Hematoporphyrin and doxorubicin co-loaded nanomicelles for the reversal of drug resistance in human breast cancer cells by combining sonodynamic therapy and chemotherapy

Abstract

Drug resistance is a main reason for the failure of chemotherapy in cancer treatments. Sonodynamic therapy (SDT) shows great potential for reversing drug resistance of chemotherapy. Here, a sonosensitizer hematoporphyrin (HP) and a chemotherapeutic drug doxorubicin (DOX) were co-loaded into Pluronic F68 nanomicelles for combining SDT and chemotherapy to reverse cancer drug resistance. This multi-functional nanosystem, called HPDF nanomicelle, had a classic “core–shell” structure and a size smaller than 100 nm. In drug-resistant human breast cancer MCF-7/ADR cells that over-express P-glycoprotein (P-gp), HPDF nanomicelles combined with a low-intensity ultrasound could effectively inhibit cell proliferation, promote cell apoptosis and arrest cell cycle at S-phase. Compared free DOX, HPDF nanomicelles significantly reversed drug resistance in MCF-7/ADR cells and the reversal index reached up to 19.0. Apparently, the synergistic effects of combination treatment of SDT and chemotherapy induced by HPDF nanomicelles played important roles in the reversal process against drug resistance. In summary, our study provides a novel strategy for overcoming drug resistance in breast cancer by combining SDT and chemotherapy.

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Introduction

Breast cancer is the most common cancer in women worldwide and seriously harms women's health. According to the statistics, nearly 2 million women are diagnosed with breast cancer each year around the world and the mortality rate is increasing year by year.¹ As is well-known, chemotherapy plays a major role in clinical breast cancer treatment. However, drug resistance either intrinsic or acquired is one of the main reasons that cause the metastasis and recurrence of breast cancer.² The drug-resistant mechanisms of breast cancer cells mainly include the decreased drug influx and increased drug efflux predominantly induced by the ATP-binding cassette (ABC) transporters, the activation of DNA repair, the metabolic modification, the detoxification, and the inactivation of apoptosis pathways with parallel activation of anti-apoptotic cellular defense modalities.^3,4 Currently, there is no effective method to overcome drug resistance in breast cancer due to its complicated drug-resistant mechanisms. The increase of drug efflux by overexpression of ABC transporters is a common mechanism for cellular resistance to anticancer agents such as doxorubicin (DOX) and paclitaxel.⁵ ABCB1 belongs to ABC transporter family and encodes a membrane protein P-glycoprotein (P-gp), which is a well-known efflux pump responsible for multiple drug resistance (MDR).⁶ Cancer cells resist DOX were found to exhibit cross-resistance to a variety of other chemotherapeutic drugs and to have elevated expression level of P-gp.⁷

Sonodynamic therapy (SDT) is a novel promising noninvasive approach derived from photodynamic therapy (PDT). SDT involves the preferential uptake and retention of a sonosensitizer in tumor tissues and the subsequent activation of sonosensitizer by local ultrasonic irradiation, which consequently causes the tissues or cell damages.⁸ In view that ultrasound has an appropriate tissue attenuation coefficiency, SDT is believed to be suitable for treatments of deep-seated solid tumors. Many investigations have shown that multiple mechanisms, including the acoustic cavitation, the generation of reactive oxygen species (ROS), the peroxidations of lipids, proteins and genes, are involved in the killing of tumors.^9,10 Interestingly, SDT exhibits strong capability for overcoming drug resistance in cancer chemotherapy through improving the cell entry of chemotherapeutic drugs, activating the mitochondrial apoptosis pathway, and reducing the expression levels of ABC transporters such as P-gp, etc.^11,12 Thus, the combination treatment of chemotherapy with SDT is promising to be used for overcoming drug resistance and achieving synergistic therapeutic effects on breast cancer. However, there are still some challenges for clinical applications of combination treatment of sonodynamic therapy and chemotherapy. For example, the different physiological fates and non-uniform distributions of sonosensitizers and chemotherapeutic drugs will influence their therapeutic activities and perhaps cause the unexpected side and toxic effects.

The emergence and rapid development of nanocarrier technology provides possibility for resolving the above challenges in cancer treatment by combining sonodynamic therapy and chemotherapy. Nanocarriers such as micelles, liposomes, nanoparticles, and dendrimers have shown many advantages for delivery of anticancer drugs, e.g., increasing their in vivo stability, enhancing their bioavailability, improving their tumor-targeted delivery via the enhanced permeability and retention (EPR) effect, and realizing their intracellular delivery, which will be benefit to reversing cancer drug resistance.^13,14 Recently, some investigations have prepared several multifunctional nanocarriers for co-delivery of chemotherapeutic drugs and other anticancer agents such as curcumin and cyclosporine, and the results show that these nanosystems can efficiently reverse cancer drug resistance both in vitro and in vivo.^15,16 Besides that, the tumor-targeted capability of nanocarriers can be further enhanced by their surface-modification with ligands or antibodies for specific receptors on tumor cells.¹⁷ In view of the above mentioned facts, we believed that co-loading of sonosensitizers and chemotherapeutic drugs inside nanocarriers would be favorable for exerting their synergistic effects against cancer drug resistance.

