Materials and Instruments. Unless otherwise stated, all the other chemicals and solvents were purchased for commercial use without further purification. ZrCl4 ( anhydrous, powder, 99.99% trace metals), 2-Aminoterephthalic acid (H2N-H2BDC, 99%), Dichloroacetic acid (DCA, ReagentPlus®, ≥ 99%), 5-Fluorouracil (5-FU, ≥99%, HPLC) and Sodium dichloroacetate (98%) were obtained from Sigma-Aldrich. HPLC was performed on an Agilent 1100 HPLC (Agilent Technologies, USA) using a Waters Symmetry Shield RP18 Column (4.6 × 250 mm, 5 mm, Waters Corporation, USA). Powder X-Ray Diffraction (PXRD) patterns were obtained on a Bruker D8 diffractometer using Cu Kα radiation with 40 mA and 40 kV, and the dates were collected within the 2θ range of 5–50°. Nuclear Magnetic Resonance Spectroscopy (NMR) were recorded on Bruker AMX 500 (1H-NMR at 500 MHz) spectrometers. UV-Vis Spectroscopy was recorded using a Thermo scientific Nanodrop 2000 spectrophotometer. Transmission electron microscopy (TEM) was performed on a JEOL JEM-1400Plus electron microscope operated at 80 kV. Fourier-transform Infrared Spectroscopy (FT-IR): FT-IR spectra of solid samples were collected using a PerkinElmer Spectrum Two FT-IR Spectrometer.
Biological studies. Cell proliferation assay Roche diagnostics reagent (WST-1), phosphate-buffered saline (PBS), Penicillin-Streptomycin, and dimethylsulfoxide (DMSO) for cell culture solution were purchased from Sigma Aldrich. Dulbecco’s modified eagle’s medium (DMEM), trypsin-EDTA (1X), and fetal bovine serum (FBS) were purchased from Lonza. Minimum essential medium non-essential amino acids (MEM NEAA, 100X) was purchased from Gibco. L-Glutamine 100X was purchased from Biowest. Breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A were used for in vitro studies. The cells were cultured in high-glucose DMEM containing 10% heat-inactivated fetal bovine serum, 0.5% penicillin-streptomycin, 1% MEM NEAA, and 1% L-glutamine at 37 °C in the sterile condition of 5% CO2 incubator with the humidified atmosphere. Cells are exponentially cultured as monolayer up to 70–80% confluency. After optimal growth, cells were detached using trypsin-EDTA. All the cell culture work was performed under sterile conditions.
Synthesis of DCA-UiO-66-NH 2 . Following these general steps to prepare DCA-UiO-66-NH2 nanoparticles: in separate vessels, ZrCl4 (1.0 g, 4.2 mmol) and H2BDC-NH2 (761.0 mg, 4.2 mmol) were dissolved in 150 mL of DMF. After mixing both precursor solutions transferring into a 250 mL vial, dichloroacetic acid (DCA, 3.0 mL, 36.0 mmol) was added to the reaction mixture, after gently stirring, it was placed in the oven at 120 ℃ for 24 hours. After cooling to room temperature, the powders were collected by centrifugation and washed 3 times with DMF and 3 times with ethanol by dispersion centrifugation cycles. The resulting MOFs were dried under vacuum for at least 12 hours before analysis.
Synthesis of 3,3′-dithiodipropionic anhydride (DTDPA). 1.0 g of 3,3′-dithiodipropionic acid (DTDP) was added to 5.0 mL of acetyl chloride and refluxed at 65 ℃ for 2 hours. The resulting solution was cooled to room temperature and the solvent was evaporated. The residue was then precipitated in diethyl ether and washed repeatedly with diethyl ether. The product 3,3′-dithiodipropionic anhydride (DTDPA) was dried in a vacuum desiccator overnight and used directly in the next step.
Postsynthetic modification of DCA-UiO-66-NH 2 by using DTDPA (as DCA-UiO-DTDP). The amine functional groups of the MOF reacted with DTDPA to form carboxylic acid-functionalized DCA-UiO-DTDP. In a typical post-synthetic modification reaction, after synthesizing of DCA-UiO-66-NH2 (ca. 4.0 mmol of -NH2), about 4.0 mmol of DTDPA (192.25 mg) was placed in the above DMF solution. After the sample was left at room temperature for 24 hours, the solution was decanted, and the crystals were washed with fresh DMF and ethanol (3 × 5 mL) and then dried under vacuum.
Synthesis of aminated folic acid. For amination, folic acid (1.0 mmol) dissolved in 30.0 ml DMSO with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 1.2 mmol) and N-Hydroxysuccinimide (NHS, 2 mmol) was reacted at 50 °C for 6 hours. The obtained folic acid-NHS was mixed with ethylenediamine (10.0 mmol) and 500.0 µg pyridine and allowed to react at room temperature overnight. The crude product was precipitated by adding an excess of acetonitrile, filtered and washed three times with diethyl ether, and then dried under vacuum.
Preparation of 5-FU-loaded DCA-UiO-DTDP. Fresh DCA-UiO-DTDP was immersed in methanol for 24 hours, and then the extract was discarded. Fresh methanol was then added, and the sample was immersed for another 24 h to remove H2O and DMF. The sample was then treated with dichloromethane to remove methanol solvates using the same procedures. After dichloromethane was removed by decantation, the sample was activated by using a dynamic vacuum at 100 ℃ for 12 hours. To load 5-FU into the pores of DCA-UiO-DTDP, dehydrated DCA-UiO-DTDP (5 mg) was dispersed in a 5- FU containing MeOH solution (5 mL) and stirred for 1 day to yield a uniform light-yellow solution. The 5-FU-DCA-UiO-DTDP was collected by centrifugation (13000 rpm for 10 min) and washed with MeOH (x2) by dispersive centrifugation cycles to ensure that no residual 5-FU remained on the particle surfaces. The resulting MOFs were dried under vacuum at least 24 hours before analysis. The amount of 5-FU adsorption in the porous solids was estimated by UV-Vis. The wavelength used for 5-FU quantification was 266 nm.
