FeCl3·6H2O and CH3COOH (Analytically pure) were purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). Pluronic F127, EDCI and NHS (98% purity) were from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). Cholesterol oxidase was purchased from Shanghai Macleans Biochemical Technology Co., Ltd. (Shanghai, China). Adriamycin hydrochloride (purity >98%) was bought from Dalian Meilun Biological Co., Ltd. (Dalian, China). Chondroitin sulfate (purity >98%) and cystamine dihydrochloride (purity >98%) were from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China).
2.2 Cells and animals.
Human breast cancer cells MCF-7/ADR were purchased from Jiangsu KGI Biotechnology Co., Ltd. (Jiangsu, China). BALB/c female nude mice (16-20g, 5 weeks, SPF grade) were provided by Beijing Sibeifu Biotechnology Co., Ltd. (Beijing, China).
2.3 Synthesis of [email protected]@CS nanoparticles.
0.32 g Pluronic F127 was dissolved in 26.68 mL ultrapure water and 0.357g FeCl3·6H2O was dissolved in 3.32 mL water. The two solutions were mixed and stirred for 1.5 h, and then 0.6 mL of acetic acid was added. After 1.5 h, 120 mg H2N-BDC was added. After the reaction mixture was stirred at room temperature for 4 h, it was transferred to an autoclave (110°C) for crystallization for 24 h. The dark brown solid product was recovered and washed with ethanol several times (at least six times) to remove the surfactant and excess reactants. The obtained solid was put into a vacuum drying oven at 50℃ for 1 day to obtain NH2-MIL-88B powder.
Cholesterol oxidase (10.0 mg) was dissolved in 10.0 mL MES buffer solution (pH 5), and 120.0 mg 1- (3-dimethylaminopropyl) -3-ethylcarbodiimine hydrochloride (EDCI) and 150.0 mg N-hydroxy-succinimide (NHS) were successively added into the enzyme solution. After being activated at 37 ℃ for 15 min in a shaker with 100 rpm, MOF powder (30.0 mg) was added into 10 mL phosphate solution and ultrasound was carried out for 15 min to make it evenly dispersed. The two solutions were mixed and reacted overnight. After that, MOF-COD nanoparticles were prepared by 8, 000 g centrifugation for 5 min. Doxorubicin hydrochloride (5.0 mg) was dissolved in 5 mL phosphate buffer. MOF-COD was evenly dispersed in 5 mL phosphate buffer solution. The two were mixed and stirred in a magnetic mixer for 24 h. Then, the solution was centrifuged at 8, 000 g for 5 min to remove the free DOX to obtain [email protected] nanoparticles.
Chondroitin sulfate (40.0 mg) was fully dissolved in 10 mL MES buffer (pH 5), EDCI and NHS were activated for 15 min, and 10.0 mg cysteamine dihydrochloride was dissolved in 10 mL phosphate buffer. The two solutions were mixed evenly and reacted overnight. After 24 h of dialysis in the dark, the white product was vacuum dried. [email protected] nanoparticles were dispersed in a phosphate buffer solution, and the CS solution was added dropwise to the above solution under dark conditions, and stirred in a magnetic stirrer for 8 h. Then the [email protected]@CS nanosystem was obtained by ultrasound.
2.4 In vitro enzyme-like properties.
3, 3', 5, 5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2, 2'-azido-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was used as an indicator. The experiment was divided into three groups: MOF + H2O2 + indicator, MOF + indicator, H2O2 + indicator. 2.94 mL NaAc-HAC buffer (pH 4) was preheated at 37℃ for 10 min, then TMB was dissolved in anhydrous ethanol. 20 µL of TMB, OPD, ABTS (1 mM), H2O2 (200 µM) and MOF (40 µg·mL-1) solutions were added sequentially. After reacting for 30 min, the three sets of solutions were scanned at full wavelength using an ultraviolet-visible spectrophotometer.
Terephthalic acid (PTA) was used as an indicator, 2.94 mL NaAc-HAC buffer (pH 4) was preheated at 37℃ for 10 min, then 20 µL PTA solution (1 mM), H2O2 (200 µM) and MOF (40 µg·mL-1) were added successively, and the reaction time was 30 min. All three experiment groups were scanned at full wavelength with a fluorescence spectrophotometer.
2.5 Cell resistance determination.
MCF-7 and MCF-7/ADR cells were seeded into 96 well plates and DOX solutions with different concentration gradients were added. The concentration gradients of DOX were 0.2 to 25.6 µg·mL-1 in MCF-7 cells, and 1.25 to 100 µg·mL-1 in MCF-7/ADR cells, respectively. The cells were incubated for 24 and 48 h, the cell survival rate was detected by SRB assay. Resistance index (RI) value could be calculated by IC50 of resistant cells/IC50 of sensitive cells.
2.6 Cell uptake.
MCF-7/ADR cells were seeded in 2×105 cells/well in a six-well plate, and incubated for 24 h. After adding 2 mL of DOX, [email protected] and [email protected]@CS nanoparticles, respectively (concentration of DOX: 10 µg·mL-1), incubation was conducted (1, 2, 4, 8 h). The culture medium was discarded, the cells in PBS were washed for 3 times, 1 mL of 4% paraformaldehyde was added and placed in the incubator for 20 min. After washing the cells for 3 times with PBS, 1 mL DAPI solution (5 µg·mL-1) was added and stained for 15 min. After washing the cells with PBS for 3-4 times, the cells were observed under confocal laser.
