Materials
Human serum albumin (HSA), 2-iminothiolane hydrochloride (Traut’s reagent, 2-IT), doxorubicin hydrochloride (DOX), dimethyl sulfoxide (DMSO), methanol, and chloroform were purchased from Sigma Aldrich (St. Louis, MO, USA). Two types of phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2k-NHS), were purchased from Nanocs Inc. (Boston, MA, USA). Chlorin e6 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RPMI 1640, fetal bovine serum (FBS), and antibiotic–antimycotic (AA) were purchased from Gibco (Waltham, MA, USA). The adriamycin resistant breast cancer cell line (MCF-7/ADR) was donated from Dr. Kwang Meyung Kim (Korea Institute of Science and Technology, Seoul, Korea).
Preparation of DOX-NPs/Ce6-MBs
First, 40 mg/mL of human serum albumin was dissolved in distilled water and the pH was adjusted to 8.5 using 0.2 N NaOH. For the thiolation of HSA, albumin solution was mixed with 2 mg/mL 2-iminothiolane hydrochloride (2-IT) in a 1:1 volume ratio and incubated for 1 h at room temperature (RT). The thiolated HSA (tHSA) solution was centrifuged at 4000 rpm for 10 min using an Amicon Ultra-30 kDa Centricon (Millipore, Billerica, MA, USA) to remove unreacted 2-IT. Then, 10 mg/mL doxorubicin (DOX) dissolved in distilled water was mixed with the tHSA solution in a 1:9 volume ratio. Ethanol was added dropwise in 1 mL/min until the solution became turbid and stirred overnight at RT. After the ethanol was almost evaporated, the tHSA solution was centrifuged at 13,200 rpm for 10 min to harvest nano-sized DOX-NPs.
Microbubbles were produced by mixing DSPC and DSPE-PEG2k-NHS in chloroform in a 9:1 molar ratio. Then, 0.5 mg/mL of Chlorin e6 dissolved in methanol was added to the lipid mixture and dried overnight under a vacuum. Dried lipid and Ce6 were dissolved in 0.5 mg/mL phosphate-buffered saline (PBS), and sonicated using a bath sonicator and incubated in hot water (over 55 °C). Then, the vial was filled with perfluoropropane gas and mixed for 45 s using VialMix™ to form the Ce6-MBs. To complete the fabrication of DOX-NPs/Ce6-MBs, DOX-tHSA-NPs and Ce6-MBs were mixed and incubated at RT for at least 1 h. The conjugation of DOX-NPs on the surface of Ce6-MBs was produced by an amide bond between the amine group of the DOX-tHSA-NPs and the NHS group of the Ce6-MBs.
Characterizations of DOX-NPs, Ce6-MBs, and DOX-NPs/Ce6-MBs
The size distributions of the DOX-NPs, MBs, and Ce6-MBs were analyzed by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Herrenberg, Germany). The in vitro release rate of DOX from the DOX-NPs was measured using high-performance liquid chromatography (HPLC) (YL9150, Younglin Anyang, South Korea). Each group was loaded into the dialysis membrane (Spectra/Por® 7, MWCO 2 kD) and then the drug-loaded dialysis membrane was placed in 10 mL of PBS (in a 15 mL tube). Dialysis membranes were maintained in a 37 °C shaking incubator. At predetermined time points, 10 mL of the PBS buffer was replaced with fresh buffer. Fluorescence imaging of the DOX-NPs/Ce6-MBs was conducted by confocal laser scanning microscopy (TCS SP8, Leica, Germany). The cavitation confdition of the microbubble was 1 MHz-US pressure waves (Sonoplus 490, Enraf–Nonius B.V., Rotterdam, Netherlands) with an intensity of 0.2 W/cm2 and a duty cycle of 50%.
Confirmation of the expression of drug efflux pump receptors
The expression levels of efflux pump receptor mRNAs in the MCF-7 cells and MCF-7/ADR cells were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Aliquots of 1 × 106 cells in 1 mL serum-free RPMI 1640 were placed in each well of a 6-well plate and incubated at 37 °C for 24 h. The total RNA was obtained using a RNeasy Mini Kit purchased from Qiagen (Venlo, Netherlands) and cDNA was synthesized using a High Capacity RNA-to-cDNA Kit purchased from Thermo Fisher Scientific (Waltham, MA, USA). RT-PCR was performed using AccuPower PCR PreMix purchased from BIONEER (Daejeon, Korea) and LightCycler FastStart DNA MasterPLUS SYBR Green purchased from Roche (Basel, Switzerland) was used for real-time PCR. ABCB1 (P-glycoprotein, P-gp), ABCC1 (multidrug resistance-associated protein 1, MRP1), and ABCG2 (ATP binding cassette subfamily G member 2) primers were purchased from BIONEER (Daejeon, Korea). Fluorescence imaging of P-glycoprotein was detected using rabbit anti-P-glycoprotein antibody and Alexa Fluor 555 goat anti-rabbit antibody purchased from Abcam (Cambridge, UK) by confocal laser scanning microscopy.
