Materials, cells and animals
All DNA sequences (the primer: 5ʹ-GTGGTGGTGTTGGTGGTGGT-3ʹ. the template: Phosphate-CCACCAACACCACCACCACCTTTGACACACTAGCGATACGCGTATCGCTATGGCATATCGTACGATATGCCAGTGTGTCTTTCCACCA), deoxy-ribonucleoside triphosphate (dNTP) and bovine serum albumin (BSA) were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Phi29 DNA polymerase was from Lucigen Co., Ltd (USA). T4 ligase was obtained from Huamaike Bio Co., Ltd (Beijing, China). Hemin and Chlorin e6 (Ce6) were purchased from Frontier Scientific Co., Ltd (Utah, USA). Tris, KCl, NaCl, ammonium molybdate and H2O2 (30%) were from Sinopharm Co., Ltd (Shanghai, China). Singlet oxygen sensor green reagent (SOSG), 2′,7′-Dichlorofluorescin (DCFH-DA), GSH Assay Kit and Calcein-AM/PI were obtained from Solarbio Co., Ltd (Beijing, China). C11 BODIPY 581/591 was purchased from Glpbio Co., Ltd (CA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from Gibco Co., Ltd. Penicillin–streptomycin solution, 0.25% (w/v) trypsin solution, methyl thiazolyl tetrazolium (MTT) and 4′,6-diamidino-2-phenylindole (DAPI) were provided by Solarbio Co., Ltd (Beijing, China). Matrigel matrix was obtained from Biosciences Co., Ltd (New Jersey, US). Anti-Glutathione Peroxidase 4 (GPX4) Rabbit polyclonal was from Servicebio Co., Ltd (WuHan, China).
A549 cells and HEK-293 cells (human embryo kidney cells) were obtained from Xiangya cell center (Changsha, China). These cells were cultured in DMEM medium supplemented with FBS (10%), penicillin (1%, 50 U/mL) and streptomycin (1%, 50 U/mL) in a 5% CO2 atmosphere (37 °C).
Female Balb/c mice (6 weeks old, ≈ 20 g) were purchased from Cavans Laboratory Animal Co., Ltd (Changzhou, China) and maintained in a sterile environment and allowed free access to food and water. All animal experiments were approved by the Experimental Animal Ethics Committee of Xiangya School of Pharmaceutical Sciences of Central South University and were carried out in accordance with the requirements the National Act on the Use of Experimental Animals (People's Republic of China).
Preparation of CH/DF
Synthesis of circular template: H2O (69.5 μL), T4 ligase buffer (10×, 10 μL), template (10 μM, 6 μL) and primer (10 μM, 12 μL) were gently mixed and incubated at 95 °C for 10 min, then cooled to room temperature. T4 ligase was added and incubated at 25 °C for 4 h to close the circular DNA gap.
Preparation of DNA Flower (DF): Circular template (100 μL), dNTPs (10 mM, 40 μL), BSA (10×, 20 μL), phi29 polymerase (10 U/μL, 20 μL) and phi29 polymerase buffer (10×, 20 μL) were gently mixed in ice-bath, and incubated at 30 °C for 3 h and 75 °C for 10 min. The DNA flower was collected by centrifugation (20,000 rpm for 20 min) and washed twice with water, then mixed with buffer (20 mM Tris, 40 mM NaCl, 40 mM KCl, pH 7.6) in equal volume and incubated at room temperature for 1 h to form G4 structure.
Preparation of Ce6, hemin-loaded DNA Flower (CH/DF): DF was mixed with appropriate Ce6, incubated at room temperature for 4 h, Ce6-loaded DNA nanoflower (C/DF) was collected by centrifuged (20,000 rpm, 20 min) and washed twice with buffer. The preparation method of CH/DF was the same as that of C/DF. The above products were stored at − 20 °C.
