Poly(ethyleneoxide)-block-poly(caprolactone) (PEG-PCL, PEG: 2000 Da, PCL: 2000 Da) and FA modified PEG-PCL (FA-PEG-PCL) were obtained from the Ruixi Biological Technology Co., Ltd (Xi’an, China). Perfluoropentane (PFP) was purchased from Strem Chemicals, Inc. (Newburyport, USA). Doxorubicin (DOX) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). DMEM medium, L-15 medium, fetal bovine serum (FBS) and penicillin/streptomycin were all provided by Life Technologies corporation (gibco®, Grand Island, NY, USA). Human breast cancer cells MDA-MB-231 were obtained from the Shanghai cell bank (Chinese Academy of Sciences, China). Fluorescent dyes DiI and Hoechst were obtained from Beyotime biotechnology Co., Ltd. (Shanghai, China).

The preparation of FA-NDs/DOX follows the process shown in Fig. 1. The FA-PEG-PCL copolymer and DOX were dissolved in Tetrahydrofuran (THF), and the solution was added to stirred deionized water. The ratio of THF and deionized water was 1:4. The concentration of FA-PEG-PCL and DOX were 3 mg/mL and 0.75 mg/mL respectively. Then the THF was removed by rotary evaporation, and the solution of FA linked micelle loaded with DOX (FA-micelle/DOX) was obtained. To remove the unloaded DOX, the micelle solution was dialyzed in deionized water using a dialysis bag (3000 Da, Viskase®, Chicago, IL, USA).

Fig. 1.

The schematic diagram of preparation process of FA-NDs/DOX.

As the liquid core of NDs, the PFP (2%, v/v) was added to the solution of FA-micelle/DOX, and the mixed solution was sonicated for 5 min by a Scientz08-Ⅱ non-contact ultrasonic cell crushing instrument (Scientz Biotechnology Co., Ltd, Ningbo, China). An ice water bath was used to maintain the temperature around the sample during sonication. The resulting emulsion was FA linked NDs loaded with DOX (FA-NDs/DOX). The non-targeted NDs was prepared by replacing the FA-PEG-PCL with PEG-PCL. Blank FA linked NDs (FA-NDs) were prepared without the addition of DOX. NDs with fluorescent dye DiI (10 µM) were prepared by adding DiI in the steps used for micelle preparation.

A Zetasizer Nano ZSE instrument (Malvern Instruments Ltd., Malvern, UK) was used to measure the particle size of the FA-micelle/DOX and FA-NDs/DOX. Transmission electron microscopy (TEM, HT7700, HITACHI, Tokyo, Japan) was used to observe the morphology of the NDs. Prior to observation, NDs dispersions were deposited onto copper grids and then stained with uranyl acetate and dried. The fluorescence of DOX loaded by FA-NDs and the morphology of the FA-NDs/DOX after incubated at 37 °C was observed through an Olympus IX73 inverted fluorescence microscope (Tokyo, Japan).

To determine the drug loading of FA-NDs/DOX, standard curves of DOX concentration and absorbance at 480 nm were plotted using an UV-1800S spectrophotometer (Macy, Shanghai, China). The DOX concentrations of FA-micelle/DOX solution and supernatant of the FA-NDs/DOX solution after centrifugation were determined, and the drug-loading content of the FA-NDs/DOX was calculated as the difference between the two concentrations.

It is assumed that the loss of very small amount of polymer and PFP during preparation is negligible, the mass concentration of the NDs could be calculated according to the total mass of the component (the mass of FA-PEG-PCL, PFP and DOX loaded) and the volume of the NDs solution. The concentration of NDs after dilution can be calculated based on the initial concentration and dilution factor.

