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. 2019 Feb 17;2(4):e1165. doi: 10.1002/cnr2.1165

Selective intracellular delivery of perfluorocarbon nanodroplets for cytotoxicity threshold reduction on ultrasound‐induced vaporization

Ayumu Ishijima 1, Satoshi Yamaguchi 2, Takashi Azuma 3,✉, Etsuko Kobayashi 4, Yoshikazu Shibasaki 2, Teruyuki Nagamune 5,6, Ichiro Sakuma 1,4,6,✉
PMCID: PMC7941471  PMID: 32721118

Abstract

Background

Phase‐change nanodroplets (PCNDs), which are liquid perfluorocarbon nanoparticles, have garnered much attention as ultrasound‐responsive nanomedicines. The vaporization phenomenon has been employed to treat tumors mechanically. However, the ultrasound pressure applied to induce vaporization must be low to avoid damage to nontarget tissues.

Aims

Here, we report that the pressure threshold for vaporization to induce cytotoxicity can be significantly reduced by selective intracellular delivery of PCNDs into targeted tumors.

Methods and results

In vitro experiments revealed that selective intracellular delivery of PCNDs induced PCND aggregation specifically inside the targeted cells. This close‐packed configuration decreased the pressure threshold for vaporization to induce cytotoxicity. Moreover, following ultrasound exposure, significant decrease was observed in the viability of cells that incorporated PCNDs (35%) but not in the viability of cells that did not incorporate PCNDs (88%).

Conclusions

Intracellular delivery of PCNDs reduced ultrasound pressure applied for vaporization to induce cytotoxicity. Confocal laser scanning microscopy and flow cytometry revealed that prolonged PCND‐cell incubation increased PCND uptake and aggregation. This aggregation effect might have contributed to the cytotoxicity threshold reduction effect.

Keywords: acoustic droplet vaporization, intracellular vaporization, perfluorocarbon, phase‐change nanodroplets, stimuli‐responsive materials, ultrasound

1. INTRODUCTION

Various therapeutic strategies have been developed against cancer. Currently, thanks to advancements in nanotechnology, nanomedicines as drug delivery systems have attracted great attention owing to their tumor‐targeting abilities and multifunctionality.1 These nanomedicines take advantage of a unique tumor characteristics—the enhanced permeability and retention effect—which allows passive transportation of drug to extravascular tumor sites.2, 3 Active targeting and external stimuli‐responsive properties can further improve targeting efficacy and reduce harmful side effects of drugs, ensuring that therapeutic effects are conferred only in the overlapping area of the drug and external stimulus.4, 5 As an external stimulus, ultrasound has benefits of noninvasiveness, deep penetration (in the order of several tens of cm), and submillimeter‐to‐millimeter–order spatial control capability that enables high spatial‐temporal control of therapeutic activation. Liquid perfluorocarbon (PFC) nanoparticles, which are phase‐change nanodroplets (PCNDs), vaporize into microbubbles following exposure to ultrasound with sufficient peak negative pressure.6, 7, 8, 9, 10, 11, 12 These PFC nanodroplets have been proposed to be imaging contrast agents and/or drug carriers.13, 14, 15, 16, 17, 18, 19 Recently, several studies have utilized mechanical effects of the vaporization phenomenon to treat tumors.15, 20, 21, 22 The conjugation of active targeting moieties to nanodroplets is a potential method to selectively destroy tumor cells with high accuracy. To avoid damage to nontarget tissues, ultrasound pressure used to induce vaporization must be sufficiently low to avoid uncontrolled nonspecific cavitation, which is not derived from PCNDs. Here, we demonstrated that the pressure threshold for vaporization to induce cytotoxicity can be significantly reduced by selective intracellular delivery of PCNDs into the targeted tumors in vitro.

To selectively introduce PCNDs into the targeted tumor cells, the monoclonal antibody 9E5 was conjugated to PCNDs.23 9E5 is an anti‐epiregulin (EREG) antibody that targets cell membrane–expressed ligands of epidermal growth factor receptors.24, 25 Previously, we have demonstrated that 9E5‐conjugated PCNDs can be selectively introduced into tumor cells via receptor‐mediated endocytosis and that exposure to ultrasound‐induced intracellular vaporization leads to a rapid disruption of cellular structure.23 In the present study, we compared cytotoxicity due to vaporization inside and outside cells and demonstrated that intracellular delivery of PCNDs reduces ultrasound pressure for vaporization to induce cytotoxicity.

