Thursday, September 29, 2022
HomeNanotechnologyA nanoreactor boosts chemodynamic therapy and ferroptosis for synergistic cancer therapy using...

A nanoreactor boosts chemodynamic therapy and ferroptosis for synergistic cancer therapy using molecular amplifier dihydroartemisinin | Journal of Nanobiotechnology

[ad_1]

Preparation of DHA@MIL-101 NRs

MIL-101 nMOF was synthesized according to an approach described in the literature with some modifications. In short, 360 mg 2-aminoterephthalic acid and 1 g FeCl3·6H2O were dissolved in 40 mL DMF. After being treated at 120 ℃ for 20 h, the brown powder was centrifuged at 10,500 rpm for 20 min and washed three times in turn with DMF and ethanol, respectively. At last, the sediment was resuspended in ethanol and stored at 4 °C. The as-synthesized MIL-101 (10.0 mg) was dispersed in ethanol (5 mL). Then DHA (5.0 mg) dissolving in 2 mL ethanol was added to the dispersion of MIL-101 and magnetically stirred for 24 h in the dark at room temperature. Finally, the nanosuspension was washed with distilled water (500 μL × 5 times) to remove the free drug. Ultrasonic suspension of the nanosuspension for one minute each time distilled water was added for washing. The obtained DHA@MIL-101 NRs was stored at 4 ℃ prior to be used. All concentrations and dosages of DHA@MIL-101 NRs normalized to DHA.

Preparation of ICG-DHA@MIL-101 NRs

30 mg DHA@MIL-101, EDC (100 mg, 0.52 mmol) and NHS (100 mg, 0.87 mmol) were dissolved in water (10 ml) and stirred for 2 h at room temperature to activate the carboxyl groups. ICG (5 mg, 6.5 μmol) was added into the solution under vigorous stirring for 24 h in the dark at room temperature and washed with water five times to obtain ICG-DHA@MIL-101 NRs. ICG- DHA@MIL-101 NRs was stored at 4 ℃ prior to be used. All concentrations and dosages of ICG-DHA@MIL-101 NRs normalized to DHA.

Characterization of DHA@MIL-101 NRs and ICG-DHA@MIL-101 NRs

We collected all the water in washing procedures and the supernatant at the beginning to measure the loading content of the drug by the UV–vis absorption spectra technique. DHA could be converted into a UV absorbing compound through incubating with NaOH (2%) at 50 °C for 30 min and detected by the characteristic UV absorbance at 290 nm. The morphology and size of DHA@MIL-101 NRs were observed using scanning electron microscopy (SEM) and Transmission electron microscopy (TEM). To assay the hydrated size and zeta potential, the DHA@MIL-101 NRs was distributed in ultrapure water, and analyzed by Malvern laser particle size analyzer. Finally, the X-ray diffraction, infrared spectroscopy and thermal weight loss analysis (TGA) were applied to detect the successful preparation of DHA@MIL-101 NRs. Furthermore, the successful attachment of ICG to DHA@MIL-101-NH2 was detected through special absorption at 790 nm using UV–Vis absorption spectroscopy.

Detecting free radical by ESR

The generation of free radical induced by Fe2+ and DHA was detected using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) by ESR. Briefly, MIL-101 NRs (0.1 mL, 5.0 mg/mL) and DHA@MIL-101 NRs (same quality of MIL-101) solution at pH 5.2 in PBS, followed by incubation at 37 ℃ for 1 h. Then DMPO (0.05 mL, 0.5 M) was added as the spin–trapping agent, and the 1:2:2:1 multiplicity characteristic peak of DMPO-OH adducts were recorded by ESR immediately. As control, DMPO group was also tested for comparison.

Analysis of PH-responsive DHA and ferric iron release

DHA@MIL-101 NRs (10 mg) were packaged into a dialysis bag (MWCO = 3500), then immersed within 20 mL of phosphate-citrate buffer (pH 5.2) and phosphate buffer solution (PBS, pH 7.4), respectively at 37 ℃ in a beaker. At different time points, 2.0 mL solution was collected to determine the concentration of DHA using UV–vis spectra, then 2.0 mL fresh PBS was added back to the beaker. After centrifugation, the released iron ions were analyzed by ICP-MS. Three independent experiments were carried out to minimize the deviations.

Cell models and treatments

Lewis lung cancer cells (LLC) were used to as a lung cancer cell model. 16HBE cells were used as a kind of normal bronchial epithelial cell model. These cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured in DMEM medium (Sigma-Aldrich, St Louis, USA) supplemented with 10% fetal bovine serum (QmSuero/Tsingmu Biotechnology, Wuhan) in a humidified incubator (5% CO2/95% air atmosphere, 37℃). In the in vitro experiments, DHA concentrations of DHA, DHA@MIL-101 NRs and ICG-DHA@MIL-101 NRs were all 8 μg/mL. The concentration of MIL-101 NRs was the same as the total concentration DHA@MIL-101 NRs.