In this study, we developed a multifunctional nanosystem with a very simple structure but highly efficiency for the reversal of cancer drug resistance by combining SDT and chemotherapy. This nanosystem, called HPDF nanomicelle, had a “core–shell” structure, in which therapeutic drug doxorubicin (DOX) was complexed with sonosensitizer hematoporphyrin (HP) to form a hydrophobic core and Pluronic F68 was coated on the surface to form a hydrophilic shell (Fig. 1a). Pluronics are a kind of triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), arranged in PEO–PPO–PEO structure. Many investigations have shown that Pluronics can selectively deplete ATP in MDR cancer cells, thus remarkably sensitize MDR cancer cells to chemotherapeutic drugs by inhibiting drug efflux transporters, abolishing drug sequestration and suppressing glutathione (GSH)/glutathione S-transferase (GST) detoxification system, etc.¹⁸ SP1049C, a Pluronic-based micellar formulation of DOX exhibits strong capability to overcome drug resistance in a number of cancers and has been granted by the US Food and Drug Administration (FDA) for the clinical treatment of gastric cancer.¹⁹ Herein, we believed that co-loading of HP and DOX inside HPDF nanomicelles would further enhance the sensitivity of drug-resistant cancer cells to DOX by combining with ultrasound. To evaluate the feasibility of HPDF nanomicelles-induced combination treatment of sonodynamic therapy and chemotherapy as a novel strategy for reversing drug resistance in breast cancer, we investigated the synergistic effects of HPDF nanomicelles combined with ultrasonic irradiation on human drug resistant breast cancer MCF-7/ADR cells that over-express P-gp, a classic ABC efflux transporter, and also preliminarily explored the possible function mechanisms.

Fig. 1 Preparation and characterization of HPDF nanomicelles. (a) Schematic illustration for preparation of HPDF nanomicelle. (b), (c) TEM and AFM images of HP nanosheets in methanol. (d), (e) TEM and AFM images of DOX/HP nanocomplex in methanol. (f) TEM image and (g) size distribution of HPDF nanomicelles in deionized water.

Materials and methods

Materials and cell lines

Doxorubicin hydrochloride (DOX·HCl) and hematoporphyrin (HP) were supplied by Meilun Biotech (Dalian, China) and J&K Scientific (Beijing, China), respectively. Pluronic F68 was purchased from BASF (Ludwigshafen, German). Cell counting kit-8 (CCK-8) was purchased from Dojindo (Beijing, China). Bovine serum albumin (BSA), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 4′,6-diamidino-2-phenylindole (DAPI), penicillin, streptomycin, and rhodamine 123 (Rh123) were purchased from Sigma-Aldrich (St Louis, MO, USA). All other chemical reagents in the study were analytical grade without further purification and obtained from commercial sources.

Two human breast cancer cell lines, DOX-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells, were obtained from Detroit Hospital (Detroit, USA). MCF-7 and MCF-7/ADR cells were cultured respectively in Dulbecco's Modified Eagle's Medium (Life Technologies, Carlsbad, USA) and in RPMI 1640 medium (Life Technologies, Carlsbad, USA) with supplements of 10% (v/v) fetal bovine serum (Gibco, Invitrogen, USA) and 1% (v/v) penicillin/streptomycin at 37 °C in an atmosphere of 5% CO₂. The cells were subcultured two to three times per week with Gibco trypsin-EDTA (0.25%). To maintain the resistance to DOX, MCF-7/ADR cells were cultured in RPMI 1640 medium containing 0.5 μmol L⁻¹ DOX.

Preparation and characterization of HPDF nanomicelles

According the previous report,²⁰ thin film hydration method was used to prepare HPDF nanomicelles. DOX·HCl was firstly desalinated in methanol containing 3.0 mol equivalents of triethylamine and then added to the HP methanol solution at DOX and HP concentration of 0.25 and 1 mg mL⁻¹, respectively. The mixture was continuously stirred for 12 h at room temperature. Pluronic F68 was dissolved in methanol at 2.5 mg mL⁻¹ and then added to the above DOX/HP mixture at a volume ratio of 2/1. After stirring overnight, the mixture solution was evaporated at 60 °C to form thin films and further dried under vacuum to remove the remaining methanol. Afterwards, the dry films were hydrated with deionized water under stirring, and followed by passing through a polycarbonate filter (pore size 0.22 μm). Finally, the filtrate was freeze-dried to obtain red powder of HPDF nanomicelles. HP-loaded Pluronic F68 (HPF) nanomicelles were also prepared using the same method but without adding DOX. All experiment steps were carried out by strictly avoiding light.

