Dual ultrasound-activatable nanodroplets for highly-penetrative and efficient ovarian cancer theranostics

Chao Yang ab, Yi Zhang ac, Yuanli Luo a, Bin Qiao a, Xingyue Wang a, Liang Zhang a, Qiaoqi Chen a, Yang Cao a, Zhigang Wang a and Haitao Ran *a
aChongqing Key Laboratory of Ultrasound Molecular Imaging & Department of Ultrasound, the Second Affiliated Hospital of Chongqing Medical University, No. 74 Linjiang Road, Yuzhong District, Chongqing, 400010, P. R. China. E-mail: ranhaitao66@126.com
bDepartment of Radiology, Chongqing General Hospital, University of Chinese Academy of Sciences, 104 Pipashan Main Street, Yuzhong District, Chongqing 400014, P. R. China
cDepartment of Ultrasound, the First Affiliated Hospital of Chongqing Medical University, No. 1 Youyi Road, Yuanjiagang, Yuzhong District, Chongqing 400016, P. R. China

Received 8th October 2019 , Accepted 8th December 2019

First published on 9th December 2019


The selective delivery and deep intertumoral penetration of nanosensitizers remain challenging in the fabrication of sonodynamic therapy (SDT) platforms. In this work, we rationally constructed dual ultrasound (US)-activatable nanodroplets (NDs)/nanoliposomes/nanosensitizers with perfluoropentane (PFP) in the core, hematoporphyrin monomethyl ether (HMME) in the phospholipid shell and folate (FA)-conjugated to the surface (collectively termed FA-H@NDs). We aimed to validate the feasibility of these FA-H@NDs for FA receptor (FR)-overexpressed ovarian cancer theranostics. The ND formulations were based on PFP that can undergo acoustic droplet vaporization (ADV) when exposed to US irradiation. The ADV phenomenon disrupts the adjacent vasculature, and the resistance to drug diffusion within the tumor can be decreased, enabling nanosensitizers to more deeply penetrate into the inner tissue far from the intertumoral vasculature. These FA-H@NDs assisted by US irradiation can also induce the production of excess reactive oxygen species (ROS) and consequently trigger tumor cell/tissue apoptosis and necrosis. Furthermore, this therapeutic process can be guided and monitored by US/photoacoustic (PA) dual-modal imaging. This work established a new paradigm for highly efficient ovarian cancer theranostics based on the rational utilization of dual US-activatable NDs.


1. Introduction

Ovarian cancer is categorized as the most prevalent and fatal cancer among women.1,2 Ovarian cancer patients are generally asymptomatic, and there is a lack of appropriate testing methods for deep-seated ovarian cancer, leading to a pressing need for doctors to diagnose and treat this disease early.3 Current therapeutic interventions predominantly include chemotherapy and cytopenic surgery.4 Of course, the side effects induced by chemotherapy are already notorious, as they severely damage the patient's quality of life.5 In contrast, surgery might be somewhat effective; however, patients treated with surgery suffer from great pain.6 Therefore, developing an effective theranostic strategy is crucial for managing deep-seated ovarian cancer.

Sonodynamic therapy (SDT), an ultrasound (US)-based therapeutic approach, has recently shown increasingly extensive applications.7–12 Similar to photodynamic therapy (PDT), SDT kills cancer cells by inducing the overproduction of reactive oxygen species (ROS), but the ROS in SDT are generated via the activation of sonosensitizers.8,13 Compared to PDT, SDT can be delivered in depth because of the intrinsic properties of US; thus, this therapy is a suitable alternative for deep-seated tumors such as ovarian cancer.7,8 A prerequisite for highly efficient SDT is the desirable accumulation of sonosensitizers in the target lesions.14,15 Therefore, it is imperative to improve the accumulation efficiency and penetration depth of sonosensitizers. It has been proposed that, by virtue of the enhanced permeability and retention (EPR) effect, nanosonosensitizers can passively accumulate in tumors with relatively low efficiency.16,17 However, the unoriented diffusion and unsatisfactory penetration depth of sonosensitizers in tumor tissues further undermine their therapeutic efficiency.18–20 Overexpression of the folate (FA) receptor (FR) was observed in approximately 90% of epithelial ovarian cancers, and the proportion can considerably exceed 90% in ovarian cancers with serous morphology.21 Therefore, engineering sonosensitizers into FA-modified nanosystems may provide an excellent tumor targeting ability and competent therapeutic efficiency against ovarian cancer.22–24

To ensure better therapeutic effects, in addition to effectively aggregating in the tumor area of fabricated nanoplatforms, it is also necessary that these nanomedicines are able to enter the deeper part of the tumor rather than remaining near the tumor vessels. Recently, permeabilized blood vessels and tissue erosion were reported as a consequence of “acoustic droplet vaporization (ADV)”,25–30 a phase-transformation process of perfluorocarbon (PFP)-based droplets with the assistance of US. Namely, efficient US stimulation could convert superheated PFP droplets into a gaseous phase, which in turn increased the penetration depth of nanodroplets.29,30 Therefore, the ADV effect has great potential for the delivery of nanocarriers, especially for treating deep-seated cancer.