The in vitro ultrasound imaging of FA-NDs/DOX was conducted in a glass container filled with degassed water to prevent the impact of air on ultrasound penetration. A sound-absorbing sponge was used to reduce the interference of echo reflection. The water in the glass container was heated and monitored by a water bath (Yushen Instruments Co. Ltd., Shanghai, China). The FA-NDs/DOX solution was diluted to the concentration of 0.75 mg/mL and 1.5 mg/mL with degassed water. Then plastic pipettes (Xinkang Medical Devices Co., Ltd., Taizhou, China) filled with FA-NDs/DOX solution (1.5 mL) was sealed and immersed into the beaker. A control group was developed using plastic pipettes with degassed water. About 10 min later, a linear array transducer (L74M, 5–13 MHz, HITACHI) was immersed into the water to radiate the pipettes with NDs, and the contrast enhancement under contrast mode was monitored and recorded by a ultrasound diagnostic system (HI VISION Ascendus, HITACHI) [10]. The frequency of the transducer was set at 13 MHz during experiments, and the single focus was placed at 2 cm. The imaging acquisition type under contrast enhanced ultrasound mode was pulse inversion. The ultrasound imaging was conducted at 25 °C and 37 °C respectively, and MIs of ultrasound transducer ranged from 0.08 to 1.0 at each temperature.

MI is a parameter directly given by the ultrasound diagnostic system in the present study, which is a measure of the power of an ultrasound transducer and can be defined as [27]:

(1)$$MI=P/\sqrt{f}$$

where P was the peak negative pressure of the ultrasound wave (MPa); f is the frequency of the ultrasound wave (MHz). When the frequency is fixed, the MI is proportional to the peak negative pressure. The peak negative pressure of the ultrasound could be measured by a hydrophone [28].

For the ultrasound diagnostic system used in this study, the MI displayed by the device is the MI at the transducer. Considering the attenuation of ultrasound in water and pipette wall (made of polyethylene), as well as the ultrasound reflection at the interface between water and pipette wall, a compensation factor for the MIs need to be calculated.

For a sound wave with initial acoustic pressure of P${}_{\mathrm{0}}$, the acoustic pressure after traveling x distance P(x) can be expressed as [29]:

(2)$$P(x)=P_0{e}^{-ax}$$

where a is the attenuation coefficient of the medium (Neper, 1 Neper = 8.686 dB/cm); x represents the distance traveled (cm); e is the base of the natural log.

When the sound wave vertically transmit from medium Ⅰ to medium Ⅱ, the reflection coefficient r${}_p$ and refraction coefficient $\tau$${}_p$ of the acoustic pressure are expressed as [29]:

(3)$$r_p=\left(Z_2-Z_1\right)/\left(Z_2+Z_1\right)$$

(4)$${\tau }_p=2Z_1/\left(Z_2+Z_1\right)$$

where Z${}_{\mathrm{1}}$ and Z${}_{\mathrm{2}}$ is the acoustic impedance (Rayl) of medium Ⅰ and medium Ⅱ respectively.

The attenuation coefficient and acoustic impedance of water is 0.002 dB/cm$\cdot$MHz and 1.48 $\times$ 10${}^6$ Rayl respectively [30]. The attenuation coefficient and acoustic impedance of polyethylene is 6.090 dB/cm$\cdot$MHz and 1.93 $\times$ 10${}^6$ Rayl respectively [31]. The acoustic impedance of the NDs solution is approximately equal to that of water due to the low concentration of NDs (0.75 mg/mL or 1.5 mg/mL). The distance from the transducer to the pipette is about 2 cm, and the thickness of the pipette wall is 0.05 cm. The ultrasound vertically transmit from water to pipette wall, and the frequency of the ultrasound is 13 MHz. According to the Eqns. 1,2,3,4, the MI inside the pipette is about 47.5% of the MI of the transducer. Therefore, the MIs inside the pipette ranged from 0.04 to 0.48 in the imaging experiments. The MIs in the following parts represent the MIs inside the pipette.

The contrast enhancement intensity was analyzed using EZU-CH9 software version Q1C-EZ1249-5 (HITACHI,) installed on the HI VISION Ascendus diagnostic system. The dynamic ultrasound imaging was loaded into the software, and ellipse regions of 0.8 cm${}^2$ were selected as the regions of interest (ROIs) within the pipettes. The location and area of the ROIs were consistent for all MIs. The acoustic signal intensity within the ROIs over time was analyzed by DAS software. The quantitative analysis technology of DAS software is based on the principle of “tracer dilution”. UCA (MBs) are used as the tracer, and the “dilution method” model of UCA acoustic video intensity is established according to the relationship of video signal intensity change over time within ROI. The time intensity curve (time intensity curve, TIC) is provided by the software, and the peak intensity (peak intensity, PI) could be obtained through fitting the TIC.