HPDF nanomicelles were dispersed in deionized water at concentration of 0.5 mg mL⁻¹. The size and size distribution of HPDF nanomicelles were measured using Malvern Zetasizer Nano ZS90 (Malvern, UK) at 25 °C. The morphologies of DOX/HP nanocomplex and HPDF nanomicelles were observed by the transmission electronic microscopy (TEM, Hitachi HT7700 Tokyo, Japan).

Detection of HP and DOX loading contents in HPDF nanomicelles

DOX and HP loading contents and encapsulation efficiencies in HPDF nanomicelles were detected using the ultra-performance liquid chromatography (UPLC) method. Briefly, the HPDF nanomicelles were dissolved in formylamine and then diluted with acetonitrile. Afterward, the determinations were performed on ACQUITY UPLC system (ACQ-BSM, Waters). C18 analytical column (50 mm × 2.1 mm, 1.7 μm, Waters) was used and the column temperature was set at 30 °C. The mobile phase consisted of methanol, acetonitrile and PBS (13 : 19 : 68 v/v/v, pH 4.0) and the flow rate was 0.2 mL min⁻¹. The detection wavelengths of DOX and HP were set at 490 nm and 395 nm, respectively. The loading contents and encapsulation efficiencies of DOX and HP were calculated according to the following formulas (LC: loading content; EE: encapsulation efficiency).

LC% = (weight of the loaded DOX or HP/weight of HPDF nanomicelles) × 100%

EE% = (weight of the loaded DOX or HP/weight of the feeding DOX or HP) × 100%

In vitro cytotoxicity assay

CCK8 assay was used to evaluate the cytotoxicities of various treatments in MCF-7 and MCF-7/ADR cells. Briefly, the cells were harvested and incubated with free DOX and HPDF nanomicelles at different DOX concentrations for 1 h, and then some of the cells were exposed to ultrasonic irradiation (1 MHz, 1.5 W cm⁻²) for 30 s using an ultrasound therapy device assembled by AG1020 ultrasound generator/amplifier (T&C Power Conversion, NY, USA) with an ultrasound transducer. Afterward, all of the cells with or without ultrasonic irradiation were seeded into 96-well plates at a density of 5 × 10³ cells/well and continually incubated for 48 h. Next, the culture media were replaced with 100 μL fresh culture media containing CCK-8 and further incubated for 3 h. Finally, the absorbance of each well was measured at 450 nm using an ELX800 absorbance microplate reader (Bio-Tek Epoch, Winooski, VT, USA) to calculate the cell survival rate. The IC₅₀, which was defined as the DOX concentration required for 50% inhibition of cell growth, was calculated by the software of Graphpad Prism.

Analysis of cell apoptosis and cell cycle distribution

MCF-7/ADR cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and cultured for 24 h. Free DOX, HPF, and HPDF nanomicelles were then added at DOX and HP concentrations respectively of 1 and 4 μg mL⁻¹, and the cells were further incubated for 1 h. After that, some of the cells were exposed to ultrasonic irradiation (1 MHz, 1.5 W cm⁻²) for 30 s and incubated for additional 48 h. For cell apoptosis analysis, the cells were processed by the apoptosis detection kit of Annexin V-APC/7-AAD (San Jian, China) according to the manufacturer's protocol and then detected by a flow cytometry. For detection of cell cycle distribution, the cells were washed with PBS solution and fixed in 70% cold ethanol overnight at −20 °C. Next, the cells were collected by centrifugation, washed with ice-cold PBS solution, and then re-suspended in propidium iodide (PI)/RNase staining solution (BD Pharmingen, USA). The cells were further incubated for 15 min at room temperature by avoiding light, and then the cell cycle distributions were analyzed by a flow cytometry.

Cellular uptakes and intracellular distributions of HPDF nanomicelles in MCF-7 and MCF-7/ADR cells

The cellular uptakes and intracellular distributions of free DOX and HPDF micelles with or without ultrasonic irradiation were observed by the confocal microscopy. Briefly, MCF-7 and MCF-7/ADR cells were separately seeded onto the 12-well glass slides at density of 5 × 10⁴ cells per well and cultured for 24 h. Then the culture media were replaced by the fresh media containing free DOX and HPDF micelles at DOX concentration of 0.5 and 3 μg mL⁻¹, respectively for MCF-7 and MCF-7/ADR cells. After 1 h, some of the cells incubated with HPDF micelles were exposed to ultrasonic irradiation (1 MHz, 1.5 W cm⁻²) for 30 s, and all the cells were further cultured for 12 h. Next, after washing with PBS solution, the cells were fixed with 4% paraformaldehyde for 10 min and stained with DAPI. Finally, the slides were collected and the cells were imaged with the confocal microscopy (FV-1000, Olympus Corporation, Tokyo, Japan).

