Bovine serum albumin (BSA) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma–Aldrich Co. Polyvinylpyrrolidone (PVP) was purchased from Alfa Aesar (China). Gallic acid (GA) and disodium terephthalate (TA) were purchased from Aladdin (Shanghai). Copper (II) chloride was purchased from Siuopharm Chemical Reagent Co. The GSH/GSSG assay kit, live/dead assay kit, calcein-AM and propidium iodide (PI) were obtained from Beyotime Biotechnology. CCK-8 was obtained APExBIO. HCl·DOX was obtained from Meilunbio.
2.2 Synthesis of nanomaterials
PVP stabilized GA-Cu nanodots (GC NDs): 60 mg PVP was dissolved in 8 mL deionized (DI) water under stirring, followed by dropwise addition of 1 mL CuCl2·2H2O (10 mg/mL) and 1 mL GA (10 mg/mL) for reaction overnight. The as-prepared GC NDs were then purified by centrifugation at 3500 rpm for 10 min using ultrafiltration Millipore tubes (10 kDa) and washed three times with DI water.
GA-Cu@BSA nanodots (GCB NDs): 70 mg BSA was dissolved in 8 mL DI water under gentle stirring for 5 min. Then, 1 mL CuCl2·2H2O (10 mg/mL) was added dropwise with constant stirring for 30 min. After that, 1 mL 10 mg/mL GA was added to the bottle with vigorous stirring for another 3 h. After the reaction, the unreacted large particles were removed by centrifugation (4000 rpm). Unreacted impurities were removed with an ultra-filtration (100 kDa, 3500 rpm) washed twice with DI water and then stored in a refrigerator at 4°C for further application.
GA-Cu@BSA-DOX nanoparticles (GCBD NPs): 10 mg DOX dissolved in 200 µL DMSO was added dropwise into 1 mL 10 mg/mL GCB solution with vigorous stirring for 12 h. After the reaction, the free DOX and DMSO were removed by ultra-filtration (100 kDa, 3500 rpm), washed with DI water for three times (until the UV absorption of DOX in the liquid below was less than 0.2) and then stored in a refrigerator at 4°C for further use.
2.3 Characterization of nanomaterials
The morphology and size of GA NDs, GCB NDs and GCBD NPs were characterized by TEM (Tecnai G2 spirit Bio Twin). Their physiological size and zeta-potential were tested by DLS (Zetasizer Nano ZS90). The absorbance spectra were tested by UV–Vis spectroscopy (GENESYS 10S Spectrophotometer). Fourier transform infrared (FT-IR) spectra were recorded by conventional Fourier infrared spectroscopy (NICOLET iS 50). XPS spectra were tested by Thermo Scientific K-Alpha. In order to test the physiological stability, GCB NDs and GCBD NPs were dissolved in PBS, water and DMEM, respectively (n=3). Every other day, the solutions were photographed, and the size and PDI value were measured by DLS for 7 days.
2.4 Drug loading and release behavior
Drug loading capacity of GCB: In order to test the ability of GCB NDs to load drugs, 10 mg GCB NDs was dissolved in 1 mL DI water. Then, DOX solutions with different concentrations (20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL and 1.25 mg/mL) were added to GCB ND solutions for reaction overnight. After the reaction, the free DOX and DMSO were removed by ultrafiltration (100 kDa, 3500 rpm).
Release of DOX: In order to test the DOX release behavior, 10 mg GCBD NPs was dissolved into 2 mL DI water in a dialysis bag, which was put into bottles containing 15 mL PBS (pH=5.6 or 7.4) for stirring (3 parallel samples were set in each group). At 30 min, 1 h, 2 h, 4 h, 6 h, 12 h and 24 h after stirring, 1 mL of the solutions outside the dialysis bag was removed to record the absorbance intensity at 500 nm by UV–Vis and then put back to the system.