MDA-MB-231 cells (10${}^5$ per well) were planted into 6-well plates and cultured in DMEM medium (10% FBS, 1% penicillin/streptomycin). DMEM medium or DMEM medium deficient in FA were used according to the following groups: DMEM medium with NDs without FA modification, DMEM medium with FA-NDs and DMEM medium deficient in FA with FA-NDs. Corresponding NDs stained by Dil were added to the 6-well plates 24 h after cell attachment, and the three groups were named NDs/DiI, FA-NDs/DiI and FA(+)-NDs/DiI, respectively. The cells were cultured with NDs for 45 min. Then the cells were washed three times with PBS, and 4% paraformaldehyde was used to fix the cells. Finally, the cells were stained by Hoechst. Targeting conjugation of NDs were investigated with inverted fluorescence microscopes. As DiI and Hoechst have different excitation and emission wavelengths, they can be observed separately by using different filters. The wavelengths of exciter filter and emission filter for observing DiI were 460 nm–550 nm and 590 nm respectively. The human breast cancer cell lines MDA-MB-231 and MCF-7 were both purchased from Cell Bank/Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cell lines used have been authenticated by short tandem repeats, and mycoplasma testing has been done. The wavelengths of exciter filter and emission filter for observing Hoechst were 330 nm–400 nm and 425 nm respectively. The fluorescence intensity was analyzed by Image J software version 1.52b (National Institutes of Health, Bethesda, MD, USA) [32].

MDA-MB-231 cells (10${}^5$ per well) were planted into 6-well plates and incubated in L-15 medium (10% FBS, 1% penicillin/streptomycin) at 37 °C for 24 h. Then FA-NDs/DOX was added (1 mg/mL) to the plates and cultured with cells for another 4 h. The NDs/DOX (1 mg/mL) without FA modification was set as control. The subsequent cell staining and cell fixation methods were similar to that in section 2.5. Cellular uptake of NDs were observed with inverted fluorescence microscopes. The wavelengths of filters for observing DOX were the same as that of DiI (section 2.5), and the wavelengths of filters for observing Hoechst were the same as that in the tumor targeting experiments (section 2.5). The fluorescence intensity was analyzed by Image J software [32].

MDA-MB-231 cells (2 $\times$ 10${}^4$ per well) were planted into 96-well plates and incubated with L-15 medium (10% FBS, 1% penicillin/streptomycin) at 37 °C for 24 h. According to the type of NDs added to the plate, three experimental groups were set: NDs without DOX, DOX loaded NDs and DOX loaded NDs combined low intensity focused ultrasound (LIFU). The NDs (3 mg/mL) was diluted to the desired concentration with fresh L-15 medium, which was used to replace the medium in 96-well plate before toxicity experiments. Each group included four different concentrations of NDs, i.e., 62.5 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL, and the corresponding concentration of DOX was 1.79 µM, 3.59 µM, 7.18 µM and 14.35 µM. Six parallel holes were set for each concentration. After addition of NDs for 2 h, the group of DOX loaded NDS combined with LIFU was irradiated with LIFU (650 KHz, Chongqing Medical University, Chongqing, China) for 1 min per well (3.5 W, 50% duty cycle). After 72 h of interaction with NDs, the Cell Counting Kit-8 (Beiren Chemical Technology Co., Ltd, Beijing, China) was added to react with the living cells for 2 h. The viability of cells was determined by measuring the reaction product of formazan with Synergy H1 enzyme plate analyzer (BioTek, Burlington, VT, USA).