The flow cytometry was used to evaluate quantitatively the cellular uptakes of free DOX and HPDF micelles. Briefly, MCF-7 and MCF-7/ADR cells were seeded into 6-well culture plates at a density of 1.5 × 10⁵ cells per well and incubated to reach 80% confluence. Then, the culture media were removed and the cells were incubated with free DOX and HPDF micelles at DOX concentration of 0.5 and 3 μg mL⁻¹, respectively, for MCF-7 and MCF-7/ADR cells. After 1 hour incubation, some of the cells were treated with ultrasonic irradiation (1 MHz, 1.5 W cm⁻²) for 30 s. After incubation for another 12 h, the cells were harvested, and followed by centrifugation at 1000 rpm. Finally, the cells were suspended in PBS and analyzed by the flow cytometry (Beckman Coulter, Brea, USA) at PE channel.

Measurement of intracellular ROS production

The intracellular ROS productions in MCF-7/ADR cells after different treatments were evaluated by the flow cytometry using DCFH-DA as a fluorescence probe. The cells were seeded in 6-well plates and treated with free DOX, HPF and HPDF nanomicelles combined with and without ultrasonic irradiation at DOX and HP concentrations of 5 and 20 μg mL⁻¹, respectively. After incubation for 24 h, the culture media were replaced with 1 mL DCFH-DA reagent and further incubated for 30 min. Next, the cells were washed with PBS solution, harvested, and finally analyzed using a flow cytometry.

Evaluation of mitochondrial membrane damage

The mitochondrial membrane damage in MCF-7/ADR cells induced by different treatments was evaluated by the flow cytometry using Rh123 as a fluorescence probe. Briefly, MCF-7/ADR cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and cultured for 48 h. Free DOX, HPF, and HPDF nanomicelles were then added at DOX and HP concentrations of 5 and 20 μg mL⁻¹, and followed by an additional incubation for 1 h. Some of the cells were exposed to ultrasonic irradiation (1 MHz, 1.5 W cm⁻²) for 30 s and further incubated for 24 h. After that, the cells were incubated with Rh123 reagent for 30 min and washed twice with PBS solution. Finally, the cells were harvested and the fluorescence intensity was analyzed with a flow cytometry.

In order to visually observe the mitochondrial damage in MCF-7/ADR cells, 5 × 10⁴ cells were seeded onto 12-well glass slides and then processed by the same method as abovementioned. After incubated with Rh123 reagent for 30 min, the cells were fixed with 4% paraformaldehyde and stained with DAPI to label nuclei. Finally, the slides were collected and then observed under a confocal microscopy.

Subcellular distribution of cytochrome c (Cyt c)

Immunofluorescence staining was used to analyze the subcellular distribution of Cyt c in MCF-7/ADR cells induced by HPDF nanomicelles at DOX and HP concentrations of 5 and 20 μg mL⁻¹. The cells with different treatment on the glass side were labeled with MitoTracker green (M7514, Invitrogen) for 30 min at 37 °C. Then, fixed with 4% paraformaldehyde for 10 min and punched with 0.2% triton X-100, and followed by blocking with 10% BSA for 1 h. For staining Cyt c, the cells were processed orderly with the diluted primary anti-Cyt c monoclonal antibody (Abcam, 1 : 1000 dilution) at 4 °C overnight and secondary Alexa-647-conjugated goat anti-rabbit antibody (Life Technologies, 1 : 300 dilution) for 1 h. The cell nuclei were stained with DAPI for 10 min and the subcellular distributions of Cyt c were finally observed by a confocal microscopy.

Statistical analysis

Each experiment was repeated at least three times and all data were expressed as mean ± standard deviation. Statistical analysis was performed using analysis of one-way ANOVA and the statistical difference between two groups of data was considered to be significant when P < 0.05.

Results and discussion

Preparation and characterization of HPDF nanomicelles

A very simple method called thin film hydration method was used to prepare HPDF nanomicelles in this study. The preparation method was illustrated in Fig. 1a. HP, as a sonosensitizer, was firstly dispersed in methanol and exhibited obvious nanosheet structure with a mean size smaller than 50 nm and a thickness of about 10 nm (Fig. 1b and c). DOX, an anthracycline antibiotic widely used in clinical cancer chemotherapy, was complexed with HP nanosheets in methanol to form DOX/HP nanocomplex, which showed a regular spherical shape and significantly increased thickness (about 30 nm) by comparison to HP nanosheets (Fig. 1d and e). Besides that, it could be distinctly observed that DOX was distributed surfacely in a punctiform manner, suggesting the successful complexation between DOX and HP nanosheets. In view of chemical structures of HP and DOX and according to the previous report,²¹ we believed the intermolecular interactions such as π–π stacking, hydrophobic interaction, and hydrogen bond involved in the formation of DOX/HP nanocomplex.