Drug uptake of ICG-DHA@MIL-101 NRs

Lewis cells were seeded onto 24-well plate or confocal dish with a density of 2 × 105 cells, then incubated with DHA, MIL-101 NRs and ICG-DHA@MIL-101 NRs for 12 h. The cells were harvested for flow cytometry (flow cytometry) analysis (Cytoflex, Beckman Coulter, USA). The cells on confocal dish were fixed with paraformaldehyde, nuclear stained with Hoechst 33,342, then imaged using a laser scanning confocal microscopy (FV3000RS, Olympus, Japan).

ROS detection

Lewis cells were plated in 24-well plates with a density of 2 × 105 cells per well and incubated with DHA@MIL-101 NRs for various times (2, 6, 12, 18, 24 h). In addition, for compared analysis, different agent (DHA, MIL-101 and DHA@MIL-101) were also performed. After treatment, the cells were incubated with 10 μM of 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA, S0033, Beyotime, Shanghai, China) at 37℃ for 40 min before being harvested and assayed by flow cytometry. H2O2 treatment was applied as the positive control.

Viability assay of cells

For assay of viability, Lewis cells or 16HBE cells were seeded onto 96-well plates with a density of 1 × 104 cells per well and treated by DHA, MIL-101 NRs, DHA@MIL-101 NRs respectively for 24 h. Viability of cells in the 96-well plates were assayed using a CCK-8 kit (HY-K0301, MCE, NJ, USA). Briefly, the CCK-8 was incubated with cells for 2–4 h. The absorbance at 450 nm of cells in 96-well plates was measured using a Multifunctional Enzyme Labeler (SpectraMax i3, Molecular devices).

Ferroptosis analyze of Lewis cells

For ferroptosis analyze of Lewis cells, the intracellular ferric irons, GSH content, expression of GPX4, COX-2, and lipid peroxidation were detected respectively. According to the manufacturer’s instruction, PGSK probe (GC40243, GLPBIO Technology Inc, USA) combined with flow cytometry was applied to detect the concentration of intracellular ferric irons. Alternatively, in light of the manufacturer’s instruction, the concentrations of intracellular GSH were measured by a biochemical assay kit (E-BC-K030-S, Elabscience). Furthermore, the expression of GPX4 and COX-2 was measured using Western blotting (WB). Finally, the production of lipid peroxidation was assayed by a C11-BODYPI probe (GC40165, GLPBIO Technology Inc, USA), whose fluorescence covert to FITC from PE indicates accumulation of lipid peroxidation in Lewis cells. Additionally, malondialdehyde (MDA) concentration in Lewis cells was measured by a MDA assay kit (E-BC-K025-S, Elabscience).

DNA and mitochondria damage assay

Comet assay were performed to evaluate the DNA double strand break (DDSB). Lewis cells were seeded in 24-well plates, treated as required. Cell suspensions in PBS were prepared and mixed with low melting point agarose (LMPA). The mixture was then dripped onto a glass slide pre-coated with agarose gel and pressed with glass, followed by electrophoresis at 25 V, 250 mA for 25 min. The mixture was then lysed in alkaline lysis solution and neutralized using tris–Hcl (PH = 6.0). Hoechst 33,342 was applied to stain nuclei. Images were acquired by fluorescence microscopy. Alternatively, proteins exacted from Lewis cells were used to measure the expression of p53 and γ-H2A.X, which are biomarkers of DNA damage response. Finally, JC-1 probe (A3516, APExBIO Technology Inc, USA) was applied to analyze the membrane potential to confirm the degree of mitochondria damage. Alternatively, the cells treated with DHA@MIL-101 NRs was prepared prepared as ultrathin sections for TEM observation.

Evaluation of in vitro anti-cancer efficacy

Lewis cells were treated by DHA, MIL-101 NRs and DHA@MIL-101 NRs for 24 h. Cell number and morphological change were observed by microscopy. In addition, the apoptosis rate of cells was assayed by FITC-Annexin-V/Propidium iodide (PI) double staining (Purchased from CHAMOT BIOTECHNOLOGY CO., LTD.) and flow cytometry. Alternatively, the expression of proliferation proteins (PCNA, CDK4 and Bcl-2), apoptosis proteins (Bax, cleaved-caspase-3) was measured using WB technique.

Flow cytometry and Annexin-V/PI staining

Lewis cells was acquired on a Beckman Cytoflex flow cytometer. FITC-Annexin-V, DCFH-DA, PGSK, C11-BODYPI-FITC fluorescence was acquired in the FITC channel. PI fluorescence was acquired in the PE channel. ICG-DHA@MIL-101 NRs fluorescence was acquired in APC-A750 channel. At least 1 × 104 cells/per sample were acquired. Geometric means (GM) were used to quantify the fluorescent intensity. For Annexin-V/PI assay, cells were incubated with Annexin-V-FITC and PI (Purchased from CHAMOT BIOTECHNOLOGY CO., LTD.) for 10 and 5 min respectively, then harvested on flow cytometry. The apoptosis of cells was assayed through calculate the Annexin-V positive cells.