2. RESULTS

2.1. Selective intracellular aggregation of 9E5‐conjugated PCNDs

Prior to ultrasound exposure experiments, we tested whether incubation time could be used to control the localization of PCNDs (inside or outside the cells) using confocal laser scanning microscopy (CLSM) and flow cytometry. To demonstrate selective targeting capability and intracellular aggregation effects of 9E5‐conjugated PCNDs on DLD‐1 cells (high EREG‐expressing cell lines), we used Alexa Fluor 647–conjugated streptavidin (SA‐AF647) as a probe. In this experiment, nontargeted PCNDs were removed by washing. Figure 1A presents the time course of 9E5‐mediated accumulation and internalization of AF647‐labeled 9E5‐conjugated PCNDs in DLD‐1 cells, as observed using CLSM. Pink fluorescent signals from AF647 were observed only from the membranes of DLD‐1 cells at 1 hour of incubation. After 1 hour of incubation, 9E5‐conjugated PCNDs were localized inside cells, and they gradually formed aggregates, which augmented the fluorescence intensity of AF647. In contrast, AGS cells (low EREG‐expressing human gastric cancer cell lines) treated with 9E5‐conjugated PCNDs showed neither accumulation nor internalization of 9E5‐conjugated PCNDs (Figure 1B). CLSM confirmed selective targeting and intracellular aggregation of 9E5‐conjugated PCNDs in DLD‐1 cells. Furthermore, a cell‐penetrating peptide (CPP) (biotin‐G3R15GYC, 3081.67 Da) was tested as a positive control for intracellular delivery of PCNDs. Polyarginine‐based CPP may be suitable for nonspecific delivery of PCNDs.26 Figure 1C shows CLSM images of CPP‐mediated accumulation and internalization of AF647‐labeled CPP‐conjugated PCNDs in DLD‐1 and AGS cells. These results confirmed that CPP‐conjugated PCNDs can be used as a positive control for intracellular delivery and aggregation of PCNDs. Notably, a weak laser beam was used to observe CPP‐conjugated PCND‐treated cells since we detected stronger fluorescence signals in these cells than in 9E5‐conjugated PCND‐treated cells. Figure 1D shows a comparison of AF647 fluorescence intensity inside and outside DLD‐1 cells. Of note, removal of nontargeted PCNDs by washing was excluded (see Section 5). Strong AF647 fluorescence signals (pink) were observed in intracellular spaces, as indicated by calcein AM fluorescence (green). These results indicate that the localization of 9E5‐conjugated PCNDs (inside or outside the cells) can be controlled by PCND‐cell incubation time.

Figure 1.

Figure 1

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Time course of confocal laser scanning microscopy (CLSM) and Alexa Fluor 647 (AF647) fluorescence intensity of cells treated with phase‐change nanodroplets (PCNDs). The pink fluorescent signal indicates AF647‐conjugated streptavidin (SA‐AF647) conjugated to PCND, while green fluorescent signal indicates cellular membranes stained with NeuroDiO. A, Images of DLD‐1 cells treated with 9E5‐conjugated PCNDs for 1 to 48 h. B, Images of AGS cells treated with 9E5‐conjugated PCNDs for 24 and 48 h. C, Images of DLD‐1 and AGS cells treated with cell‐penetrating peptide (CPP)–conjugated PCNDs for 24 h. Scale bars represent 10 μm. D, Comparison of AF647 fluorescence intensity inside and outside the cells. The pink fluorescent signal indicates SA‐AF647 labeled with 9E5‐conjugated PCNDs, the green fluorescent signal indicates cytoplasm stained with calcein AM, and the blue fluorescent signal indicates nuclei stained with Hoechst dye. The scale bar represents 50 μm. E, Representative AF647 fluorescence intensity histograms of DLD‐1 cells treated with 9E5‐ and CPP‐conjugated PCNDs. F, Median values of AF647 fluorescence intensity calculated based on histograms at various time points (blue circle: DLD‐1 cells treated with 9E5‐conjugated PCNDs; red circle: DLD‐1 cells treated with CPP‐conjugated PCNDs; black circle: only DLD‐1 cells; blue triangle: DLD‐1 cells treated with free 9E5‐ and 9E5‐conjugated PCNDs; blue square: AGS cells treated with 9E5‐conjugated PCNDs; and red square: AGS cells treated with CPP‐conjugated PCNDs)