Release of copper ions: The release of Cu2+ from GCBD NPs was detected by Copper Determination Kit (Beyotime Biotechnology). Generally, 10 mg GCBD NPs was dissolved in 2 mL DI water, which was then put into dialysis tube with PBS (pH 5.6 and pH 7.4) (3 parallel samples were set in each group) outside, 10 µL was taken at intervals of 5 min, 30 min, 1 h, 2 h, 6 h, 12 h, 24 h. The same volume of PBS solution was then added to the reaction system.
2.5 Labeling Cy5.5 and 125I onto GCBD NPs
Cy5.5 labeling: 10 mg GCB NDs and GCBD NPs were dissolved in 1 mL of DI water. Subsequently, 10 µL aminated Cy 5.5 was added into the solution and stirred for 2 h under dark conditions. Excess Cy5.5 was removed with 10 kDa (for Cy5.5-GCB) and 100 kDa (for Cy5.5-GCBD) ultra-filtration tube and the products were stored at 4°C in a dark environment for further use.
125 I labeling: 2.21 mg 1,3,4,6-Tetrachloro-3α,6α-diphenyl-glycouril (Idongen) was dissolved into 600 µL chloroform with chloroform and later blow-dried with nitrogen. Then, 2 mg/mL GCBD NPs were mixed with 500 µCi 125I, and then the mixture was mixed with blow-dried Idongen for 20 min with shaking. After the reaction, the mixture was washed three times with 100 kDa ultra-filtration tube.
Radiolabeling stability: The radiolabeling stability of 125I labeled GCBD NPs was then evaluated by incubating 125I @GCBD NPs in serum or PBS for 48 h. The unlabeled radionuclides were washed with 100 kDa ultra-filtration tube for several times. The residual nuclide dose in the solution at different time points was detected by gamma counter (PerkinElmer) for radiostability.
2.6 Detection of hydroxyl radical and GSH
Generation of ·OH catalysed by GCBD NPs: GCBD NPs (100 µg/ml for GCB NDs) were incubated with disodium terephthalate (TA) (purchased from Aladdin) and H2O2 on a shaking table at 350 rpm and 37°C for 3 h. The final concentrations of TA and H2O2 in the reaction system were 1.5 mM and 2 mM, respectively. TA+H2O2, TA+GCBD NPs, and H2O2+GCBD NPs were used as controls. The fluorescence emission peaks at 435 nm (λex=315 nm) were detected by fluorescence spectra of all samples.
Depletion of intracellular GSH: To detect the depletion of GSH by GCB NDs and GCBD NPs at the cellular level, 4T1 cells were cultured in 6-well plates for 24 h. Then, cells were cocultured with GCB NDs (n=3) and GCBD NPs (n=3). The blank group without nanomaterials was used as a control, respectively. After 24 h, cells were collected by centrifugation at 1000 rpm. The GSH content in each group was detected according to the instructions of the GSH/GSSG assay kit (Beyotime Biotechnology).
2.7 Cellular experiments
Cytotoxicity: CCK-8 assay was used to detect the biosafety and cytotoxicity of NPs. Generally, 4T1 cells and human umbilical vein epithelial cells (HUVECs), which were cultured in high glucose DMEM medium with 10% fetal bovine serum and 1% penicillin–streptomycin in a humidified atmosphere containing 5% CO2 at 37°C, were placed in 96-well plates and cultured in a cell incubator for 24 h. For biosafety, GCB NDs with different concentration gradients were added to the HUVEC plates. For cytotoxicity, GCB NDs, DOX, GCBD NPs with different concentration gradients and blanks were cocultured with 4T1 cells. After 24 h of culture, the relative cell viability was tested by CCK-8 assay.
Detection of cellular ·OH: 4T1 cells were treated with DCFH-DA (10 µM, dispersed in DMEM medium) for 20 mins. After washing with PBS for three times, samples were then separated into three groups: blank group cultured only with DMEM medium, experimental group incubated with GCB NDs (20 µg/mL) and GCBD NPs (6 µg/mL for DOX) for 3 h. Then, all samples were imaged by fluorescence microscope (OLYMPUS IX73).