Imaging systems could be used during the therapeutic process to trace the distribution of the sonosensitizer and to guide therapeutic administration, improving the accuracy and efficiency of SDT.31–35 US imaging is a common examination technique because of its unique advantages of excellent penetration capability and real-time imaging.36 As a reliable US-activatable reagent that is also known for its phase-translation capability, PFP has been widely studied for enhancing US imaging.34,37 Importantly, upon US irradiation, the irradiation force converts nanosized droplets to microbubbles,35,38–40 simultaneously driving the NDs toward the inner tumor region.20,29 In addition to US imaging, photoacoustic (PA) imaging, a new nonionizing imaging technology, has achieved deep tissue penetration by converting photon energy into acoustic pressure waves and has been incorporated into a multifunctional theranostic nanosystem.41,42 Molecular imaging and targeted therapy, which combines nanosystems with drugs and imaging contrast agents, aim to achieve targeted delivery and evaluate the therapeutic efficiency. With the assistance of dual-imaging modalities (US and PA imaging), improvements in the accuracy and precision of targeted SDT can be expected.43

In this study, for highly efficient ovarian cancer theranostics, we rationally constructed dual US-activatable NDs with PFP-encapsulated in the core, hematoporphyrin monomethyl ether (HMME)-loaded in the phospholipid shell and FA conjugated to the surface (FA-H@NDs) (Fig. 1a). These FA-H@NDs can readily accumulate in ovarian tumor regions via both the passive EPR effect and FA-induced active targeting. HMME endowed NDs with PA imaging capability and SDT performance. Phospholipid shell-stabilized acoustic phase-changing PFP droplets can undergo ADV when exposed to US irradiation, further enhancing US imaging and the penetration of the NDs. In addition, these FA-H@NDs assisted by US irradiation can generate ROS to induce ovarian cancer cell/tissue apoptosis and necrosis. Both in vitro and in vivo systematic experiments were performed in this study to demonstrate the highly efficient theranostics of the constructed FA-H@NDs against ovarian cancer.


image file: c9tb02198a-f1.tif
Fig. 1 (a) Schematic illustration of the theranostic functions of dual US-activatable FA-H@NDs, including active targeting capability via FA, ADV-augmented deep penetration of sonosensitizers, SDT-induced tumor cells/tissue apoptosis/necrosis and guidance/monitoring by US/PA dual imaging. (b) Schematic illustration of the synthetic procedure of FA-H@NDs.

2. Results and discussion

2.1 Design, synthesis, and characterization of FA-H@NDs

FA-H@NDs, unique US-activatable NDs with a core/shell structure (PFP as the core and HMME/lipid as the shell) and FA modified on the surface, were constructed through a simple one-step emulsion method (Fig. 1b and Fig. S1 and S2, ESI). The successful loading of HMME in the FA-H@NDs turns the aqueous solution reddish brown in color, whereas the FA@ND solutions appear white (Fig. S3, ESI). These fabricated FA-H@NDs were found to have a well-defined spherical morphology (Fig. 2a). The average diameter of the FA-H@NDs was 337.1 ± 69.63 nm, as measured by dynamic light scattering (DLS, Fig. 2b). The desirable size support FA-H@NDs can accumulate into the tumor through leaky vasculature.44–46 The zeta potential of these NDs was −32.1 ± 4.11 mV, a favorable negative potential (Fig. 2c). The UV-vis spectrum showed that HMME exhibited a strong absorption peak at 398 nm and four small specific absorption peaks between 450 nm and 650 nm (Fig. 2d). The absorption of HMME behaves in a concentration dependent manner, according to which the standard curve of HMME is plotted and shown in Fig. 2e. Considering the poor solubility of HMME among almost all pharmaceutical solvents, the loading of HMME into biocompatible liposomes was conducted. Based on the formula in Fig. 2e, the loading efficiency of HMME in FA-H@NDs was calculated to be 82.67%. To further prove the efficient loading of HMME, the UV-vis spectrum of FA-H@NDs was compared with that of NDs and pristine HMME. It was found that FA-H@NDs had a similar peak absorption at approximately 390 nm, similar to that of HMME, while the ND spectrum showed a smooth curve without characteristic absorption (Fig. 2f). The results showed the successful loading efficiency of HMME. Considering the insolubility of HMME in all pharmaceutically acceptable solvents, within the FA-H@NDs, HMME quenched significantly more slowly than free HMME, which allows it to function in a difficult biological environment. As shown in Fig. S4 (ESI), after one day in the dark, FA-H@NDs maintained approximately 80.8% of the maximum absorbance. In contrast, free HMME was severely quenched. We measured the sizes of FA-H@NDs in fetal blood serum within a prolonged time of 7 d to monitor the colloidal stability of FA-H@NDs under simulated in vivo conditions (Fig. S5, ESI). It was found that the fluctuation of the size was negligible, indicating that FA-H@NDs were colloidally stable under certain physiological conditions.
image file: c9tb02198a-f2.tif
Fig. 2 Characterization of FA-H@NDs. (a) TEM image of FA-H@NDs. (b) The average diameter and (c) surface zeta potential of FA-H@NDs as detected by DLS. (d) UV-vis absorption spectra of HMME at elevated concentrations. (e) The standard curve of HMME by UV-vis absorption at 398 nm. (f) UV-vis absorption spectra of FA-H@NDs, FA@NDs and HMME.