Pluronic F68, also known as poloxamer 188, has very low toxicity compared with other polymeric surfactants, thus led it to be approved by the FDA for oral, topical, ophthalmic, periodontal, subcutaneous, and intravenous administrations with relatively high administration doses (10% oral and 0.60% intravenous injection). Pluronic F68 possesses 75 subunits of PEO in each tail and 29 subunits of PPO in the central block, and has a high HLB value (about 29).²² Pluronic F68 can form micelles with nanoscale size in aqueous media. These micelles can effectively escape the serum-dependent opsonization, thus significantly enhance the blood circulation time of the cargos.²³ Recently, many investigations have shown that Pluronic F68 and other Pluronics can reverse cancer drug resistance by selective ATP depletion.²⁴ Here, Pluronic F68 was coated on the surface of DOX/HP nanocomplex to form HPDF nanomicelles, thus hoping to realize the efficient reversal of cancer drug resistance. As shown in Fig. 1f, HPDF nanomicelles had a classical “core–shell” structure with a shell thickness of 10–20 nm, suggesting that Pluronic F68 was successfully coated on the surface of DOX/HP nanocomplex. Moreover, HPDF nanomicelles had a small size about 77.8 ± 17.5 nm with relatively uniform distribution (Fig. 1g), and the polydispersity index was 0.13 ± 0.021. The loading contents of DOX and HP, measured by the UPLC method, were 3.0% and 12.6%, respectively, and their encapsulation efficiencies were 69.2% and 86.7%, respectively.

Reversal effect of HPDF nanomicelles combined with ultrasonic irradiation on drug resistance in MCF-7/ADR cells

Many novel treatment methods e.g., anti-angiogenesis therapy, immunotherapy, monoclonal antibody therapy, and gene therapy, have recently emerged and some of them have showed satisfying effects on breast cancer in clinic.²⁵ However, chemotherapy still plays an important role in clinical treatments for breast cancer patients especially at middle-advanced stage. Drug resistance either intrinsic or acquired is one of the main reasons to cause the failure of chemotherapy on breast cancer. Therefore, exploring effective strategies to reverse drug resistance has become an urgent task in breast cancer treatments. In this study, we hoped to use HPDF nanomicelles as a novel therapeutic agent to overcome drug resistance of breast cancer by combining chemotherapy and SDT. To evaluate the reversal capability of HPDF nanomicelles, we firstly detected the cytotoxicities of free DOX and HPDF nanomicelles combined with and without a low-energy ultrasonic irradiation (1 MHz, 1.5 W cm⁻²) in two human breast cancer cell lines, DOX-sensitive MCF-7 and DOX-resistant MCF-7/ADR cell lines, using CCK-8 assay.

The results are shown in Fig. 2. Obviously, free DOX exhibited strikingly different cytotoxicities in MCF-7 (Fig. 2a) and MCF-7/ADR cells (Fig. 2b), respectively responding to the IC₅₀ values of 0.15 and 23.8 μg mL⁻¹. The resistance index, defined as the ratio of IC₅₀ values of DOX in MCF-7/ADR and MCF-7 cells, was high to 158.7, which indicated that MCF-7/ADR cells had strong resistance to DOX. In MCF-7 cells, HPDF nanomicelles combined ultrasonic irradiation had significantly higher cytotoxicity than both free DOX and HPDF nanomicelles (Fig. 2a). The IC₅₀ values of free DOX, HPDF nanomicelles alone and combined with ultrasonic irradiation in MCF-7 cells were 0.15, 0.10 and 0.02 μg mL⁻¹ DOX, respectively. It suggested that HPDF nanomicelles exerted synergistic effects derived from sonodynamic efficiency of HP and chemotherapeutic activity of DOX under the ultrasonic irradiation. In MCF-7/ADR cells, these synergistic effects became even more significant. As shown in Fig. 2b and c, HPDF nanomicelles combined with ultrasonic irradiation displayed remarkably enhanced cytotoxicity in MCF-7/ADR cells compared to free DOX, and the IC₅₀ value was only about 1.25 μg mL⁻¹ DOX. This result demonstrated that HPDF nanomicelles reversed the resistance of MCF-7/ADR cells to DOX by combining SDT and chemotherapy. The reversal index, calculated as the ratio between IC₅₀ values of free DOX and HPDF nanomicelles combined with irradiation, reached up to 19.0. In addition, the treatment of HPDF nanomicelles alone also exhibited chemosensitization efficiency to some extent in both MCF-7 and MCF-7/ADR cells. According to the previous report,^26,27 we believed it was perhaps because that Pluronic F68 micelles efficiently delivered DOX into these breast cancer cells to exert its cytotoxicity effects and Pluronic F68 itself played a certain inhibitory effect on the cell proliferation via the ATP-depletion.

Fig. 2 HPDF nanomicelles efficiently reversed drug resistance of MCF-7/ADR cells. (a) Cytotoxicities of free DOX, HPDF nanomicelles alone and combined with ultrasonic irradiation (HPDF/U) in MCF-7 cells. (b) Cytotoxicities of free DOX in MCF-7/ADR cells. (c) Cytotoxicities of HPDF nanomicelles alone and combined with ultrasonic irradiation in MCF-7/ADR cells.