The flow cytometry assay was carried out to assess cell apoptosis. MCF-7 cells or MDA-MB-231 cells were seeded into 6-well plates with L-15 medium (10% FBS, 1% penicillin/streptomycin) at 37 °C for 24 h. Then, the two types of cells were each divided into five groups: control (PBS), FA-NDs without DOX (FA-NDs), LIFU, FA-NDs/DOX and FA-NDs/DOX combined LIFU (FA-NDs/DOX-LIFU). Three parallel holes were set for each group. The specific treatment method of the cells was similar to that previously described, and the concentration of NDs was 250 µg/mL for all the groups. After addition of FA-NDs for 2 h, the group of FA-NDs/DOX-LIFU was irradiated with LIFU for 3 min per well (650 KHz, 3.5 W, 50% duty cycle). After 72 h of interaction with FA-NDs, the cell apoptosis was measured by applying the Annexin V-FITC Apoptosis Detection Kit (Solarbio, Beijing, China) and PI Flow Cytometry Kit (Solarbio) to treat each group of cells in line with the specifications. The apoptotic rates were recorded under flow cytometry. The experimental procedure to obtain the cytotoxicity is shown in Fig. 2.

Fig. 2.

The flow chart of cell apoptosis measurement of FA-NDs/DOX combined LIFU on breast cancer cells.

To evaluate the cavitation effect of LIFU on MBs, CEUS experiments of the FA-NDs/DOX irradiated by LIFU was performed. The FA-NDs/DOX solution (3 mg/mL) was added to the 96-well plate and incubated at 37 °C for 2 h. Then the FA-NDs/DOX were divided into 4 groups, each group was irradiated with LIFU (650 KHz, 3.5 W, 50% duty cycle) for 0 s (i.e., without LIFU irradiation), 30 s, 1 min and 2 min, respectively. The CEUS and intensity analysis for each group of NDs solution were conducted using the method and parameters in section 2.4.

The mean $\pm$ standard deviation (SD) of at least three independent samples was used to represent the results, and the SD is represented by error bars in each graph. Independent-samples T test was used to compare the experimental data of two groups, and one-way ANOVA was used to compare the experimental data of three or more groups. A value of p 0.05 was considered significant, and p 0.01 was considered highly significant.

The particle size distribution of FA-micelle/DOX (Fig. 3A) and FA-NDs/DOX (Fig. 3B) measured through dynamic light scattering (DLS) is shown in Fig. 3. The particle size distribution were obtained in intensity, volume and number. The particle size of FA-micelle/DOX in intensity, volume and number were 139.9 $\pm$ 1.6 nm, 115.3 $\pm$ 1.4 nm and 79.0 $\pm$ 1.4 nm, respectively. For FA-NDs/DOX, the particle size in intensity, volume and number were 448.0 $\pm$ 8.9 nm, 582.3 $\pm$ 36.4 nm and 272.2 $\pm$ 12.5 nm, respectively. The particle dispersion index (PDI) of micelle and NDs were 0.12 and 0.21 respectively.

Fig. 3.

Particle size distribution and zeta potential of FA-micelles/DOX and FA-NDs/DOX. Particle size distribution of FA-micelles/DOX (A) and FA-NDs/DOX (B) presented in intensity, volume and number. Zeta potential of FA-micelle/DOX (C) and FA-NDs/DOX (D).

The larger particle size of NDs compared with that of the micelle indicates the successful encapsulation of PFP. The differences among the particle size in intensity, volume and number may be due to the fact that the uniformity of the particles is not very high. For a single particle, the volume and intensity of large particle is much larger than that of small particle, so the ratio of volume to intensity could not directly represent the number of particles. The particle size distribution in number indicated that the number of particles with particle size below 300 nm accounts for the majority, which is beneficial for the NDs to penetrate blood vessels at the tumor site. The zeta potential of FA-micelle/DOX and FA-NDs/DOX were 27.5 $\pm$ 0.3 mV (Fig. 3C) and 30.43 $\pm$ 0.32 mV (Fig. 3D) respectively. The high zeta potential could ensure their dispersibility and stability.