2.2 In vitro ADV, US/PA imaging, and ROS generation of FA-H@NDs

Based on the phase-transformation capability of PFP, PFP loaded NDs can be converted into microbubbles upon US irradiation, which is known as ADV.25–30 This specific functionality endowed these fabricated FA-H@NDs with high stability as well as excellent US imaging performance. By virtue of the ADV effect, the phase transition of the FA-H@NDs immediately occurred upon US irradiation and could be visualized under optical microscopy. As depicted in Fig. 3a, a substantial number of FA-H@NDs were converted into microbubbles after US irradiation. Time dependent phase-transformation optical and fluorescence images of FA-H@NDs under US irradiation were also captured (Fig. S6, ESI). In addition, with prolonged irradiation time, contrast-enhanced US imaging (contrast mode) showed a marked enhancement feature, which was consistent with the time dependent bubble generation (Fig. 3b). The echo intensities in the region of interest were determined, and were consistent with the US imaging findings (Fig. 3c). These results confirmed that the PFP core endowed the FA-H@NDs with ADV capability and good US imaging performance. The combination of multimodality imaging is desirable and urgently needed. In addition to excellent US imaging performance, the FA-H@NDs also exhibited enhanced PA imaging capacity. In vitro PA imaging was performed at excitation wavelengths ranging from 680 nm to 970 nm to detect the optimal excitation wavelength. As shown in Fig. S7 (ESI), an evident peak at 685 nm was detected; therefore, 685 nm was selected as the best excitation wavelength in the following PA imaging experiments. In addition, with increasing FH-H@ND concentrations, the corresponding PA signal was enhanced (Fig. 3d). The PA images were also in accordance with the quantitative analysis.
image file: c9tb02198a-f3.tif
Fig. 3 In vitro ADV capability, US/PA imaging and SDT performance. (a) Phase-transformation optical images of FA-H@NDs before or after US irradiation. (b) US imaging of FA-H@NDs with prolonged US irradiation time. (c) The corresponding echo intensities of FA-H@NDs in B mode and contrast mode. (d) In vitro PA images and corresponding PA intensities of FA-H@NDs. (e) In vitro ROS evaluation in the presence of DPBF probe. (f) Time dependent ROS generation of FA-H@NDs as detected by SOSG with prolonged US irradiation.

Multifunctional FA-H@ND-assisted SDT was characterized by the amount of ROS generated to kill the target cells. The ROS probes 1,3-diphenylisobenzofuran (DPBF) and singlet oxygen sensor green (SOSG) were used in this study to detect the ROS generation in vitro. Compared to the three control groups, the FA-H@ND group exhibited an obviously increased ROS generation efficacy after US irradiation (Fig. 3e). The fluorescence intensity of SOSG increased with the US irradiation time at a fixed concentration of FA-H@NDs, revealing the generation of a large amount of ROS (Fig. 3f). These results suggested the potential of FA-H@NDs as a sonosensitizer to assist SDT against cancer cells.

2.3 Intracellular uptake efficiency of FA-H@NDs in vitro

To facilitate the accumulation of more NDs in the tumor site, it was necessary to modify the NDs with an active targeting molecule. In this work, considering that SKOV3 cells express a large amount of FR, we developed FR-targeting multifunctional NDs. To verify the efficient uptake of FA-H@NDs with the guidance of FA in vitro, H@NDs were used as a control. Both confocal laser scanning microscopy (CLSM) and flow cytometry were used to compare the uptake efficiency between the FA-H@NDs and H@NDs after different incubation times. The results in Fig. 4a show that an increasing amount of red fluorescence from the FA-H@NDs can be observed in SKOV3 cells, especially 4 h postincubation. Comparatively, only negligible red fluorescence from the H@NDs accumulated in cells. The flow cytometry analysis is in accordance with the CLSM observation (Fig. 4b), suggesting that FA favored active accumulation in SKOV3 cells.
image file: c9tb02198a-f4.tif
Fig. 4 In vitro endocytosis of FA-H@NDs by SKOV3 cells. (a) Intracellular uptake of FA-H@NDs and H@NDs by SKOV3 cells detected by CLSM after different incubation times (0.5 h, 1 h, 2 h, 3 h and 4 h). (b) Flow cytometry analysis of cell phagocytosis of FA-H@NDs and H@NDs. The numbers in the texts indicate the percentages of cells which have phagocytized NDs.

2.4 Intracellular ROS generation, in vitro cytotoxicity and sonotoxicity against SKOV3

After successfully demonstrating the efficiency of ROS production by the FA-H@NDs via DPBF and SOSG, we further tested ROS generation at the cell level. 2,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was assigned as an ROS probe that specifically labels intracellular ROS. Both the H@NDs and FA-H@NDs generated ROS intracellularly after US stimulation, as indicated by the obvious green fluorescence in SKOV3 cells (Fig. 5a). Nevertheless, the FA-H@NDs showed a stronger green fluorescence intensity than H@NDs under the same conditions, which was ascribed to the FA-mediated active targeting capability. Furthermore, flow cytometry showed that the proportion of positive cells (green fluorescence) treated with FA-H@NDs was 1.68-fold higher than cells treated with H@NDs after US irradiation (Fig. 5b). Efficient ROS generation in cells ensures the intracellular SDT effect against SKOV3 cells. Based on the above in vitro ROS generation results, we wondered whether FA-H@NDs could produce sufficient amounts of ROS to induce cytotoxicity and further cause necrosis or apoptosis.
image file: c9tb02198a-f5.tif
Fig. 5 Intercellular ROS detection. CLSM images (a) and flow cytometry analysis (b) of ROS generation in SKOV3 cells after various treatments. The green fluorescence represents a positive result of ROS generation after DCFH-DA staining. The numbers in the texts indicate the percentages of cells with ROS generation.

