Materials
Distearoylphospha-tidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)−2000 (DSPE-PEG2000), 3-(N-(N′,N′-dimethyl-laminoethane)-carbamoyl-cholesterol (DC-CHOL), and Perfluoropentane (PFP) were purchased from Avanti Polar Lipids (Alabaster, USA). Tannic acid (TA) and Iron (III) chloride hexahydrate (FeCl3·6H2O) were from Sigma-Aldrich (St. Louis, MO, USA). Glucose oxidase (GOx), glutathione (GSH), methylene blue (MB), and hydrogen peroxide (H2O2, 30 wt%) were from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Liproxstatin-1 (Lip-1) and deferoxamine mesylate (DFOM) were from Med Chem Express (Shanghai, China). Cell counting kit-8 (CCK-8) assay, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), Bicinchoninic acid (BCA) assay kit, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), Calcein-AM, propidium iodide (PI), Glutathione (GSH) assay kit, and Malondialdehyde (MDA) assay kit were from Beyotime Biotechnology Co., Ltd. (Shanghai, China). GPX4 and GAPDH antibodies were from Proteintech Group, Inc. (Wuhan, China). 4T1 cells (triple negative breast cancer cells), MCF-10 A cells (breast normal cells), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were from Pricella Biotechnology Co., Ltd. (Wuhan, China).
Preparation of PND and PND@GOx
The phase-transition nanodroplets (PND) were prepared by using thin-film hydration and ultrasonic emulsification method [34]. DSPE-PEG2000, DC-CHOL and DSPC were mixed together at weight ratio of 2:2:5. Next, the lipid mixture was dissolved in chloroform, and then transferred into a rotary evaporator. The solvent was evaporated at 50 °C with continuous rotation for 2 h, resulting in formation of a lipid thin film. Then, the lipid film was hydrated in 3 mL PBS. Subsequently, 100 µL perfluoropentane (PFP) was added into the lipid film solution, and emulsified with a sonicator (125 W) for 5 min. Finally, PND was obtained after centrifugation. To prepare PND@GOx, 6 mg GOx was also added into the lipid solution before emulsification. The synthesis of Cy5.5-loaded nanoparticles followed the same procedure as for PND@GOx, except that DSPE-PEG2000 was replaced with Cy5.5-DSPE-PEG2000.
Preparation of PND@Fe-TA and PND@GOx@Fe-TA
1 mg PND was dispersed into 2 mL ultrapure water and followed by adding 10 µL tannic acid (TA) solution (40 mg/mL) and 20 µL FeCl3 solution (10 mg/mL), and then under vortex. Afterward, NaOH solution (0.1 mol/L) was added into the solution to neutralize. Subsequently, the mixed solutions were centrifuged to acquire PND@Fe-TA. Finally, the as-prepared PND@Fe-TA was rinsed with ultrapure water for future use. The preparation of PND@GOx@Fe-TA was similar, except for the replacement of PND with PND@GOx.
In vitro Fe release assay
To measure pH-triggered release of Fe, 1 mg PND@GOx@Fe-TA was dispersed in 1 mL PBS under different pHs (6.0 and 7.4). The suspension was dialyzed in 10 mL corresponding buffer medium for 24 h (cut-off 500 Da MW). After that, 1 mL dialysis solution was removed at the selected time interval, and measured by inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer, USA), meanwhile an equal volume of fresh buffer medium was added into dialysis solution.
In vitro GOx release assay
4 mL PND@GOx@Fe-TA solution (1 mg/mL) was divided into two groups, one group was irradiated with 808 nm laser (1 W/cm2, 5 min) at the selected time interval. Then, the solution was centrifuged after each irradiation, and the content of released GOx in supernatant was quantified through UV-vis spectrophotometer (PerkinElmer, USA) and calculated on the basis of the standard curves. The other group went through the same treatment without laser irradiation.
Detection of gluconic acid generation
Since glucose could be catalyzed to produce gluconic acid and lower pH, we measured the pH of system to detect the gluonic acid generation with or without glucose. First, 2 mL PND@GOx@Fe-TA solution (1 mg/mL) was irradiated with 808 nm laser (1 W/cm2, 5 min). Subsequently, 5 mL glucose solution (1 mg/mL) was added to the above solution, and the pH of system was measured at regular time interval using a pH meter (INESA, China).