The morphology of FA-micelle/DOX and FA-NDs/DOX were observed by TEM. Both micelle (Fig. 4A) and NDs (Fig. 4B) appeared spherical under TEM, but NDs showed a clear shell-core structure due to the introduction of PFP. The particle size of micelle and NDs observed by TEM were about 100 nm and 500 nm respectively, which were in good agreement with that measured by DLS. The red fluorescence of DOX, shown in Fig. 4C, indicates that the DOX is successfully encapsulated, and the drug loading of FA-NDs was 15.6 µg/mg.

Fig. 4.

Morphology of FA-micelle/DOX and FA-NDs/DOX. Morphology of FA-micelle/DOX (A) and FA-NDs/DOX (B) observed by TEM. (C) Image of FA-NDs/DOX observed by the fluorescence microscope. Optical image of FA-NDs/DOX at 25 °C (D) and heated at 37 °C for 30 min (E). (F) Optical image of vaporized MBs in the upper layer of FA-NDs/DOX solution after heated at 37 °C for 30 min. (G) Particle size distribution (in intensity) of FA-NDs/DOX heated at 37 °C for 30 min.

The morphology of the FA-NDs/DOX heated at 37 °C was further observed. Before heating, the NDs were uniformly spherical particles under the microscope (Fig. 4D). A small amount of particles with larger size (5–10 µm in diameter) appeared after heating the NDs at 37 °C for 30 min (Fig. 4E). These larger particles and NDs distributed in different layers of the solution, because the shape of the particles was not clear in Fig. 4E. However, these larger particles can be clearly seen when the microscope was focused on the upper layer of the solution (Fig. 4F), and the smaller NDs in Fig. 4E almost disappeared from the image at this time, indicating that the larger particles should be vaporized MBs rather than large NDs formed by coalescence/fusion of small NDs. Though the vaporized MBs represent the phased transition induced by heating, the large amount of NDs that did not vaporize. Fig. 4G showed the particle size distribution of FA-NDs/DOX (37 °C, 30 min) in intensity. Compared with the average particle size of FA-NDs/DOX before heating (Fig. 4B, 448.0 $\pm$ 8.9 nm), the average particle size of FA-NDs/DOX in Fig. 4G only increased by 10.9%, indicating the stability of FA-NDs/DOX at 37 °C. Therefore, other stimulations such as ultrasound may be needed to induce the vaporization of more NDs.

The results of the CEUS of FA-NDs/DOX in vitro is shown in Fig. 5. The ultrasound imaging of FA-NDs/DOX was conducted under MIs ranging from 0.04 to 0.48. For degassed water, no acoustic signal was observed at all of the MIs under 25 °C and 37 °C. For NDs (25 °C, 0.75 mg/mL), no acoustic signal was observed until the MI reached 0.48 at 25 °C, and the contrast enhancement at MI of 0.48 was very weak. At 37 °C, the contrast enhancement of NDs with concentration of 0.75 mg/mL and 1.5 mg/mL both began to appear when MI reached 0.19, and the contrast enhancement was stronger for NDs under ultrasound with higher MIs. The duration of ultrasound imaging was also monitored. The contrast enhancement of FA-NDs/DOX decayed with time, but an obvious acoustic signal could still be observed at 30 min.

Fig. 5.

Contrast-enhanced ultrasound imaging of water at 37 °C, FA-NDs/DOX (25 °C, 0.75 mg/mL), FA-NDs/DOX (37 °C, 0.75 mg/mL) and FA-NDs/DOX (37 °C, 1.5 mg/mL) under MIs of 0.04, 0.19, 0.29 and 0.48.

Signal intensity of contrast-enhanced ultrasound under different MIs was analyzed to describe the ultrasound imaging of NDs (Fig. 6). The ROIs selected within the pipettes is shown in Fig. 6A. The results in Fig. 6B indicated that the signal intensity of NDs (37 °C, 0.75 mg/mL) were stronger than NDs (25 °C, 0.75 mg/mL) at MI of 0.19, 0.29 and 0.48 (p 0.01). NDs with the concentration of 1.5 mg/mL showed stronger acoustic signal than NDs with concentration of 0.75 mg/mL under the same MIs (p 0.01). Moreover, the signal intensity increased with MI for both NDs (37 °C, 0.75 mg/mL) and NDs (37 °C, 1.5 mg/mL), and the differences in signal strength between any two MIs is significant (p 0.05). The intensity analysis of contrast enhancement of NDs within 30 min under MI of 0.48 suggests that the signal intensity of NDs stayed above 60% and 20% of the initial intensity for the first 5 minutes and 30 minutes respectively (Fig. 6C). The acoustic signal of FA-NDs/DOX lasted much longer than that of MBs used in clinical practice.