Synergistic effects of HPDF nanomicelles combined with ultrasonic irradiation on cell apoptosis and cell cycle progression in MCF-7/ADR cells

As previously reported,²⁸ DOX can induce DNA double-strand breaks by intercalating into DNA and blocking the activity of topoisomerase II enzymes, thus inhibits the biosynthesis of DNA and RNA, and further induces the cell death (cell apoptosis and cell necrosis) and cell cycle arrest. In addition, the generation of a large amount of intracellular ROS induced by SDT can cause the damages of biomembranes, organelles and DNA through the peroxidations of lipids, proteins and genes, thus further leading to cell apoptosis and cell cycle arrest.²⁹ In view of the above described, we believed that HPDF nanomicelles could combine chemotherapy and SDT to exert synergistic effects on cell apoptosis and cell cycle progression. Therefore, we evaluated these synergistic effects in MCF-7/ADR cells using the flow cytometry.

Fig. 3a shows the apoptosis evaluation of MCF-7/ADR cells with various treatments for 48 h. Compared to the control, the ultrasonic irradiation at a low-intensity did not induce the apoptosis of MCF-7/ADR cells. Free DOX alone and combined with ultrasonic irradiation also did not induced the cell apoptosis at DOX concentration of 1 μg mL⁻¹ due to drug resistance of MCF-7/ADR cells. HPF nanomicelles combined with ultrasonic irradiation significantly induced the early and late apoptosis of MCF-7/ADR cells at HP concentration of 4 μg mL⁻¹ and the apoptosis ratio was about 6.3%, which was obviously higher than that of MCF-7/ADR cells only treated with HPF nanomicelles. By contrast with the other treatments, HPDF nanomicelles displayed significantly stronger induction effect on the apoptosis of MCF-7/ADR cells with an apoptosis ratio of 13.1% at the same DOX and HP concentrations. After ultrasonic irradiation, induction effect of HPDF nanomicelles on the apoptosis of MCF-7/ADR cells was further enhanced, e.g., an apoptosis ratio of 19.9% and even a dead ratio of 6.44%. Fig. 3b shows the cell cycle distributions of MCF-7/ADR cells with various treatments for 24 h. Compared to the control, all treatments except ultrasonic irradiation alone induced the more or less cell cycle arrest at S phase. However, HPDF nanomicelles combined with ultrasonic irradiation also exhibited significantly stronger induction effect on cell cycle arrest in MCF-7/ADR cells than the other treatments at DOX and HP concentrations respectively of 1 and 4 μg mL⁻¹. All above results demonstrated that HPDF nanomicelles successfully exerted synergistic effects of chemotherapeutic activities of DOX and sonodynamic efficiency of HP under ultrasonic irradiation on cell apoptosis and cell cycle progression in MCF-7/ADR cells.

Fig. 3 Analysis of cell apoptosis (a) and cell-cycle distributions (b) of MCF-7/ADR cells at 48 h after treatments of ultrasonic irradiation (U), DOX alone and combined with ultrasonic irradiation (DOX/U), HPF nanomicelles alone and combined with ultrasonic irradiation (HPF/U), and HDPF nanomicelles alone and combined with ultrasonic irradiation (HPDF/U). Q1: dead cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: live cells.

Cellular internalization of HPDF nanomicelles in MCF-7 and MCF-7/ADR cells

The above results showed that HPDF nanomicelles combined with ultrasonic irradiation successfully reversed drug resistance of MCF-7/ADR cells and displayed significantly enhanced synergistic effects on the cell apoptosis and cell cycle arrest. In MCF-7/ADR cells used in this study, the over-expression of P-gp is a main drug-resistant mechanism.²⁴ As shown in Fig. S1a and b,† MCF-7/ADR cells expressed a significantly higher level of P-gp than MCF-7 cells. P-gp, as a classic ABC efflux transporter, can use the energy released from ATP hydrolysis to pump a variety of anticancer drugs such as DOX out of the cells.³⁰ As previously reported,³¹ Pluronic F68 can inhibit the efflux functions of P-gp via ATP depletion. Thus, we detected the cellular internalization of DOX delivered by HPDF nanomicelles in MCF-7/ADR cells using the confocal microscopy and the flow cytometry to evaluate the inhibitory effect of HPDF nanomicelles on P-gp. As shown in Fig. 4a, free DOX and HPDF nanomicelles efficiently entered into MCF-7 cells almost without P-gp expression and the red fluorescence of DOX were mainly located in the cell nuclei at DOX concentration of 0.5 μg mL⁻¹ after 12 hour incubation. The results of flow cytometry showed that the fluorescence intensities in MCF-7 cells had no significant difference between treatment groups of free DOX and HPDF nanomicelles (Fig. 4b). Due to drug resistance of MCF-7/ADR cells, only a small amount of free DOX entered the cells and wholly located in the cytoplasm after 12 hour incubation even at a relatively high DOX concentration of 3 μg mL⁻¹; by contrast, HPDF nanomicelles delivered a large amount of DOX into MCF-7/ADR cells and some of DOX began to enter the cell nuclei at the same DOX concentration (Fig. 4c and d). In addition, the ultrasonic irradiation did not change the cellular internalization and intracellular distribution of DOX in both MCF-7 and MCF-7/ADR cells treated with HPDF nanomicelles. From above results, we deduced that HPDF nanomicelles could efficiently deliver DOX into drug resistant MCF-7/ADR cells by inhibiting the efflux function of P-gp.

