All DNA oligonucleotide sequences were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China). PI3Kγ siRNA were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). All oligonucleotide sequences were showed in Table S1. Antibodies against PI3Kγ was purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Antibodies for iNOS, Arg-1, NF-κB p65 and phospho-NF-κB p65 (Ser536) were obtained from Cell Signaling Technology (Danvers, USA). Antibodies against GAPDH and β-tubulin were purchased from Bioworld Technology Co., Ltd. (Nanjing, China). The anti-CD16/32, anti-CD86 and anti-CD206 were obtained from BioLegend (San Diego, CA, USA). The horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibodies were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All ELISA kits were obtained from Bopei Biotechnology Co., Ltd. (Chongqing, China). Total RNA extraction kit was obtained from Promega (Madison, USA). The caspase-3 activity kit was purchased Beyotime Biotechnology Co., Ltd. (Shanghai, China). A PrimeScript™ RT reagent kit and a SYBR® Premix Ex Taq™ reagent kit were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). All ssDNAs were dissolved in sterile water and stored at -20°C. PI3Kγ siRNA were dissolved in DEPC water (Sangon, Shanghai, China).
The mouse breast cancer cell lines 4T1 and mouse macrophage cell line RAW264.7 were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin. All cell lines were incubated in an atmosphere of 5% CO2 at 37°C according to ATCC guidelines.
Preparation of tFNA, CpG-tFNA and CpG-RNAi-tFNA
DNA self-assembly structures were prepared as reported previously. For tFNA, six different ssDNAs mixtures of component oligonucleotides (S1, S2, S3, S4, S5 and S6) were combined in TM buffer (10 mM Tris, 5 mM MgCl2), heated to 95°C for 2 minutes, then rapidly cooled to 4°C in a T100TM Thermal Cycler (Bio-Rad, Hercules, CA, USA). Each ssDNAs strand final concentration was 1mM. The CpG-tFNA and CpG-RNAi-tFNA were prepared in the same way.
Agarose gel electrophoresis was performed to confirm the successful assembly of tFNA, CpG-tFNA and CpG-RNAi-tFNA. The experiments were operated in 1 × TBE running buffer under the condition of 100 V voltage. After nucleic acid stain (gold view) disposed, the gel was visualized with imaging system (Bio-Rad, Hercules, CA, USA).
Stability analysis of tFNA
To test the nanostructures stability, tFNA and dsDNA were separately incubated with fetal bovine serum of equal volume at 37°C for different time periods (0, 3, 6, 12 and 24h). The mixtures were then subjected to 2% agarose gel.
RAW264.7 cells were grown on glass cover slips in 24-well culture plates at a density of 5 ´ 105 cells/mL and incubated at 37℃ for 24 h. They were then washed with sterile phosphate-buffered saline (PBS) and changed into fresh RPMI 1640 medium. Next, cells were separately incubated with fluorescently labeled 100 nM CpG and CpG-tFNA at 37℃. After culturing for 1 h, all cells were washed with pre-cold phosphate-buffered saline (PBS) three times and then fixed with 4% formaldehyde (Solarbio, Beijing, China) for 20 min at room temperature. And cells nuclei were treated with DAPI for 10 min. Finally, all confocal images were captured using a laser confocal microscope (Leica TCS SP8, Germany) and a fluorescence microscope (Nikon ECLIPSE Ti-s, Japan).
Cellular cytotoxicity assays
RAW264.7 cells were seeded in 96-well plate with 5 ´ 103 cells per well in 100 μL and cultured for cell adhesion. The medium was replaced by 100 μL fresh medium containing CpG-tFNA or CpG-RNAi-tFNA at different concentrations. The cells were further incubated for 48 h at 37°C. Then the medium was replaced by fresh culture medium and CCK-8 solution was added and incubated for 1 h. The absorbance at 450 nm was measured using a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA).
Total RNA was harvested from RAW264.7 cells and reverse transcribed into cDNA. Relevant inflammatory cytokines gene (Il-6, Il-12, Tnfa) expression were determine according to the manufacturer’s instruction. The sequences of primers were provided in Table S2. The reactions were performed in a volume of 10 μL using CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).
RAW264.7 cells were seeded in a 6-well plate and cultured for cell adhesion. Then, the medium was replaced by 2 mL fresh medium containing different concentrations CpG-tFNA or CpG-RNAi-tFNA for 24h. The supernatants were collected and the levels of cytokines were determined by an ELISA assay as the manufacturer’s protocol.
Whole protein from RAW264.7 cells were harvested with RIPA buffer containing proteinase and phosphatase inhibitor cocktail. The protein concentration was measured by BCA assay. Protein samples were separated on SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h followed by incubation at 4̊C overnight with the primary antibodies. HRP anti-rabbit IgG were used as the secondary antibodies. The signals were analyzed using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).
RAW264.7 cells and 4T1 cells were co-culture using the Transwell system (Millipore, Bedford, MA, USA) with 3 μm pore-size. Then CpG-tFNA and CpG-RNAi-tFNA were added to the upper chamber. After incubation for 48 h, the cell viability of 4T1 cells in the lower chambers was determined by a CCK-8 assay.
Caspase-3 activity assay
A transwell system (3μm-pore polycarbonate filter membrane) was performed to explore the antitumor effect of CpG-RNAi-tFNA. RAW264.7 cells and 4T1 cells were respectively added to the upper and lower chambers and cultured in RPMI 1640 medium containing 10% FBS for 24h. Then CpG-tFNA and CpG-RNAi-tFNA were added to the upper chamber. After incubation for 24h, the 4T1 cells were collected and the activity of caspase-3 was determined by the caspase-3 activity kit.
4T1 cells were harvested with EDTA-free trypsin and washed with cold PBS for three times. Subsequently, the cells were resuspended in 500 μL PBS and stained with 5 μL Annexin V-fluorescein isothiocyanate (FITC) and 5 μL propidium iodide (PI) for 15 min in the dark at room temperature. Finally, the apoptosis of 4T1 cells were evaluated by FACScan flow cytometer (BD Biosciences).
Briefly, RAW264.7 cells were treated with CpG-siRNA-tFNA for 24h. Then, the cells were resuspended in 500 μL PBS and blocked with anti-CD16/32 for 10min at 4 °C. After washed with cold PBS, the cells were incubated with anti-CD86 and anti-CD206 for 30 min in darkness. Finally, the expression of CD86 and CD206 were evaluated by flow cytometry.
Student’s t-test or one-way analysis of variance (ANOVA) was used to assess the differences between treated and control groups. with GraphPad Prism 8. All quantitative results are presented as mean ± SD (standard deviation) with at least three independent repetitive experiments.