The fraction of bound DLD‐1 cells was quantitatively measured using flow cytometry. Figure 1E,F shows the quantitative time course of AF647 fluorescence intensity of cells treated with PCNDs, as measured by flow cytometry. Figure 1E shows representative AF647 fluorescence intensity histograms of DLD‐1 cells treated with 9E5‐ and CPP‐conjugated PCNDs, and Figure 1F shows the median values of AF647 fluorescence intensity calculated from the histograms at various time points (N = 5). Fluorescence intensity of DLD‐1 cells treated with 9E5‐conjugated PCNDs increased until 24 hours and then plateaued, while fluorescence intensity of AGS and EREG‐blocked DLD‐1 cells remained weak. High fluorescence intensity values were detected for both DLD‐1 and AGS cells treated with CPP‐conjugated PCNDs. Taken together, results of CLSM and flow cytometry supported the fact that selective intracellular delivery of PCNDs can lead to their aggregation specifically inside the targeted cells.

2.2. Cytotoxicity threshold for extracellular and intracellular vaporization

Next, we quantitatively evaluated the negative pressure that elicited vaporization‐induced cytotoxic effects on intracellular aggregated and extracellular PCNDs. Ultrasound was applied to cultured cells using the experimental setup illustrated in Figure 2A. The ultrasound pressure required to produce vaporization‐induced cytotoxic effects on intracellular aggregated PCNDs was lower than that required to produce this effect on extracellular PCNDs. Figure 2B shows DLD‐1 cell viability at the peak negative pressure of applied ultrasound. Of note, removal of nontargeted PCNDs by washing was excluded. Blue squares indicate the viability of PCND‐treated DLD‐1 cells exposed to ultrasound immediately after the treatment of 9E5‐conjugated PCNDs (N = 5; outside: 0 h). Green squares indicate the viability of cells treated with 9E5‐conjugated PCNDs for 1 hour (outside: 1 h) and black circles for 24 hours (inside: 24 h). Red circles indicate the viability of cells treated with CPP‐conjugated PCNDs for 24 hours (inside: 24 h). For all treatments, significant decrease in cell viability was observed once the ultrasound pressure exceeded a certain value. When the applied pressure exceeded 2.8 MPa, cell viability decreased due to both extracellular and intracellular vaporization. However, at 2 MPa, cell viability significantly decreased due to intracellular vaporization alone (N = 5, P < 0.05). These results indicate that intracellular delivery of PCNDs significantly reduced the ultrasound pressure for vaporization to induce cellular damage (cytotoxicity threshold reduction effect). Next, we tested whether this threshold reduction effect was highly selective for DLD‐1 cells. Figure 2C shows viability of DLD‐1, AGS, and EREG‐blocked DLD‐1 cells treated with 9E5‐conjugated PCNDs (for 24 h without washing) following ultrasound exposure. Viability of DLD‐1 cells exposed to ultrasound at a peak negative pressure of 2.0 MPa (57 ± 10%, N = 8, P < 0.001) was significantly reduced compared with that of AGS (82 ± 7%) and EREG‐blocked DLD‐1 (86 ± 3%) cells.

Figure 2.

Figure 2

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Evaluation of cytotoxicity threshold for extracellular and intracellular vaporization. A, Experimental setup for ultrasound exposure. Schematic diagram of the scanning system used for ultrasound exposure on 35‐mm culture dishes and acoustic profiles inside the dish measured by a hydrophone. B, Cytotoxicity pressure threshold for extracellular and intracellular vaporization. Cell viability after extracellular (0‐ and 1‐h incubation, square plots) and intracellular vaporization (24‐h incubation, circle plots). C, Cell viability of DLD‐1 (white), AGS (black), and epiregulin (EREG)–blocked DLD‐1 cells (blue) treated with 9E5‐conjugated phase‐change nanodroplets (PCNDs) for 24 h following ultrasound exposure