Characterization of CH/DF
CH/DF was examined by the dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments, UK) to monitor the particle diameter, ζ-potential and polydispersity index. The morphological characteristics of CH/DF were evaluated using transmission electron microscopy (TEM, FEI, Oregon State, US) and scanning electron microscope (SEM, JSM-7900F, Tokyo, Japan). The encapsulation efficiency of drugs was calculated as follows: encapsulation efficiency = (weight of loaded drugs) / (weight of initially added drugs) × 100%. The encapsulation efficiencies of Ce6 and hemin were measured by Microplate Reader (Infinite M200, Tecan, Switzerland) and Visible–UV spectrophotometer (UV-2600, Shimadzu, Japan), respectively.
Catalytic activity test
The catalytic activity of CH/DF was determined by the Góth method. H2O2 (0.5 mL, 1 mM) and CH/DF (0.1 mL, 0.5 μM) were mixed and reacted at room temperature for 1 min. Then ammonium molybdate solution (0.5 mL, 32.4 mM) was added to form a yellow complex. After standing for 10 min, the catalase activity of CH/DF was determined by measuring the absorbance at 350 nm.
In vitro O2 production and enhanced 1O2 generation
To study the self-producing O2 performance of CH/DF or CH/G4, the O2 production was monitored the portable dissolved oxygen meter (JPBJ-609L, INESA Scientific Instrument Co., Ltd., China) every 10 s for 90 s. When O2 level did not change, laser irradiation (660 nm, 0.75 W/cm2) was added to study the dynamic change of O2 of CH/DF. The 1O2 production was tested by singlet oxygen sensor green (SOSG) probe after laser irradiation. Briefly, CH/DF (0.8 mL, 0.5 μM) was mixed with SOSG solution (0.1 mL, 25 μM). Then, H2O2 (0.1 mL, 100 mM) was added, and a continuous laser at 660 nm was applied with a power of 0.75 W/cm2 every 10 s for 50 s. The fluorescence intensity of SOSG was measured by a fluorescence spectrophotometer (Ex = 490 nm, Em = 525 nm). To study the biological stability of the DNAzyme, the CH/DF or CH/G4 were pretreated with 10% FBS for 10 h, followed by the treatments as described above.
Cellular uptake study
A549 cells were seeded in 24-well plate at a density of 2 × 105 cells per dish overnight. Subsequently, CH/DF was added to incubated for 1 h, 2 h or 4 h. After washing three times with PBS, the cells were stained with DAPI, and the fluorescence was observed by fluorescence imaging system (Model No. CYTATION5, BioTek). Moreover, A549 cells and HEK-293 cells were utilized to investigate the specific uptake of CH/DF for tumor cells. To study the cell uptake mechanism of DF, a variety of inhibitors (chlorpromazine: the clathrin inhibitor; colchicine: the macropinocytosis inhibitor; nystatin: the caveolin inhibitor; NaN3: ATP inhibitor) were used to intervene the endocytosis pathway. A549 cells were seeded with 2 × 105 cells per well in 24-well plate and incubated overnight. The cells were treated with chlorpromazine (10 µg/mL), colchicine (5 µg/mL), nystatin (15 µg/mL) or NaN3 (1 mg/mL) for 30 min, and then CH/DF was added and incubated for 2 h. Fluorescence was observed and quantified by fluorescence imaging system (Model No. CYTATION5, BioTek).
In vitro cytotoxicity study
MTT assay was used to evaluate the cytotoxic effects of CH/DF to A549 cells. A549 cells were seeded with 5 × 103 cells per well in 96-well plate and incubated overnight, and then treated with a series of concentration dilutions of DF, C/DF and CH/DF (Ce6: 0.45, 0.9, 1.8, 3.6 and 7.2 μM; Hemin: 0.02, 0.04, 0.08, 0.16 and 0.32 μM) for 48 h. The C/DF and CH/DF groups were irradiated with laser (0.75 W/cm2, 1 min) after incubation for 24 h. After that, the cells were washed twice with PBS and treated with the MTT reagent (5 mg/mL, 10 μL) for 4 h. Subsequently, the medium was removed and dimethyl sulfoxide (DMSO, 150 μL) was added. Finally, the UV–vis absorbance of each well was measured by Microplate Reader, and the cells viability was computed using the following formula:
Cell viability = (Asample/Acontrol) × 100%, where A represents the absorbance at 570 nm.