Fig. 4 HPDF nanomicelles efficiently delivered DOX into MCF-7/ADR cells. Confocal images (a and c) and flow cytometry profiles (b and d) of MCF-7 cells (a and b) and MCF-7/ADR cells (c and d) at 12 h after treatments of free DOX, HPDF nanomicelles alone and combined with ultrasonic irradiation. DOX treatment concentrations in MCF-7 and MCF-7/ADR cells were 0.5 and 3 μg mL⁻¹, respectively.

Intracellular ROS production and mitochondrial damage induced by HPDF nanomicelles combined with ultrasonic irradiation

It is well known that the intracellular production of ROS induced by sonosensitizer under the ultrasound was one of the main mechanisms of SDT to cause severe toxicity on cancer cells. The low-intensity ultrasound can activate sonosensitizer from the ground state into the excited state. As the activated sonosensitizer returns to the ground state, the released energy can be transferred to the circumambient oxygen to produce a large amount of ROS, including oxygen ion, peroxide and singlet oxygen, which subsequently induce the fatal damages on target cells and organelles through the peroxidations of lipids, proteins and genes.³² To evaluate the sonodynamic efficiency of HPDF nanomicelles combined with ultrasonic irradiation, DCFH-DA was used as a fluorescence probe to detect the intracellular generation of ROS in MCF-7/ADR cells after various treatments by using the flow cytometry. The stronger intracellular fluorescence signal of DCFH-DA indicated the higher ROS production level. The flow cytometry profiles of intracellular DCFH-DA are shown in Fig. 5a and S2a,† and the comparison of their fluorescence intensities is shown in Fig. 5b. Compared to the control, the treatments of ultrasonic irradiation, free DOX combined with and without ultrasonic irradiation almost did not induce the ROS production in MCF-7/ADR cells at DOX concentration of 5 μg mL⁻¹. By contrast, HPF and HPDF nanomicelles both induced the significant ROS production in MCF-7/ADR cells at HP concentration of 20 μg mL⁻¹, and ultrasonic irradiation further enhanced the intracellular ROS levels. In addition, a significantly larger amount of ROS was detected in MCF-7/ADR cells treated with HPDF nanomicelles than HPF nanomicelles after combination with ultrasonic irradiation. All above results demonstrated that HPDF nanomicelles had relatively strong sonodynamic efficiency by combining with ultrasonic irradiation.

Fig. 5 Production of intracellular ROS and damage of mitochondrial membrane in MCF-7/ADR cells using DCFH-DA and Rh123 as the fluorescence probes, respectively. (a) Flow cytometry analysis of DCFH-DA in MCF-7/ADR cells at 24 h after various treatments. (b) Mean fluorescence intensity (MFI) of intracellular DCFH-DA. (c) Confocal images of MCF-7/ADR cells at 48 h after various treatments. Nuclei (blue fluorescence) were stained with DAPI and mitochondrial trans-membrane potentials were detected by Rh123 (green fluorescence). (d) Flow cytometry analysis of Rh123 in MCF-7/ADR cells at 24 h after various treatments. (e) Mean fluorescence intensity (MFI) of Rh123.

According to the previous reports,^33,34 HP has natural targeting property to the mitochondria after internalization by cancer cells. Thus, the ROS induced by HP can cause the irreversible damage of mitochondria via the peroxidation of mitochondrial membrane. Once the mitochondria collapses, the cell apoptosis or necrosis can be initiated. Here, we detected the mitochondrial membrane damage using Rh123, a fluorescence probe for detecting mitochondrial trans-membrane potential. Fig. 5c shows the confocal images of MCF-7/ADR cells with Rh123 staining after various treatments. HPF and HPDF nanomicelles combined with ultrasonic irradiation displayed evidently stronger fluorescence signals compared to the other treatment groups. It meant that the ROS induced by SDT could cause the severe damage of mitochondrial membrane. Moreover, we also quantitatively assessed the mitochondrial damage in MCF-7/ADR cells with various treatments. The flow cytometry profiles of intracellular Rh123 are shown in Fig. 5d and S2b,† and the comparison of their fluorescence intensities is displayed in Fig. 5e. MCF-7/ADR cells with all treatments except ultrasonic irradiation alone exhibited significantly increased fluorescence intensities compared to the control, indicating that these treatments could induce the damage of mitochondrial membrane. By contrast, HPDF nanomicelles combined with ultrasonic irradiation displayed the highest induction effect.

