Hydralazine loaded nanodroplets combined with ultrasound-targeted microbubble destruction to induce pyroptosis for tumor treatment | Journal of Nanobiotechnology
Materials
Hydralazine was acquired from GlpBio (Shanghai, China). Lactate dehydrogenase (LDH) Release Assay Kit and Reactive Oxygen Species Assay Kit were bought from Beyotime (Shanghai, China). N-acetyl-Lcysteine (NAC), a ROS inhibitor was bought from Macklin (Shanghai, China). Anti-DFNA5/GSDME was bought from Abcam (Cambridge, UK) and Proteintech (Wuhan, China). Cleaved Caspase-3 (Cleaved-CASP3) antibody was acquired from Cell Signaling Technology (Danvers, USA).
Cell culture
The American Type Culture Collection provided 4T1 (mouse breast cancer) cells. The cell lines were cultivated at 37 °C in a humid environment with 5% CO2 in RPMI 1640 medium (BasalMedia, Shanghai, China) supplied with 10% fetal bovine serum (FBS, Gibco, Carlsbad, USA) and 1% penicillin-streptomycin (Solarbio, Beijing, China). The study’s cell lines were verified through the use of short tandem repeats (STRs) profiling.
Animal model
The Qilu Hospital of Shandong University’s Laboratory Animal Ethics Committee authorized the protocols for the animal experiments. Female BALB/c mice (4–5 weeks) were purchased from charles river (Zhejiang, China). A suspension of 4T1 cells (1 × 106 cells) was subcutaneously injected into each mouse’s right flank to create the tumor model.
Preparation of (HYD-)NDs
Both NDs and HYD-NDs were produced through emulsion homogenization. Lecithin, PFH and Tween were added to deionized water and treated with an ultrasonic crusher (UP250, Scientz, China) at 150 w under ice bath conditions for 5 min (alternating between working for 10 s and resting for 10 s). After that, O-CMC solutions with or without HYD were added drop by drop while continuing the ultrasonic treatment for 5 min. Next, mixture was centrifuged at 300 rpm for 5 min. After being collected, the intermediate layer was centrifuged for 15 min at 12,500 rpm. The resulting precipitate was washed three times using PBS as purified NDs or HYD-NDs. For later use, purified NDs and HYD-NDs were re-suspended in PBS.
Characterization of (HYD-)NDs
The size distribution and zeta potential of HYD-NDs were examined by dynamic light scattering (DLS, Malvern Zetasizer Nano, UK). HYD-NDs were analyzed using transmission electron microscope (TEM, Tokyo, Japan) to confirm their morphological and dispersive properties. The stability of HYD-NDs was assessed from tracking the change in size at predetermined intervals within 50% FBS over a 48-hour period.
The ultraviolet absorption spectra of HYD-NDs, NDs, and HYD were obtained using an ultraviolet-visible spectrophotometer (DeNovix, Wilmington, USA). The standard curves of HYD drawn, and HYD’s loading efficiency (LE) and entrapment efficiency (EE) in HYD-NDs were calculated. Through dialysis at 37 °C, the drug release curve of HYD-NDs was established. A 2 mL solution of HYD-NDs was put in a dialysis bag and cultured in PBS buffers with various pH values on a shaker at 37 °C and 100 rpm in order to test the pH responsiveness of HYD-NDs. Then 1 mL aliquots were taken out and repeated with equal volumes of PBS at predetermined intervals. We measured the amount of HYD released with an ultraviolet spectrophotometer. To assess its ultrasonic sensitivity, HYD-NDs was enclosed within a dialysis bag, with or without exposure to ultrasonic irradiation, followed by the aforementioned treatment steps to generate an ultrasonic response release profile. A WED-300 focused ultrasound therapeutic instrument (Shenzhen, China) was applied, with the following main parameters: transducer size (irradiation area) of 6.0 cm2, working frequency of 1.0 MHz, intensity of 1.0 W/cm2, and irradiation time of 30 s.
In vitro cytotoxicity
The cytotoxicity to 4T1 cells was assessed via Counting Kit-8 (CCK-8) assay. The microplate reader (Infinite M200, TECAN, Switzerland) was applied to monitor the optical density (OD) at 450 nm after 4T1 cells were treated with NDs at diffrent concentrations.
Hemolysis assay
The hemolysis reaction was employed to examine the blood compatibility of nanodroplets. The blood cells resuspended in PBS were added to different concentrations of NDs, incubated at 37℃ for 1 h, and the supernatant was added to a 96-well plate. The OD value of every well was determined at 545 nm by the microplate reader.
In vivo biodistribution assessment
Upon the tumor volume reaching 200 mm3, Dil-marked HYD-NDs were injected intravenously into the mice. Subsequently, the mice underwent various time intervals of sacrifice (1, 2, 4, 8, and 24 h), during which tumors and primary organs were extracted. The study employed a Small Animal in vivo Imager (IVIS, PerkinElmer, Waltham, MA, USA) for both fluorescence imaging and quantification.