Dead and live cell staining: In order to display the cytotoxicity of the materials more intuitively, live and dead staining was used to further verify apoptosis. 4T1 cells were cocultured with DOX (10 µg/ml), GCB NDs (200 µg/ml), and GCBD NPs (10 µg/ml in DOX concentration) for 24 h, respectively. Afterwards, cells in different groups were stained with Calcein-AM (2 µM) and PI (4.5 µM) for 15 mins and then observed by fluorescence microscope (OLYMPUS IX73).
Cellular uptake of DOX: 4T1 cells were incubated with GCBD NPs at 1 µg/mL doxorubicin and free DOX (1 µg/mL) in 24-well plates with high glucose medium. After incubation at different time points for 1, 6, 12, and 24 h, the medium was removed. Then, the 4T1 cells were washed with PBS for 3 times and fixed with 4% paraformaldehyde for 15 min. Then, the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 15 min. After that, the cells were washed with PBS for 3 times and finally photographed with confocal laser microscope (FV1200).
Cell apoptosis: 4T1 cells were first incubated in 6-well plates for 24 h. Then, cells were incubated with GCB NDs or GCBD NPs for 24 h, and the group without materials was used as the control. Cells were collected by centrifugation at 1000 rpm and then stained with Annexin V-FITC and PI at 20-25°C for 20 min. Finally, the apoptosis of 4T1 cells was detected by flow cytometry (BD FACSVERSE).
2.8 Blood circulation and biodistribution
Blood circulation of GCBD NPs: GCBD NPs labeled with 125I (125I@GCBD NPs) were injected into tumor-bearing BALB/c mice (n=3) through the tail vein for blood circulation and tissue distribution, respectively. For blood circulation, 10 µL of blood was collected from the orbit of mice, and the blood weight was recorded. The radiation intensity of blood was measured by gamma counter (PerkinElmer) at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 10 h and 24 h.
Biodistribution of GCBD NPs: GCBD NPs were injected into BALB-c mice (n=3) through the tail vein to study the distribution of the material in vivo by measuring the intensity of radioactivity of 125I. The radiation intensity in the heart, liver, spleen, lung, kidney and tumors of the sacrificed mice was measured by gamma counter (PerkinElmer) after the injection of 125I@GCBD NPs for 4 h and 24 h, respectively.
In vivo imaging: In order to reflect the enrichment of materials in mice more intuitively, GCB NDs and GCBD NPs were labeled with Cy5.5 and then injected into mice that were randomly divided into 2 groups (n=5) through the tail vein. Then, the mice in each group were photographed by IVIS (PerkinEimer) at 0 h, 1 h, 4 h, 12 h and 24 h. After injection for 24 h, all mice were sacrificed to collect their hearts, livers, spleens, lungs, kidneys and tumors.
2.9 Combined chemo/chemodynamic therapy of tumor
In order to verify the effect of tumor therapy in vivo, 4T1 cells were injected into the back of BALB-c mice to establish 4T1 tumor-bearing mouse models. When the tumor volume reached approximately 50-100 mm3, the mice were randomly divided into 4 groups (n=6): (1) PBS, (2) DOX (5 mg kg-1), (3) GCB NDs (18.72 mg kg-1), and (4) GCBD NPs (5 mg kg-1 of DOX) and respectively injected with specific solutions through the tail vein. On day 7 post treatment, one mouse in each group was randomly sacrificed, and the tumor was subjected to TUNEL staining to test the therapeutic effect of the drugs. One mouse in each group was randomly sacrificed for H&E-stained slices of its heart, liver, spleen, lung, kidney and tumor after the treatment to test whether drugs have obvious toxic and side effects on normal tissues and 4T1 tumors of mice. The tumor volume and the weight of the mice were recorded every other day for 14 days. All the mice were sacrificed after the treatment, and the 4T1 tumors were removed, weighed and photographed.