Detection of O2 consumption
The O2 consumption was detected by an O2 content meter in 10 mL Hepes buffer. The concentrations of glucose and PND@GOx@Fe-TA were set at 0.1 mg/mL and 0.2 mg/mL, respectively. Prior to measuring the O2 content, PND@GOx@Fe-TA solution was irradiated with 808 nm laser (1 W/cm2, 5 min).
Detection of GSH consumption
PND@GOx@Fe-TA (200 µg/mL) was dispersed in PBS solution (pH 6.0) containing GSH solution (1 mM), and the supernatant was removed at predefined time points (0, 1, 2 and 4 h). After mixing with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) solution, the absorbance of the sample was measured by using UV-vis spectrophotometer.
Inspection of •OH generation
The degradation of MB was detected via classical colorimetry to evaluate the hydroxyl radical (•OH) generation ability of PND@GOx@Fe-TA. Firstly, PND@GOx@Fe-TA was dispersed into 5 mL phosphate buffer (pH 6.0) containing H2O2 (10 mM) and MB (5 µg/mL), and incubated at 37 ◦C. After centrifugation, the UV-vis spectrophotometer was used to determine the absorbance of supernatant at 665 nm with different time intervals. Subsequently, the •OH generation ability of PND@GOx@Fe-TA was studied ulteriorly under different pHs (6.0 and 7.4). The PND@GOx@Fe-TA nanocomplexes were similarly dispersed into 5 mL phosphate buffer with different pHs (6.0 and 7.4) containing H2O2 (10 mM) and MB (5 µg/mL), followed by incubation at 37 ◦C for 2 h. Then, the •OH-induced MB degradation was detected by UV-vis spectrophotometer. As a contrast, the MB solution and MB/H2O2 mixture solution were also monitored under the same conditions. Thereafter, PND@GOx@Fe-TA was suspended in 5 mL phosphate buffer (pH 6.0) containing H2O2 (10 mM) and MB (5 µg/mL), and incubated at either 37–43 °C. The absorbance of MB was subsequently determined using a UV-vis spectrophotometer. Finally, we investigated the •OH generation ability of PND@GOx@Fe-TA with glucose. Glucose (1 mg/mL) and MB (10 µg/mL) were added into the PND@GOx@Fe-TA solution and incubated at 37 °C. After centrifugation, the absorbance of supernatant at 665 nm with different time intervals was measured by UV-vis spectrophotometer.
In vitro photothermal property
An 808 nm laser (2 W/cm2) was used to irradiate 200 µL aqueous dispersion of PND@GOx@Fe-TA with different concentrations (0, 75, 150, 300, and 600 µg/mL) for 10 min. Water was used as control. Similarly, PND@GOx@Fe-TA solution at a concentration of 300 µg/mL was irradiated using an 808 nm laser with different irradiation intensities (0.5, 1, 1.5, and 2 W/cm2) for 10 min. The temperature was recorded by a thermal infrared camera (E6, Inc, USA) every 30 s. After turning off the laser, the sample was naturally cooling for another 10 min. Three cycles of laser on/off were employed to study the photothermal stability of PND@GOx@Fe-TA. Photothermal conversion efficiency (PCE) of PND@GOx@Fe-TA was calculated according to previously report [35].
Intracellular GSH, MDA, and GPX4 detection
4T1 cells in six-well plates were incubated with (1) Control, (2) Laser, (3) PND@Fe-TA, (4) PND@GOx@Fe-TA, and (5) PND@GOx@Fe-TA + Laser (PND@GOx@Fe-TA + L) for 12 h. For laser groups, an 808 nm laser (1 W/cm2) was employed to irradiate cells for 5 min. The GSH amount was detected using GSH assay kit and malondialdehyde (MDA) amount was evaluated using MDA assay kit. The glutathione peroxidase 4 (GPX4) protein expression was examined by western blot assay.