To study the effect of ferroptosis inhibitors or inducers t, the cells were co-treated with CH/DF (Ce6: 1.6 μM) plus ferrostatin-1 (1 μM), glutathione (GSH, 1 mM), glutamic acid (1 mM), or erastin (10 μM), respectively. After culturing for 48 h, MTT assay was performed to measure the cell viability.
Live/Dead Staining assay
A549 cells were seeded with 2 × 105 cells per well in 12-well plate and treated with different formulations. The cells without any treatment were used as control. Subsequently, the cells were stained with both Calcein AM and PI, and observed by inverted fluorescent microscope (NIKON, Ti-S, Japan).
Intracellular ROS and LPO generation
DCFH-DA was used to evaluate the generation of intracellular ROS. A549 cells were seeded with 2 × 105 cells per well in 24-well plate and treated with different formulations (Ce6: 5 μM) for 6 h. The cells were washed with PBS three times and incubated with DCFH-DA (10 μM) for 30 min, and then irradiated for 1 min (0.75 W/cm2). Finally, the fluorescence of DCFH-DA of cells was observed by fluorescence imaging system (Model No. CYTATION5, BioTek). C11 BODIPY 581/591 probe was used to detect LPO, and the detection method was similar to ROS.
Intracellular GSH level measurement
GSH assay kit was used to quantify the intracellular GSH level. A549 cells were seeded with 2 × 105 cells per well in 24-well plate and treated with different formulations (Hemin: 2 μM) for 24 h. Then, the C/DF and CH/DF groups were irradiated with laser and incubated for another 1 h. All cells were collected and lysed by liquid nitrogen, and the supernatants were collected by centrifugation (12,000 rpm, 10 min). Then, the supernatants were treated GSH assay kit and the absorbances were measured at 412 nm by Microplate Reader (Infinite M200, Tecan, Switzerland).
Western blot analysis of HIF-α protein expression
A549 cells were seeded with 4 × 106 cells per well in 6-well plate and treated with different formulations for 24 h. Cells were treated by RIPA buffer to collect total protein. Protein concentrations were determined by a BCA Protein Assay Kit (Dingguo changsheng, China). Then, proteins were run on polyacrylamide gel and were transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with HIF-α polyclonal antibody and β-actin antibody overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the proteins were visualized by the ChemiDoc MP Imaging System (Bio-Rad).
Construction of A549 xenograft tumor model
Six-week-old female BALB/c nude mice were used to establish the A549 xenograft tumor model. Briefly, A549 cells were collected and dispersed in PBS at a density of 2 × 107/mL, and then injected into the skin of mice subcutaneously (100 μL per mouse).
To study biodistribution, free Ce6 or CH/DF (Ce6: 2 mg/kg) was injected to A549 tumor-bearing mice intravenously. After 24 h administration, the mice were sacrificed and their primary organs (heart, liver, spleen, lung and kidney) and tumors were collected for ex vivo imaging with the IVIS Lumina XRMS Series III system (PerkinElmer, Waltham, MA).
In vivo antitumor efficacy and histological analysis
The A549 tumor-bearing mice with tumor volumes of approximately 100 mm3 were randomly divided into 6 groups (n = 6 per group): (1) PBS group as the control; (2) DF; (3) C/DF; (4) C/DF + L; (5) CH/DF; (6) CH/DF + L (Ce6: 2.5 mg/kg, respectively). The preparations were injected through a tail vein, and tumors were irradiated with a laser irradiation (0.75 W/cm2, 5 min) after 24 h of administration. Tumor sizes and body weights were recorded every other day after injection. Tumor volumes were calculated as follows: Volume = (length × width2)/2.
On the 16th day, all mice were sacrificed, tumors were collected for the hematoxylin and eosin (H&E) staining, TdT-mediated dUTP nick-end labeling (TUNEL) staining, immunofluorescence (caspase-3 and GPX4) staining. All major organs were collected for H&E staining to evaluate the safety of formulations.
The data were expressed as mean ± SD on the basis of at least three independent experiments. One-way ANOVA analysis of variance was used to determine the statistical significance of the difference group. P value < 0.05 was considered statistically significant.