Fig. 6.

Signal intensity of contrast-enhanced ultrasound for FA-NDs/DOX. (A) Schematic diagram of ROI with area of 0.8 cm${}^2$ for intensity analysis. (B) The intensity of contrast enhancement within ROI for NDs (25 °C, 0.75 mg/mL), NDs (37 °C, 0.75 mg/mL) and NDs (37 °C, 1.5 mg/mL) under MI of 0.04, 0.19, 0.29 and 0.48 (mean $\pm$ SD, n = 3; **p 0.01 vs NDs (25 °C, 0.75 mg/mL); ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs NDs (37 °C, 0.75 mg/mL). The difference in signal strength between any two MIs for NDs (37 °C, 0.75 mg/mL) and NDs (37 °C, 1.5 mg/mL) was significant (p 0.05). (C) The intensity variation of contrast enhancement for NDs (37 °C, 1.5 mg/mL) within 30 min under MI of 0.48.

In order to improve the tumor targeting of NDs, FA was used to modify the NDs. MDA-MB-231 cells with high expression of FR were selected to conduct the targeting experiment. In Fig. 5, the NDs and FA-NDs were stained by DiI (red), and the nucleus of MDA-MB-231 cells were stained by Hoechst (blue). The blue in Fig. 7 are the nucleus, and the red around the nucleus are mainly FA-NDs that bind to the cells after 45 min of co-incubation. The unbound NDs have been removed during the experimental operation. In Fig. 7A, there was nearly no red fluorescence around the nucleus for the non-targeted NDs group, implying that the NDs without modification of FA could not recognize and bind to cells. In Fig. 7B,C, the red around the nucleus should be FA-NDs binding to the membrane or entering the cytoplasm, indicating the targeted recognition of FA-NDs to cells. The intensity ratio of red fluorescence (DOX) to blue fluorescence (nucleus) was used to estimate the tumor targeting of NDs (Fig. 7D). The higher ratio of FA-NDs/DiI than NDs/DiI (p 0.01) indicated that more NDs were combined with tumor cells after modification of FA. The highest ratio for the group of FA(+)-NDs/DiI (FA-NDs with cells cultured without FA) provided further evidence of the function of FA in tumor targeting.

Fig. 7.

Targeted recognition of NDs by MDA-MB-231 cells observed by fluorescence microscope. (A) NDs/DiI. (B) FA-NDs/DiI. (C) FA (+)-NDs/DiI, i.e., FA-NDs/DiI and cells cultured without FA. The nucleus of MDA-MB-231 cell were stained with Hoechst and shown in blue, whereas NDs or FA-NDs were stained with DiI and shown in red. (D) Fluorescence intensity ratio of red to blue in (A–C) (mean $\pm$ SD, n = 3; **p 0.01 vs NDs/DiI; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs FA-NDs/DiI).

The cellular uptake of FA-NDs/DOX was compared with that of NDs/DOX (without modification of FA) by fluorescent microscopy, and the results was shown in Fig. 8. Both the NDs (Fig. 8A) and the FA-NDs (Fig. 8B) entered cells after 4 h of co-incubation. However, the red fluorescence of DOX appeared in cells was significant higher for FA-NDs/DOX than that of NDs/DOX, and most of the DOX had been entered cell nucleus for FA-NDs/DOX. The higher ratio of red fluorescence (DOX) to the blue fluorescence (nucleus) for FA-NDs/DOX (Fig. 8C) indicated that modification of FA could promote tumor cells to endocytosis of FA-NDs/DOX.

Fig. 8.