Intracellular LPO assay
4T1 cells were plated into confocal culture dishes and incubated overnight. Then, the cells were divided into five groups and subjected to the following treatments: (1) Control, (2) Laser, (3) PND@Fe-TA, (4) PND@GOx@Fe-TA, and (5) PND@GOx@Fe-TA + L. Group (2) and (5) were exposed to an 808 nm laser (1 W/cm2) for 5 min. After these treatments, the cells were stained with C11-BODIPY581/591 fluorescent probe for 30 min prior to imaging.
Cellular ferroptosis Inhibition assay
4T1 cells were seeded in a 96-well plate and cultured for 24 h. Then, the cells were co-incubated with PND@GOx@Fe-TA (600 µg/mL) and two types of ferroptosis inhibitors including Liproxstatin-1 (Lip-1) (2, 4, and 8 µM) and deferoxamine mesylate (DFOM) (50, 80, and 100 µM). After another 24 h incubation, the cell viability was measured by CCK-8.
Cellular uptake assay
The cellular uptake of PND@GOx@Fe-TA was confirmed by analyzing the Fe content using ICP-MS. 4T1 cells were seeded in a 12-well plate and incubated overnight. PND@GOx@Fe-TA was added to the cells and incubated for different time periods. After incubation, the cells were rinsed three times with PBS and digested using trypsin. Next, cells were counted and mixed with aqua regia (a mixture of nitric acid and hydrochloric acid in a 1:3 volume ratio) for 24 h. Subsequently, ICP-MS was conducted to quantify the Fe content within each sample. PBS treatment group was used as control.
Cell cytotoxicity assay
Breast cancer cell (4T1) and breast normal cell (MCF-10 A) were used to investigate the cytotoxicity of PND@GOx@Fe-TA. The cells were seeded into 96-well plate and cultured for overnight. Then the fresh medium containing PND@Fe-TA or PND@GOx@Fe-TA at different concentrations (0, 75, 150, 300, and 600 µg/mL) were added to the well and incubated for 24–48 h. Finally, CCK-8 assay was used to determine the cell viability.
To study the therapeutic efficacy of PND@GOx@Fe-TA under laser irradiation, 4T1 cells were seeded in a 96-well plate. Then, the cells were treated with (1) Control, (2) Laser, (3) PND@Fe-TA, (4) PND@GOx@Fe-TA, and (5) PND@GOx@Fe-TA + L. Group (2) and (5) were exposed to an 808 nm laser (1 W/cm2) for 5 min. After different treatments, the cell viability was analyzed by CCK-8.
Intracellular ROS detection
4T1 cells were seeded into 24-well plate and cultured overnight. Subsequently, the cells were divided into five groups: Group 1 incubated with 4T1 cells only; Group 2 incubated with Laser; Group 3 incubated with PND@Fe-TA; Group 4 incubated with PND@GOx@Fe-TA; Group 5 incubated with PND@GOx@Fe-TA + L. Group 2 and 5 were exposed to an 808 nm laser (1 W/cm2) for 5 min. After different treatments, the cells were dyed with 2.7-dichlorofluorescin diacetate (DCFH-DA) for 30 min. The fluorescence of DCF was captured by fluorescence microscope. ImageJ software was used to analyze fluorescence intensity.
In vitro Live/dead cell staining
4T1 cells were seeded into 24-well plate and cultured overnight. Then the cells underwent the same treatment as described in the intracellular ROS Assay and stained by Calcein-AM and PI. Finally, the cells were washed with PBS and observed under fluorescence microscope.
Animal treatment
All animal studies were conducted in accordance with the standards outlined in China’s National Regulations for the Care and Utilization of Laboratory Animals and after approval by the ethical committee of Harbin Medical University Cancer Hospital.
In vitro and in vivo PA, MR, and CEUS imaging
To assess the potential of PND@GOx@Fe-TA as a contrast agent for PA imaging in vitro, PND@GOx@Fe-TA with various concentrations ranging from 25 µg/mL to 250 µg/mL were analyzed using the Vivo LAZR PA imaging system (VisualSonics, Canada). Additionally, BALB/c mice bearing 4T1 tumors were chosen to evaluate the PA imaging performance in vivo. The mice received PND@GOx@Fe-TA via tail vein injection, and PA signals from the tumors were recorded at different time points (0, 2, 6, and 12 h).
