2.1 Chemicals
Polyvinyl alcohol (PVA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), levofloxacin (LEV), isopropanol, dichloromethane (DCM), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), and Tween-80 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly (lactide-co-glycolic) acid (PLGA) polymer material with a molecular weight of 21 kDa (ratio of lactide to glycolic acid molar ratio of 50:50) was purchased from Rui Jia Biological (Xi’an, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), trypsin, and phosphate buffered saline (PBS) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Penicillin-streptomycin solution, a reactive oxygen species (ROS) assay kit, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), and 4,6-Diamidino-2-phenylindole (DAPI) were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Middlebrook’s 7H9 broth medium, Luria-Bertani (LB) broth, and oleic acid-albumin-dextrose-catalase (OADC) were purchased from BD Biosciences (New York, USA).
2.2 Cell and bacterial culture experiments
Mouse peritoneal macrophages RAW264.7 were purchased from the Shanghai Institute of Cells, Chinese Academy of Sciences and cultured in a humidified incubator under a setting of a partial pressure of 5% CO2 at 37 °C in DMEM, which was supplemented with 1% penicillin/streptomycin and 10% FBS. The RAW264.7 cells were generally seeded in a cell culture flask (Corning, USA) for 12 h to adhere. Then, they were harvested using a 0.25% trypsin-EDTA solution for 2 min for the following experiments.
The bacterial strain used in this study was M. smegmatis mc2155 (purchased from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China), an acid-fast bacterial species, which is considered to be a model organism for researching Mtb in the laboratory [29]. The bacteria were grown in Middlebrook’s 7H9 broth medium supplemented with 10% OADC and 0.05% Tween-80 at 37 °C for 24 h with agitation (180 rpm). The bacteria were allowed to reach the exponential phase with an optical density (OD600) of 0.6–0.8, and were harvested and re-suspended using PBS to a concentration of 106 CFU/mL for the following experiments. The minimally inhibitory concentration (MIC) of LEV against M. smegmatis was determined using the micro-broth dilution method.
2.3 Preparation and characterization of the nanoparticles
The LEV-loaded PLGA nanoparticles (LEV-NPs) were prepared using the double emulsification method by sonication as previously described [28, 33]. Briefly, PLGA was dissolved completely in DCM. A pre-weighed amount of LEV was dissolved in acetic acid that was miscible with water (20:80%, v/v). The dissolved PLGA polymer material and the drug were mixed for the first ultrasonic sonication using an ultrasonic processor (XL2020, USA) in an ice bath at 100 W ultrasonic power for 2 min. Next, the 1% PVA cooled was added to the polymeric mixture for the second ultrasonic sonication in an ice bath at 100 W ultrasonic power for 5 min. After that, 2% isopropanol was added to the suspension, followed by magnetic stirring in an ice bath in the fume hood for at least 4 h until there was no pungent smell to complete the removal of DCM from the prepared nanoparticles. The purity of the prepared nanoparticles was further confirmed using proton nuclear magnetic resonance (1H-NMR) spectroscopy (Varian, Palo Alto, CA, 400 MHz) with no residual peak of the DCM solvent. The LEV-NPs were washed and collected using centrifugation at 8000 g for 10 min. After that, the LEV-NPs were lyophilized in a freeze dryer (Christ ALPHA 2–4 LSC plus, Osterode, Germany) for the following study. Plain PLGA nanoparticles (NPs) and Dil loading PLGA nanoparticles (DiI-NPs) were prepared following a similar method, except that the drug was exchanged for an equal amount of deionized water or DiI (final concentration of 10 µM).
The average diameter, zeta potential, and polydispersity index (PDI) were determined by dynamic light scattering (DLS) using a Malvern laser particle size analyzer (Zeta SIZER 3000HS, USA). The morphological characterization of the nanoparticles was observed using transmission electron microscopy (TEM, Hitachi High-Technologies, Tokyo, Japan) and scanning electron microscopy (SEM, Hitachi High-Technologies).
2.3.1 Determination of the LEV-NPs loading content and encapsulation efficiency
A total of 2 mg of freeze-dried nanoparticles was resuspended in 1 mL of dimethyl sulfoxide (DMSO) after the nanoparticles were destroyed. Then the drug concentration was determined using a UV−Vis spectrophotometer (UV-2600 SHIMADZU) at 290 nm. The drug loading content (LC) and encapsulation efficiency (EE) were calculated using the following equations:
LC (%) = [weight of the drug in nanoparticles / weight of the nanoparticles] × 100%, and
EE (%) = [weight of the drug in nanoparticles / weight of the feeding drug] × 100%.