Fabrication of 5-FU-DCA-UiO-DTDP-FA. Same as encapsulating 5-FU in DCA-UiO-DTDP, dehydrated DCA-UiO-DTDP (5 mg) was dispersed in a 5- FU MeOH solution (3 mg/mL, 5 mL) and stirred for 1 day to yield a homogenous light-yellow solution. Then according to the ration 1:1.2:2, EDC and NHS were added to activate the carboxyl groups of DCA-UiO-DTDP. After 6 hours, 5 mg of aminated FA was added to the solution. 5-FU-DCA-UiO-DTDP-FA were collected by centrifugation (12000 r/min for 20 min) and washed with MeOH (x2) by dispersive centrifugation cycles to ensure that there is no residual 5-FU on the particle surfaces. The resulting MOFs were dried under vacuum at least 24 hours before analysis. The amount of 5-FU adsorption in the porous solids was estimated by UV-Vis. The wavelengths used for 5-FU quantification was 266 nm.
In vitro drug release kinetics. The cumulative levels of 5-FU released from 5-FU-DCA-UiO-DTDP-FA nanoparticles were characterized using the dialysis method. Briefly, 15 mg of drug-loaded 5-FU-DCA-UiO-DTDP-FA was dispersed in 1.5 mL of PBS buffer solution (pH = 7.4), and placed in a dialysis bag (MWCO = 500D), and then immersed in 50 mL of PBS with or without DTT (10 mM), and resuspended in a 37 °C constant temperature water bath with horizontal shaking. Here, DTT was used as a reducing agent that mimics the action of GSH, which provides a reducing environment in the microenvironment of cancer cells. At each time interval, 1 mL of the incubation solution was taken out and replenished with an equal volume of corresponding PBS. The content of 5-FU in the removed sample was monitored by HPLC, where the detection wavelength was 265 nm. The flow rate of the mobile phase was 1.0 mL·min− 1, and the temperature was set at 30 °C.
In vitro cellular assay
Cell viability assay. 5-FU acts as a thymidylate synthase (TS) inhibitor, so it needs to reach the nucleus of cancer cells to be effective, while DCA inhibits pyruvate kinase and therefore acts on mitochondria. The cytotoxicity of the free DCA, 5-FU, 5-FU + DCA, DCA-UiO-DTDP, DCA-UiO-DTDP-FA, and 5-FU-DCA-UiO-DTDP-FA were measured against breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A, using the WST-1 cell proliferation assay Roche diagnostics reduction assay.
First, breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A were cultivated in a 96-well plate, with 4000–5000 cells per well, and incubated for 24 hours with CO2 (5%, 37 ℃). Thereafter, the DCA suspension was diluted to concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 mg·mL− 1. 5-FU suspension was diluted to concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 µg·mL− 1. 5-FU + DCA suspensions based on the concentrations of 5-FU and the ratio of DCA:5-FU in the final formulation was diluted to 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 µg·mL− 1. DCA-UiO-DTDP and DCA-UiO-DTDP-FA suspensions were diluted to concentrations of 10, 20, 50, 100, 200, 500 and 1000 µg·mL− 1. 5-FU-DCA-UiO-DTDP-FA suspensions based on 5-FU concentrations in the final formulation was diluted to 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 µg·mL− 1. Then the solutions were added to the wells and incubated for another 24 hours. As blank controls, only eight wells were left with culture, and then DMSO (10.0 µL) was added to each well. Subsequently, WST-1 cell proliferation assay solution (10.0 µL) was added to each well, and then incubated at 37 ℃ for another 2 hours. Finally, the absorbances of the solutions at 490 nm were measured by using a microplate reader.
Detection of cellular uptake and kinetics using confocal analysis. Cellular uptake: The cellular uptake of breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A were measured by confocal laser scanning microscopy (CLSM). Generally, MDA-MB-231 and MCF-10A cells (2 × 105 per well) were first seeded in confocal dishes and grown for 12 hours before the uptake study, and then incubated with the prepared DCA-UiO-DTDP and DCA-UiO-DTDP-FA suspensions (2 mL, 500 mg·mL− 1) at 37 ℃ for different times (1, 6 and 16 hours). At different time points, the cells were washed three times with PBS. Thereafter, the cells were fixed with 4% paraformaldehyde (2 mL/well) at 37 ℃ for 10 min and further rinsed three times with PBS. For nucleus labeling, the nucleus were stained with DAPI solution (20 mg·mL− 1 in PBS, 1 mL/well) for 5 minutes, and the cells were then washed three times with PBS again, and the samples were examined with a Zeiss LSM880 with air scan instrument.
Flow cytometric detection of cellular uptake and kinetics. The cellular uptake kinetics was studied using flow cytometer. 1–5 × 105 density of breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A were detached using trypsin and resuspended in 2.00 mL cell culture serum-free media containing DCA-UiO-DTDP and DCA-UiO-DTDP-FA at the concentration 500 mg·mL− 1 at different periods up to 24 hours. After incubation at 37 ℃ cells suspension was diluted with 2 mL of PBS and centrifuged at 800 × g. The cell pellet was washed twice with PBS by centrifugation and resuspended in 500 µL of serum-free media. Fluorescence analysis was performed with BD LSRFORTESSA using Alexa Fluor 700-A channels and a 640 nm emission filter.