To investigate the potential of PND@GOx@Fe-TA as a contrast agent for MRI in vitro, different concentrations of PND@GOx@Fe-TA were tested using a Bruker 9.4T MR scanner (Bruker, Ettlingen, Germany). 4T1 tumor-bearing BALB/c mice were utilized to evaluate the MR imaging property in vivo. The mice were injected with PND@GOx@Fe-TA via the tail vein, and MRI signals from the tumor sites were captured at different time intervals (0 and 6 h).
To study the function of PND@GOx@Fe-TA as a contrast agent for in vitro ultrasound imaging, Contrast enhanced ultrasound (CEUS) images of PND@GOx@Fe-TA were obtained at various concentrations (50, 100, 200, 300, and 400 µg/mL) using a Resona R9 device (Mindray, China). Following exposure of PND@GOx@Fe-TA to an 808 nm laser at 1 W/cm² for 5 min, CEUS images were captured for each concentration group. For in vivo ultrasound imaging, the mice were injected intravenously with PND@GOx@Fe-TA, and tumor images were acquired before and after laser irradiation (808 nm, 1 W/cm2, 10 min). Meanwhile, SonoVue was utilized as a positive control.
In vivo antitumor therapy
4T1 cells were subcutaneously administered into BALB/c mice to establish a tumor model. Once the tumor volume had reached approximately 100 mm3, the mice were randomly assigned to five different groups, including PBS, PBS + L, PND@Fe-TA, PND@GOx@Fe-TA, PND@GOx@Fe-TA + L. Both PND@Fe-TA and PND@GOx@Fe-TA at an equivalent dose of 100 µL were administered via the tail vein with a concentration of 1 mg/mL. After 6 h of injection, the mice were irradiated with an 808 nm laser (1 W/cm2, 10 min). A thermal infrared camera was used to record the photothermal images and temperatures at various points. The tumor volumes and body weights were recorded every other day for 14 days. The tumor volume (mm³) was calculated using the formula: V = (L × W2)/2, where W represents the minimum diameter and L represents the maximum diameter. The relative tumor volume was determined by calculating the ratio V/V0, where V0 denotes the tumor volume prior to treatment and V denotes the tumor volume following treatment.
In vivo distribution and excretion study
To assess the biodistribution, tumor-bearing BALB/c mice were injected with Cy5.5-PND@GOx@Fe-TA via tail vein and imaged at 2, 4, 6, 12, and 24 h. The mice were then euthanized at 24 h for ex vivo imaging of organs and tumor tissue.
To evaluate the excretion of PND@GOx@Fe-TA, the mice were intravenously administered PND@GOx@Fe-TA and then housed in metabolic cages to facilitate sample collection. Urine and feces were collected, digested with aqua regia, and analyzed by ICP-MS.
In vivo histological and hematological examinations
At the end of treatment, the tumor-bearing mice were euthanized at 14 days post-injection, and mouse tumors were then collected for photographing and subsequent staining with Hematoxylin and Eosin (H&E) as well as GPX4. The staining intensity of GPX4 immunohistochemical was graded as negative (score 0), weak (score 1), moderate (score 2), or strong (score 3). The H‑score for each slide was computed as ∑pi × i, where “pi” denoted the percentage of cells at intensity level “i”, and “i” was the corresponding staining intensity. Additionally, the vital organs, including the heart, liver, spleen, lung, and kidney, were sectioned and stained with H&E to assess the histological alterations induced by the various treatments.
Fresh blood collected from post-treated mice on the 14th day was subjected to routine hematological analysis using an automated hematology analyzer. Additionally, the hemolysis study was performed to evaluate biosafety. Briefly, mice fresh blood was centrifuged, and the red blood cells were resuspended in either normal saline (negative control), distilled water (positive control), or varying concentrations of PND@GOx@Fe-TA. All samples were incubated for 2 h and then centrifuged at 1500 g for 10 min. The supernatant’s absorbance at 545 nm was measured using a UV-vis spectrophotometer, and the hemolysis rate was calculated using the following formula: hemolysis rate (%) = (sample absorbance − saline absorbance)/(water absorbance − saline absorbance) × 100%.
Statistical analysis
All data were described as mean ± standard deviation (SD). A one-way ANOVA was used to analyze the significant difference among multiple groups. Besides, paired t-test was conducted for comparison with pairs of groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