Next, the sonotoxicity of FA-H@NDs against SKOV3 cells was assessed by the standard CCK-8 assay and CLSM. First, the cells were incubated with various concentrations of FA-H@NDs for 4 h, 12 h, and 24 h in the absence of US irradiation. As shown in Fig. 6a, there was no evident cytotoxicity, even when the FA-H@ND concentration was as high as 0.4 mg mL−1. In contrast, the cell viability drastically decreased after treatment with FA-H@NDs at concentrations of 0.2 mg mL−1 and 0.4 mg mL−1 along with US irradiation. Notably, with the assistance of US irradiation, the cell viability in the FA-H@NDs + US group (6.15%) was significantly lower than that in the H@NDs + US group (38.53%) at the same concentration (0.4 mg mL−1) (Fig. 6b). According to CLSM, FA-H@NDs induced cell apoptosis (red fluorescence) after US irradiation, which was consistent with the CCK-8 results (Fig. 6c). As a result, cell damage was mainly induced by the FA-H@ND-mediated SDT effect.


image file: c9tb02198a-f6.tif
Fig. 6 In vitro SDT. (a) Relative cell viabilities of SKOV3 cells coincubated with FA-H@NDs at different concentrations for 4 h, 12 h and 24 h. (b) Relative cell viabilities of SKOV3 cells after various treatments. (c) CLSM images of live/dead cell staining after different treatments.

2.5 Biosafety assessment of FA-H@NDs

The high biocompatibility and biosafety of FA-H@NDs are prerequisites to ensure their subsequent in vivo application. Therefore, the blood of healthy mice was collected for blood cell analysis and biochemical examination to detect the influence of FA-H@NDs on mice. The blood cell indicators of the mice revealed almost no fluctuation in mice exposed to FA-H@NDs compared with that in healthy mice (Fig. 7a). In addition, there were no meaningful changes in indicators of hepatotoxicity and nephrotoxicity (Fig. 7b), showing the relative biosafety of FA-H@NDs, which was a desirable feature for further applications.
image file: c9tb02198a-f7.tif
Fig. 7 Biosafety assay in vivo. (a) Hematological indexes of the mice and (b) H&E staining of major organs 0 (control), 3, 7, 14 and 30 d after intravenous administration of FA-H@NDs.

2.6 In vivo targeting capability and biodistribution of FA-H@NDs

Considering the active targeting of FA-H@NDs to SKOV3 cells, fluorescence imaging was further performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR)-labeled FA-H@NDs as a contrast agent to observe its targeting capability and biodistribution in vivo. Initially, SKOV3 tumor-bearing mice were intravenously injected with DiR-labeled FA-H@NDs or H@NDs emulsion. After varied time points, fluorescence images were captured, and the corresponding fluorescence intensities in the tumor areas were recorded. As depicted in Fig. 8a, the tumor regions were highlighted with a fluorescence signal at 3–24 h postinjection, and the fluorescence signal reached a peak at 3 h postadministration in the FA-H@ND group. After 24 h postinjection, an obvious fluorescence signal was detected, demonstrating that an amount of FA-H@NDs still remained in the tumor regions. Comparatively, there was a relatively weak fluorescence signal in the tumor after the injection of H@NDs, suggesting that some NDs accumulated in the tumor via the EPR effect. The fluorescence intensities in the FA-H@ND group were much higher than that in the H@ND group at 3 h postinjection (Fig. 8b). To further observe the biodistribution of the fabricated NDs, the major organs and tumor tissues were harvested for ex vivo fluorescence imaging (Fig. 8c). The images revealed that both FA-H@NDs and H@NDs were extensively accumulated in the liver and spleen as a result of phagocytosis by the reticuloendothelial system, whereas in the group of FA-H@NDs, a substantial number of NDs accumulated in the tumor region (Fig. 8d). In addition to fluorescence imaging, the above tumor tissues were made into frozen sections. The data showed the same accumulation tendency as that seen in the fluorescence imaging results (Fig. S8, ESI). These results demonstrated the efficient accumulation of NDs in the tumor under the guidance of the FA targeting effect, which played an important role in further tumoral distribution.
image file: c9tb02198a-f8.tif
Fig. 8 Biodistribution of FA-H@NDs. (a) In vivo fluorescence imaging of SKOV3 tumor-bearing mice after intravenous injection of DiR-labeled H@NDs or FA-H@NDs at different time points. (b) Corresponding tumor fluorescence intensities at different time points. (c) Ex vivo fluorescence imaging of tumor tissues and major organs dissected from above mice 3 h postinjection and (d) the corresponding average fluorescence intensities.

