Sonoporation based on repeated vaporization of gold nanodroplets

Abstract

Background: Gold nanodroplets (AuNDs) have been proposed as agents for photothermal therapy and photoacoustic imaging. Previously, we demonstrated that the sonoporation can be more effectively achieved with synchronized optical and acoustic droplet vaporization. By applying a laser pulse at the rarefactional phase of the ultrasound (US) pulse, the vaporization threshold can be reached at a considerably lower laser average power. However, a large loading quantity of the AuNDs may increase the risk of air embolism. The destruction of phase-shifted AuNDs at the inertial cavitation stage leads to a reduced drug delivery performance. And it also causes instability of echogenicity during therapeutic monitoring.

Purpose: In this study, we propose to further improve the sonoporation effectiveness with repeated vaporization. In other words, the AuNDs repeatedly undergo vaporization and recondensation so that sonoporation effects are accumulated over time at lower energy requirements. Previously, repeated vaporization has been demonstrated as an imaging contrast agent. In this study, we aim to adopt this repeated vaporization scheme for sonoporation.

Methods: Perfluoropentane NDs with a shell made of human serum albumin were used as the US contrast agents. Laser pulses at 808 nm and US pulses of 1 MHz were delivered for triggering vaporization and inertial cavitation of NDs. We detected the vaporization and cavitation effects under different activation firings, US peak negative pressures (PNPs), and laser fluences using 5- and 10-MHz focused US receivers. Numbers of calcein-AM and propidium iodide signals uptake by BNL hepatocarcinoma cancer cells were used to evaluate the sonoporation and cell death rate of the cells.

Results: We demonstrate that sonoporation can be realized based on repeatable vaporization instead of the commonly adopted inertial cavitation effects. In addition, it is found that the laser fluence and the acoustic pressure can be reduced. As an example, we demonstrate that the acoustic and optical energy for achieving a similar level of sonoporation rate can be as low as 0.44 MPa for the US PNP and 4.01 mJ/cm² for the laser fluence, which are lower than those with our previous approach (0.53 MPa and 4.95 mJ/cm² , respectively).

Conclusion: We demonstrated the feasibility of vaporization-based sonoporation at a lower optical and acoustic energy. It is an advantageous method that can enhance drug delivery efficiency, therapeutic safety and potentially deliver an upgraded gene therapy strategy for improved theragnosis.

Keywords: acoustic droplet vaporization; gold nanodroplets; inertial cavitation; optical droplet vaporization; sonoporation.

FIGURE 1

Experimental setup. (a) System setup and (b) time sequence for synchronizing laser and ultrasound pulses. Optical parametric oscillator (OPO) laser indicates pulsed optical parametric oscillator laser. RF signal indicates radio frequency signal. FG indicates the function generator

FIGURE 2

Vaporization and inertial cavitation dose. (a) Representative received signals (b) Spectra of the received vaporization signals. (c) Spectra of the received inertial cavitation signals. Gray boxes indicate the selected regions for data analysis. The baseline signals were received from phosphate‐buffered saline (PBS) only

FIGURE 3

Sonoporation and cell death of the cells. Red fluorescence denotes the propidium iodide (PI) dye. Green fluorescence denotes the calcein‐AM viability dye. Blue fluorescence denotes the cell nuclei. Red arrows indicate examples of sonoporated, live, or dead cells. Scale bar: 20 μm

FIGURE 4

Characterization of gold nanodroplets (AuNDs). (a) The optical absorbance of gold nanorods. The dashed line indicated the peak absorbance of gold nanorods was 818 nm. (b) Size distribution of AuNDs. (c) Flow cytometry results. AuNDs with FITC fluorescence were gated in the M1 region. (d) Fluorescence image of cells attached with FITC‐CD54‐conjugated AuNDs. Green fluorescent dots indicate the FITC‐CD54‐conjugated AuNDs. Blue fluorescence indicates the cell nuclei

FIGURE 5

Differential vaporization dose (dVAP) and differential inertial cavitation dose (dICD) values as a function of different activation firing numbers. Blue and red lines indicate the dVAP and dICD values, respectively. The laser pulse repetition frequency (PRF) was set as 20 Hz and the US cycle was set as 10 cycles in all experiments. Symbols and error bars denote the mean and SD from six individual experiments

FIGURE 6

Vaporization and cavitation effect in different activation firing sections. (a) differential vaporization dose (dVAP) as a function of differential inertial cavitation dose (dICD) in different activation sections. (b, c) Reduction of dVAP and dICD as a function of different activation firing sections within 5000 firings. Symbols and error bars denote the mean and standard deviation (SD) from six individual experiments. The student's t‐test was applied for the determination of the significant difference between two data sets. *, p < 0.05; **, p < 0.01; ***, p < 0.001

FIGURE 7

Sonoporation as a function of activation firing and ultrasound peak negative pressure (US PNP). (a) Sonoporation rate and cell death rate grouped with different activation firings. (b) Sonoporation rate and cell death rate grouped with different US PNPs. The laser pulse repetition frequency (PRF) was set as 20 Hz and the number of US cycles was set at 10 cycles in all experiments. Each column indicates the mean and standard deviation (SD) from six individual experiments. The student's t‐test was applied for the determination of the significant difference between two data sets. *, p < 0.05; **, p < 0.01; ***, p < 0.001

FIGURE 8

differential vaporization dose (dVAP) and differential inertial cavitation dose (dICD) as a function of laser fluence and ultrasound peak negative pressure (US PNP). The activation number was fixed at 1000, laser pulse repetition frequency (PRF) was set as 20 Hz, and the number of US cycles was set at 10 in all experiments. Each column indicates the mean and SD from six individual experiments. The student's t‐test was applied for the determination of the significant difference between two data sets. ns, no significance. Except for the data set labeled with no significant difference and data sets with no US or laser treatment, all other data sets showed a significant difference

FIGURE 9

Sonoporation rate as a function of (a) ultrasound peak negative pressure (US PNP) or (b) laser fluence. The activation number was fixed at 1000, laser pulse repetition frequency (PRF) was set as 20 Hz, and the number of US cycles was set at 10 cycles in all experiments. Each column indicates the mean and standard deviation (SD) from six individual experiments. The student's t‐test was applied for the determination of the significant difference between two data sets. *, p < 0.05; **, p < 0.01; ***, p < 0.001

FIGURE 10

Correlation of sonoporation rate and differential vaporization dose (dVAP)/ differential inertial cavitation dose (dICD) values. The correlation coefficient (r), the p‐value of the Pearson's correlation test, and the equation of the linear regression were shown below the lines. Symbols and error bars denote the mean and standard deviation (SD) from six individual experiments

FIGURE 11

Correlation of sonoporation rate and differential vaporization dose (dVAP) values from conditions without significant inertial cavitation. The correlation coefficient (r), the p‐value of the Pearson's correlation test, and the equation of the linear regression were shown below the line. Symbols and error bars denote the mean and standard deviation (SD) from six individual experiments