Figure 3A shows the association between threshold reduction and PCND concentration (N = 5). Following exposure to ultrasound at a peak negative pressure of 2.0 MPa, decrease in viability was observed for DLD‐1 cells treated with 9E5‐ or CPP‐conjugated PCNDs at a concentration of 1011 droplets/mL as well as for AGS cells treated with CPP‐conjugated PCNDs at a concentration of 1011 droplets/mL. The trends were similar for DLD‐1 and AGS cells, such that cell viability decreased after intracellular delivery of PCNDs at a concentration of 1011 droplets/mL. These results indicate that cells can be damaged by intracellular vaporization even at a low negative pressure when sufficient PCNDs are delivered inside the cells. Figure 3B shows viability of DLD‐1 and AGS cells treated with 9E5‐conjugated PCNDs for 24 hours (N = 5) following ultrasound exposure at a peak negative pressure of 5.1 MPa. Significant differences in cell viability were observed at a concentration of 1010 droplets/mL. This result indicates that cells can be damaged by intracellular vaporization even at a low PCND concentration when sufficient negative pressure is applied. Figure 3C shows viability of cells treated with 9E5‐conjugated PCNDs at a concentration of 1010 droplets/mL following ultrasound exposure at a peak negative pressure of 2.8 MPa. These results indicate that cell viability decreased even at the lower concentration of PCNDs, which formed aggregates to some degree inside the cell.

Figure 3.

Figure 3

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Effect of phase‐change nanodroplet (PCND) concentrations on the cytotoxicity threshold reduction effect. Cell viability after ultrasound exposure at a peak negative pressure of A, 2.0 MPa and B, 5.1 MPa (blue circle: DLD‐1 cells treated with 9E5‐conjugated PCNDs; red circle: DLD‐1 cells treated with cell‐penetrating peptide [CPP]–conjugated PCNDs; blue square: AGS cells treated with 9E5‐conjugated PCNDs; and red square: AGS cells treated with CPP‐conjugated PCNDs). C, Cell viability after treatment with 1010 droplets/mL of 9E5‐conjugated PCNDs and ultrasound exposure at 2.8 MPa

3. DISCUSSION

In this study, we demonstrated that intracellular delivery of PCNDs into targeted cells can reduce the ultrasound pressure for vaporization to induce cytotoxicity. CLSM and flow cytometry revealed that prolonged PCND‐cell incubation time increased PCND uptake and aggregation. A portion of the aggregated individual PCNDs may have increased in size via coalescence and/or Ostwald ripening.27, 28 Increase in droplet size decreases the vaporization threshold due to reduction in the Laplace pressure applied to droplets.27, 29 This aggregation might have contributed to the cytotoxicity threshold reduction effect.

In ultrasound exposure experiments, a significant decrease in cell viability was observed in cells that incorporated PCNDs (35%) but not in cells that did not incorporate PCNDs (88%). With this experiment, we assessed the cytotoxicity threshold reduction effect via decreased cell viability; however, the mechanism underlying this threshold reduction effect remains unknown. Several factors may contribute to the cytotoxicity threshold reduction effect. Specifically, reduction in vaporization pressure threshold is assumed to play a major role since extracellular PCNDs did not vaporize although intracellular PCNDs did. A previous study has shown that aggregated PFC nanoparticles behave as a single particle, leading to increase in the pressure amplification effect.28 This pressure amplification effect is the key mechanism of vaporization; nonlinear effects of acoustic waves generate superharmonic components and focus these superharmonic components inside the droplets.9, 11, 12 Therefore, we assumed that intracellular aggregation of individual droplets reduced the vaporization threshold, which, in turn, is related to cytotoxicity. Direct observations of instantaneous intracellular vaporization at a nanosecond‐to‐microsecond range would be necessary to clarify the mechanism underlying the cytotoxic threshold reduction effect induced by vaporization via intracellular delivery of PCNDs.

Although cytotoxic threshold reduction occurred under in vitro conditions, further investigations are imperative to determine whether this effect occurs in vivo and whether adequate PCNDs can be delivered to extravascular tumor sites to induce internalization and aggregation. In particular, in cases where PCNDs are locally injected at tumor tissue sites, higher concentration of PCNDs could potentially be achieved.30, 31 Moreover, use of low–boiling point PFCs as internal composites of droplets7, 32, 33, 34 and application of increased ultrasound frequency8, 9, 35, 36, 37 would reduce the vaporization threshold. This combination of conventional methodologies has the potential to further decrease the cytotoxicity threshold via intracellular vaporization of PCNDs.