2.3.2 In vitro ultrasound-triggered LEV release from the LEV-NPs
The kinetic release of LEV from the LEV-NPs in vitro with sonication was investigated. A sample of LEV-NPs lyophilized powder was diluted in PBS by sonication (fixed frequency of 42 kHz) at an intensity of 0.13 W/cm2 for 10 min. After sonication, the samples were then individually transferred into dialysis bags (34 mm flat width, MWCO: 7000 Da, Biosharp, Hefei, China), which were then incubated in 50 mL of PBS and shaken at 100 rpm. At each predetermined time point, dialysate samples (1 mL) were individually collected for determination of the LEV concentration using UV-vis spectrophotometry (UV-2600 SHIMADZU, Japan) at 290 nm. Then the samples were returned into the original solution to maintain the total volume of the dialysate constant. Dialysate samples from the LEV-NPs that did not undergo sonication were used as controls. The cumulative drug release (%) was calculated using the following equation:
Cumulative release (%) = [weight of LEV released from LEV-NPs / initial weight of the drug in LEV-NPs] × 100%.
2.4 Ultrasonic irradiation method
The LFLIU system device used in this experiment was developed by the Chongqing Medical University Institute of Biomedical Engineering. It had a transducer diameter of 45 mm, a fixed frequency of 42 kHz, and an adjustable ultrasonic intensity output of 0.13 W/cm2 to 0.33 W/cm2. The acoustic field was measured using a hydrophone (Onda Corp, Sunnyvale, CA, USA). The medical ultrasonic couplant was uniformly coated on the top of the transducer. The 35 mm cell culture dish was placed directly above the transducer and gently squeezed to expel the air. This was followed by ultrasound irradiation (as shown in Fig. 1) with a working mode of a continuous-wave. In this study, the dose of ultrasound at an intensity of 0.13 W/cm2 with irradiation for 10 min was selected due to little effect on macrophage activity (based on the preliminary data). Prior to the experiment, all of the samples were equilibrated at room temperature (25 °C) using air conditioners. The temperatures of the cell suspensions were monitored using needle thermo-sensors with digital displays (batch 119, No. 02810232; Yuyao Temperature Instrument Factory Co., Ltd, China).
2.5 Cytotoxicity assay
The cytotoxicity of the LEV and the LEV-NPs against the RAW264.7 cells was investigated using a MTT assay. Briefly, the RAW264.7 cells (1 × 105 cells/mL) were grown in a Petri dish for 24 h to allow cell adhesion. They were then treated with the LEV and LEV-NPs containing the final drug concentrations of 0 µg/mL, 2 µg/mL, 4 µg/mL, 8 µg/mL, 16 µg/mL, 32 µg/mL, 64 µg/mL, 128 µg/mL, and 256 µg/mL for 4 h. After that, the cells were continuously irradiated for 10 min using 0.13 W/cm2 ultrasound (the ultrasound dose used was based on the preliminary data), and then the cells were cultured for another 24 h. The MTT experiment was then conducted. The control group consisted of the same procedure, but no ultrasound treatment. The relative cell viability was calculated as follows: cell viability (%) = OD570 (treatment)/OD570 (control) × 100%. The values are presented as averages of the three independent experiments.
2.6 Phagocytosis of macrophages on the nanoparticles
In this study, DiI-NPs was used as a model to study the phagocytic effect of macrophages on the LEV-NPs under ultrasound. The nucleus of RAW264.7 cells were stained with DAPI (blue fluorescence, 10 µg/mL). DiI-NPs (red fluorescence, 4 µg/mL) were added to the culture dish of RAW264.7 cells, and then irradiated with 0.13 W/cm2 ultrasound for 10 min. Those cells without ultrasonic irradiation were used as the control. After incubation for another three hours, the plates were washed three times to remove the extracellular DiI-NPs. RAW264.7 cells were observed by laser confocal microscopy (CLSM, A1 + R, Nikon, Tokyo, Japan) at excitation/emission wavelengths of 364/454 nm for DAPI and excitation/emission wavelengths of 549/565 nm for Dil. In addition, the relative fluorescence intensity of the intracellular DiI-NPs was quantified by flow cytometry (CytoFLEX, Beckman Coulter, Inc. CA, USA).