The damage of mitochondria can lead to the release of Cyt c from the mitochondria to the cytoplasm, and subsequently activate the apoptotic cascade in cancer cells. Thus, the intracellular translocation of Cyt c is a major indicator of the mitochondrial damage and the initiation of cell apoptosis. The above results showed that HPDF nanomicelles combined with ultrasonic irradiation significantly induced the damage of mitochondrial membrane and the apoptosis in MCF-7/ADR cells. Here, we further detected the intracellular locations of Cyt c in MCF-7/ADR cells after various treatments using the immunofluorescence technique. Fig. 6a displays the subcellular location of Cyt c. The blue, green and red fluorescence signals represented the nucleus, mitochondria and Cyt c, respectively. In the merged images, the yellow fluorescence indicated that Cyt c was located in the mitochondria and the red fluorescence demonstrated that Cyt c was released from the mitochondria to the cytoplasm. Fig. 6b shows the comparison of relative fluorescence intensities of Cyt c released from the mitochondria in MCF-7/ADR cells with various treatments. The results were basically consistent with the above results of mitochondrial membrane damage. Obviously, the combined treatment of HPDF nanomicelles and ultrasonic irradiation also triggered a larger amount of Cyt c to be released from the miochondria than the other treatments, demonstrating the synergistic effects of chemotherapeutic activity of DOX and sonodynamic efficiency of HP, and also suggesting the efficient activation of mitochondria-mediated cell apoptosis.

Fig. 6 Analysis of Cyt c released from mitochondria in MCF-7/ADR cells with various treatments. (a) Confocal images of MCF-7/ADR cells at 48 h after treatments. Nuclei and mitochondria were stained respectively with DAPI (blue fluorescence) and Mito-Tracker Green FM (green fluorescence). Cyt c was detected by immunofluorescence technique, which exhibited red fluorescence. (b) Comparison of MFI of Cyt c released from mitochondria.

Illustration of reversal mechanisms of HPDF nanomicelles combined with ultrasonic irradiation against drug resistance in MCF-7/ADR cells

All above results indicated that HPDF nanomicelles prepared in this study could efficiently combine chemotherapy and SDT to reverse the resistance of MCF-7/ADR cells to DOX. The possible reversal mechanisms are illustrated in Fig. 7. First, HPDF nanomicelles can efficiently deliver DOX and HP into MCF-7/ADR cells via endocytosis and conspicuously improve the intracellular accumulation of DOX, which is contributed to shutting off the efflux function of P-gp by the ATP-depletion of Pluronic F68. Second, the released HP in MCF-7/ADR cells can trigger the production of a large amount of ROS after activation by the ultrasound. The high intracellular ROS level can further lead to the damage of mitochondrial membrane and the release of Cyt c from mitochondria, thus induce the apoptosis of MCF-7/ADR cells. Third, the released DOX can enter the nucleus to exert chemotherapeutic effects such as the DNA damage and the synthesis inhibition of biomacromolecules, and thereby further induce the cell apoptosis and cell cycle arrest. From description above, we can see that it is the synergistic effects of chemotherapy and SDT mediated by HPDF nanomicelles to efficiently reverse drug resistance in MCF-7/ADR cells.

Fig. 7 Schematic illustration of reversal mechanisms of HPDF nanomicelles combined with ultrasonic irradiation on drug resistance of MCF-7/ADR cells.

Conclusion

In this study, HPDF nanomicelles were prepared and their capability for reversing cancer drug resistance was evaluated in human drug resistant MCF-7/ADR cells, in which P-gp was over-expressed. The results showed that HPDF nanomicelles combined with a low-intensity ultrasound efficiently reversed drug resistance in MCF-7/ADR cells, which was evidenced by the enhanced cytotoxicity, the promoted cell apoptosis and cell cycle arrest compared to both free DOX and HPF nanomicelles mediated SDT. Several mechanisms were involved in the reversal effects of combination treatment of HPDF nanomicelles and ultrasonic irradiation on MCF-7ADR cells. HPDF nanomicelles efficiently delivered DOX and HP into MCF-7/ADR cells and conspicuously improved the intracellular accumulation of DOX. Moreover, HPDF nanomicelles triggered the production of a large amount of ROS in MCF-7/ADR cells, and subsequently induced the damage of mitochondrial membrane and the release of Cyt c from mitochondria, thus activated the mitochondria-dependent cell apoptosis pathway. In summary, this study put forward a novel strategy for reversing cancer drug resistance through the combination treatment of chemotherapy and SDT mediated by HPDF nanomicelles.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 81573005, 81371671 and 81572655).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra22724d

‡ These authors contributed equally.

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