4. CONCLUSIONS

Selective intracellular delivery of PCNDs to targeted cells can reduce required ultrasound pressure for vaporization to induce cytotoxicity. CLSM and flow cytometry revealed that prolonged PCND‐cell incubation time increased PCND uptake and aggregation. This aggregation effect contributed to the cytotoxicity threshold reduction effect. Although the mechanisms underlying this cytotoxic threshold reduction effect warrant further explanation, our approach of using a combination of intracellular delivery of PCNDs and low‐intensity ultrasound may be a powerful ultrasound‐targeted cancer therapy.

5. MATERIALS AND METHODS

5.1. Preparation of AF647‐labeled 9E5‐conjugated PCNDs

The anti‐EREG antibody (9E5) was selected as the active targeting agent due to its potential to induce PCNDs' internalization. 9E5 hybridoma cells were intraperitoneally implanted in BALB/c nude mice, and ascites were obtained and purified on a Protein G column. 9E5 monoclonal antibody was prepared and biotinylated as described previously.23, 24, 25 All animals were maintained in accordance with the regulations set by the University of Tokyo, and all animal experiments were conducted following institutional guidelines. The animal study protocol was approved by the University of Tokyo (#RAC120101).

Biotinylated PCNDs (internal composition: 1:1 mixture of perfluoropentane and perfluorohexane), provided by the Central Research Laboratory Hitachi (Tokyo, Japan), were prepared as described previously.7, 35 The composition ratio of 1:1 was selected to ensure thermal stability (boiling point: 40°C) and sensitivity to activation by ultrasound.7 The particle size distribution of PCND suspensions was assessed using a laser diffraction particle analyzer (LS13320, Beckman Coulter, USA). Biotinylated 9E5 was conjugated to PCNDs via SA‐AF647 (Life Technologies, USA), and AF647‐labeled 9E5‐conjugated PCNDs were prepared as described previously.23 Briefly, biotinylated 9E5 and SA‐AF647 were mixed in phosphate‐buffered saline (PBS) at concentrations of 0.4μM each at a total volume of 50 μL and incubated for 15 minutes at room temperature to form AF647‐labeled 9E5‐SA conjugates. The biotinylated PCND dispersion was mixed with a solution containing AF647‐labeled 9E5‐SA conjugate at a volume ratio of 1:1. The mixture was incubated for 30 minutes, and unconjugated 9E5 and SA‐AF647 were removed by centrifugation.

CPP (biotin‐G3R15GYC, 3081.67 Da; TORAY Research Center, Inc, Japan) was used as a positive control for intracellular delivery of PCNDs. Polyarginine‐based CPP has the potential to deliver PCNDs nonspecifically.26 Biotinylated CPP was alternatively added at a concentration of 0.8μM to biotinylated 9E5 to form CPP‐conjugated PCNDs.

5.2. Cell culture

The human colonic adenocarcinoma cell line DLD‐1 and the human gastric cancer cell line AGS were selected as the high and low EREG‐expressing cancer cell lines, respectively. The conjugation of 9E5 to PCNDs permits selective targeting of PCNDs to high EREG‐expressing cells and induces internalization by endocytosis. DLD‐1 and AGS cells were cultured at an initial density of 2 × 105 cells per dish in 35‐mm cell culture dishes containing RPMI medium supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified atmosphere with 5% CO2. All experiments were conducted 1 day after inoculation. The same procedure was used for cells cultured in 35‐mm glass bottom dishes.