Western blotting (WB) measurement

Lewis cells treated as mentioned before in 6-well plates were lysed in RIPA buffer with protease inhibitor. Cell lysates were cleared by centrifugation and protein concentration determined using a BCA assay kit. Equal protein aliquots (10 μg) were separated by SDS-PAGE electrophoresis and transferred to a PVDF membranes. The membranes were blocked with 5% bovine serum albumin in TBST and then incubated with antibodies of PCNA (bs-2006R, Bioss, Beijing, China), Caspase-3 (19677-1-AP, Proteintech, Wuhan, China), Bax (50599-2-Ig, Proteintech, Wuhan, China), Bcl-2 (26593-1-AP, Proteintech, Wuhan, China), CDK4 (11026-1-AP, Proteintech, Wuhan, China), p53 (bs-2090R, Bioss, Beijing, China), γ-H2A.X (bs-3185R, Bioss, Beijing, China), GPX4 (14432-1-AP, Proteintech, Wuhan, China), COX-2 (A1253, Abclonal, Wuhan, China), and GADPH (PMK053C, BioPM, Wuhan, China) overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies were used to bind to primary antibodies as mentioned above. Protein bands were imaged using a ECL luminescent liquid (PMK003, BioPM, Wuhan, China). The bands were exposed using a Bio Imaging system (170-8265, Bio-Rad).

RT-PCR analysis

The mRNA was quantified to 1 μg, then reverse transcripted to cDNA using a transcriptor cDNA synthesis kit (PC5801, TRUEscript RT MasterMix, Beijing, Aidlab). Fluorescence real-time quantitative PCR was performed using an SYBRGreen real time PCR Master Mix kit (PC3301, Beijing, Aidlab). The Primers (5′ to 3′) sequences were as follows:

Mouse GAPDH Forward: AGGTCGGTGTGAACGGATTTG.

Mouse GAPDH Reverse: TGTAGACCATGTAGTTGAGGTCA.

Mouse TfR1 Forward: CTGGCTCTCACACTCTCTCAGCTTT.

Mouse TfR1 Reverse: GCATTTGCGACTCCCTGAATAGTCC.

Lewis cells-bearing mouse model and treatments

Female C57 mice at 5–6 weeks of age (18 ~ 20 g) were purchased from Laboratory Animal Center at the Hubei University of Medicine (Hubei, China). Animal handling and experimental procedures were in line with protocols approved by the Animal Care Committee at the Hubei University of Medicine. Mice were housed in a temperature-controlled environment with fresh water and rodent diet available at all times. All inoculations and administrations were performed under Nembutal anesthesia. Each mouse was subcutaneously injected at the right haunch with Lewis cells (2 × 106 cells/200 μL in PBS). The animals were randomly grouped (5 mice per group) when the tumor growth reached roughly 500 mm3. Injections of DHA, MIL-101 NRs and DHA@MIL-101 NRs, each in 200 μL of PBS per mouse, were administered through the caudal vein. For DHA and DHA@MIL-101 NRs, dosages were normalized to 5 mg/kg of DHA. The concentration of MIL-101 NRs was the same to MIL-101 in DHA@MIL-101 NRs. The fluorescence distribution of nanoagent was detected using in vivo imaging at 12 h after administration. The mice were treated with the various agent a total of 3 times, every 24 h. Finally, the mice were sacrificed and vital organs were harvested and imaged for drug fluorescence. Cryosections (5 μm) of tumor tissues and vital organs were prepared for fluorescent microscopy and immunohistochemical staining.

HE, immunohistochemical, tunel and immunofluorescent staining

Briefly, paraffin sections were dewaxed, rehydrated, antigen repaired with sodium citrate for 20 min. For HE staining, the paraffin sections were stained by Eosin and hematoxylin. According to the manufacturer’s instruction, tunel and PI were used to label the apoptotic cells and necrotic cells firstly, then DAPI was applied to stain the nuclei. For IHC staining, the paraffin sections were then incubated in 3% hydrogen peroxide for 12 min at room temperature. The paraffin sections were blocked with 5% BSA for 40 min, stained with primary antibodies overnight at 4 ℃, then stained with secondary antibody (PV-9000, ZSGB-BIO, Beijing, China) for 1 h at 37 ℃. Diaminobenzidine (DAB, ZLI-9018, ZSGB-BIO, Beijing, China) was applied for coloration for 1–3 min at room temperature. Hematoxylin was used to stain the nucleus. Primary antibodies included PCNA (bs-2006R, Bioss, Beijing, China), Caspase-3 (19677-1-AP, Proteintech, Wuhan, China), Bax (50599-2-Ig, Proteintech, Wuhan, China), p53 (bs-2090R, Bioss, Beijing, China), γ-H2A.X (bs-3185R, Bioss, Beijing, China), GPX4 (14432-1-AP, Proteintech, Wuhan, China), COX-2 (A1253, Abclonal, Wuhan, China). For nanagent fluorescent microscopy, cryosections were dewaxed, rehydrated, then stained by DAPI for labeling of the nucleus.

Statistical analysis

Statistical differences between groups were analyzed using One-way analysis of variance (ANOVA). Statistical significance was determined by Turkey Test. The p value less than 0.05 was considered to be statistically different.

[ad_2]

Source link

RELATED ARTICLES

LEAVE A REPLY

Please enter your comment!
Please enter your name here

Most Popular

Recent Comments