2.7 Investigation of cholesterol content.
MCF-7/ADR cells were seeded in a six-well plate at 2×106 cells/well and incubated overnight. MOF-COD, [email protected]@CS (concentration of DOX: 10 µg·mL-1) were added and diluted with culture medium. M-β-CD was the positive control group (5 mM). After incubation for 24 h, the medicated media were discarded, the cells were washed 3 times with PBS, and the cholesterol concentration of each group was determined by the cholesterol kit.
2.8 Fluidity measurement of cell membrane.
The concentration of MCF-7/ADR cell suspension was adjusted to 2×105/well and inoculated in the six-well plate. The cells were cultured overnight in an incubator, and M-β-CD (positive control, 5 mM), MOF-COD and [email protected]@CS nanoparticles (DOX concentration was 10 µg·mL-1) were diluted in medium. After incubation for 2 h, the medium was discarded, the cells were washed with PBS for 3 times, and 1 mL trypsin was added to make cell suspension. The cell suspension was incubated with 1-pyridinedienoic acid (final concentration: 2 µM) for 5 min in darkness. In the fluorescence spectrophotometer, the excitation wavelength of 380 nm and the emission wavelength of 380-580 nm were used to scan the fluorescence spectra, and the fluidity of each group was compared according to the ratio of excimer 475 nm to pyrene monomer 397 nm.
2.9 Membrane lipid raft structure determination.
MCF-7/ADR cells were seeded into 6-well plates with 2 × 105 cells per well and cultured for 24 h. The medium was discarded, and M-β-CD (5 mM), MOF-COD and [email protected]@CS nanoparticles (DOX concentration was 10 µg·mL-1) were diluted with the medium, the control group was set, and incubated for 24 h. The culture medium was discarded, the cells were washed with PBS for 3 times, and 1mL AF488-CTB (5 µg·mL-1) was added to each well. The cells were placed on ice and incubated in the dark. The cells were washed three times with PBS and fixed with 1 mL 4% paraformaldehyde for 15 min. After the cells were washed 3 times with PBS, 1mL DAPI was added for staining (15 min, 37°C), washed 3-5 times with PBS, observed and photographed under a confocal laser microscope.
2.10 Western blotting analysis.
MCF-7/ADR cells were treated with [email protected], [email protected]@CS nanoparticles for 24 h, washed with PBS, added lysate to lyse cells on ice and extracted cell protein. The contents of Bcl-XL, COX-2 and P-gp were quantitatively determined by Western blot.
2.11 Biodistribution of [email protected]@CS nanoparticles in nude mice.
MCF-7/ADR cells were inoculated into the axilla of the right forelimb of 5-week-old nude mice in a quantity of 1×107 cells/100 µL, and the tumor growth and status were regularly observed. If the tumor volume reaches 60 mm3, a nude mouse model of tumor-bearing mice was successfully established. IR783 was encapsulated into the carrier as a fluorescent dye to prepare [email protected]@CS nanoparticles. Two groups of nude mice were injected through the tail vein, and the distributions of [email protected]@CS nanoparticles and free IR783 in the body were observed at different time points (1, 2, 4, 8, 12, 24 and 36 h) using a near-infrared imager. The anesthetic pentobarbital sodium solution (7 mg kg-1) was intraperitoneally injected and fixed on a live imaging device to take pictures and record the fluorescence distribution.
In addition, the fluorescence distributions of the isolated tissues and tumors were investigated. Nude mice were sacrificed after 12 h of injection, and the organs (spleen, liver, lung, kidney, heart) and tumors were collected. The fluorescence distributions in tissues and organs in vitro were photographed by in an imaging system.
2.12 Tumor inhibition study.
When the tumor volume reached 100 mm3, the nude mice were randomly divided into 7 groups with 6 mice in each group. The groups were as follows: (1) Saline, (2) free DOX, (3) MOF, (4) MOF-COD, (5) [email protected], (6) [email protected], (7) [email protected]@CS (DOX dose: 5 mg kg-1). The preparation of each group was injected via tail vein every other day, and the treatment ended 14 days later.
During the treatment, the normal feeding and growth of nude mice should be ensured. The nude mice were weighed and recorded before each administration. The tumor volume was calculated according to the formula: volume = (width)2 × length/2. After treatment, nude mice of each group were dissected, the heart, liver, spleen, lung, and kidney were soaked in 10% formalin and stained with hematoxylin and eosin. The tumor weight of nude mice was taken and the tumor inhibition rate was calculated according to the following formula: tumor inhibition rate (%) = (M0-M)/M, where M is the average tumor weight of different preparation groups. M0 represents the average tumor weight in the saline group.
2.13 In vitro safety.
After the treatment, blood was collected from the orbit of nude mice and placed in heparin sodium anticoagulant tube to detect and quantify the indexes of hemoglobin and creatinine. The heart, liver, spleen, lung and kidney of nude mice in each group were washed with normal saline, dried with filter paper, the weight of organs was weighed with electronic balance, and the organ index was calculated according to the following formula: organ index = organ weight (g)/nude mouse weight (g).
2.14 Statistical analysis
All experimental data are analyzed by GraphPad Prism 8. Statistical analysis adopts t test and One-way ANONA Analysis. *P <0.05, **P <0.01, ***P <0.001.