2.7 Intracellular killing ofM. smegmatis and the TEM observations
The killing efficiency of ultrasound combined with nanoparticle treatment on M. smegmatis in macrophages was investigated. A total of 105 RAW264.7 cells per well were seeded in a Petri dish and allowed to grow for 24 h. After washing three times with an antibiotic-free medium, freshly cultured M. smegmatis (ratio of bacteria/cells:10:1) were added into the Petri dish to infect the cells for 2 h. They were then washed three times to remove the extracellular bacteria. The RAW264.7 cells infected with M. smegmatis alone without ultrasound treatments were used as the controls. Subsequently, the infected macrophages were incubated in DMEM to expose them to the LEV-NPs (where the LEV was at a final concentration of 4 µg/mL) and the free LEV (at a final concentration of 4 µg/mL). After this, the macrophages were irradiated using an ultrasonic intensity of 0.13 W/cm2 for 10 min, cultured for another 3 h at 37 °C in an atmosphere of 5% CO2, and then the extracellular free LEV and LEV-NPs were washed using PBS. After treatment for 12 h, the cells were washed and collected. One portion of the cell samples after treatment were made into ultrathin sections for observation of the internal structure of the macrophages by transmission electron microscopy (TEM, JEM-1400PLUS, Hitachi High-Technologies). The other portion of the cell samples were lysed using distilled water containing 0.25% SDS for the observation of the intracellular bacteria and an evaluation of bacterial activity. The survival of the intracellular bacteria was estimated by plating serially diluted cultures on 7H10 plates, and the colony-forming units (CFUs) were enumerated after 48 h. All of the samples were plated in triplicate, and the values were averaged from three independent trials. Similarly, the intracellular bacteria were also made into ultrathin sections for TEM observation.
2.8 Quantification of the intracellular reactive oxygen species
The intracellular ROS were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter, Inc. CA, USA) with a ROS reagent kit and fluorescent probe DCFH-DA. The DCFH-DA itself has no fluorescence, and after entering the cell, it was hydrolyzed by the esterase in the cell to form dichlorofluorescein (DCFH). The intracellular ROS oxidizes non-fluorescent DCFH to produce fluorescent dichlorofluorescein (DCF), which is impermeable to the cell membrane. Therefore, the level of ROS in the cells can be known by detecting the fluorescence of the DCF. Briefly, infection RAW264.7 cells were incubated with DCFH-DA (final concentration of 10 µM) for 30 min. The nucleus was blue stained with DAPI (10 µg/mL) for 10 min. After this, the cells were washed and incubated in DMEM for exposure to the LEV-NPs (drug concentration of 4 µg/mL) and the free LEV (4 µg/mL) and treated with ultrasound at an intensity of 0.13 W/cm2 for 10 min. Then the treated cells were collected, resuspended in serum-free medium, and measured using the flow cytometer with the excitation setting at 488 nm. The obtained data were analyzed using Cell Lab Quanta SC MPL Analysis software (CytoFLEX, Beckman Coulter, CA, USA). In addition, the level of intracellular ROS production was observed using a laser confocal microscope (CLSM, A1 + R, Nikon) at the excitation/emission wavelengths of 364/454 nm for the DAPI and the excitation/emission wavelengths of 488/525 nm for the DCF. Without ultrasound irradiation, the others were treated with the same method as a control. The experiments were repeated independently three times.
2.9 Apoptosis and necrosis of the RAW264.7 cells
The Annexin V-FITC/PI double staining kit was used to detect the apoptosis and necrosis of RAW264.7 cells under the different treatments: control (no drug, no ultrasound), free LEV(only LEV), ultrasound (US), ultrasound combined with free LEV (US + LEV), LEV-NPs, and ultrasound combined with LEV-NPs (US + LEV-NPs). The drug concentrations in the LEV group and the LEV-NPs group were 4 µg/mL based on the MIC. The ultrasonic dose used was 0.13 W/cm2 for 10 min (based on preliminary data). After the treatment was completed, the cells were incubated for another 24 h. The treated cells were collected and resuspended in 1 mL PBS while adding 5 µL of Annexin V-FITC and 10 µL of PI. The dye was mixed and incubated at room temperature for 15 min in a dark environment. The resulting samples were detected using flow cytometry (CytoFLEX, Beckman Coulter, Inc. CA, USA). The obtained data was analyzed using Cell Lab Quanta SC MPL Analysis software (CytoFLEX, Beckman Coulter). The experiments were repeated independently three times.
2.10 Statistical analysis
The results were analyzed using a one-way ANOVA in SPSS 17 statistical software (IBM, Chicago, USA). The data were expressed as mean ± standard deviation. A P < 0.05 was considered to be statistically significant.