2.7 Deep penetration of FA-H@NDs assisted by ADV

The anticancer therapeutic efficacy can be extensively hindered by the uneven distribution and undesirable deep diffusion of drugs resulting from the pathophysiological characteristics of tumors.47–49 The passive accumulation induced by the EPR effect cannot meet the penetration depth objective of nanomedicine.37,48 To promote drug dissemination to the deep tissue of a tumor mass, US was applied to stimulate the PFP in the core of the FA-H@NDs (termed the ADV effect). The penetration depth of the FA-H@NDs in the SKOV3 tumor tissue was determined by comparing the intertumoral florescence intensities with or without US treatment. After 3 h of intravenous administration, red fluorescence was found to diffuse throughout the irradiated tumor, while only a small region of the unirradiated tumor was highlighted with fluorescence signals, indicating that the US endowed the FA-H@NDs with a deep penetration capability (Fig. 9a). In addition to the distribution of FA-H@NDs in different sections of the tumor tissue, we further studied the extravascular influx of FA-H@NDs around the tumor vessels stained by CD31. As shown in Fig. 9b, merged images of FA@NDs and CD31 were acquired to reveal the locations of the FA-H@NDs and tumor vessels. The FA-H@NDs combined with US irradiation (ADV) group showed extensive FA-H@ND distribution around the tumor vessels, while the EPR group showed only a small number of FA-H@NDs within the vessels. These results validated that the ADV effect induced greater accumulation of NDs than the single EPR effect, showing great promise for overcoming the penetration barrier of the tumor microenvironment. We further detected the penetration performance of H@NDs assisted by ADV. As shown in Fig. S9 (ESI), without the guidance of FA, the accumulation of H@NDs is much less than that of FA-H@NDs; thus, relatively few NDs undergo ADV, and even fewer NDs penetrated into the deep core.
image file: c9tb02198a-f9.tif
Fig. 9 ADV-assisted sonosensitizer penetration. (a) FA-H@NDs distribution in different tumor sections with or without ADV. (b) Extravascular distribution of FA-H@NDs (red fluorescence) with or without ADV. Blood vasculature was stained in green. The white arrows indicated the extravascular distribution of the FA-H@NDs.

2.8 In vivo US/PA imaging performance of FA-H@NDs

Because of simultaneous precise therapy delivery/guidance and therapeutic monitoring, image-guided therapy is beneficial for anticancer therapy. Taking in vitro US/PA imaging performance into consideration, we next verified the in vivo US/PA imaging capability of FA-H@NDs. First, the FA-H@NDs solution was intravenously injected into SKOV3 tumor-bearing mice followed by US irradiation. Three hours later, the US imaging system was used to acquire enhanced US images of the tumor masses. As a control, US images of the mice were recorded before the FA-H@ND injection. As shown in Fig. 10a, upon US stimulation, the tumor regions were highlighted with a very large US echo signal after the injection of the FA-H@NDs. The echo intensity within tumor areas was also higher after the injection than before injection (Fig. 10b). Due to its unique absorbance, HMME has the potential to be a PA contrast agent, which was also confirmed by in vitro experiments. Then, the tumor-bearing mice were treated with FA-H@NDs for in vivo PA imaging performance. As shown in Fig. 10c, the PA signal within the tumors reached a peak at 3 h postinjection; after that, the FA-H@NDs gradually moved from the tumor regions, which was the same as the results obtained by fluorescence imaging. In addition, the intertumoral PA intensities were also in accordance with the PA images (Fig. 10d).
image file: c9tb02198a-f10.tif
Fig. 10 In vivo dual-modal imaging performance. (a) In vivo US imaging before and after the injection of FA-H@NDs assisted by US irradiation. (b) The corresponding quantitative analysis of US imaging. (c) In vivo PA imaging at different time points after administration of FA-H@NDs. (d) Corresponding quantitative analysis of the PA images.

2.9 In vivo therapeutic efficacy

The strong ROS generation ability, efficient in vitro cellular sonotoxicity, FA-based active targeting effect and deep penetration capability potentially guarantee the in vivo therapeutic efficacy of FA-H@NDs. To demonstrate this hypothesis, SKOV3 tumor-bearing mice were used for in vivo FA-H@ND-based therapeutic assessment. When the tumor volume reached approximately 80 mm3, the mice received an injection of FA-H@NDs or H@NDs solution, and control mice were injected with saline. Based on the in vivo fluorescence imaging and PA imaging data, a substantial number of FA-H@NDs accumulated in the tumors at 3 h postinjection; thus, this time point was adopted for US irradiation after the administration of the two types of agent. During the therapeutic period, the tumor volumes and body weights were monitored and the therapeutic protocol is shown in Fig. 11a. At the end of the treatment, the tumor nodes in each group were excised and are shown in Fig. 11b. According to digital photographs and tumor growth curves, the FA-H@NDs or US by themselves exhibited almost no therapeutic effect. For the H@NDs + US group, the tumor growth was slightly suppressed; compared to the original tumor volumes, the volumes increased nearly 2.04-fold. Comparatively, the combination of the FA-H@ND + US group greatly inhibited tumor growth, demonstrating the high efficiency of SDT (Fig. 11c). The tumor mass weights showed the same trends as the tumor volume (Fig. 11d). Moreover, the tumor inhibition rate was considerably higher in the therapeutic group (87.68%) than in the control group (Fig. 11e). The body weight (Fig. 11f) and major organs pathology (Fig. S10, ESI) of the mice remained unchanged, indicating the high biosafety of this therapeutic modality.
image file: c9tb02198a-f11.tif
Fig. 11 In vivo SDT. (a) Schematic illustration of the SDT protocol. (b) Photographs of SKOV3 tumor tissues collected from five groups at the end of treatment. (c) Tumor volumes of mice in five groups during the therapeutic period. (d) Tumor weights in each group at the end of treatment. (e) Tumor inhibition rates in each group according to the tumor weights. (f) Body weights of mice during the treatment period.