Cellular uptake of NDs by MDA-MB-231cells observed by fluorescent microscopy. (A) NDs/DOX without modification of FA. (B) FA-NDs/DOX. The nucleus of MDA-MB-231 cell were stained with Hoechst and shown in blue, whereas NDs/DOX or FA-NDs/DOX were shown in red. (C) Fluorescence intensity ratio of red to blue in (A,B) (mean $\pm$ SD, n = 3; **p 0.01 vs NDs/DOX).

As LIFU was introduced to the anti-tumor experiments, the cavitation effect of LIFU on FA-NDs/DOX was estimated by CEUS. The contrast-enhanced ultrasound imaging and signal intensity of FA-NDs/DOX irradiated by LIFU (3.5 W, 50% duty cycle) for 0 s, 30 s, 1 min and 2 min are shown in Fig. 9A,B. The enhancement intensity of FA-NDs/DOX irradiated by LIFU for 30 s, 1 min and 2 min was about 42%, 26% and 16% of that without irradiation by LIFU, respectively. The results proved that the irradiation of LIFU could induced effective cavitation and destruction of MBs, and the number of MBs decreased with the extension of irradiation time.

Fig. 9.

The CEUS of FA-NDs/DOX irradiated by LIFU and viability of MDA-MB-231 cells cultured with different NDs. (A) CEUS of FA-NDs/DOX irradiated by LIFU for 0 s, 30 s, 1 min and 2 min (37 °C, 0.75 mg/mL, MI = 0.48). (B) The intensity of contrast enhancement of FA-NDs/DOX irradiated by LIFU for 0 s, 30 s, 1 min and 2 min (mean $\pm$ SD, n = 3; **p 0.01 vs LIFU-0s. The difference in intensity between any two groups was significant (p 0.01). (C) Viability of MDA-MB-231 cells cultured with FA-NDs, FA-NDs/DOX and FA-NDs/DOX combined LIFU (FA-NDs/DOX-LIFU) when the concentration of NDs were 62.5 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL (mean $\pm$ SD, n = 6; **p 0.01 vs FA-NDs; ${}^{\mathrm{\#}}$p 0.05 vs FA-NDs/DOX; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs FA-NDs/DOX).

The cytotoxicity of FA-NDs/DOX with different concentrations to MDA-MB-231 cells were investigated. In Fig. 9C, the cell viability was above 90% at all concentrations for blank NDs, which indicated the well biocompatibility of FA-NDs as UCA. For FA-NDs/DOX, the cell viability decreased with the increase of concentrations (p 0.01 vs NDs group), which was mainly due to the toxicity of DOX loaded in FA-NDs. For FA-NDs/DOX combined LIFU (3.5 W, 1 min, 50% duty cycle), the cell viability was lower than that of single FA-NDs/DOX at the concentration of 125 µg/mL (p 0.05) and 250 µg/mL (p 0.01), implying that the LIFU enhanced the toxicity of FA-NDs/DOX.

The apoptosis of MDA-MB-231 cells and MCF-7 cells were also detected by flow cytometry after treatment with FA-NDs, LIFU, FA-NDs/DOX and FA-NDs/DOX combined with LIFU (FA-NDs/DOX-LIFU). In Fig. 10, we found that FA-NDs treatment had no effect on apoptosis of MDA-MB-231 cells (Fig. 10A) and MCF-7 cells (Fig. 10B). The cell apoptosis MCF-7 cell and MDA-MB-231 were significant after irradiation by LIFU alone or treated with FA-NDs/DOX alone (p 0.01). Following combined treatment with FA-NDs/DOX and LIFU, cell apoptosis was observed to be increased versus the LIFU and FA-NDs/DOX groups (p 0.01). These results indicate that only by combining irradiation by LIFU with FA-NDs/DOX can the targeted cell death effect be maximized.

Fig. 10.

The apoptotic rate of MDA-MB-231 cells (A) and MCF-7 cells (B) detected by flow cytometry after treated with FA-NDs, LIFU, FA-NDs/DOX and FA-NDs/DOX combined with LIFU (FA-NDs/DOX-LIFU). Representative images of cell apoptosis. Data are presented as the mean $\pm$ SD. n = 3. **p 0.01 vs the control group, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs the FA-NDs/DOX-LIFU group.