Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles

Abstract

Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. In this study, we conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome, and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmids coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9%±2.2% (n=9), comparable with lipofection (7.5%±0.8%, n=9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection.

Keywords: Gene transfection; High speed videomicroscopy; Intracellular delivery; Microbubbles; Sonoporation; Ultrasound.

Fig. 1

Experimental setup of ultrasound excitation of targeted microbubbles attached to cells for sonoporation. Fluorescence imaging was used to detect intracellular delivery and cell viability, while high speed videomicroscopy was used to monitor ultrasound-induced bubble activities.

Fig. 2

(A) A typical bright filed image of cells with attached bubbles. (B) Distribution of microbubbles per cell obtained from 5 typical experiments. (C) Distribution of bubble radii obtained from 5 typical experiments.

Fig. 3

Characteristic bubble dynamics induced by pulsed ultrasound exposures. (A) Selective images showing stable cavitation of microbubbles with minimal translational movement. The cell was outlined in yellow. Acoustic pressure was 0.06 MPa, duty cycle 20%, and PRF 20 Hz. A schematic illustration of the pulsed ultrasound exposure was plotted above the images. No PI uptake was observed, and cell viability indicated by calcein retention. (B) Coalescence and translation of microbubbles. Acoustic pressure 0.4 MPa, duty cycle 20%, and PRF 20 Hz. PIfluorescence indicates cell membrane disruption. The absence of calcein indicated cell death. (C) Inertial cavitation with minimal displacement showing shrinkageof microbubbles after each ultrasound pulse before eventually disappeared. Acoustic pressure was 0.4 MPa, duty cycle 0.016%, and PRF 20 Hz. PI uptake was observed and calcein AM assay showed cell survived. (D) Characterization of bubble dynamics by the average rate of active bubble size change and total displacement of microbubbles. Data include 257 microbubbles. (E) Cell viability. (F) PI delivery rate (n = 6 for each group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Bubble dissolution after stable and inertial cavitation. (A) Selected time-lapse images of a bubble undergoing stable cavitation. (B) Radius–time curve of the bubble in (A) after the 1st and 2nd ultrasound pulse and fitting using the diffusion model. Acoustic pressure was 0.06 MPa, duty cycle 20%, PRF 20 Hz. (C) Selected time-lapse images of a bubble undergoing inertial cavitation. (D) Radius–time curve of the bubble shown in (C) after 1st and 2nd ultrasound pulses and fitting with diffusion model. Acoustic pressure 0.4 MPa, duty cycle 0.016%, PRF 20 Hz.

Fig. 5

Effects of number of pulses on sonoporation delivery. (A) Microbubbles in experiments using 20 ultrasound pulses (n = 6). Grouping of microbubbles into cohorts based on their effect on cells: cell death (red curves, 32 bubbles), PI delivery (blue curves, 20 bubbles), and no effect (green curves, 18 bubbles). (B) Microbubbles in experiments using 2 pulses (n = 6). (C) Microbubbles in experiments using 1 pulse (n = 5). Acoustic pressure 0.4 MPa, pulse duration 8 µs, and PRF 20 Hz. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Effects of acoustic pressure amplitude of single ultrasound pulse (pulse duration 8 µs) application on sonoporation delivery. (A) Viability, (B) delivery rate, (C) Delivery efficiency (average PI intensity per cell). n = 5 for each acoustic pressure. (D, F, H, and J) Distribution of intracellular PI intensity. (E, G, I and K) Size distribution of different cohorts of microbubbles grouped based on their roles in sonoporation: viable cells with PI uptake (blue bars), non-viable cell (red), viable cell without PI uptake (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 7

Effects of number of bubbles per cell on sonoporation delivery in terms of percentage of cells that were non-viable (red curves), or viable without PI uptake (green curves), or viable with PI uptake (blue curves). (A) Experiments (n = 5) using 1 pulse ultrasound exposure with duration8 µs, acoustic pressure 0.6 MPa. (B) Experiments (n = 5) using 1 pulse ultrasound exposure with duration 8 µs, acoustic pressure 1.6 MPa.

Fig. 8

(A) Intracellular transport of PI. (B) Total PI fluorescence after sonoporation. (C) Sizes of pores generated in sonoporation using different ultrasound conditions: 1 pulse (0.4 MPa, 8 µs), 2 pulses with equal amplitude (0.4 MPa, 8 µs, pulse interval 0.05 s), and 2 pulses with ramped amplitude (0.4 MPa and 0.6 MPa, pulse duration 8 µs, pulse interval 0.05 s). n = 140 for each condition. (D) Distribution of intracellular PI uptake for the three ultrasound conditions. (E) Representative radius–time curve of the bubbles (n = 10 for each condition). (F) Cell viability. (G) Delivery rate. n = 6 for each group.

Fig. 9

(A) Bobo/plasmid confined in patterned DEX droplets using a DEX/PEG ATPS. (B) GFP transfection in RASMCs. (C) Superimposed image of bright filed image with images of GFP expression. (D) Gene transfection efficiency. For experiments using 1 pulse (n = 6), acoustic pressure was 0.4 MPa. For 2 pulse exposure (n = 6) with equal amplitude, acoustic pressure was 0.4 MPa and pulse interval 0.05 s. For 2 pulses with ramped amplitude, the acoustic pressure was 0.4 MPa for the 1st pulse and 0.6 MPa for the 2nd pulse. The time interval was either 0.5 s (n = 6) or 0.05 s (n = 9). Pulse duration was 8 µs for all pulses. (E) The radius–time curves for bubbles exposed to 2 ramped pulses with 0.5 s and 0.05 s time interval. n = 10.

Fig. 10

(A) Time lapse images of intracellular delivery of BOBO–plasmid (middle panel) and GFP expression after sonoporation (bottom). (B) Live cell imaging of BOBO–plasmid and expression after lipofection. (C) 24 h after sonoporation. (D) 24 h after lipofection. The left panels in (C and D) are fluorescent images showing GFP expression. The middle panels show intracellular BOBO–plasmid complexes. The right panels are phase contrast images superimposed with fluorescent images of GFP and BOBO–plasmid.