Determination of the intracellular uptake and retention of DOX and Ce6
The intracellular uptake of DOX and Ce6 was determined by confocal laser scanning microscopy. Aliquots of 2 × 105 MCF-7/ADR cells in 1 mL RPMI 1640 were seeded into each well of a collagen-coated 12-well plate and incubated at 37 °C for 24 h. US was irradiated immediately after treatment with DOX-NPs, Ce6-MBs, and DOX-NPs/Ce6-MBs (0.2 W/cm2, 50% duty cycle, 30 s/well) and the cells were incubated for an additional 3 h. Each group was treated with 2 mg/mL DOX and 1 µg/mL Ce6. After another incubation, the cells were washed with cold DPBS (Dulbecco’s Phosphate-Buffered Saline) and fixed with 4% paraformaldehyde. The cells were mounted using a mounting solution and stained with DAPI (4′,6-diamidino-2-phenylindole) (Vectashield, Vector Laboratories. Inc., Burlingame, CA, USA).
To verify the intracellular retention of DOX, each group was treated with DOX-NPs/Ce6-MBs. The groups included control (no treatment), verapamil-treated (positive control), and laser-treated groups. Each group was treated with DOX-NPs/Ce6-MBs and US and incubated for 3 h. After 3 h, each group was washed with PBS and incubated further for different times (in 0.5 h increments for confocal microscopy and l h increments for flow cytometry). At each time point, the cells were fixed with 4% paraformaldehyde and examined using confocal microscopy and flow cytometry.
Measurement of ROS generation
For the detection of ROS generation by the DOX-NPs, Ce6-MBs, and DOX-NPs/Ce6-MBs, the DCFDA kit (Cellular Reactive Oxygen Species Detection Assay Kit, Abcam, Cambridge, UK) was used. MCF-7/ADR cells in 100 µL RPMI 1640 were cultured in 96-well plates to over 80% density and incubated overnight. Then, all groups were changed to DCFDA solution, incubated for 45 min at 37 °C, and the DOX-NPs/Ce6-MBs were treated with 2 mg/mL DOX and 1 µg/mL Ce6. The cells were immediately irradiated with US using the same conditions as in the cellular uptake experiment, incubated for 3 h, and laser-irradiated (671 nm wavelength, 1.0 J/cm2, 100 mW). The intracellular ROS levels were measured at 488 nm by a microplate reader (Bio-Tek, Winooski, VT, USA).
Side Population assay
For detection of the side population, 3 × 105 MCF-7/ADR cells in 1 mL RPMI 1640 were cultured in 6-well plates. The cells were incubated at 37 °C for 90 min with Hoechst 33342 dye (Sigma Aldrich) alone, or with 50 µM verapamil (Sigma Aldrich). The cells were washed with DPBS twice, centrifuged, and re-suspended in 2% FBS/PBS solution. The cells were analyzed with image cytometry based on the intracellular levels of Hoechst 33342 dye.
In vitro cell viability assay
To confirm the cytotoxic effect of the DOX-NPs/Ce6-MBs with laser via sonoporation an MTT assay was performed in the MCF-7/ADR cells. The cells (1 × 104) were seeded in each 96-well plate and cultured at 37 °C for 24 h. The groups used in the experiment were negative control (non-treated), free DOX, DOX-NPs, and DOX-NPs/Ce6-MBs (N = 6) which were further divided into irradiation with US (0.3 W/cm2, 50% duty cycle, 10 s/well) and laser (671 nm wavelength, 1.0 J/cm2, 50 mW) groups. After a 3 h incubation, all groups were changed to fresh media and cultured for a further 48 h. To perform the MTT assay, MTT solution (0.5 mg/mL) was added to each well and the cells were incubated at 37 °C for 2 h. Then, 100 µL of DMSO was added to dissolve the formazan crystals in the living cells. The absorbance intensity was measured at 570 nm by a microplate reader (Bio-Tek).
Statistical analysis
All experimental data are presented as the mean ± standard error of at least three independent experiments. Statistical analysis was performed using Student’s t-test and one-way ANOVA were used for multiple comparisons. A p-value less than 0.05 was considered statistically significant.