Intracellular uptake
The ability of ultrasound to enhance cellular uptake of HYD-NDs was evaluated. 24-well plates were seeded with 4T1 cells overnight, and the cells were subsequently treated with media containing Dil-marked HYD-NDs for two hours with or without ultrasonic irradiation. The intracellular uptake of HYD-NDs was photographed by fluorescence microscope (Eclipse Ti2, Nikon, Tokyo, Japan).
The pH responsiveness of cells to HYD-NDs uptake was assessed. Using a fluorescent microscope, Dil-marked HYD-NDs were examined and photographed after being co-incubated with cells in a serum-free media at pH 7.4 and pH 6.5 for two hours.
Liquid-gas phase transition
To detect the thermotropic phase transition, HYD-NDs was dropped on a glass slide inside the heating plate, the temperature of the heating plate was adjusted, and the phase transition of HYD-NDs was observed under an oil microscope.
To detect the acoustic phase transition, HYD-NDs were added into the well plate for ultrasonic irradiation (1.0 W/cm2, 30 s), and the irradiated HYD-NDs were dropped on a slide, and the phase transition was observed and compared under an oil microscope.
Ultrasound imaging capability
The HYD-NDs solution was pipetted into the specially built examination model made of pipette drips for in vitro imaging. The model was then submerged in 37 °C aqueous solution and assessed using the ultrasound system (LOGIQ E9, GE, USA) with 9 L linear transducer to determine its ultrasound imaging capability.
Mice were anesthetized and injected with either 200 µL NDs, HYD-NDs or PBS via their vein for contrast enhanced ultrasound (CEUS) imaging in vivo. An ultrasound probe was then positioned over the tumor area and ultrasound imaging was performed according to the aforementioned parameters.
LDH release assay
4T1 cells were planted and given several treatments in 96-well plates. Amounts of LDH seeping through injured cell membranes were used to assess the integrity of the cellular membrane, and then the OD value at 490 nm was recorded.
Measurement of ROS
After 24 h of treatment in different ways, proceed according to the instructions to determine ROS using the fluorescent probe DCFH-DA. Photographs were taken by fluorescence microscope.
Western blot
4T1 cells were subjected to various treatments, followed by cell harvesting and lysis using RIPA buffer supplemented with PMSF for 30 min. Protein samples from different experimental groups were used for subsequent experiments. The protein bands were analyzed by the chemiluminescence instrument (Tanon-4800, Shanghai, China).
TEM imaging
Following a 24-hour period of varying treatments, 4T1 cells underwent digestion, were gathered, treated with 2.5% glutaraldehyde fixative. Following the fixative’s removal, 2% osmium tetroxide was used instead. The sections were then visualized using TEM.
In vitro anti-tumor effect
4T1 cells were cultured with diverse treatments (PBS, NDs, Free HYD, HYD-NDs, NDs + US, HYD-NDs + US (1.0 W/cm2, 30 s, 1.0 MHz)), PBS served as control.
The CCK-8 assay was applied for assessing cell viability. Remove the old medium, add prepared CCK-8 solution and continue incubation for 1.5 h. Using a microplate reader, the OD values at 450 nm were determined.
The cell proliferation ability was evaluated utilizing the EdU-567 cell proliferation kit. The procedure was performed according to the kit procedure and then viewed under the fluorescence microscopy.
The transwell assay was applied to evaluate the capacity of cell invasion. 4T1 cells were treated differently and seeded at 4 × 104 cells/well in a chamber containing matrigel, and the lower layer of the chamber was filled with media containing 15% FBS. Twenty-four hours after cultivation, the transell chambers were fixed, stained. Then viewed and photographed using a microscope.
In vivo anti-tumor effect
Female BALB/c mice were utilized as an experimental model to observe the inhibitory effect of each treatment on tumor growth. Once the tumor reached a volume of 60 mm3, the mice were randomly divided into 6 groups (n = 5), the same group as in vitro. The dosage of HYD administered was 15 mg/kg, and ultrasound irradiation (1.5 W/cm2, 60 s, 1.0 MHz) was performed four hours after injection. The treatment duration lasted for twelve days during which the tumor volume and body weight of mice were recorded at two-day. At the end of observation period, all mice were sacrificed and tumor specimens were collected for HE staining, TUNEL staining, and immunohistochemical analysis (IHC). Furthermore, biological safety analyses were performed by means of tissue sections from primary organs.
Statistical analysis
Every experiment has been performed independently at least three times. The statistical information was displayed as mean ± SD. Using GraphPad Prism 9 software, statistical analysis was carried out in accordance with the student’s t-test or one-way ANOVA. Statistics were considered significant when p < 0.05. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.