5.3. CLSM and flow cytometry of targeted cells

To evaluate selective internalization and intracellular aggregation of 9E5‐conjugated PCNDs, the targeted cells were observed by CLSM and the number of targeted PCNDs and the fraction of bound DLD‐1 cells were quantitatively assessed by flow cytometry. First, cultured DLD‐1 and AGS cells were treated with 9E5‐conjugated PCNDs. All culture medium in the dish was aspirated after 24 hours of inoculation. The cells were washed with PBS, and 2 mL of RPMI‐diluted 9E5‐conjugated PCND solution (1011 droplets/mL) was added. After incubation with 9E5‐conjugated PCNDs, all solution was removed. Incubation times were set at 1, 3, 6, 24, or 48 hours. Nontargeted PCNDs were removed via an “inverted” washing procedure, as described previously.23 After washing twice, cells were assessed by either CLSM or flow cytometry. Cells without PCNDs (cells only) and CPP‐PCNDs were incubated in the same manner and used as additional negative and positive controls, respectively. We considered that 9E5‐conjugated PCND uptake might be inhibited by the addition of free 9E5 to DLD‐1 cells because free 9E5 may bind to EREGs and block the binding of 9E5‐conjugated PCNDs.23 Hence, 1 mL of 1.0 μM 9E5 antibody solution was added to DLD‐1 cells before adding 9E5‐conjugated PCNDs. After incubation for 3 hours, 1 mL of RPMI‐diluted 9E5‐conjugated PCNDs was added at a final concentration of 1011 droplets/mL.

For CLSM, cellular membranes were stained with NeuroDiO cell‐labeling solution (30021, Biotium Inc, USA) following the manufacturer's recommended protocols. The cells were observed by CLSM (LSM510 META‐ConfoCor 3, Carl Zeiss, USA). For flow cytometry, 1 mL of PBS was added to the dish and removed in the tube, and then 100 μL of 0.25% EDTA‐trypsin solution was added to the dish. After 3 minutes of incubation, 2 mL of PBS was added, and all media were removed to the same tube. The tube was centrifuged at 1,400 rpm for 3 minutes, and the supernatant was aspirated. The pellet was resuspended in 200 μL of cold PBS and incubated on ice until measurement using a flow cytometer (BD FACSCalibur, BD Biosciences, USA). Cells without PCNDs (cells only) and CPP‐PCNDs were incubated in the same manner and assessed by CLSM and flow cytometry as additional negative and positive controls, respectively.

Intracellular aggregations were evaluated by comparing AF647‐labeled PCND‐derived fluorescence intensity inside and outside DLD‐1 cells. After 24 hours of incubation with 9E5‐conjugated PCNDs, as described above, 2‐μL calcein AM solution (341‐07901, Dojindo Laboratories, Japan) and 10‐μL Hoechst solution (H347, Dojindo Laboratories) were added to the dish to stain the cytoplasm and nuclei, respectively. Cells treated with 9E5‐conjugated PCNDs were observed by CLSM without washing.

5.4. Ultrasound exposure and flow cytometry analysis of cell viability

The cytotoxicity thresholds for the peak negative pressure for intracellular and extracellular vaporization were evaluated. A silicon ring (inner diameter = 21 mm; outer diameter = 26 mm; thickness = 15 mm) was placed on a 35‐mm culture dish to avoid dish‐edge culturing because of difficulty in coherent focusing at the dish edge. The cells were seeded at an initial density of 104 cells per dish in RPMI medium supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified atmosphere with 5% CO2. After overnight incubation, the cells were washed with PBS. Then, 2 mL of RPMI‐diluted 9E5‐conjugated PCND solution (1011 droplets/mL) was added to the cells, and cells were incubated for 0, 1, or 24 hours. For incubation times less than 24 hours, 2 mL of RPMI‐diluted 9E5‐conjugated PCNDs was incubated in a 35‐mm dish for a certain amount of time to make the total incubation time 24 hours, and cells were treated with incubated PCNDs. Cells without PCNDs (cells only) and CPP‐PCNDs were incubated in the same manner as additional negative and positive controls, respectively. For DLD‐1 cells treated with 9E5‐conjugated PCNDs, EREG blocking was performed in the same manner as described above. AGS cells were also tested.