After the various treatments, the FA-H@ND-induced sonotoxic effect was further assessed by pathological analysis (Fig. 12a). The tumor tissues in the H@NDs + US group and the FA-H@NDs + US group were dramatically damaged, while the cells in the other control groups remained unaffected without obvious chromatic agglutination, karyopyknosis, or nuclear fragmentation. The representative apoptosis index and the proliferative index showed the same trends as the above treatment outcomes (Fig. 12b). Based on the aforementioned in vivo therapeutic results, we conclude that the combination of FA-H@NDs with US irradiation could exert a formidable therapeutic effect against SKOV3 ovarian cancer.


image file: c9tb02198a-f12.tif
Fig. 12 Pathological analysis of tumor tissues in different groups. (a) Hematoxylin & eosin (H&E), terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) staining of tumor tissues in different groups. (b) The corresponding apoptotic index and (c) proliferative index after various treatments.

3. Conclusion

In conclusion, we successfully constructed dual US-activatable NDs for US/PA imaging guided SDT. These FA-H@NDs readily accumulated in the ovarian tumor regions from blood circulation via both the passive EPR effect and selective binding to FA receptor-positive SKOV3 tumor cells, which can be monitored by PA imaging. Upon US irradiation, these FA-H@NDs undergo ADV and converted into bubbles, further enhancing US imaging and the penetration capability of the NDs. In addition, these FA-H@NDs assisted by US irradiation generated excess ROS to trigger ovarian cancer cell/tissue apoptosis and necrosis. Evaluations were performed and showed both high SDT efficiency in vitro, and significant tumor inhibition in vivo. The satisfactory biosafety of these FA-H@NDs further facilitated their development toward clinical applications. The combination of on-demand US irradiation and the as-constructed FA-H@NDs presents a promising paradigm for ovarian tumor theranostics.

4. Experimental section

4.1 Materials

HMME was purchased from Shanghai D&B Biotechnology Co., Ltd (Shanghai, China). Phospholipids including 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (PEG2000-DSPE)], FA-PEG2000-DSPE and 1,2-dipalmitoyl-sn-glycero-3-phospha-tidylcholine (DPPC) were synthesized by Xi’an Ruixi Biological Technology Co., Ltd (Xi’an, China). PFP, DCFH-DA, and cholesterol were obtained from Sigma-Aldrich (USA). Chloroform (CHCl3) and absolute alcohol were obtained from Chongqing Chuandong Chemical Co. Ltd (Chongqing, China). CCK-8 kits, calcein AM, and pyridine iodide (PI) were purchased from Dojindo (Japan). The nuclear dyes 4,6-diamidino-2-phenylindole (DAPI) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) were purchased from Beyotime Biotechnology (Shanghai, China). DiR was purchased from Amy Jet Scientific Inc. (Wuhan, China). DPBF was purchased from Shanghai McLean Biochemical Technology Co., Ltd (Shanghai, China). Singlet oxygen sensor SOSG was purchased from Thermo Fisher Scientific (Invitrogen).

4.2 Synthesis and characterization of FA-H@NDs

The preparation of FA-H@NDs was performed according to our previous study.13,50 Briefly, 2.5 mg of FA-PEG2000-DSPE, 6 mg of DSPC, 1.5 mg of cholesterol and 1 mg of HMME were dissolved in CHCl3. Then, the solution was placed in a rotary evaporation for 2 h to form a film. Next, the film was peeled and dissolved in phosphate-buffered saline (PBS) assisted by a sonicator. Subsequently, 100 μL of PFP was added to the above solution. Then, the solution was subjected to sonication in an ice bath (100 W, total on 3 min, on 5 s, off 5 s). Next, FA-H@NDs were collected after hypothermal centrifugation three times (5 min, 1468 × g). For the fabrication process of H@NDs, the difference lies in the replacement of FA-PEG2000-DSPE with PEG2000-DSPE.

The structure of the FA-H@NDs was characterized using a transmission electron microscope (TEM) (Hitachi 7500, Tokyo, Japan). The size distribution and zeta potential of the FA-H@NDs were determined using a Malvern Zetasizer Nanoseries instrument (Malvern Instruments, Malvern, UK). We measured the sizes of FA-H@NDs in fetal blood serum within a prolonged time of 7 d. A UV-vis spectrophotometer was employed to evaluate the optical absorption properties of HMME (in ethyl alcohol) and calculate the loading efficacy of HMME loaded in FA-H@NDs. The formula is as follows:

Loading efficacy of HMME = (initial weight of HMME − unloaded HMME)/(initial weight of HMME) × 100%
In addition to characterizing the spectrum of HMME, the spectra of FA-H@NDs and NDs were also characterized. To compare the stability of HMME in FA-H@NDs and free HMME, the spectra of FA-H@NDs (peak absorbance: A01) and free HMME (A02) were collected. After storing in the dark for one day, the spectrums were collected again (A1 and A2), and the normalized absorption were calculated (A/A0).

