (c) DLS particle size distributions and (d) PXRD results for ZIF-8, DOX@ZIF-8, and FBSA@ZIF-8 nanoparticles. (e, f) UV-Vis absorbance spectrum distributions for FBSA@ZIF-8 and DOX@ZIF-8 nanoparticles. Bright-field and fluorescence images for (g, j) an original glass capillary wall without coating, (h, k) a DOX@ZIF-8 coated glass capillary wall, and (i, l) an FBSA@ZIF-8 coated glass capillary wall (scale bars: 150 μm). 2θ: the diffraction angle.

Fig. 3.

Acoustic wave-induced cell movement and permeability changes. (a) Finite element simulation result showing the displacement field of a glass capillary at the acoustic excitation frequency of 710 kHz. (b–d) Simulation results for the acoustic pressure field, the acoustic radiation force field, and the cell movement trajectories in the fluid domain, respectively. Max: maximum; Min: minimum. (e) Microscopy image for showing randomly distributed U937 cells in a capillary without acoustic waves. (f) Stacked microscopy images illustrating the trajectories of acoustic wave-induced cell movements. (g) Microscopy image demonstrating that cells can be pushed to the top capillary wall within 4 s by the acoustic waves. (h, i) Fluorescence microscopy images of U937 cells treated without and with acoustic waves. The cells were mixed with DOX@ZIF-8 nanoparticles in a solution and then loaded into clean capillaries without nanoparticle coating. The treated cells were collected from the capillaries, washed with PBS, and then dispensed on microscope slides for imaging. Scale bars: 100 μm.

Fig. 4.

Testing delivery of different types of cargo. (a, b) Microscopy images (left: bright-field, right: fluorescence) showing the delivery of FBSA into U937 cells with acoustic waves off and on, respectively (scale bars: 100 μm). In these experiments, the glass capillaries were coated with FBSA@ZIF-8 nanoparticles. (c, d) Microscopy images (left: bright-field, right: fluorescence) showing the delivery of DOX into U937 cells with acoustic waves off and on, respectively (scale bars: 100 μm). In these experiments, the glass capillaries were coated with DOX@ZIF-8 nanoparticles. (e) Results of viability studies for three different cases. This data indicate that the used acoustic waves and nanocarriers have low cytotoxicities. (f) Results of viability studies for delivering DOX@ZIF-8 nanoparticles with and without acoustic waves. In the case of active acoustic waves, the cell viability gradually decreases over time because the successfully delivered DOX@ZIF-8 nanoparticles gradually release cytotoxic DOX, which induces cell apoptosis. The column heights and error bars in (e, f) represent the mean values and standard deviations, respectively.

Fig. 5.

Acoustofluidic delivery of cargo-encapsulated nanoparticles into HeLa cells. (a) Time-lapse microscopic images for showing the movement of a cell (in yellow circles) along a capillary wall coated with DOX@ZIF-8 nanoparticles (scale bars: 100 μm). (b) Fluorescent images of a coated capillary before and after an intracellular delivery experiment (scale bar: 100 μm). (c) Bright-field microscopy image acquired 5 min after the experiment showing HeLa cells with attached DOX@ZIF-8 nanoparticles marked with red arrows (scale bar: 20 μm). (d) Fluorescence microscopy image acquired 6 h after the experiment showing DOX molecules in cells (scale bar: 20 μm, red: DOX, blue: DAPI for nucleus). These DOX molecules, which are released from the successfully delivered DOX@ZIF-8 nanoparticles, gradually cause cell apoptosis, as confirmed by the cell morphology changes and the viability decreases shown in Fig. S4. (e, f) Flow cytometry results for delivering DOX@ZIF-8 nanoparticles into HeLa cells (N = 3).