Ultrasound was applied to cultured DLD‐1 and AGS cells using the experimental system illustrated in Figure 2A. An ultrasound imaging probe (EUP‐L73S, Hitachi, Ltd, Japan) connected to a programmable ultrasound imaging system (V1, Verasonics, USA) was used to deliver vaporization pulses to PCNDs. Cell culture dishes were positioned at 36 mm from the transmission surface of the ultrasound imaging probe using a custom‐made dish holder connected to a single‐axis motorized stage (ALZ‐115‐E1P, Chuo Precision Industrial, Japan). The pulse length was set to five cycles at 5 MHz, with peak negative pressures ranging from 1.2 to 5.1 MPa (measured inside the dish where cells are seeded, see Section 5.5). The focus of transmitted ultrasound was set at 38 mm from the transmission surface of the ultrasound imaging probe. Such short pulses were used to avoid unintended cavitation that could damage the cells nonspecifically. The acoustic profile measured inside the dish is shown in Figure 2B. The focus was electronically scanned from −1.20 to 1.20 mm (the center of the probe was set to 0 mm), with a 0.20‐mm pitch in the lateral direction of the probe; the pulse repetition frequency was set to 0.5 kHz. These sonication conditions achieved successful vaporization of PCNDs.23 The probe was positioned by a two‐axis motorized stage (ALD‐604‐E1P, Chuo Precision Industrial) with a 0.30‐mm pitch in the elevational direction and a 1.20‐mm pitch in the lateral direction, both at an interval of 100 milliseconds. Trigger signals were transmitted to the programmable ultrasound imaging system at each position to apply six sets of the above‐described vaporization pulses. The water tank was filled with degassed water, and the temperature was maintained at 37°C.

Immediately after sonication, 2 μL of calcein AM (341‐07901, Dojindo Laboratories) was added to the dishes, resulting in a final concentration of 1 μL/mL. Calcein AM is used to distinguish viable and nonviable cells. After 5 minutes of incubation, all media were moved to a tube following ultrasound exposure and calcein AM addition. Cell viability was quantitatively assessed using flow cytometry following the procedure described above.

5.5. Acoustic profile measurement

The acoustic pressure profile inside the dish was measured using a calibrated needle hydrophone (type: 80‐0.5‐4.0, Imotec Meßtechnik, DE). The hydrophone was positioned by a three‐axis motorized stage (ALS‐906‐E1P; ALD‐301‐HM, Chuo Precision Industrial) and scanned along the lateral and elevation axes of the ultrasonic probe with a pitch of 0.1 mm. The water tank was filled with degassed water.

5.6. Statistical analysis

Unpaired two‐tailed Student t test was used to establish the significance of differences between the experimental groups. A P value <0.05 was considered statistically significant. All analyses were performed using Microsoft Excel 2013 (Microsoft, USA).

CONFLICT OF INTEREST

This research is partly supported by Olympus Corporation in the framework of Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program: Translational Systems Biology and Medicine Initiative funded by the Ministry of Education, Culture, Sports and Technology of Japan.

ACKNOWLEDGEMENTS

The authors thank Dr Kenichi Kawabata and Rei Asami for their kind consultations and for providing PCNDs. We thank Miho Kisaka, Kenta Nakamae, Dr Mariko Iijima, Dr Kousuke Minamihata, Dr Shinya Yamahira, Dr Keiichi Nakagawa, Dr Yuki Akagi, and Prof. Shu Takagi for participating in the discussions and assisting with experiments. This work was partially supported by a grant for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program: Translational Systems Biology and Medicine Initiative funded by the Ministry of Education, Culture, Sports and Technology of Japan and Grant‐in‐Aid from Olympus Corporation. A.I. was partially supported by a Grant‐in‐Aid from the Japan Society for the Promotion of Science (JSPS) Research Fellows.

AUTHORS' CONTRIBUTIONS

All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization, A.I., S.Y., T.A., E.K., Y.S., T.N., and I.S.; Methodology, A.I., S.Y., T.A., E.K., Y.S., T.N., and I.S.; Investigation, A.I.; Formal Analysis, A.I., S.I., and T.A.; Resources, A.I., S.Y., T.A., E.K., Y.S., T.N., and I.S.; Writing‐Original Draft, A.I.; Writing‐Review & Editing, A.I., S.Y., T.A., E.K., Y.S., T.N., and I.S.; Visualization, A.I.; Supervision, I.S.; Funding Acquisition, A.I., T.N., and I.S.

Ishijima A, Yamaguchi S, Azuma T, et al. Selective intracellular delivery of perfluorocarbon nanodroplets for cytotoxicity threshold reduction on ultrasound‐induced vaporization. Cancer Reports. 2019;2:e1165. 10.1002/cnr2.1165

Contributor Information

Takashi Azuma, Email: azuma@bmpe.t.u-tokyo.ac.jp.

Ichiro Sakuma, Email: sakuma@bmpe.t.u-tokyo.ac.jp.

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