4.3 In vitro ADV, US/PA imaging, and ROS generation ability of FA-H@NDs

The time dependent phase-transformation process of the FA-H@NDs upon US irradiation was observed by optical microscopy and fluorescence microscopy. Subsequently, enhanced US imaging of FA-H@NDs was conducted on an US system. Typically, FA-H@NDs were exposed to US irradiation (frequency, 650 kHz; 50% duty cycle; pulsed-mode) for 1 min, 2 min, 3 min, 4 min or 5 min at an intensity of 1.5 W cm−2. US images in both B mode and contrast mode were collected using a US system (MyLab 90, Esaote, Italy) equipped with a high frequency linear array probe (LA523, frequency: 12 MHz; mechanical index (MI): 0.06). The corresponding echo intensity was analyzed using US imaging analysis software.

For in vitro PA imaging, FA-H@NDs were suspended in distilled water at a concentration of 5 mg mL−1, and used for PA imaging with the excitation wavelength ranging from 680 nm to 970 nm. Then PA images of FA-H@NDs at varied concentrations (1, 2, 3, 4, and 5 mg mL−1) were recorded and the corresponding PA intensities were measured for further analysis.

The ROS generation ability of FA-H@NDs after US stimulation was monitored by DPBF. Briefly, the FA-H@ND aqueous solution (1 mg mL−1) was treated with US irradiation in the presence of DPBF. Immediately afterwards, the absorbance of the mixture was recorded using a UV-vis spectrophotometer. As control groups, US only and FA-H@NDs only were also analyzed for comparison. In addition to DPBF, another ROS probe, SOSG was also employed. FA-H@NDs (0.1 mg mL−1) mixed with SOSG were irradiated with US for different periods of time (0 s, 15 s, 30 s, 60 s, 90 s, 120 s, and 180 s).

4.4 Intracellular uptake efficiency of FA-H@NDs in vitro

Human ovarian cancer SKOV3 cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences. The cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) in a humidified environment (5% CO2, 37 °C).

The intracellular uptake of FA-H@NDs was observed by CLSM. SKOV3 cells in the logarithmic growth phase were used. Then, the cells were seeded into CLSM-specific dishes and were cultured for 24 h. Then, the cells were incubated with FA-H@NDs or H@NDs at a concentration of 0.4 mg mL−1 for 0.5 h, 1 h, 2 h, 3 h, or 4 h, respectively. Ultimately, the cells were observed by CLSM after staining with DAPI. In addition, flow cytometry was carried out to analyze phagocytosis. After incubation with FA-H@NDs or H@NDs, the cells were harvested and suspended in 200 μL of PBS. Then the uptake efficiency was determined by flow cytometry.

4.5 Intercellular ROS generation ability, in vitro cytotoxicity and sonotoxicity against SKOV3

The potential of FA-H@NDs as a sonosensitizer for producing ROS was monitored and explored. Five groups were established for comparison: control, FA-H@NDs, US Only, H@NDs + US and FA-H@NDs + US. SKOV3 cells seeded in dishes were administered the different treatments mentioned above. The US power intensity was set as follows: 1.5 W cm−2, 60 s, 1 MHz. In particular, DCFH-DA (100 μL, 300 μM) was added to the dishes before US irradiation. Thirty minutes later, the cells were observed under CLSM. Additionally, the fluorescence intensity of 7′-dichlorofluorescein (DCF) which indicates ROS generation was detected by flow cytometry. The process was similar to that mentioned above except that the cells were harvested by a 0.25% trypsin solution and suspended in PBS.

The cytotoxicity of FA-H@NDs was determined in SKOV3 cells. First, SKOV3 cells were transferred to 96 wells plate at a density of 1 × 104 cells per well. Twenty-four hours later, the medium in the plates was replaced with fresh medium containing FA-H@NDs at various concentrations (0.1, 0.2, 0.3, and 0.4 mg mL−1) for an incubation time of 4 h, 12 h or 24 h. Then, the CCK-8 assay was used to evaluate the relative cell viability. To elucidate the sonotoxicity of FA-H@NDs against SKOV3 cells, different groups were established: control, FA-H@NDs, US Only, H@NDs + US and FA-H@NDs + US. SKOV3 cells cultured in 96 wells plate were treated with the above mentioned protocols. The US irradiation parameters were set as follows: 1.5 W cm−2, 1 min. Then, the CCK-8 assay was carried out to determine the cell viability of each group. To further illustrate the cell-killing effect of SDT, the cells in CLSM-specific dishes received the same treatments. Then, 10 μL of calcein/PI was used to stain the cells to distinguish between the live cells (green fluorescence) and dead cells (red fluorescence).

4.6 Animal model

Six-week-old female BALB/c nude mice were purchased from Chongqing Medical University. All animal studies were in compliance with licensed procedures approved by the Institutional Animal Care of Chongqing Medical University. To establish SKOV3 tumor-bearing mice, cells (5 × 106) suspended in 100 μL of FBS free medium and implanted into the back flank of mice. When the tumor mass reached a volume of nearly 80 mm3, the mice were used for the following experiments.

4.7 Biosafety assessment of FA-H@NDs

The biosafety of the FA-H@NDs was implemented with healthy male Kunming mice. The mice were intravenously administered FA-H@NDs (50 mg kg−1). The blood of the mice was collected at various time points (3 d, 7 d, 14 d and 30 d) for biochemical assays and blood cell analysis. The untreated, heathy mice were used as controls. Meanwhile, the major organs of mice were collected and then subjected to H&E staining.

4.8 In vivo targeting capability and biodistribution of FA-H@NDs

To evaluate the biodistribution of FA-H@NDs, SKOV3 tumor-bearing mice were randomly divided into two groups (n = 5 per group): FA-H@NDs group and H@NDs group. The mice were intravenously administered DiR-labeled FA-H@NDs or H@NDs (50 mg kg−1). The mice were imaged by a Living Imaging System before administration and at different postadministration time points (1 h, 3 h, 6 h, 8 h and 24 h). Quantitative analysis of the tumor regions was performed with Living Image software. Another 10 SKOV3 tumor-bearing mice were intravenously administered DiR-labeled FA-H@NDs or H@NDs (50 mg kg−1). The mice were sacrificed and major organs were dissected for ex vivo fluorescence imaging 3 h postinjection. Similarly, the Living Image System was utilized to measure the fluorescence intensities of the regions of interest. To further observe the biodistribution of the FA-H@NDs, the tumor tissue and major organs were made into frozen sections 3 h after FA-H@NDs administration. Then, the sections were observed using fluorescence microscopy.

4.9 Deep penetration of FA-H@NDs assisted by ADV

The SKOV3 tumor-bearing mice were randomly divided into three groups: the control group (intravenous administration of DiI-labeled FA-H@NDs without US irradiation), the ADV group (intravenous administration of DiI-labeled FA-H@NDs + US irradiation at the tumor region) and the untargeted ADV group (intravenous administration of DiI-labeled H@NDs + US irradiation at the tumor region). Three hours after intravenous administration of DiI-labeled FA-H@NDs or H@NDs, the mice were treated with US irradiation at the tumor region for 10 min. Then the tumor tissues in the three groups were sliced, and an anti-CD31 antibody was applied to stain blood vessels for fluorescence microscopy observation. The slices were scanned using a slide scanner (Pannoramic MIDI, 3D HISTECH, Hungary).

4.10 In vivo US/PA imaging performance of FA-H@NDs

To characterize the US imaging performance of FA-H@NDs, five SKOV3 tumor-bearing mice were treated with FA-H@NDs emulsion (50 mg kg−1) followed by US irradiation. US images were captured and analyzed using an US imaging system in B mode and contrast mode. Quantitative data were analyzed by mapping the region of interest and comparing the echo intensity obtained after FA-H@NDs administration with that recorded prior to administration. For in vivo PA imaging, five SKOV3 tumor-bearing mice were intravenously injected with FA-H@NDs emulsion (50 mg kg−1). PA images of the tumor regions were recorded at various time points (1 h, 2 h, 3 h, 6 h, 8 h, and 24 h) using a PA imaging system. The corresponding PA signals in the tumor regions were simultaneously measured.

4.11 In vivo SDT efficacy

Twenty-five SKOV3 tumor-bearing mice were randomly separated into five groups (n = 5 per group) as follows: (a) control group (injection of saline), (b) FA-H@NDs group (treated FA-H@NDs, 50 mg kg−1), (c) US only group (subjected to only US irradiation without administration of NDs), (d) H@NDs + US group (injection of H@NDs (50 mg kg−1) combined with US irradiation), (e) FA-H@NDs + US (injection of FA-H@NDs (50 mg kg−1) combined with US irradiation). Three hours after administration, US was used to execute the above experimental protocols. The treatments were conducted every five days over 30 d. During the therapeutic period, the body weight and tumor volume of the mice were monitored. The tumor masses were dissected and sliced for pathological analysis including H&E, TUNEL and PCNA staining to observe the cell structure and status. The apoptotic index and proliferative index were further analysed. Typically, three images were captured randomly on each pathological slice. Each vision was viewed under consistent background luminance. The green fluorescent nucleus and blue nucleus (the apoptotic cells) were chosen as the criterion to assess positive cells and total cells, respectively, using Image-Pro Plus 6.0 software. The apoptotic rate was calculated with the following formula: apoptotic index (%) = (positive cells/total cells) × 100%. Similarly, the yellow brown nucleus (the proliferative cells) was chosen as the criterion to judge positive cells, and proliferative rate was calculated as follows: proliferative index (%) = (positive cells/total cells) × 100%.

4.12 Statistical analysis

All statistical data were compared with one-way analysis of variance (ANOVA): *P < 0.05.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81630047, 31630026, 81401503, 81471713), China Postdoctoral Science Foundation funded projects (2015T80963, 2016M590869) and Chongqing Postdoctoral Science Foundation funded project (Xm2015089).

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Footnotes

Electronic supplementary information (ESI) available: Photographs of FA-H@NDs and FA@NDs; PA intensity of FA-H@NDs; the frozen section of tumor tissues; H&E staining of major organs of mice after different treatments. See DOI: 10.1039/c9tb02198a
Chao Yang and Yi Zhang contributed equally to this work.

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